Virtual Library

Abstract:
Small cell lung cancer, which accounts for 10-15% of all lung cancers, is a biologically and clinically virulent malignancy that has a propensity to disseminate systemically and therefore is often initially diagnosed at an advanced incurable stage. Although typically associated with heavy tobacco use, there have been recent clinical observations of histologic evolution from adenocarcinoma to a SCLC phenotype, particularly in tumors harboring activating mutations in the epidermal growth factor receptor (EGFR) gene that had been treated with EGFR inhibitors. Due to its high proliferative rate, SCLC is known to be highly responsive – at least initially - to cytotoxic chemotherapy. Tumor response rates of 50-70% following platinum-based chemotherapy are usually expected. Intracranial metastases, a common feature of ES-SCLC, have been also shown to respond at a similar rate to cytotoxic therapy as that of metastases to other visceral organs. The standard frontline chemotherapy for ES-SCLC, unchanged for the past three decades, has been platinum (either cisplatin or carboplatin) plus etoposide. No other regimen has convincingly been demonstrated to be superior to platinum-etoposide in the frontline setting. Neither dose intensification approaches nor the incorporation of other cytotoxic agents into the platinum backbone have yielded any palpable or tangible survival benefits. In recent years, only prophylactic cranial irradiation and (in highly selected patients) consolidative thoracic irradiation have been shown to marginally improve survival outcomes. Furthermore, despite the high initial response rates to chemotherapy, drug resistance and subsequent tumor progression are universal events. Following failure of frontline platinum-based therapy, therapeutic options are limited and generally are of very modest clinical benefit. Current investigations into optimizing cytotoxic therapy include the development of novel cytotoxics (e.g., alisertib), sequential/combination strategies that involve novel “targeted” therapies and immunotherapeutics such as checkpoint inhibitors, or conjugating a cytotoxic payload onto a monoclonal antibody directed against an antigen expressed on SCLC, among others. A critical appraisal of the current status and future directions of cytotoxic therapy in ES-SCLC will be presented.

Abstract:
Limited stage Small cell lung cancer (SCLC) comprises 10-15% of all lung tumors and is associated with an aggressive clinical behavior. Two out of three patients presents with hematogenous metastases at diagnosis (extensive stage (ES)). For patients without hematogenous metastases (limited stage (LS)), chemoradiotherapy is the standard treatment. Although radiotherapy after chemotherapy has the theoretical benefit of treating smaller target volumes with less toxicity, concurrent chemoradiotherapy has shown to be superior. Moreover, earlier use of radiotherapy during chemotherapy leads to better results. The absolute benefit of early versus late radiotherapy was about 10% for patients who had received cisplatinum-based chemotherapy [1]. Turrisi et al. [2] demonstrated that twice daily radiotherapy starting with a first course of chemotherapy resulted in improved survival rates. Median survival was 23 months for patients who received twice-daily radiotherapy (45Gy/30fractions/3weeks) versus 19 months for once daily treated patients (45Gy/25fractions/5weeks). The corresponding 5 years survival rates were 26% and 16%. However, more Grade 3-4 oesophagitis was seen during twice-daily treatment (32% versus 16%). Only a minority of patients in the US and Europe receive twice daily radiotherapy. Recently the results of the CONVERT trial, in which once-daily radiotherapy (70Gy) was compared with twice daily radiotherapy (45 Gy) were presented [3]. Radiotherapy was initiated at the 2nd course of 4-6 cycles of cisplatin/etoposide. There was no statistically significant difference in overall survival between the two arms. Overall survival at 2 years was 56% for patients treated twice-daily versus 51% for patients treated once-daily (p= 0.15) [3]. There was also no significant difference in time to progression. There were no differences in a acute toxicity, except for Grade 3-4 neutropenia, which occurred more often in the twice-daily treatment arm (74% versus 65%). There were no differences in Grade 3-5 oesophagitis (19%) and pneumonitis (2%). The authors concluded that survival in both study arms was higher than reported previously and that radiation related toxicities were lower than expected, probably related to the use of modern radiotherapy techniques. The results of the study support the use of either twice daily or once daily as standard of care for patients with limited stage disease and in good performance score. In RTOG0538 study, which also compares 70 Gy once-daily and 45 Gy twice-daily radiotherapy, radiotherapy commences with the first or second course of chemotherapy. The results of this study are eagerly awaited [4]. Extensive stage In the EORTC study on prophylactic cranial irradiation (PCI) for ES-SCLC, it was noted that the vast majority of patients still had intrathoracic disease after completion of chemotherapy. After on the positive effects of PCI which not only reduced the risk of symptomatic brain metastases (40 versus 15%) but also improved overall survival (1 year: 27 versus 13% (P= 0,003)) [5], the next logical step was to investigate the use of thoracic radiotherapy in ES-SCLC as well. Evidence for a possible role of thoracic radiotherapy (TRT) in ES-SCLC also comes from the results of a trial published by Jeremic et al. in 1999 [6] in which patients with ES-SCLC and good prognosis with a complete response outside the thorax were randomized between TRT plus PCI during chemotherapy versus chemotherapy and PCI only. Overall survival was 17 months for the patients who received thoracic radiotherapy versus 11 months for those who did not. In the CREST trial, patients with ES-SCLC and any response after 4-6 cycles of platinum based chemotherapy were randomized between PCI plus TRT (30Gy/10 fractions) or PCI only. Overall survival at one year, the primary endpoint of the study, was not statistically significant between the groups (p=0.066) but with longer follow up the survival curves diverged and at 2 years, survival was 13% in patients who received TRT versus 3% in the controls (p=0,004). There was also significant difference in progression free survival. In an additional analysis of patients with and without residual intrathoracic disease, which was one of the stratification factors of the study, it was demonstrated that there was no significant benefit of TRT in patients with a CR in the thorax. However, in patients with residual intrathoracic disease after chemotherapy, TRT led to a significant improvement in overall survival [7]. In patients who received thoracic radiotherapy the risk of intrathoracic recurrence was reduced from 80% to 44%. In patients who received thoracic radiotherapy the most recurrences occurred outside the brain and the thorax and at a later stage. The next logical step after demonstration of a beneficial effect of PCI and TRT would be the use of higher doses for TRT and possibly also treatment of extrathoracic metastatic sites. This topic was addressed in the NRG-RTOG0937 study and was presented at ASTRO 2015 [8]. patients with ES-SCLC and a CR or PR after chemotherapy and 1-4 metastatic lesions were randomized between PCI or PCI plus TRT plus radiotherapy of metastatic sites. The study which accrued very slowly was closed early due to observed toxicities. The study did not show a survival difference between the two groups, but included only 86 patients over a five years period and had imbalance groups with worse prognostic factors in the experimental arm. There were many, partly unrelated, Grade 4-5 toxicities in the experimental arm. To define which patients are most likely to benefit from a more aggressive approach we have performed an additional analysis for patients from the top accruing centers from the CREST trial. An evaluation of 260 patients showed significantly better outcome in patients with 0 to 2 metastases versus and without liver metastases [10]. These patients are believed to be best candidates for future studies. References 1. Fried DB, Morris DE, Poole C, et al. Systematic review evaluating the timing of thoracic radiation therapy in combined modality therapy for limited-stage small-cell lung cancer. J Clin Oncol 2004. 22, 4837-45. 2. Turrisi AT 3rd, Kim K, Blum R, et al. Twice-daily compared with once-daily thoracic radiotherapy in limited small-cell lung cancer treated concurrently with cisplatin and etoposide. N Engl J Med. 1999 340, 265-71. 3. Faivre-Finn C, Snee M, Ashcroft L. CONVERT: An international randomised trial of concurrent chemo-radiotherapy (cCTRT) comparing twice-daily (BD) and once-daily (OD) radiotherapy schedules in patients with limited stage small cell lung cancer (LS-SCLC) and good performance status (PS). ASCO Meeting abstracts J Clin Oncol 2016, 8504. 4. Slotman BJ, Senan S; Radiotherapy in small-cell lung cancer: Lessons learned and future directions. Int J Radiat Oncol Biol Phys 2011, 79, 998-1003. 5. Slotman BJ, Faivre-Finn C, Kramer G. Prophylactic cranial irradiation in extensive small-cell lung cancer. N Engl J Med 2007, 357, 664-72. 6. Jeremic B, Shibamoto Y, Nikolic N, et al. Role of radiation therapy in the combined- modality treatment of patients with extensive disease small-cell lung cancer; A randomized study. J Clin Oncol 1999,17, 2092-9. 7. Slotman BJ, van Tinteren H, Praag JO, et al., Use of thoracic radiotherapy for extensive stage small-cell lung cancer: a phase 3 randomised controlled trial. Lancet 2015, 385, 239-44. 8. Slotman BJ, van Tinteren H. Which patients with extensive stage small-cell lung cancer should and should not receive thoracic radiotherapy? Transl Lung Cancer Res. 2015, 4, 292-4. 9. Gore EM, Hu C, Sun A, et al. NRG Oncology/RTOG 0937: Randomized phase II study comparing prophylactic cranial irradiation (PCI) alone to PCI and consolidative extra-cranial irradiation for extensive disease small cell lung cancer (ED-SCLC). Proc ASTRO, Int J Radiat Oncol Biol Phys 2016, 94, 5.

Abstract:
Background: A previous study has shown that prophylactic cranial irradiation (PCI) reduced the risk of brain metastases (BM) and prolonged the overall survival (OS) of patients (pts) with extended disease small cell lung cancer (ED-SCLC). However Japanese trial to reconfirm these results was stopped at first interim analysis (n=163 pts) because of futurity. According to this study protocol, final follow up was done. Materials and methods: From March 2009 pts with ED-SCLC who had any response to first-line chemotherapy (platinum agent plus irinotecan or etoposide) were randomized to either PCI (25Gy/10 fractions) or observation (Obs) alone. The patients were required to prove the absence of BM by MRI prior to enrollment. The primary endpoint was OS and a planned sample size of 330 was determined to detect the hazard ratio (HR) of 0.75 at a significance level of 0.05 and a power of 80%. Secondary endpoints included time to BM (evaluated every 3 months by imaging), progression-free survival (PFS), and adverse effects (AEs) and mini mental status examination (MMSE). Results: In Apr 2014, follow up analysis was conducted for the survival data of 224 all enrolled pts. One hundred fourth-five deaths were observed. The median OS was 11.6 and 14.1 months for PCI (n=112) and Obs (n=111), respectively (HR=1.28, 95%CI= 0.95-1.72; stratified log-rank test, P=0.107). PCI significantly reduced the risk of BM as compared to Obs (33.6% vs 59.7% at 12 months; Gray’s test, P<0.001), whereas PFS was comparable between the two arms (median, 2.3 vs. 2.4 months; HR=0.98, 95%CI=0.75-1.28). No significant difference in AEs greater than Grade 2 was observed between the two arms. At the MMSE there was no statistical difference between two arms, however in pts age 70 and older pts in PCI tended to be worse over time. Conclusion: PCI after response to chemotherapy could not show the OS impact in pts with ED-SCLC even in this follow up data.

Abstract:
The role of surgical treatment in the management of patients with small-cell lung cancer (SCLC) remains controversial. Although in the past, two randomized studies have failed to show any survival benefit by adding surgery to chemotherapy, different retrospective and prospective reports including the recently published studies using the database of Cancer Institute Surveillance Epidemiology and End Results (SEER), showed, that surgery offers a reasonable overall survival in a subset of patients with SCLC stage I and II disease. Two important recommendations have been introduced regarding the histology of SCLC as a high grade aggressive neuroendocrine tumor and the use of TNM classification in staging of SCLC and in clinical trials. Patient’s selection is important including extensive radiologic staging and biopsy of the mediastinal nodes. The use of PET scanning is likely to improve the accuracy of staging. Surgery can be performed with a curative intent in patients with SCLC and stage I or II disease or significant nodal response after chemotherapy. Weksler has used the SEER database and analyzed the outcomes of 3566 patients with SCLC stage I and II from 1988 to 2007. The surgical treatment was performed in 895 patients (25.1%); the median survival was 34 months in the surgical group versus 16 months in the nonsurgical group. In a similar report by Yu and colleagues, 21 the 5-year overall survival was 21.1%, but it was 50.3% for those patients who received a resection (45.7% after pneumonectomy and 33.7% after sublobar one). This analysis confirmed the acceptable survival rates in a subset of patients with stage I SCLC. By primary surgery or after induction chemotherapy complete tumor resection and systematic mediastinal lymphadenectomy should be undertaken. Adjuvant chemotherapy is recommended also for stage I patients; prophylactic cranial irradiation prolongs survival in those patients who achieve a complete or partial response to initial treatment. Until now, the standard systemic treatment of patients with LD-SCLC remains the combination of platinum and etoposide. The following groups of patients could potentially benefit from surgical resection: a. Patients with small lesion unexpectedly identified as SCLC at the time of thoracotomy. Complete resection and systematic lymph node dissection should be undertaken. Chemotherapy is recommended postoperatively and PCI should be considered. b. For stage I and II disease after chemotherapy and tumor response, surgery can improve local control and increase cure rates and long term survival. Complete resection and mediastinal lymph node resection should be performed. If possible, rather than pneumonectomy sleeve lobectomy should be preferred. c. In patients with mixed histology initial treatment should be chemotherapy to control the small cell component and after that surgery to control the non-small cell part of the tumor. d. For patients with initial failure to chemotherapy or patients with localized late relapse after treatment for pure small cell tumors salvage operations may be considered on individual basis. e. In patients with second primary small cell or non-small cell lung cancer who achieved cure from primary SCLC, surgery should be considered in the course of an multidisciplinary approach f. Patients with synchronous ispilateral or bilateral small and non small cell tumors could be potential candidates for surgery in a diagnostic or therapeutic intention g. In selected patients with IIIA N2 disease and complete histological regression of tumor tissue in the mediastinal lymph nodes after induction chemotherapy or chemoradiortherapy, surgery can improve local control and survival. Taking into account the TNM use in SCLC and the encouraging SEER results for patients submitted to surgery, a reconsideration of the role of surgery seems to be mandatory. Finally, to improve current management strategies for SCLC, surgeons should participate in the evaluation of SCLC patients together with oncologists and radiotherapists and common guidelines for indications and therapy concepts should be adopted. Interdisciplinary approaches should be employed in the context of controlled clinical trials. Fox W, Scadding JG. Medical Research Council comparative trial of surgery and radiotherapy for primary treatment of small-celled or oat-celled carcinoma of the bronchus. Ten-year follow-up. Lancet 1973;2(7820):63-65 Lad T, Piantadosi S, Thomas P, et al. A prospective randomized trial to determine the benefit of surgical resection of residual disease following response of small cell lung cancer to combination chemotherapy. Chest 1994;106:320-323 Waddell TK, Shepherd FA. Should aggressive surgery ever be part of the management of small cell lung cancer? Thorac Surg Clin 2004;14:271-281 Eberhardt W, Stamatis G. Stuschke M, et al. Prognostically orientated multimodality treatment including surgery for selected patients of small-cell lung cancer patients stage Ib to IIIB: long-term results ofc a phase II trial. Br J Cancer 1999;81:1206-12 Shepherd FA, Crowley J, Van Houte P, Postmus PE, Carney D, Chansky K, Shaokh Z, Goldstraw P. International Association for the Study of Lung Cancer International Staging Committee and Participating Institutions. The International Association for the Study of Lung Cancer lung cancer staging project: proposals regarding the clinical staging of small cell lung cancer in the forthcoming (seventh) edition of the tumor, node, metastasis classification for lung cancer. J Thorac Oncol 2007;2:1067-77 Valliéres E, Shepherd FA, Crowley J, Van Houte P, Postmus PE, Carney D, Chansky K, Shaokh Z, Goldstraw P. International Association for the Study of Lung Cancer International Staging Committee and Participating Institutions. The IASLC Lung Cancer Staging Project: proposals regarding the relevance of TNM in the pathological staging of small cell lung cancer in the forthcoming (seventh) edition of the TNM classification for lung cancer. J Thorac Oncol 2009;4:1049-59 Yu JB, Decker RH, Detterbeck FC, et al. Surveillance Epidemiology and End Results Evaluation on the Role of Surgery for Stage I Small Cell Lung Cancer. J Thorac Oncol 2010; 5:215–9. Weksler B, Nason KS, Shende M, et al. Surgical resection should be considered for stage I and II small cell carcinoma of the lung. Ann Thorac Surg 2012; 94:889–93. Stamatis G. Neuroendocrine tumors of the lung: the role of surgery in small cell lung cancer Thorac Surg Clin. 2014 Aug; 24(3):313-26.

Abstract:
Immunotherapy of Small Cell Lung Cancer Nevin Murray MD, British Columbia Cancer Agency, Vancouver, Canada The general principles of cytotoxic chemotherapy for advanced SCLC and NSCLC have many similarities and have advanced minimally over the past two decades.(1) The success of cancer genomics research in changing the care of patients with NSCLC with a driver mutation suitable for targeted treatment has been a powerful incentive to discover such molecular targets in SCLC. Although comparative genomic profiling shows some similarities between SCLC and NSCLC, for SCLC, the abnormalities identified to date are mainly tumor suppressor genes.(2) These loss-of-function alterations do not provide the clear opportunity for rapid clinical translation offered by an activating mutation in a known receptor tyrosine kinase. A considerable number of targeted agents have already been tried in SCLC clinical trials without notable success.(3) In contrast, there is a growing body of evidence for immunotherapy as a promising new treatment for both SCLC and NSCLC. Immunotherapy investigated for SCLC includes interferon, vaccines, antibody-drug conjugates and immune checkpoint inhibitors. Interferon and vaccines have been studied in phase II and III trials without sufficient activity to change practice. Although preliminary, the data emerging from trials of antibody-drug conjugates and immune checkpoint inhibitors has generated more excitement and are the focus for this abstract. Antibody-Drug Conjugates The components of an antibody-drug conjugate include an antibody directed at a defined antigen on cancer cells, a linker, and a cytotoxic agent. This package represents an effective mechanism of targeted drug delivery potentially resulting in decreased toxicity and an improved therapeutic index. Rovalpituzumab teserine targets the Notch pathway with a monoclonal antibody portion directed against the cell surface available Notch ligand delta-like protein 3 (DLL3), which is over-expressed in SCLC tumor-initiating cells but not in normal tissue. Rudin et al.(4) have reported a phase I study including 74 patients with previously treated SCLC. The confirmed response rate in 56 evaluable patients was 16%, but in 26 that showed high DLL3 expression, the response rate was 31%. Response rate was 13% in second-line and 25% in the third-line setting with some durable responses observed. A phase II trial for third-line treatment of patients with DLL3 expressing tumors has begun and if positive will be the first approved agent in this setting. Sacituzumab govitecan is another antibody-drug conjugate of a topoisomerase I inhibitor linked to an antibody to the Trop-2 epithelial antigen.(5) In a phase I/II clinical trial enrolling 33 evaluable SCLC patients with a median of 2.5 previous chemotherapies, the response rate was 24% and the median overall survival was 8.1 months. Dose limiting neutropenia was 34% and grade 3+ diarrhea was seen in 9%. Immune Checkpoint Inhibitors Since immune checkpoint blockade is more active in hyper-mutated tumors, SCLC should be a good candidate disease for this treatment because of a strong association with tobacco carcinogenesis and a high frequency of somatic mutations.(2) The most advanced trial evidence is available for a cytotoxic T-cell antibody (CTLA4) and data for two programmed death (PD-1) immune checkpoint inhibitors is emerging. After a randomized phase II trial of ipilimumab and phased chemotherapy showed a modest improvement in progression-free survival as first-line treatment of advanced SCLC,(6) a large phase III placebo controlled trial was performed in which 1,132 previously untreated patients were randomly assigned to receive either etoposide and platinum for four cycles alone or together with the CTLA-4 antibody ipilimumab.(7) The trial was negative with similar response rates and no difference in the primary end point of overall survival (hazard ratio 0.94; 95% CI 0.81-1.09). Immune checkpoint blockade with PD-1 or dual CTLA-4 and PD-1 inhibition may be a more effective strategy. In a large phase I/II trial including 180 previously treated SCLC patients, Antonia et al.(8) reported a response rate of 13% (7/80) with nivolumab 3 mg/kg and 31% (14/45) in a cohort of nivolumab 1 mg/kg plus ipilimumab 3 mg/kg. The activity of nivolumab alone or combined with ipilimumab was seen regardless of PD-L1 expression and not related to platinum sensitivity or line of therapy. The responses were durable with one-year overall survival of 27% for nivolumab alone and 48% for the combination arm. These results have led to two phase III studies among patients with SCLC evaluating nivolumab, nivolumab/ipilimumab versus placebo in the maintenance setting after first-line therapy and nivolumab versus placebo in the second-line setting. As part of a phase IB multi-cohort study (KEYNOTE-028), pembrolizumab was evaluated among patients with relapsed SCLC with PD-L1 positive tumors.(9) Of the 135 SCLC patients screened, 37 (27%) had PD-L1 positive tumors. The response rate was 29% in 24 evaluable patients. The median duration of response was 29 weeks and durable responses were observed. There is an ongoing phase II study of this agent as maintenance therapy after the completion of standard first-line therapy in extensive stage disease. A phase I trial is evaluating pembrolizumab with conconcurrent chemoradiation. Adverse events associated with checkpoint inhibitors is greater with CTLA-4 combined with the PD-1 antibody combination but were generally manageable. The proportion discontinuing therapy for toxicity was usually less than 10%. The literature contains anecdotes of autoimmune syndromes such as limbic encephalitis.(8) Immune para-neoplastic syndromes are expected in a small proportion of patients with SCLC and an increase in their occurrence with immunotherapy requires close monitoring. However, this concern is currently insufficient to impede further trials with these promising agents. Conclusion Over the past 20 years, almost all phase III trials of systemic therapy for SCLC have failed to improve outcome and advances have been restricted to improved application of radiotherapy. Like squamous carcinomas, the SCLC molecular battlefield is complex and bleak with little opportunity of even temporary respite by identification of mutually exclusive oncogenic drivers that can be treated for patient benefit. Ironically, this hyper-mutated genome and greater neo-antigen expression may enhance the probability of success with immunotherapy. One senses that the likelihood is high for approval of antibody-drug conjugates and immune checkpoint inhibitors for SCLC after the current roster of clinical trials are reported. References 1. Murray N, Lam S. Contrasting Management of Small Cell Lung Cancer and Non-Small Cell Lung Cancer: Emerging Data for Low-Dose Computed Tomography Screening. J Thorac Oncol. 2016 Feb;11(2):139-41. 2. Pietanza MC, Ladanyi M. Bringing the genomic landscape of small-cell lung cancer into focus. Nat Genet. 2012 Oct;44(10):1074-5. 3. Murray N, Noonan K. Can we expect progress of targeted therapy of small cell lung cancer? In: Dingemans A, Reck M, Westeel V, editors. Lung cancer. Sheffield: European Respiratory Society; 2015. p. 234. 4. Rudin CM, Pietanza MC, Bauer TM, Spigel DR, Ready N, Morgensztern D, et al. Safety and efficacy of single-agent rovalpituzumab tesirine (SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody-drug conjugate (ADC) in recurrent or refractory small cell lung cancer (SCLC). ASCO Meeting Abstracts. 2016 June 21;34(18_suppl):LBA8505. 5. Starodub A, Camidge DR, Scheff RJ, Thomas SS, Guarino MJ, Masters GA, et al. Trop-2 as a therapeutic target for the antibody-drug conjugate (ADC), sacituzumab govitecan (IMMU-132), in patients (pts) with previously treated metastatic small-cell lung cancer (mSCLC). ASCO Meeting Abstracts. 2016 May 31;34(15_suppl):8559. 6. Reck M, Bondarenko I, Luft A, Serwatowski P, Barlesi F, Chacko R, et al. Ipilimumab in combination with paclitaxel and carboplatin as first-line therapy in extensive-disease-small-cell lung cancer: results from a randomized, double-blind, multicenter phase 2 trial. Ann Oncol. 2013 Jan;24(1):75-83. 7. Reck M, Luft A, Szczesna A, Havel L, Kim SW, Akerley W, et al. Phase III Randomized Trial of Ipilimumab Plus Etoposide and Platinum Versus Placebo Plus Etoposide and Platinum in Extensive-Stage Small-Cell Lung Cancer. J Clin Oncol. 2016 Jul 25. 8. Antonia SJ, Lopez-Martin JA, Bendell J, Ott PA, Taylor M, Eder JP, et al. Nivolumab alone and nivolumab plus ipilimumab in recurrent small-cell lung cancer (CheckMate 032): a multicentre, open-label, phase 1/2 trial. Lancet Oncol. 2016 Jul;17(7):883-95. 9. Ott PA, Callahan MK, Odunsi K, Park AJ, Pan LS, Venhaus RR, et al. A phase I study to evaluate the safety and tolerability of MEDI4736, an anti- programmed cell death-ligand-1 (PD-L1) antibody, in combination with tremelimumab in patients with advanced solid tumors. ASCO Meeting Abstracts. 2015 May 18;33(15_suppl):TPS3099.

Abstract not provided

Abstract:
Thymic epithelial tumors Thymic epithelial tumors are the most common malignancy among mediastinal tumors according to Japanese thoracic surgery survey (1). Surgical resection is generally the treatment of choice for thymic epithelial tumors. Thymic epithelial tumors are classified into thymoma, thymic carcinoma (TC), and thymic neuroendocrine carcinoma (TNEC). Retrospective surgical database of Japanese Association for Research of the Thymus (JART) revealed that recurrence free 10-year survival after macroscopic complete resection was 88% in thymoma, 51% in TC, and 11% in TNEC. Thymomas are further classified mainly into 5 pathological subtypes, WHO type A, AB, B1, B2 and B3. Pathological subtype of thymoma has been shown to reflect the oncological behaviors, and post-operative recurrence rate increases in this order. JART database study revealed that nearly 3 quarters of thymoma surgical cases have Masaoka stage I or II disease. Pleural dissemination is often encountered either before or after resection in thymoma while hematogenous or lymphatic spread seldom occurs. On the other hand, TC is often associated with metastasis to distant organs as well as nodal involvement in the mediastinum and cervical region. Approximately 3 quarters of surgically treated TC have Masaoka Stage III or IV disease in surgical cases. While most thymomas are treated by surgical resection, a considerable portion of TC are judged unresectable at initial presentation. TNEC often has nodal involvement. Initial resection is indicated when clinical diagnosis is a thymic epithelial tumor with Masaoka stage I or II. The standard procedure is extended thymectomy through median sternotomy even for tumors with Masaoka stage I or II disease because of the possibility of post-thymectomy myasthenia gravis, intrathymic metastasis and multiple foci of tumor. JART database study, however, revealed that recurrence rate in thymoma with T1N0M0 by UICC was not significantly different between two procedures, thymothymomectomy (1.4%) and thymomectomy (2.8%) (p = 0.192) (2). Furtheremore, systematic dissection of mediastinal lymph nodes is not supposed essential in thymoma because incidence of nodal involvement is negligible. Advancement in video-assisted thoracic surgery (VATS) has prompted endoscopic operation also for thymoma, and currently, partial resection of the thymus by VATS seems accepted for less-invasive thymoma when myasthenia gravis is not associated, but careful observation by annual examination by CT scan is recommended after partial thymectomy. Highly invasive thymomas should be treated by preoperative induction chemotherapy to reduce the tumor size. Pathological diagnosis by biopsy is required before chemotherapy to differentiate between invasive thymoma and TC. Resection of the pericardium, lung, great vessels, and thoracic wall is sometimes required. JART database study revealed that invasion of the thoracic wall was the independent factor of recurrence after complete resection. (3) Even subtotal resection sometimes results in long-term survival. If complete resection is not achieved, radiotherapy is supposed to control the remaining tumor. Surgery for thymoma with pleural or intrapericardial dissemination can be indicated. JART database study revealed that the number of the disseminated lesions is a prognostic factor and that patients with less than 10 lesions had better survival. (4) Operative procedure varies from partial pleurectomy to extrapleural pneumonectomy with resection of the primary lesion. The recommended procedure depends on the spread of disseminations. Although intrapericardial implantation is commonly thought to be hard to resect, resection can be achieved in some cases because thymomas usually do not invade into the heart muscle severely. Preoperative chemotherapy is supposed to enable complete resection of intrapericardial implantations through reduction of the tumor volume. Most of the hematogenous metastases of thymoma occur in the lung probably because the neoplastic cells can directly enter the blood stream through thymic veins. Surgical treatment for thymomas with lung metastasis is feasible, but indication of surgery for thymoma with extrathoracic distant metastasis should be determined carefully. Recurrence often occurs on the pleural surface followed by the lung metastasis. Surgical resection of the recurrent lesions in the intrathoracic cavity is generally thought to contribute to survival. (5) Preoperative induction therapy is almost mandatory in highly invasive TC and poorly-differentiated NEC. Concurrent chemoradiotherapy is effective in reducing the tumor size. Resection and reconstruction of even the ascending aorta under cardiopulmonary bypass can be attempted. Systematic mediastinal and cervical lymph node dissection is recommended because of high incidence of nodal involvement. Malignant germ cell tumors (GCT) Malignant GCT is a highly aggressive neoplasm arising in young males. Chemotherapy is recommended without pathological diagnosis when serum tumor marker is extraordinarily elevated. In case of non-seminomatous GCT, complete resection of the tumor after normalization of tumor marker value by chemotherapy should be achieved, or otherwise, tumor recurrence is highly possible. Resection and reconstruction of the great vessels under cardiopulmonary bypass is often necessary. Liposarcoma Mediatinal liposarcoma is a rare neoplasms and sometimes appears as a huge tumor. This neoplasm is supposed to be resistant to chemotherapy, and complete surgical resection is required. Local recurrence occurs frequently because obtaining safe surgical margin is difficult. Radiotherapy could be a treatment of choice for recurrent tumors. Lymphoid malignancies Role of surgery is limited. Surgical biopsy is sometimes required when ML is suspected by imaging and high value of serum sIL-2 receptor. When tumor remains after chemotherapy, surgical resection is sometimes indicated. Low-grade malignancy including MALT and Castleman’s disease can be exceptionally treated by initial surgery. References Committee for Scientific Affairs, The Japanese Association for Thoracic Surgery. Thoracic and cardiovascular surgery in Japan during 2013: Annual report by The Japanese Association for Thoracic Surgery. Gen Thorac Cardiovasc Surg. 2015 ;63:670-701. Nakagawa K, et al. Is thymomectomy alone Appropriate for stage I (T1N0M0) thymoma? Results of a propensity-score analysis. Ann Thorac Surg. 2016;101:520-6. Yamada Y, et al. Surgical outcomes of patients with stage III thymoma in the Japanese nation-wide database. Ann Thorac Surg 2015;100:961–7. Okuda K, et al. Thymoma Patients With Pleural Dissemination: Nationwide Retrospective Study of 136 Cases in Japan. Ann Thorac Surg 2014;97:1743–9. Mizuno T, et al. Surgical management of recurrent thymic epithelial tumors. A retrospective analysis based on the Japanese nationwide database. J Thorac Oncol. 2015;10:199–205.

Abstract:
Radiation therapy (RT) plays an important role in the multimodality management of thymic malignancies. It can be employed in the neoadjuvant, adjuvant, definitive or palliative setting. Adjuvant RT is the most extensively studied setting for RT in thymic malignancies. After complete resection there is likely no role for adjuvant RT for patients with stage I thymomas, a possible role for patients with stage II thymomas, and likely a survival benefit in patients with stage III and IV thymomas. Several recent large database and population-based studies have detected a survival benefit for advanced thymomas, while the results for stage II thymomas have been mixed. For thymic carcinomas the impact of adjuvant RT appears more significant. Several large database and population-based studies have consistently reported a survival benefit with adjuvant RT for thymic carcinoma across various disease stages. For incompletely resected thymic tumors there is a stronger rationale for adjuvant RT based on emerging data and general oncologic principles. Neoadjuvant RT has been mostly explored in thymic carcinoma and demonstrated high response and operability rates. Definitive RT is an excellent treatment option for patients with unresectable thymic malignancies. While most thymic tumors are resectable, a subset of patients is technically or medically inoperable, due to invasion of critical structures or comorbidities. In general, thymic malignancies are radiosensitive, allowing for long-term local control rates. Palliative RT should be considered even in the recurrent or metastatic setting. Image-guided hypofractioned ablative RT may be used for oligometastatic disease as an alternative to surgical resection and has been shown to be a highly effective treatment modality with >90% long-term local control rates and minimal morbidity. Conventional palliative RT is an important modality to improve quality of life by alleviating pain, treating SVC syndrome, airway compression and other symptoms. Modern radiation therapy techniques such as 3D conformal radiation therapy or intensity-modulated radiation therapy should be used to minimize morbidity from treatment. Proton therapy may have advantages in certain clinical scenarios and is currently under investigation.

Abstract:
Thymic malignancies represent a heterogeneous group of rare thoracic cancers. The histopathological classification distinguishes thymomas from thymic carcinomas. Thymomas are further subdivided into different types (so-called A, AB, B1, B2, and B3) based upon the atypia of tumor cells, the relative proportion of the associated non-tumoral lymphocytic component, and resemblance to the normal thymic architecture. Thymic carcinomas are similar to their extra-thymic counterpart, the most frequent subtype being squamous cell carcinoma. The management of thymic epithelial tumours is a paradigm of multidisciplinary collaboration. The treatment strategy is primarily based on the resectability of the tumour. If complete resection is deemed not to be achievable upfront based on imaging studies, what is the case in Masaoka-Koga stage III/IVA tumors (classified as stage IIIA/IIIB/IVA in the 2015 IASLC-ITMIG TNM proposed system), after a biopsy is performed, primary/induction chemotherapy is administered, part of curative-intent sequential strategy integrating subsequent surgery or radiotherapy. Cases not eligible for local treatment receive definitive chemotherapy. Primary/induction chemotherapy is then standardin non-resectable advanced thymic epithelial tumors. Cisplatin-based combination regimens should be administered; combinations of cisplatin, adriamycin, and cyclophosphamide, and cisplatin and etoposide are the most usually used. Primary chemoradiotherapy with platin and etoposide is an option, especially for thymic carcinomas. Usually 2-4 cycles are administered before imaging is performed to reassess resectability of the tumor. Surgery should be offered to patients for whom complete resection is thought to be ultimately achievable; extended resection may be required. Hyperthermic intrapleural chemotherapy, as well as extra-pleural pneumonectomy may be discussed in case of stage IVA tumor. Postoperative radiotherapy is usually delivered. When the patient is not deemed to be a surgical candidate - either because R0 resection is not thought to be achievable, or because of poor performance status or co-existent medical condition, definitive radiotherapy is recommended part of a sequential chemoradiotherapy strategy. Combination with chemotherapy (including cisplatin, etoposide chemotherapy and a total dose of radiation of 60 Gy) may be considered as well. Chemotherapy should be offered as the single modality treatment in advanced, non-resectable, non-irradiable or metastatic (stage IVB) thymic epithelial tumor to improve tumor-related symptoms the aim is to improve tumor-related symptoms through obtention of tumor response, while prolonged survival is uncertain. Cisplatin-based combination regimen should be administered. No randomized studies have been conducted, and it is unclear which regimens are best; multi-agent combination regimens and anthracycline-based regimens appear to have improved response rates compared to others, especially the etoposide, ifosfamide and cisplatin combination. Combinations of cisplatin, adriamycin, and cyclophosphamide is preferred. Combination of carboplatin and paclitaxel is an option for thymic carcinoma. Surgery or radiotherapy is possible in rare and selected cases with unknown survival benefit. Recurrences of thymic epithelial tumors should be managed according to the same strategy as newly diagnosed tumors. Complete resection of recurrent lesions represents a major predictor of favorable outcome, and surgery is then recommended in case of resectable lesion. In non-resectable recurrences, several consecutive lines of chemotherapy may be administered when the patient presents with tumor progression. The re-administration of a previously effective regimen has to be considered, especially in case of previous response, late occurring recurrence, and for anthracyclins, a patient in a good medical condition and not having received cumulative doses precluding the safe delivery of at least 3 additional cycles. Preferred regimens for second-line treatment include carboplatin plus paclitaxel, and platin plus etoposide; capecitabine plus gemcitabine is an option. These regimens were evaluated in dedicated phase II trials. Options for subsequent lines include pemetrexed, oral etoposide. In patients with octreoscan-positive thymoma, not eligible to receive additional chemotherapy, octreotide alone or with prednisone may represent a valuable option. The use of targeted agents may be done in an off-label setting in advanced thymic malignancies. While KIT is overexpressed in 80% of thymic carcinomas, KIT gene mutations are found only in 9% of cases, consisting of mutations observed in other malignancies (V560del, L576P) or mutations unique to thymic carcinomas (H697Y, D820E). Responses and possibly prolonged survival was reported with the use KIT inhibitors - imatinib, sunitinib, or sorafenib - , mostly in single-case observations. Non-pretreated reported KIT mutants are not uniformly sensitive to imatinib, based on the clinical and/or the preclinical evidence in thymic carcinoma and/or other KIT-mutant malignancies. KIT sequencing (exons 9-17) is an option for refractory thymic carcinomas in the setting of possible access to off-label use of such inhibitors. KIT inhibitors also potently inhibiting other kinases, including Vascular Endothelial Growth Factor Receptors and Platelet-Derived Growth Factor Receptors activated in thymic malignancies. A phase II trial recently demonstrated the efficacy of sunitinib in terms of response and disease control rate in thymic epithelial tumors, including thymic carcinomas (ORR 26%; DCR: 91%) and, to a lesser extent, thymomas (ORR:6%; DCR:81%). Sunitinib may then represent an option as second-line treatment for thymic carcinomas, independantly from KIT status. There is no clinical data reporting on antitumor efficacy of other antiangiogenic drugs. mTOR is emerging as a potential target in thymic epithelial tumors, following tumor responses observed in phase I trials. Everolimus (10 mg daily) was evaluated in thymic epithelial tumors in a recently reported phase II trial reporting on a 22% response rate, as well as a 93% disease control rate. Everolimus may then represent an option for refractory tumors. Several trials assessing the efficacy of PD-1 checkpoint inhibitors are currently ongoing. A phase II study of pembrolizumab, a fully humanized IgG4 Ab that targets the PD-1 receptor, was recently reported; the study has accrued 30 patients. Four serious autoimmune disorders developed. Out of 30 patients evaluable for response so far the response rate is 24%. The off-label use of checkpoint inhibitors is currently not recommended. Overall, a dramatic improvement in our knowledge of the management of thymic tumors has occurred in the last few years. This improvement has primarily resulted from an increased interest in these rare tumors at some dedicated centers, and from the development of international efforts that succeed in putting together large-volume, top-quality centers all over the world, for databases, translational research, and clinical trials.

Abstract:
Use of Inspiratory Muscle Training (IMT) in Managing Dyspnoea in Lung Cancer Patients Lung cancer, the most common cause of cancer-related death in men and women, is responsible for 1.3 million deaths worldwide annually. Lung cancer (LC) patients face many symptoms throughout the cancer trajectory and these often co-occur. Among the most prevalent (ranging from 21–90%), burdening and debilating symptoms that patients face is dyspnoea. Although this symptom tends to become more frequent and persistent towards end-of-life, evidence show that even in early stage NSCLC patients who are most likely to be cured may also be faced with debilitating dyspnoea that results in poor QOL during survivorship (Sarna et al 2008). Dyspnoea in the setting of lung cancer has a complex aetiology that includes: direct involvement of lung tissue by cancer, indirect respiratory complications related to the cancer, treatment related complications (fibrosis secondary to chemotherapy or radiation), respiratory co-morbidities (pulmonary embolism) and other co-morbid conditions (i.e. COPD)(Kvale et al 2007). Due to its complex aetiology, dyspnoea also has a multimodal management strategy including both pharmacological and non-pharmacological interventions (Kloke & Cherny 2015). Pharmacological management options include bronchodilators, corticosteroids, anxiolytics, antidepressants, opiods and oxygen (Ferrell et al 2011; Kloke & Cherny 2015). The non-pharmacological interventions include patient’s education on measures for ameliorating the symptom, such as opening windows, using small ventilators, adequate positioning, respiratory training and relaxation techniques (Galbraith et al 2010; Molassiotis et al 2015). A non-pharmacological intervention that has been widely used for the management of respiratory symptoms in asthma and COPD but not lung cancer is Inspiratory Muscle Training (IMT). This method can reduce dyspnoea mainly through two distinct ways. Firstly, by strengthening the inspiratory muscles therefore lessening the effort during a given task (dyspnoea) and secondly by providing a means for controlled breathing. An improved inspiratory muscle strength and endurance can lead to the better management of dyspnoea and therefore facilitate the increase in the level of activity and improving the quality of life for patients. Despite the wealth of data on the effect of IMT on inspiratory muscle strength and endurance, exercise capacity, dyspnoea and quality of life for adults with COPD, there are no available data for lung cancer patients. Whilst the literature shows that COPD and lung cancer are correlated (Sekine et al 2012), this is not sufficient to advocate towards the use of IMT in lung cancer patients experiencing dyspnoea. Despite the scarcity of evidence, the fact that both COPD and lung cancer patients face many common problems such as increased resistance to airflow, air trapping and hyperventilation of the lung , increases the likelihood that IMT can also have a positive effect on lung cancer patients’ dyspnoea. Aim This randomised study aimed to assess the feasibility and effectiveness of inspiratory muscle training in patients with lung cancer regarding their dyspnoea, psychological distress and quality of life. Design The trial is a two-arm, non-blinded, randomised controlled, proof-of-principle study. Patients were randomly assigned to IMT or a control group. The IMT group received standard care and additionally included the intervention with home follow-up every month for 3 months. Patients were recruited from the outpatients’ clinics of two large cancer centres in the UK and one in Cyprus. Participants were eligible if they a) were adults with histological diagnosis of primary LC or mesothelioma; b) had refractory dyspnoea not responding to current treatment for the past 2 weeks (breathlessness daily for 3 months at rest or on minimal exertion where contributing causes have been treated maximally); c) expected prognosis of >3 months as judged by the clinicians and d) had oxygen saturation above 85 % at rest. Patients were excluded if: they suffered from unstable COPD with frequent or acute exacerbations, had rapidly worsening dyspnoea requiring urgent medical intervention, they received palliative radiotherapy to the chest received within 4 weeks or chemotherapy within 2 weeks, they were experiencing intractable cough, and those having unstable angina or clinically significant pleural effusion needing drainage. Intervention A pressure threshold device was used to deliver IMT, which controls a constant inspiratory pressure training load that is maintained unless the patient drastically alters his/her breathing pattern. Based on the literature on COPD patients, the intervention protocol included five sessions weekly for 12 weeks for 30 mins/day, divided over two sessions (the actual intervention had duration of 3-5 min for each session and progressively the time was increased to 30mins/day). The IMT resistance level was set to a low level (baseline) that allowed the patient to inhale comfortably. Progressively, the resistance level was increased according to the patient’s performance. Outcome measures Outcome measures were completed at baseline and monthly for 3 months, and included: physiological parameters (FEV1,FVC); perceived severity of breathlessness using six 10-point NRS; modified Borg Scale; quality of life using the short form Chronic Respiratory Disease Questionnaire; Hospital Anxiety and Depression Scale, and safety. Results The final sample included 46 patients (M=37, F=9) at a mean age of 69.5 years old and a mean of 16 months post-diagnosis mainly with NSCLC and advanced disease (70%). Statistical and clinically important differences were seen with regard to distress from breathlessness (p=0.03), ability to cope with breathlessness (p=0.01), satisfaction with breathlessness management (p=0.001), fatigue (p=0.005), emotional function (p=0.011), breathlessness mastery (p=0.015) and depression (p=0.028). Changes were more evident in the 3-month assessment where the effect of the intervention came to its peak. Discussion This trial showed that the IMT is feasible and potentially effective in patients with lung cancer. A larger trial will provide more concrete conclusions on the usefulness of IMT in the management of dyspnoea in lung cancer patients with relatively stable disease, relatively higher performance status and life expectancy of >3 months. However, those patients with acute or severe dyspnoea should be treated according to established protocols rather than IMT. References Sarna L, Cooley ME, Brown JK, Chernecky C, Elashoff D, Kotlerman J (2008). Symptom Severity 1 to 4 Months After Thoracotomy for Lung Cancer. Am J Crit Care; 17(5):455–467. Simon ST, Müller-Busch C, Bausewein C (2011). Symptom management of pain and breathlessness. Internist (Berl); 52: 28, 30–35. Galbraith S, Fagan P, Perkins P et al (2010). Does the use of a handheld fan improve chronic dyspnea? A randomized, controlled, crossover trial. J Pain Symptom Manage; 39: 831–838 Kloke M & N. Cherny N (2015). Treatment of dyspnoea in advanced cancer patients: ESMO Clinical Practice Guidelines. Annals of Oncology 26 (Supplement 5): v169–v173, 2015 doi:10.1093/annonc/mdv306. Kvale PA, Selecky PA, Prakash UB (2007). Palliative care in lung cancer: ACCP evidence-based clinical practice guidelines (2nd edition) Chest; 132(3 Suppl):368S–403S Ferrell B, Koczywas M, Grannis F, Harrington A. (2011) Palliative Care in Lung Cancer. Surg Clin North Am. 91(2): 403–ix. Molassiotis A, Charalambous A, Taylor P, Stamataki Z, Summers, Y (2015). The effect of resistance inspiratory muscle training in the management of breathlessness in patients with thoracic malignancies: a feasibility randomised trial. Support Care Cancer, 23:1637–1645.

Abstract not provided

Abstract:
In this presentation some of the existing beliefs and ideas concerning survivorship in the context of a diagnosis of lung cancer will be briefly considered. The changing face of cancer in the 21[st] century will be highlighted using data from the United Kingdom (UK) as an exemplar to show survival patterns. Consideration will then be given to the commonalities of the concerns of people with lung cancer and people with other cancers common in the developed world where survivorship services currently prioritised. Finally a challenge will be offered to the lung caner community for the coming year.

Background:
As one form of tumor invasion, cancer cells can invade the extracellular matrix (ECM) through tracks that have been physically remodeled by cancer-associated fibroblasts (CAFs). However, CAFs are a heterogeneous population with diverse matrix-remodeling capacities. The purpose of this study is to investigate how CAFs with various matrix-remodeling capacities influence cancer cell invasion.

Methods:
We established single-cell derived clones from 3 primary cultures of CAFs from lung adenocarcinoma patients (Case 1, 5 clones; Case 2, 5 clones; Case 3, 7 clones). Using a co-culture model, we evaluated the correlations between the number of invaded cancer cells and the remodeling areas generated by CAF clones in each case.

Results:
When A549 lung adenocarcinoma cells and CAF clones were co-cultured, both the numbers of invaded cancer cells and the remodeling areas generated by the CAF clones varied greatly. The number of invaded cancer cells was moderately and strongly correlated with the remodeling areas generated by each CAF clone originating from Cases 1 and 2 (R[2] value = 0.53 and 0.68, respectively), suggesting that the remodeling areas in the ECM may determine the number of invaded cancer cells. In contrast, the number of invaded cancer cells was not correlated with the remodeling areas generated by CAF clones originating from Case 3, suggesting that factors other than the remodeling areas might determine the number of invading cancer cells.

Conclusion:
These findings showed two types of fibroblast-dependent cancer cell invasion that are dependent on and independent of the remodeling areas generated by CAFs.

Background:
Spread through air spaces (STAS) is identified as a newly invasive pattern in lung adenocarcinoma. It contributes to the significantly increased recurrence rate for patients with small adenocarcinoma. But the presence of STAS and its clinical impact has remained uncertain in squamous cell carcinoma (SQCC.) The purpose of this study is to analyze whether STAS happens in surgically resected pathological Stage I (pStage I) lung SQCC.

Methods:
We retrospectively reviewed 141 pStage I patients of SQCC (Female/Male, 13/128; Smoker/Never smoker, 135/6; pStage IA/IB, 93/48). Tumor STAS was defined as tumor cells within the air spaces in the lung parenchyma beyond the edge of the main tumor. Statistical analyses were conducted to investigate the relationship between its presence and the clinicopathological background factors, including the clinical outcome.

Results:
STAS was identified in 23 of 141 patients (16.3%) with limited (7.1%) and extensive (9.2%) feature, respectively. Both disease-free survival (DFS) and overall survival (OS) were significantly worse in the patients with STAS in comparison with the patients without STAS (5-year DFS, 35.1% vs. 65.6%, p<0.01; 5-year OS, 41.7% vs. 71.2%, p<0.01, respectively). In multivariate analyses adjusting for sex, year, smoking history and pStage, the presence of STAS was found to be an independent predictive factor of both DFS (HR=3.154, 95%CI: 1.592-6.249; p=0.001) and OS (HR=3.07, 95%CI: 1.595-5.911; p=0.0008). The 141 tumors were divided into patients who underwent limited resection and those who underwent standard resection in order to examine whether the surgical procedure affected the DFS and OS of patients with and without STAS. In the standard resection group, both 5-year DFS and 5-year OS were worse in the patients with STAS in comparison with the patients without STAS (44.1% vs.68.3%, p=0.03; 53.8% vs. 72.3%, p=0.048, respectively). In the limited resection group, both 5-year DFS and 5-year OS were worse in the patients with STAS in comparison with the patients without STAS (0% vs.57.5%, p=0.001; 0% vs. 66.4%, p=0.001, respectively).

Conclusion:
STAS happened in lung SQCC and was found to be an independent predictive factor of both DFS and OS. Both 5-year DFS and 5-year OS were worse in the patients with STAS regardless of surgical procedure.

Background:
Thyroid transcription factor-1 (TTF-1) is useful as an immunohistochemical marker of the high specificity of the primary lung adenocarcinoma. Whereas immunohistochemistry (IHC) is emerging as a powerful tool in undetermined diagnosis case, it was difficult to achieve intraoperative quickly IHC due to time constraints. We previously developed and reported a device that enables us to complete IHC analyses within 20 minutes. The purpose of rapid-IHC analysis during surgery is more accurate tissue diagnosis. Because of lung adenocarcinoma discrimination, we were examined the usefulness of TTF-1 rapid IHC.

Methods:
Eighty-two patients with lung tumor were enrolled in the study between May 2011 and September 2013. Resected samples from each patient were sectioned immunohistochemically labeled with anti-TTF-1 antibody using the rapid-IHC procedures.

Results:
Using the rapid-IHC procedure, IHC analyses were completed within 20 min, and TTF-1-positive tumors were detected by the pathologist within about 30 min. Intraoperative diagnosis using the rapid-IHC procedure was resulting in 97.6% accuracy (80/82). Of the 47 primary lung adenocarcinoma eligible for analysis, 31 (66%) were positive for TTF-1 by intraoperative diagnosis using the rapid-IHC procedure, and the positive predictive value (PPV) of primary lung adenocarcinoma was 100% (31/31).

Conclusion:
Our experience demonstrates that intraoperative IHC investigation of TTF-1 using the rapid-IHC in proper consequences could be a very useful tool for lung tumors. Further investigation in multicenter studies　will be needed to confirm the utility of this method. With refinement, this technology may prove to be an important supplement to standard pathology for routine practice.

Background:
to describe an exceptional case of malignant transformation of benign metastasizing leiomyomas (BML) of the lung.

Methods:
a 62-year-old woman presented for radiological finding of multiple bilateral pulmonary nodules. Nine years before, she underwent hysterectomy for uterine leiomyoma. After pulmonary biopsy, a diagnosis of BML was made. Subsequently, a surgical resection of the right pulmonary nodules was performed; histopathological examinations confirmed the diagnosis of BML (Ki67=1%; strong expression of estrogen and progesterone receptor as depicted in Fig.1C-E). The patient was put on anastrazole therapy but an increase of the left pulmonary nodules was observed. Therefore, a surgical resection of the left nodules was carried out; final pathological examination confirmed BML. Three years later, a chest CT-scan showed bilateral pulmonary relapses; tamoxifen was started but it was ineffective. Then, a surgical resection of the right pulmonary nodules was performed (Fig.1A-B). The patient was discharged uneventfully on postoperative day 5.

Results:
macroscopically, the nodules had whitish and yellowish colour, smooth margins, with tense-elastic consistency. Microscopically, an intersecting bundles of spindle cells with moderate nuclear atypia organized in a fascicular pattern were clearly evident (Fig.1F). The mitotic activity was more pronounced than previous histological samples (up to 10 mitoses/10HPF). Immunohistochemical studies showed positivity for smooth-muscle actin, desmin, and negativity for HMB-45, CD34, and TTF-1; Ki-67=20% (Fig.1G). Estrogen and progesterone receptor were weakly positive (Fig.1H). Based on the current criteria, a diagnosis of low-grade leiomyosarcoma was made. The patient denied the contralateral surgical resection. Eighteen months later the chest CT-scan revealed bilateral pulmonary nodules; she is currently under megesterol acetate treatment. Figure 1



Conclusion:
BML of the lung is a rare pathological condition with a usually indolent clinical course. Although it’s exceptional, an evolution towards a low grade leiomyosarcoma should be considered in the natural history of the disease.

Background:
This article reviews the pathologic classification of lung cancer based on the WHO classification of lung tumors and the IASLC/ATS/ERS classification in our population, we don’t use 2015-WHO classification because at ongoing of this review wasn’t release yet.

Methods:
Between January 2004 and December 2013, all patients diagnosed with a pathology of SCLC and NSCLC at National Institute of Oncology at Paraguay were analyzed retrospectively. Demographic information were recorded. SPSS 20 was used to analyze.

Results:
We studied 478 subjects. The histological subtypes found were SCC(Squamous cell carcinoma) : 48.7% and prevalence grade III: 87.3%(P=0.000), Adenocarcinoma: 21%.Prevalence grade II: 50%(P=0.000), Small cell carcinoma 14%, Large cell carcinoma 75 Prevalence grade III: 100%(P=0.000), Unclassified 6% Prevalence grade III:100%(P=0.000), Carcinoid tumors 1.3%, Adenosquamous Carcinoma 0.8%, Carcinomas of salivary gland type 0,8% prevalence grade II: 100%(P=0.000),carcinomas with pleomorphic sarcomatoid or sarcomatous elements 0.5%. Gender prevalence at women was SCC: 37.5% and Squamous cell carcinoma at men: 50.3%(P=0.000), Group ages prevalence 18 to 44 years old: Squamous cell carcinoma 39.1%, 45 to 65: Squamous cell carcinoma 46.3%.>65 years: Squamous cell carcinoma 55.5% (P=0.018).At relation with prevalence at rural area living place SCC: 51.9% and Urban SCC: 43.7% both (P>0.05).First motive of consultation was dyspnea Carcinomas of salivary gland type: 66,7%, carcinomas with pleomorphic sarcomatoid or sarcomatous elements: 50.7%, Small cell carcinoma: 44,4%, Unclassified: 43.5% Large cell carcinoma: 32.0%, Adenocarcinoma: 30%, Adenosquamous Carcinoma:30.0%. Coug to Carcinoid tumors 40%, SCC: 32.4% (P>0.05).Clinical severity correlation to pathologic classification predominance at stage IV was to carcinomas with pleomorphic sarcomatoid or sarcomatous elements: 100%, Carcinomas of salivary gland type: 100%, Adenocarcinoma: 64,9%,Large Cell Carcinoma 60%, Unclassified: 50%. SCC: 43.7%. At stage IIIB Adenosquamous Carcinoma: 100%, Unclassified: 50%. At IB Carcinoid tumors 50%. Small cell carcinoma advance stage:69.2%(P=0.000)

Conclusion:
We found statistical significance relation between severity grade and histopathology of lung cancer also with gender prevalence. We hadn't found statistical significance relation with first motive of consultation or living place. We had statistical relation at our population with age groups as bibliography references mentions that is rare to find lung cancer in young patients, but we found prevalence at 18 to 44 years old of Squamous cell carcinoma at 39.1% in this age group. Also we found statistical relation at histopathology type ad clinical severity stage, in our population IV was predominant. This is the first review of relation between histopathology with clinical and demographic variables in our Institution.

Background:
Tumor spread through air space (STAS) is proposed as a new factor of lung cancer invasion, according to the new World Health Organization (WHO) classification. The aim of this study is to elucidate the prognostic impact and conduct a histopathological evaluation of STAS in primary lung cancer patients who underwent limited resection.

Methods:
We retrospectively collected 508 samples from p-Stage I primary lung cancer patients who underwent limited resection between 2004 and 2013. Hematoxylin and eosin stained tumor slides were reviewed to evaluate pathological features, including the presence or absence of STAS, and the morphological pattern in cases with STAS. We defined the pattern of STAS as single cell (SG), small cluster (SM), or large cluster (LG). Clinicopathological characteristics and patient outcome data were collected from medical records. SPSS statistical software (IBM Corporation, Somers, NY, USA) was used for statistical analysis.

Results:
Histological diagnoses were 440 adenocarcinomas (Ad) (including 107 Adenocarcinoma in situ and 144 Minimally invasive adenocarcinoma), 44 squamous cell carcinomas (Sq), and 24 other types of cancer. Seventy-six cases (15.0%: 60 Ad, 9 Sq, and 7 other types of cancer) were positive for STAS. The morphological STAS patterns were 12 SG, 45 SM, and 19 LG, respectively. There was no significant relationship between recurrence rate and morphological STAS pattern. The STAS-positive group was associated with the presence of micropapillary and/or solid components in Ad, and with lymphovascular and pleural invasion, compared to the STAS-negative group (p < 0.01). The median follow-up was 51 months. Eight local recurrences (1.6%), 16 locoregional (lung parenchyma, hilum, mediastinum) recurrences (3.1%), and 10 distant recurrences (2.0%) were recorded. In multivariate analysis, the risk of local (hazard ratio [HR]: 12.75; p < 0.01) and locoregional (HR: 4.12; p = 0.01) recurrence was significantly higher in the STAS-positive group than in the STAS-negative group. However, in a multivariate Cox model the presence of STAS was not associated with distant recurrence (p = 0.58).

Conclusion:
Our results indicated that the presence of STAS is a significant risk factor for local and locoregional recurrence, but not distant recurrence, in p-Stage I lung cancer following limited resection.

Background:
Glomus Tumor is almost a benign tumor that often develops in the nail bed of the extremities, and accounts for 1.6% of all soft tissue tumors. However, they developed in the other organs, such as gastrointestinal, bone, adrenal gland, central nerve system, uterus and vagina. We here reported a rare case of a pulmonary glomus tumor with some literature.

Methods:
Case A 36–year–old female was admitted to our hospital with an abnormal shadow in the left lung field. She has no major disease, and nonsmoker. The laboratory data and physical examination are normal. A chest computed tomography scan showed a nodal lesion of 1.0 cm in diameter in the left lower lobe. Thoracoscopic partial resection was performed.

Results:
The tumor was well-circumscribed lesion consists of solid sheets of tumor cells interrupted by capillaries and vessels of varying sizes in the pulmonary tissue. In the pathological findings, the tumor cells have relatively uniform, rounded to oval nuclei, indistinct nucleoli, and ill-defined cytoplasmic borders. In the immunohistochemical examination, the tumor cells were positive for αSMA, HHF-35, desmin and vimentin, but negative for EMA, cytokeratinAE1/AE3, TTF-1, surfactant apoproteinA, CD34 and Factor VIII. Some tumor cells were positive for Ki-67. Those features were consistent with pulmonary glomus tumor.

Conclusion:
Glomus tumor of the lung is rare tumor and only a few cases have been reported in the literature. The behavior of this neoplasm is uncertain, so the methods of diagnosis and treatment, includes of surgical approach will demand to do careful observation and further examination.

Background:
We performed the analysis to clarify the differences of lung invasive mucinous adenocarcinoma (IMA) based on computed tomography (CT) finding, in clinicopathological and molecular features, as well as prognosis.

Methods:
On the basis of CT findings, we divided lung IMA into three subtypes; solid, bubbling, and pneumonic type. We investigated differences in clinicopathological characteristics, prognosis, and expressions of well-identified biomarkers, including Cox-2, ERCC1, RRM1, Class III beta-tubulin, TS, SPARC, PD-L1and EGFR mutation, among the three subtypes.

Results:
A total of 29 patients of resected lung IMA were analyzed. Compared with the solid or bubbling type, the pneumonic type had a higher proportion of a symptom, large tumor size, a higher pathological stage, and a significantly worse prognosis. Immunohistochemical findings tended to be high expression in RRM1, Class III beta tubulin, Cox-2 in tumor and SPARC in stroma, but not ERCC1, TS, and PDL-1 in tumor. All biomarkers in tumor were not prognostic biomarker, however, only SPARC expression in stroma was worse prognostic biomarker.

Conclusion:
Clinical and pathological features in conjunction with molecular data indicate that IMA should be divided into different subgroups. In our results, pneumonic type had significantly worse prognosis, and could guess to considered to be effective to cisplatin, pemetrexed, or nab-paclitaxel, and/or Cox-2 inhibitor. Further studies should be performed to confirm our conclusion and explore its molecular functions.

Background:
An extraneous tissue contaminant on a slide is called a floater. Spread Through Air Spaces (STAS) is in the WHO classification considered as a form of invasion in lung adenocarcinoma. The artifactual spread of tissue fragments during lung specimen sectioning was recently described and termed Spreading Through A Knife Surface (STAKS).1 The purpose of this study was to prospectively examine lung resection specimens for the presence and frequency of STAKS.

Methods:
A prospective, multi-institutional study of NSCLC lobectomy and pneumonectomy resection specimen was performed from January 1 –July 1, 2016. Prosection, sampling and scoring of displaced fragments was undertaken in a systematic manner. The first cut was made with a clean long knife, the second cut was made in a parallel plane to the first cut, without cleaning the knife. Four tissue blocks were sampled: Block 1: first cut, upper part; Block 2: first cut, lower part; Block 3: second cut, upper part; Block 4: second cut, lower part. From these formalin fixed and paraffin embedded tissue blocks a superficial complete H&E stained slide was examined for the presence of displaced tissue fragments at 10x or 20x. A displaced fragment was scored as STAKS if the tissue fragment was at least 0.5 mm from the tumor or if it was on the pleural surface in the plane of the second cut. Benign and malignant STAKS were separately noted.

Results:
A total of 41 resection specimen were included in this study. The mean number of malignant STAKS for blocks 1-4 was 0.36, 1.44, 1.86 and 1.95, respectively and for benign STAKS the mean number was 0.11, 0.11, 0.13 and 0.25, respectively. Almost all STAKS were intra-alveolar. Comparison of malignant STAKS in block 1 (before the tumor was reached) with blocks 2-4 (containing tumor) was significant with p-values (p=0.003 Friedman’s test and post-hoc comparisons p=0.031, p=0.002 and p=0.005, respectively). For benign STAKS no difference was identified (p=0.23). The chance of malignant STAKS seemed to be higher when tumor was cut fresh than when cut after formalin fixation.

Conclusion:
The morphologic definition of STAKS is not different from STAS. This prospective study confirms the presence of benign and malignant STAKS. The presence of malignant STAKS is an artifact and increases with each and every knife cut during tissue sectioning. 1) Thunnissen et al. ArchPatholLabMed2016,140(212-220)

Background:
Giant cell carcinoma (GCC) of the lung is a subtype of sarcomatoid carcinoma (2015 WHO classification) traditionally associated with a highly aggressive clinical behavior. The histology consists in giant cells without differentiated carcinomatous elements. The aim of this study is to analyze the clinical, pathological and molecular features of seven GCC cases diagnosed in our hospital.

Methods:
Twenty-nine sarcomatoid carcinoma diagnosed in our hospital during the years 2009-2016 were reviewed and 7 cases with GCC histology were selected for the study. Immunohistochemical staining with antibodies targeting TTF1, napsinA, p40, and β-HCG were performed. ALK and MET status were assessed by FISH. EGFR mutations were performed using real-time PCR.

Results:
The patients were 4 men and 3 women with a mean age of 61 years (range 45-79). At the moment of diagnosis three patients were current smokers and 4 former smokers. Five cases were peripheral tumors, six in the left lung, and one in the right lung. Complete resection was achieved in all patients. Tumor staging showed 3 cases pT1; 3 with pT2 stage and one case pT3. Histopathologically, all were pure GCC and immunohistochemical stains revealed that the giant cells were negative for β-HCG in all cases except one who could not be analyzed. Two cases showed null phenotype (TTF1 and p40-negative), two cases were TTF1-negative and p40-positive, two cases co-expressed TTF1 and p40 and one case was TTF1-positive and p40-negative. NapsinA was positive in two cases. Molecular analysis was done in 6 cases and no EGFR mutation was detected. FISH results for c-MET probe showed a MET/CEN7 ratio <2 in all cases, polysomy with ≥5 MET signals without amplification was found in 5 cases. No ALK rearrangement was observed in the series. Five cases showed ALK copy number gain (3 to 5 fusion signals) and one case had two fusion signals. With a median follow-up of 38 months (5-130 months), two patients died due to brain metastases (both with vascular-lymphatic invasion and nodal metastases at the time of surgery), and five patients are alive at the moment of analysis.

Conclusion:
Pure GCC is a very rare lung cancer subtype and there are few series reported. Lymphovascular invasion and lymph node involvement at diagnosis can predict a worse outcome in this subtype. GCC in our series do not have a specific immunohistochemical profile. Neither EGFR nor ALK were potential molecular targets, nevertheless c-MET status could be an interesting biomarker in GCC tumors.

Background:
The clinico-pathological profile of lung cancer has changed considerably over the time in India. The histologic type also has changed from a predominant squamous histology to adenocarcinoma.We performed a prospective evaluation of primary lung cancers (PLCs) on the basis of clinical characteristics, histopathology and immunohistochemistry (IHC).

Methods:
The clinicohistopathological features and IHC characteristics of all PLCs(as per 2015 WHO classification) were described prospectively over a period of two years (2014-2016).The antibodies that were used were TTF-1, napsin A, P40, CK7, CK20, vimentin, synaptophysin, chromogranin, BCL-2, CD34, LCA, CD99, AFP & βHCG.

Results:
We have studied 140 PLCs (78.6% male and 21.4%females) with age ranging from 25 to 85 years.Most common symptoms were cough and chest pain observed in 65% of our cases. In 14.2% cases the patients primarily presented with metastasis. The most common site was brain (40 %), cervical nodes (45%) and skin (15%) in our record. There were 84cases of NSCLC and 5 cases of small cell carcinoma. 95.2% of NSCC could be further classified with the help of TTF-1,napsin A, CK7 & P40 into adenocarcinoma (71.4%), 23.8% cases of squamous cell carcinoma, and 4.8% of cases could not be subtyped further. TTF-1 was seen in all the cases of adenocarcinoma, where as p40 waas seen in all the cases of squamous cell carcinoma.Only in 4.8% of cases neither the morphology nor the staining pattern supported adenocarcinoma or squamous and hence was diagnosed as NSCC-NOS. In addition to these usual types,other unusual morphological variants seen were3 cases each of carcinoid, large cell neuroendocrine carcinoma, & synovial sarcoma. There were also few rarer ones such as lymphoepithelioma like carcinoma, choriocarcinoma and yolk sac tumor. In 39 cases the biopsy was inadequate and hence could not be opined.

Conclusion:
Accurate categorization of primary lung tumors, has both therapeutic and prognostic significance. TTF1 and P40 are very sensitive markers for differentiating adenocarcinoma and squamous cell carcinoma of the lung. Addition of napsinA contributes to a higher sensitivity for adenocarcinomas

Background:
p40, one of the two isomers of p63, is nowadays widely used for diagnosis of squamous cell carcinoma, especially in subtyping non-small cell carcinoma on lung biopsies.

Methods:
We describe a case in which lung tumour was misdiagnosed as squamous cell carcinoma due to p40 immunopositivity.

Results:
A 36-year-old lady presented with cough and left sided chest pain for 2 months duration. Chest imaging revealed a lesion in left lower lobe of lung and biopsy was suggestive of squamous cell carcinoma (Fig1). However, past history revealed amputation of great toe for non-healing discharging ulcer which on histopathology was diagnosed as choriocarcinoma. She developed similar nodules and ulcers over the left arm, followed by a gradually worsening dry cough and progressive shortness of breath. On imaging, she was found to have a septated left sided pleural effusion. A positron emission tomography–computed tomography (PET-CT) revealed a large hypermetabolic soft tissue mass in left lower lobe with bilateral lung metastases and multiple liver deposits. On reviewing obstetric history, she also had a history of hysterectomy five years ago, details of which were not available. Post-amputation β-hCG levels were high and she had been treated with multimodality chemotherapy for choriocarcinoma. She had good response to chemotherapy initially, however became resistant later on. Review of lung biopsy in the light of the past history along with extensive literature review led to the final diagnosis of metastatic trophoblastic tumour to lung. Figure 1- The lung biopsy shows an invasive tumour (A) (H&E, 10x); composed of polygonal cells with moderate amount of eosinophilic cytoplasm, round to oval nuclei and inconspicuous nucleoli (B) (H&E, 20x). Hyaline eosinophilic material is seen amid tumour cells with mitotic activity (C) (H&E, 20x). These tumour cells show strong nuclear immunopositivity for p40 in approximately half of the tumour cells (D) (IHC, 20x).

Conclusion:
Hence, awareness that p40 immunopositivity can be seen in trophoblastic tumours is essential to avoid misdiagnosis, especially in sites like lung where squamous cell carcinoma is common.

Background:
Melanotic schwannomas (MS) are tumors associated with the Carney complex of hyperpigmentation, myxomas, and endocrine overactivity. They most frequently arise from spinal nerve roots and present a diagnostic challenge due to their lack of characteristic pathologic features. We present the case of an otherwise healthy 35-year-old man who presented with nocturnal dyspnea and ptosis. Imaging identified a large 8.1 x 9.2 x 8.4 cm mass in the right apical posterior mediastinum. Core biopsy was consistent with melanoma, although no primary site could be identified. The patient underwent complete R0 resection of an encapsulated posterior thoracic inlet mass adherent to the sympathetic chain and apical parietal pleura. Surgical pathology showed nests of large pleomorphic epithelioid cells with prominent nucleoli and abundant intracytoplasmic pigment consistent with the initial diagnosis of melanoma. The actual diagnosis of melanontic schwannoma was made only when the tumor was sent for molecular testing and a rare mutation was identified.

Methods:
Oncogene sequencing (UCSF-Syapse) was performed on surgically resected formalin-fixed and paraffin embedded tumor. Single nucleotide variations, copy number changes, and rearrangements were detected using a hybridization-based enrichment assay of approximately 500 oncogenes commonly implicated in the development of neoplasia. Of the genes assayed, entire coding regions were analyzed in 429 genes with additional analysis of selected introns in 42 genes.

Results:
Based on standard hematoxyalin and eosin (H&E) stains as well as S-100 and Melan-A positivity on immunohistochemistry (IHC) stains, the specimen was originally diagnosed as melanoma. The initial diagnosis was also supported by a Ki-67 proliferative index of 15%. Molecular testing uncovered a rare PRKAR1A mutation inconsistent with melanoma and consistent with melanotic schwannoma. No other mutations were identified. PRKAR1A mutations are known to occur in up to 70% of Carney complex patients but have never been known to occur in melanoma.

Conclusion:
Standard techniques of H&E and IHC staining with their potential to misdiagnose two similar tumor histologies are outdated in the context of 21st century technology. Modern precision medicine and molecular diagnostics enable the clear distinction of histologically similar tumors. The speed and low cost of sequencing technology has advanced to recommend its frequent use in cases such as this where a diagnosis is not entirely clear.

Background:
The current lung cancer classification from the IASLC/ATS/ERS integrates lung invasive adenocarcinoma subtypes accounting for the clinical, radiological, molecular and prognostic differences with its implications within the clinical practice. We analyzed the differences in genetic expression of the adenocarcinoma subtypes according to the new WHO 2015 classification.

Methods:
A cohort of 29 NSCLC patients treated at the Instituto Nacional de Cancerología of Mexico from 2008 to 2011. All patients had an available biopsy sample and were classified in four different subtypes of adenocarcinoma (2015 WHO classification). All the tissue samples were analyzed by microarrays to characterize the different expressed genes. IPA Software was used to identify biological processes, functions and biomarkers.

Results:
Lepidic predominant adenocarcinoma subtype was the only pattern that showed a marked gene expression difference against all predominant histologic patterns, revealing genes with significant (p < 0.01) expression. For all the histological predominant pattern subtype comparisons the top functional networks were related to eight different biological categories as follows: DNA replication; Recombination and Repair; Cell Cycle; Cell Death and Survival; RNA Post-Transcriptional Modification; Cancer; Organismal Injury and Abnormalities; Cellular Development. Moreover, we observed 13 genes with specific differential expression in the Lepidic predominant adenocarcinoma subtype.

Conclusion:
Lepidic predominant histological pattern subtype has a differential gene expression profile when compared against all predominant histological patterns subtypes. Moreover, we found a gene expression signature of 13 genes that has a unique behavior in the Lepidic histologic pattern subtype that could be used as a specific gene expression signature, biomarker or therapeutic target.

Background:
In the 2015 WHO classification, histologic factors that are associated with invasion in primary lung adenocarcinoma (AdCa) include the presence of non-lepidic histologic subtypes (invasive subtypes) and the presence of cancer-associated myofibroblasts (CAFs). The prognostic significance of CAFs in combination with each invasive subtype has not been well assessed. We conducted this study to clarify the prognostic impact of CAFs in the absence of architectural remodeling.

Methods:
We retrospectively collected data and re-evaluated samples from 1052 patients with pathological stage 0 or IA pulmonary AdCa who underwent complete resection at our hospital between 2007 and 2012. HE and elastica van Gieson stains were used for histological evaluation. We defined two invasive subtypes: those with (INV-1) and without (INV-2) architectural remodeling of lung parenchyma. The postoperative recurrence of tumor was analyzed in each group.

Results:
Our reviewed diagnoses were 172 Stage 0 and 880 Stage IA AdCa. Of the 880 stage IA cases, 706 (80.2%) and 174 (19.8%) were categorized as INV-1 and INV-2, respectively. CAFs were observed in all cases in the INV-2 group, but were not always present in the INV-1 group. In the INV-2 group, the median diameter of the invasive component was 6 mm (range: 1-16), the median postoperative follow-up period was 60 months (range: 2-105), and none of the cases developed recurrence. In the INV-1 group, the median postoperative follow-up period was 55 months (range: 1-104) and the estimated 5-year recurrence-free probability by the Kaplan-Meier method was 93.0%. All cases with postoperative recurrence were categorized in the INV-1 group.

Conclusion:
The INV-2 group AdCa had a low risk of recurrence. These findings suggest that certain subtypes of invasive AdCa, which are classifiable based on the architectural remodeling pattern and the presence of CAF, can be considered to have a good prognosis.

Background:
Lung adenocarcinoma is heterogeneous, characterized by various histological subtypes. Determination of the predominant histological subtype (lepidic, papillary, acinar or solid-predominant) has been shown to correlate with genetic abnormalities and clinicopathological features. Although subtyping using small biopsy samples is important for tailored approaches to clinical management, limited data exist on the concordance of predominant subtype between resected specimens and biopsy specimens.

Methods:
We compared the diagnosed predominant subtypes in resected specimens and matched biopsy specimens in a series of 327 lung adenocarcinomas. Histological subtyping of preoperative material was made by review of archived hematoxylin and eosin stain slides that had originally been prepared for diagnosis before surgery. The histological subtype of surgically resected tumors was obtained from the pathological case records for each surgical resection specimen. The accuracy of preoperative diagnosis by biopsy and the factors that influence concordance with resected specimen analysis were examined.

Results:
In 211 of the 326 patients (66.0%), the predominant adenocarcinoma subtype diagnosed from biopsy matched the findings of resection analysis. Concordance rate was highest in papillary pattern (82%), followed by lepidic pattern (75%), solid pattern (66%), and acinar pattern (39%). Overall, the concordance rate in biopsy samples with larger tumor areas (≥0.7 mm[2]) was significantly higher than in those with smaller tumor area (<0.7 mm[2]; 71% vs 58%, respectively; p = 0.02). Other factors in biopsy samples, such as number of biopsies, or the small biopsy type, did not have significant influence on the concordance between preoperative and postoperative diagnosis. In the biopsy samples with smaller tumor areas, the concordance rate was 77% in lepidic subtype, 71% in papillary subtype, 60% in solid subtype, and 40% in acinar subtype. Concordance rate in the biopsy samples with larger tumor area was higher in papillary and solid subtypes (88% and 76%, respectively), but remained low in acinar subtype (37%).

Conclusion:
These results indicate that accuracy of adenocarcinoma subtyping based on small biopsy samples is influenced by tumor area. Our study also suggests that subtyping of acinar histology using biopsy specimen is particularly error-prone.

Background:
Primary lung enteric adenocarcinoma is a rare histologic type sharing morphologic features with colorectal adenocarcinoma. Few reports are described in literature, and no specific indications are available to address treatment.

Methods:
We retrospectively collected primary lung adenocarcinomas defined as enteric according to the 2011 International Association for the Study of Lung Cancer classification and analyzed clinical, immunohistochemical and molecular data. Immunohistochemistry (IHC) for CDX2, CK20, CK7 and TTF1 were performed and EGFR, RAS and ALK status was determined as standard procedures.

Results:
The series included 18 patients diagnosed and treated at our Institution between 2012 and 2015. Gastrointestinal primitive lesions were excluded using [18]FDG-CT-PET and endoscopic examination. Median age was 60.5 years, patients were predominantly males (M:F 12:6). More than a half of patients (56%) were never or former smokers. IHC characterization identified 14 cases expressing at least one intestinal differentiation marker (CDX2 and/or CK20), while TTF1 was expressed in five cases. At time of diagnosis, 15 cases (83%) were stage IV, while 3 patients were stage II and underwent systemic progression within one year from radical surgery. Most frequent metastatic sites were bone (44%), adrenal gland (32%) and pleura (28%). Exon 18-19-20-21 EGFR mutations were assessed in 15 patients, resulting in 3 (20%) rare mutations (exon 19 I745insKIPVAI; exon 18 G719A; exon 20 S768R) and no common sensitizing EGFR mutations. No RAS or ALK alterations were found. For metastatic disease, 15 patients were able to receive first-line treatment: 12 patients received platinum-based doublet (with the addition of bevacizumab in two cases), one capecitabine (n: 1), two patients received EGFR inhibitors. Eight patients were able to receive second-line systemic treatment and one patient was treated with fluoruracil, oxaliplatin and bevacixumab. Three patients obtained radiological response following chemotherapy and two of them received fluoropyrimidine. Median overall survival from metastatic diagnosis was 10 (95%CI: 8-NA) months and median progression-free survival was 6 (95% CI: 2-NA) months, but great heterogeneity in outcome was noticed and three EGFR, RAS wild-type patients live more than 30 months from diagnosis of metastatic disease. The presence of rare EGFR mutations was associated with no smoking history and worse outcome; best radiological response to EGFR inhibitors was progression.

Conclusion:
Primary lung enteric adenocarcinoma has heterogeneous clinical behavior and is mainly refractory to standard chemotherapy. It presents specific epidemiological features and deeper genetic characterization is ongoing to define different subgroups and try to improve therapeutic approach.

Background:
The 2015 WHO classification for lung adenocarcinoma (ACA) provides criteria for diagnosis of adenocarcinoma in-situ (AIS), minimally invasive adenocarcinoma (MIA), and invasive adenocarcinoma (INV). Differentiating these entities can be difficult, and as understanding of prognostic significance increases, inconsistent classification is problematic.

Methods:
Sixty cases of lung ACAs (<2cm) were reviewed by an international panel of 6 lung pathologists. One slide reflecting overall morphology of each case was digitally scanned to an internet browser-based viewer. In round one, the panel independently reviewed each case to assess predominant pattern, invasive component size, and final diagnosis (AIS, MIA or INV). After a consensus conference among participants, a second round of independent review of the cases was performed. Additionally, a discussion on interpretation of elastic stain for evaluation of invasion will precede a third round of review with assessment of a concomitant elastic stain for each case. Statistical analysis was performed for each round.

Results:
In round one, the overall kappa value for AIS versus MIA and INV was 0.34 (fair agreement), and that for AIS and MIA versus INV was 0.44 (moderate agreement). The raters had 100% agreement on final diagnosis in 10 cases (AIS, n=2; MIA, n=2; INV, n=6). In 28 cases with poor agreement on final diagnosis and invasive measurement, inconsistent measurement of multifocal invasion led to wide variance in 5 cases, and subjectivity in pattern recognition led to variance in 23 cases. Misinterpretation of the WHO criteria for MIA resulted in 18 instances of misclassification across all raters. A case with a predominant mucinous lepidic pattern had a range of diagnoses (AIS, n=1; MIA, n=1; INV, n=4). In round two, the overall kappa value for AIS versus MIA and INV is 0.40 (fair agreement), and that for AIS and MIA versus INV is 0.36 (moderate agreement). The raters had 100% agreement on final diagnosis in 12 cases (AIS, n=3; MIA n=4; INV, n=5). Misinterpretation of the WHO criteria for MIA was seen in 6 instances. The intraobserver kappa coefficient ranged widely from 0.259 to 0.859.

Conclusion:
Interobserver agreement on diagnosis of small lung ACAs between raters was fair to moderate, with minimal improvement after a consensus conference. Inconsistent measurement of multifocal invasion, subjectivity in pattern recognition, misinterpretation of the WHO criteria, and subjective interpretation of mucinous ACA have contributed to interobserver discordance. A third round of evaluation is currently ongoing to assess for improvement and the utility of elastic stains.

Background:
Cancer-associated fibroblasts (CAFs) are known to influence tumor development, progression and metastasis. Their characteristics and prognostic role in non-small cell lung cancer (NSCLC) patients have been recognized. However, the functional heterogeneity of CAFs between patients and its genetic basis is less understood.

Methods:
Two pathologists scored for desmoplasia on Hematoxylin-Eosin stained sections of resected lung tumors from two patient cohorts: one consisting of 171 NSCLC patients (128 adenocarcinoma, 43 squamous carcinoma) and the second of 24 primary cultures of CAFs. Percent area of desmoplasia among total tumor stroma was used to define high desmoplasia (HD) versus low desmoplasia (LD). The desmoplasia and survival analysis were assessed for 171 NSCLC patients. Gene expression data on RNA extracted from CAFs in contracted gels following 24 hours incubation was obtained using Illumina Human HT-12v4 Bead Chips array and was preprocessed and normalized using RMA and values were log2 transformed. Significant genes whose expression is strongly correlated (Spearman correlation coefficient and p value) with percent of desmoplasia were identified in both cohorts. The gene set enrichment analysis (GSEA) was applied to test for the enrichment of CAF cohort significant genes in 171 NSCLC cohort. Additionally, CAF significant genes were subjected to pathway enrichment analysis using Pathway Data Integration Portal ver. 2.5 (http://ophid.utoronto.ca/pahtDIP).

Results:
The prognosis of adenocarcinoma patients with HD had poorer outcome in comparison to the patients with LD (disease free survival at 3 years 34% vs. 75% p=0.00045 and relapse rate 41% vs. 14%, p=0.0051). In the CAF cohort, the number of genes that are significantly associated with desmoplasia for enrichment are 356. Using GSEA, these genes were enriched in 171 NSCLC cohort (with a p value of 0.045). Protein-protein interaction (PPI) partners of these 356 genes were acquired using Integrated Interaction Database – IID (version 2016-03, http://dcv.uhnres.utoronto.ca/iid/). Obtained genes were then ranked according to their degree, i.e., number of PPIs. Top 44 (top 1%) of the genes were then selected to pathway enrichment analysis using pathDIP version 2.5. 245 pathways that significantly enriched by these 44 genes (FDR < 0.01) were obtained. Many of these pathways are known to be involved in lung cancer.

Conclusion:
We demonstrated that the prognosis of lung adenocarcinoma patients with HD had poorer outcome in comparison to the patients with LD. Furthermore, PPI analysis of CAF genes associated with HD reveals enrichment of many cancer-related pathways, suggesting their high relevance to lung cancer.

Background:
The Danish Lung Cancer Registry has since 2003 reported all cases of lung cancer in Denmark including the pathology. We present the trends over time in the distribution of subgroups of pathology.

Methods:
All Danish lung cancer patients are ascertained based on coded information in the National Patient Register. Supplementary information for each patient is obtained from the clinical units as well as from the National Pathology Register (NPR). Based on SNOMED coding the patients is categorized in 12 subgroups of lung cancer.

Results:
Table 1. Distribution of pathology subgroups, n = 56,554: Figure 1 Figure 1. Trends in lung cancer pathology (%) Figure 2 The increased number of lung cancer falls mainly in the adenocarcinoma group. Moreover, there is a significant relative increase of adenocarcinomas corresponding with a decrease of patients with NOS and NSCC. The occurrences of the other categories, including small cell carcinoma and squamous carcinoma, have remained largely unchanged.





Conclusion:
The trend of adenocarcinoma as the predominate type of lung cancer is in accordance with the global evolution. The high frequency is partly due to the need for specific subtyping and the agreement of diagnostic criterias which has resulted in a shift from NOS and NSCC categories to adenocarcinoma.

Background:
Multiple tumor nodules (MTNs) are being encountered, with increasing frequency with the 8[th] TNM staging system recommending classification as separate primary lung cancers (SPLC) or intrapulmonary metastases (IM). Pathological staging requires assessment of morphological features, with criteria of Martini and Melamed supplanted by comprehensive histologic assessment of tumour type, predominant pattern, other histologic patterns and cytologic features. With publication of the 2015 WHO classification of lung tumours, we assessed the reproducibility of comprehensive histologic assessment and also sought to identify the most useful histological features.

Methods:
We conducted an online survey in which pathologists reviewed a sequential cohort of resected multifocal tumours to determine whether they were SPLC, IM, or a combination. Specific histological features for each nodule were entered into the database by the observing pathologist (tumour type, predominant adenocarcinoma pattern, and histological features including presence of lepidic growth, intra-alveolar cell clusters, cell size, mitotic rate, nuclear pleomorphism, nucleolar size and pleomorphism, nuclear inclusions, necrosis pattern, vascular invasion, mucin content, keratinization, clear cell change, cytoplasmic granules¸ lymphocytosis, macrophage response, acute inflammation and emperipolesis). Results were statistically analyzed for concordance with submitting diagnosis (gold standard) and among pathologists. Consistency of each feature was correlated with final determination of SPLC vs. IM status (p staging) by chi square analysis and Fisher exact test.

Results:
Seventeen pathologists evaluated 126 tumors from 48 patients. Kappa score on overall assessment of primary v. metastatic status was 0.60. There was good agreement as measured by Cohen’s Kappa (0.64, p<0.0001) between WHO histological patterns in individual cases with SPLC or IM status but proportions for histology and SPT or IM status were not identical (McNemar's test, p<0.0001) and additional histological features were assessed. There was marked variation in p values among the specific histological features. The strongest correlations (<0.05) between p staging status and histological features were with nuclear pleomorphism, cell size, acinus formation, nucleolar size, mitotic rate, nuclear inclusions, intra-alveolar clusters and necrosis pattern. Correlation between lymphocytosis, mucin content, lepidic growth, vascular invasion, macrophage response, clear cell change, acute inflammation keratinization and emperipolesis did not reach a p value of 0.05.

Conclusion:
Comprehensive histologic assessment shows good reproducibility between practicing lung pathologists. In addition to main tumour type and predominant patterns, nuclear pleomorphism, cell size, acinus formation, nucleolar size, and mitotic rate appear to be useful in distinguishing between SPLC and IM.

Background:
Approximately 15% of patient with non-small cell lung cancer (NSCLC) is present with stage III (N2) disease. The patient prognosis after complete resection for pathological N2 NSCLC remains a significant concern. Currently, the new World Health Organization classification of lung cancers was revised and newly prescribed to describe the presence of each histologic subtype in adenocarcinoma (ADC) and the Spread Through Air Spaces (STAS). The purpose of this study is to examine the relationship between histologic subtype and patient outcome, especially for metastatic lymph node, and clinicopathologic features of STAS in stage III (N2) lung ADC according to new WHO classification retrospectively.

Methods:
All available tumor slides from patients with pathological N2, surgically resected lung ADC (1998-2013) were reviewed. Each tumor was evaluated by comprehensive histologic subtyping according to new WHO classification, and the percentage of each histologic component was recorded in 5% increments. We reviewed the histologic subtype in the N2 lymph nodes and relationship between main tumor and N2 lymph nodes. Recurrence-free probability (RFP) and overall survival (OS) were estimated using the Kaplan-Meier method.

Results:
78 patients met inclusion criteria (55% men; median age: 68yrs; 76 stageIIIA/ 2 stageIIIB, 77 lobectomy). The 5-year RFP and OS in N2 lung ADC were 27.8%, 66.8%, respectively. The histologic subtypes such as acinar, micropapillary and solid components in the main tumor were significantly seen in the N2 lymph nodes (P < 0.001, P < 0.05, P < 0.05, respectively). STAS was identified in 48 patients (61.5%) and significantly associated with recurrence (5-year RFP: 18.4% vs. 43.8%, P < 0.05). STAS was significantly associated with presence of micropapillary component (≥ 5%) and lymphatic invasion in the main tumor (P < 0.001).

Conclusion:
Presence of acinar, micropapillary and solid component in the main tumor are associated with metastasizing to lymph nodes. Presence of STAS was significantly associated with increased risk of recurrence in stage III (N2) lung adenocarcinoma.

Background:
Most patients treated against molecular targets eventually develop resistance even after an initial dramatic response. Although rebiopsy of tumors at progression provide information for next-line therapy, it is expected that the tumor tissues would be modified by the therapy.

Methods:
We retrospectively examined histologic features in the resampled specimens in lung cancer patients with resistance to the initial therapy. Furthermore, we also analyzed the differences of tumor cell contents and molecular testing performance according to each biopsy site.

Results:
A total of 315 resampled specimens were submitted to pathology department from 260 patients. Of 315 samples, 116 (37%) were obtained from the lung and 96 (30%) from pleural effusion, 42 (13%) from lymph node, 16 (5%) from liver, 12 (4%) from cerebrospinal fluid (CSF), 10 (3%) from pleura and pericardial effusion, 7 (2%) from bone and 6 (2%) from other biopsy sites. When we compared 48 paired lung tissues between initial and rebiopsies, rebiopsy specimens had significantly less extents of tumor cells and more fibrosis than those in initial biopsy, and these differences were statistically significant with digital quantitation. Resampled sites affect the tumor cell extents and those were high in the order of liver, subcutaneous tissue, lymph node and lung biopsy, whereas pleura and bone samples had a tendency to contain a less number of tumor cells. Molecular testing was performed in 272 samples (from 222 patients). Of 272 samples, 223 (82%) were successfully analyzed, whereas 49 samples were unsuitable for the testing due to low tumor-cell content or complete absence of tumor cells. Higher success rates for molecular testing were seen in the liver and lymph nodes and the value of bone was lowest. Resistant T790M mutations were also differently detected and the higher detection rates were seen in liver, pleura and pericardial effusions.

Conclusion:
Resampled specimens had different property in terms of tumor extents, which differed among the biopsy sites. For molecular testing using resampled specimens, the difference should be taken into account.

Background:
The expansion of micrometastatic tumors to macrometastatic ones is thought to be tightly regulated by several microenvironmental factors. The aim of this study was to elucidate the morphological and phenotypical differences between micrometastatic and macrometastatic tumors.

Methods:
We first examined the morphological characteristics of 66 lymph node (LN) micrometastatic tumors (less than 2 mm in size) and 51 macrometastatic tumors (more than 10 mm in size) in 42 lung adenocarcinoma cases. Then, we evaluated the expression level of E-cadherin, S100A4, ALDH1, and Geminin in cancer cells and the number of smooth muscle actin (SMA), CD34, and CD204 (+) stromal cells in the primary tumors, matched micrometastatic tumors, and macrometastatic tumors (n = 34, each).

Results:
Tumor budding reflects the process of EMT, and stromal reactions were observed more frequently in macrometastatic tumors (P < 0.001). E-cadherin staining score for the micrometastatic tumors was significantly higher than that for the primary tumors (P < 0.001). In contrast, the E-cadherin staining score for the macrometastatic tumors was significantly lower than that for the micrometastatic tumors (P = 0.017). As for the stromal cells, the numbers of SMA (+) fibroblasts, CD34 (+) microvessels, and CD204 (+) macrophages were significantly higher for the macrometastatic tumors and primary tumors than for the micrometastatic tumors (P < 0.001, all).

Conclusion:
The present study clearly showed that dynamic microenvironmental changes (e.g., EMT-related changes incancer cells and structural changes in stromal cells) occur during the growth of micrometastases into macrometastases.

Background:
Primary pulmonary sarcomas are very rare with an incidence rate of <0.5% of all lung malignancies. Their low incidence has impeded comprehensive evaluation of their association with smoking, definitive diagnostic and treatment-regimes. They are often misdiagnosed, both on radiology as well as on fine-needle-aspirate/small-biopsies. We present a series of primary pulmonary sarcomas diagnosed over the last two and a half years.

Methods:
All cases of primary pulmonary sarcomas (2014-2016) were retrieved and reviewed.

Results:
A total of 21 sarcomas were identified. The most common was synovial sarcoma. Four exceptionally rare cases included pulmonary-artery intimal sarcoma, primary pulmonary myxoid sarcoma, malignant peripheral nerve-sheath tumor and follicular dendritic-cell sarcoma. The clinical and pathology details of which are provided in table1. The patients were distributed over a wide-age range (range:9-65 years, median:34 years) with a male-preponderance (M:F=2.2:1). Radiological features were non-specific except in case1(table1). Histopathology revealed spindle-cell tumor in all cases(figure1) and an extensive immunohistochemical-panel and cytogenetic testing was required to clinch the diagnosis. Figure 1 Figure 2





Conclusion:
This is a series of primary thoracic sarcomas with a highlight on four extremely rare cases which bring to light their unique clinical, radiological, histopathological and immunohistochemical findings. Awareness of such entities is essential for proper diagnosis, appropriate molecular-testing and treatment.

Background:
Lymphoepithelioma-like carcinoma (LELC) is a rare form of lung cancer, usually encountered in Chinese patients. Similar to nasopharyngeal carcinoma which is strongly associated with Epstein-Barr virus (EBV) infection, LELC is defined as a poorly differentiated carcinoma reveals EBER-positive neoplastic cells and marked lymphocyte infiltrate by 2015 WHO classification; however, EBER-negative carcinomas showing LELC-like morphology are present and such cases might be classified as adenocarcinoma or squamous cell carcinoma based on the results of immunohistochemistry staining, such as p40 or TTF-1.

Methods:
We retrospectively reviewed the medical records of 5 LELC patients who underwent pulmonary resection in Kyoto University Hospital between 2005 and 2016. All five cases were primary lung tumors with histologic features of carcinoma characterized by poorly differentiated morphology admixed with heavy lymphocyte infiltrates which fit the criteria for the diagnosis of LELC as morphologic findings.

Results:
There were 4 men and 1 woman who ranged in age from 65 to 78 years, with a median age of 70. Three patients had lymph node metastasis and underwent surgical resection, followed by adjuvant chemotherapy.One patient died of second primary lung cancer (small cell carcinoma) but four patients were alive without tumor recurrence 4 months to 8 years and 11 months.Four patients (80%) were negative for EBV, suggesting no association between EBV and LELC in our institution study group.In immunohistochemistry staining, 4 cases were positive for p40 and one case was for TTF-1.

patient	Age	Sex	Smoking	Location	pStage	Treatment	EBER	TTF-1	p40	Outcome
1	78	M	Yes	RUL	T1aN0M0	Surg＋adjuvant rad	negative	negative	positive	3y6m dead
2	68	M	Yes	LLL	T1aN2M0	Surg＋adjuvant chemo	negative	negative	positive	8y11m alive
3	71	F	None	LLL	T3N1M0	Surg＋adjuvant chemo	positive	negative	positive	6y2m alive
4	65	M	Yes	RUL	T1bN1M0	Surg＋adjuvant chemo	negative	positive	negative	5y11m alive
5	73	M	Yes	RUL	T1aN0M0	Surg only	negative	negative	positive	0y4m alive

Conclusion:
Our reexamination revealed that most LELCs were negative for EBER and were classified as squamous cell carcinoma by IHC study. This results might imply that EBER is not a requisite factor in the lung carcinoma with LELC-like morphology.

Background:
Lung cancer is the leading cause of cancer-related mortality. Adenocarcinoma (AC) representing 50% of diagnosed lung cancer. At diagnosis, 25% of pulmonary AC present as multicentric lesions and an half are considered synchronous AC (SLA) while the remaining represents intrapulmonary metastases (MAC) from a primary lung AC. Surgical resection is the treatment of choice for SLA and the outcome of the patients is generally good. On the other hand, intrapulmonary metastases (MAC) are related lesions associated with a poor prognosis and generally not amenable to surgical therapy. There is currently no way to distinguish SLA from MAC without analyzing a surgical specimen from each lesion, which is rarely possible. It is then likely that a significant proportion of patients with multiple AC do not get the appropriate treatment. There is therefore an urgent need to develop molecular tools to classify multicentric lesions. Genomic instability is one of the drivers of metastases, and the alteration of telomeric nuclear organization (TNO) is a predictor of genomic instability and tumor progression. Our hypothesis is that the profile of TNO can discriminate SLA from MAC.

Methods:
We assessed the parameters defining 3D-TNO using 3D quantitative fluorescence in situ hybridization, 3D imaging and 3D-TNO analyses on formalin-fixed paraffin-embedded tissue sections from 10 patients with SLA or MAC. For each patient, were analyzed two lesions: primary and metastatic lesions for MAC and two different primary tumors for SLA. The following 3D-TNO parameters were evaluated: 1) number of telomere (telomere signals), 2) telomere length (telomere signal intensities), 3) number of telomere aggregates (telomere clusters), 4) telomere distribution within a nucleus and 5) nuclear volume.

Results:
Firstly, we compared 3D-TNO of cancer cells between MAC and SLA and found that four of the five parameters defining 3D-TNO showed statistical difference between the two pathological groups. Secondly, for each patient, we did pairwise comparison of parameters defining 3D-TNO between the two lesions. For the patients presenting MAC, we found that metastatic lesions had higher telomere aggregates than primary lesions. The comparison of the number of telomere aggregates did not display statistical difference between the two primary tumors from SLA.

Conclusion:
This study shows that the number of telomere aggregates is a powerful discriminative parameter that can reliably distinguish patients with SLA from patients with MAC. Our results suggest that 3D-TNO signature has the potential to provide a molecular tool that can eventually be implemented in a clinical setting.

Background:
There have been some reports on transbronchial biopsy (TBB) through endobronchial ultrasonography with a guide sheath (EBUS-GS) for diagnostic sampling of small-sized tumors which showing ground-glass opacity (GGO) on chest CT. However, technique such as EBUS-GS is limited in their ability to diagnose such small lung tumors. The discussion about the cytological features of small tumors with GGO in detail is necessary. We evaluated about the association of the cytological features with the histological examination using the surgically resected specimen. 140 patients, age between 23–86 years old, who showed clinical and radiological signs of peripheral lung tumors below 3.0cm in diameter, underwent surgical resection at our institution between 2013 and 2015.

Methods:
Imprints or touch preparation and squash smears preparation were prepared from the unfixed, fresh sample in 140 cases. Papanicolaou's stain was employed in all cases. To make the squash smears preparation, the slides are drawn apart away from each other, in the direction of the long axis of the slide. Tissue fragments taken from surgical specimen were fixed with 10% neutral buffered formalin and stained with hematoxylin and eosin (H&E).

Results:
By histological examination (in the 140 cases), the diagnostic of lung cancer was given with the establishing of the histological type. In 110 cases (78.6%) of the cases diagnosed as adenocarcinoma, in 21 cases (15%) squamous cell carcinoma, in 4cases (2.9%) was neuroendocrine tumors, and one case each of adenosquamous carcinoma, pleomorphic carcinoma and pleomorphic sarcoma. In 84 of the 110cases (76.3%), the result of imprint cytological examination was adenocarcinoma. In the 110 pathological diagnosed as adenocarcinoma cases, 52 patients (47.2%) are below 2.0cm in size. Tumor stamps of small sized adenocarcinoma are characterized by moderate cellularity and are composed of atypical cells arranged in small flat sheets. The nuclei are generally round, slightly hyperchromatic with small nucleoli.

Conclusion:
Our data indicate the fact that the cytological examination on stamps from surgical material offers a very high percentage of positive results, close to the histological one. But in the tumor size less than 1.0cm, the establishing of the histological type of lung cancer is more difficult by cytological examination. Despite this, the cytology may be extremely useful in diagnose of the small peripheral tumors. The cytological characteristics of small peripheral adenocarcinoma were little reference to the differentiation at the cellular level. Our findings indicated that the presence of several nucleoli and granular chromatin densely are the factors of adenocarcinoma.

Background:
STAS is defined as a pattern of tumor cell spread in the lung parenchyma beyond the edge of a lung cancer. It has been postulated that this is an ex vivo artifact due to the force of knife with the premise that STAS is clinically unimportant and it should be ignored like true artifacts.

Methods:
We present three cases providing evidence that STAS is not an artifact and is clinically relevant.

Results:
Case 1: 68F underwent wedge resection of a left upper lobe (LUL) lung adenocarcinoma. During the surgical procedure the surgeon did not cut across the tumor, but sent a separate wedge biopsy as an additional margin. The latter wedge contained an 8 mm focus of adenocarcinoma consisting almost entirely of a STAS pattern with a 1mm area of acinar growth. Case 2: 66M underwent RUL wedge resection in August 2013 for a 1.3 cm lung adenocarcinoma. The resection margin was positive with only STAS in the margin. In the absence of any clinical sign of recurrence or metastases, a completion right upper lobectomy was performed revealing three separate foci of residual adenocarcinoma including 1.5 and 1.0 mm acinar areas and a 0.5 mm focus of STAS with N1 and N2 lymph node metastases. Adjuvant chemotherapy and radiation were given. In 2014, the patient developed multiple bilateral nodules and in November underwent LUL wedge resection that showed three foci of adenocarcinoma with a STAS predominant pattern. In July 2016, the patient remains on chemotherapy with slowly growing bilateral nodules. Case 3: A 77M presented with pneumonia and bilateral ground glass opacities with focal consolidation. A biopsy, originally interpreted as benign, showed diffuse involvement by adenocarcinoma with a STAS predominant pattern. The morphology does not explain the consolidation seen on CT indicating the surgeon did not cut across the main tumor area.

Conclusion:
We present three cases which provide evidence that STAS is not an artifact that should be ignored. In two cases the extensive STAS predominant pattern was not a knife cutting artifact because the main tumor was not cut either by the surgeon or pathologist. In the third case, STAS was the only pattern of tumor identified at a wedge resection margin. If this had been ignored, the residual and metastatic tumor would not have been identified delaying introduction of chemotherapy. These findings support the concept that STAS is a clinically important invasive pattern and not an artifact.

Background:
Comparison of radiographic parameters and histologic sub-types of lung adenocarcinoma(ADC) proposed by the IASLC/ATS/ERS in 2011 may help to direct surgical procedures and evaluate prognosis. To analyze the relationship between them,we conducted our study.

Methods:
The architectural patterns of 197 completely resected lung ADCs were analyzed in 5% increments, and classified and graded according to their predominant patterns. Preoperative CT imaging characteristics, including lesion site, diameter, shape, margins, attenuation, cavitation, et al, were also collected.

Results:
Low-grade group(including lepidic predominant ADC, LPA) was more likely linked with vague boundary, irregular shape, vascular clusters and GGO or sub-solid nodules(SSN) , while high-grade group(including solid predominant ADC,SPA and micro-papillary predominant ADC,MPA) were vice versa (p values were 0.003, 0.037, 0.037, 0.011, respectively). More proportion of lepidic growth pattern were detected in GGOs or SSNs (p<0.001), and in those lesions characterized by the following CT features, such as vague boundary, non-lobulated margins, cavitation, irregular shape and vascular clusters(p=0.040、0.009、0.040、0.001,respectively). While the higher ratio of acinar and solid growth patterns were associated with solid lesions on CT (p=0.006, 0.020 , respectively). More proportion of Solid growth pattern were detected in spherical tumors (p=0.016).

Conclusion:
We conclude that CT imaging characteristics are associated with histo-morphological patterns of ADC to some extent. It may offer some clues for the diagnosis of ADC and predicting its survivals as well.

Background:
Incomplete retrieval of intrapulmonary lymph nodes and missed nodal metastasis are associated with worse-than-expected survival after (NSCLC) resection. We tested the nodal yield from a novel gross dissection method.

Methods:
multi-institutional prospective cohort study of intrapulmonary (stations 11-14) lymph node yield from lobectomy/greater NSCLC resection specimens from 11 US hospitals from 2009-2016. A novel gross dissection protocol was used in 2 hospital pathology departments from June 2012 onwards. Intrapulmonary lymph node yields from all lobectomy or greater resections before and after the new protocol in the intervention hospitals were compared to yields from 9 non-intervention hospitals over the same time-span, using Wilcoxon-Mann-Whitney. From February 2015, some randomly selected discarded remnant lung specimens in the intervention hospitals were re-dissected for inadvertently discarded lymph nodes as a quality control measure.

Results:
Intrapulmonary lymph node yields in the 2 groups of hospitals was similar at baseline, followed by a significant increase in the intervention hospitals with the novel dissection protocol (Table 1). Subsequently, in 112 specimens re-dissected for independent quality control after application of the novel dissection protocol, discarded lymph nodes were found in 30 (27%), down from 90% historically; discarded lymph nodes with metastasis in 6 (5%), down from 29% historically; and missed N1 nodal metastasis was found in 1 of 67 (1.5%) pN0 patients, down from 12% historically. The median number of missed intrapulmonary lymph nodes was 0 (down from 6 historically), the mean (standard deviation) was 0.88 (2.58). The gross dissection protocol required a median of 15 minutes (range 10 – 24). Figure 1



Conclusion:
A novel gross dissection protocol significantly improves the thoroughness of intrapulmonary lymph node retrieval and can be successfully implemented in community-level pathology departments, providing a pathway for quality improvement in pathologic nodal staging of resected NSCLC.

Background:
Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein which functions as a coregulator of several transcription factors and nuclear receptors. It has a histone-binding domain and plays essential roles in several pathways including hormonal signaling. PELP1 is a coregulator of estrogen receptor (ER) and has been shown to be deregulated, contributing to therapy resistance and is a prognostic biomarker in breast cancer survival, as well as in other hormone-dependent cancers. Estrogens are also known to enhance lung tumorigenesis by estrogen receptor pathway[1, 2]. In this study, we investigated the expression of PELP1 in molecularly classified lung adenocarcinomas, specifically those with known EGFR and KRAS mutations.

Methods:
Tissue microarray (TMA) was created using 0.6 mm tissue cores in triplicates from 62 resected lung adenocarcinoma cases (26 with EGFR mutation and 36 with KRAS mutation). PELP1 antibody (Clone EPR15212; ABCAM) immunostaining was performed after heat induced epitope retrieval. Nuclear immunoreactivity for PELP1 was scored for staining intensity as 0 (negative), 1+ (weak, nucleolar), 2+ (moderate, nucleolar/nuclear) and 3+ (strong nucleolar/nuclear). For statistical analysis, binary split was done as negative (scores 0 and 1+) and positive (scores 2+ and 3+). These TMAs were also stained for estrogen receptor (ER) with SP1 rabbit monoclonal antibody and scored similarly.

Results:
In our study, 61 cases had evaluable tissue cores with tumor. Positive PELP1 expression was noted in 14/25 (56%) EGFR mutated and 30/36 (83%) in KRAS mutated lung adenocarcinomas (p<0.05). From the EGFR-mutated group, 4/25 (16%) reveal weak (1+) and focal staining for ER while one case revealed strong ER staining. From the KRAS-mutated group, 2/36 (5%) cases revealed weak (1+) staining for ER. All these cases with ER positivity (7/61; 11.5%) (weak/strong) were also positive for PELP1.

Conclusion:
Our study has demonstrated that apart from PELP1 expression in ER positive lung adenocarcinomas, it can also be seen in estrogen receptor negative molecularly classified lung adenocarcinomas. It is more often seen in KRAS mutated lung adenocarcinomas. Estrogen receptor independent PELP1 expression in lung adenocarcinoma suggests presence of alternate pathways for tumorigenesis or tumor progression, which needs further investigation; especially in KRAS mutated lung adenocarcinomas. References: 1. Slowikowski BK, Gatecki B, Dyszkiewicz W et al. Increased expression of proline-, glutamic acid- and leucine-rich protein PELP1 in non-small cell lung cancer. Biomed Pharmacother 73:97-101; 2015. 2. Sareddy GR, Vadlamudi RK. PELP1: Structure, biological function and clinical significance. Gene. 585(1):128-34; 2016.

Background:
Metastasis and growth in neoplastic lesions requires the multi-step regulation of microenvironmental factors. We aimed to elucidate the microenvironmental changes in the process of lymphatic metastasis of lung squamous cell carcinoma.

Methods:
We examined the morphological characteristics of 102 cases of Primary Tumor (PT), 50 of intralymphatic tumor (ILT), 51 of lymph node (LN) micrometastasis (LN-Mic; less than 2 mm in size) and 82 of LN Macrometastasis (LN-Mac; greater than 10 mm in size). Afterwards we evaluated the expression of nine molecules (EGFR, FGFR2, CD44, ALDH1, Podoplanin, E-cadherin, S100A4, geminin and ezrin) in matched PT, ILT, LN-Mic and LN-Mac from 23 of these cases.

Results:
The number of smooth muscle actin α-positive fibroblasts, CD34-positive microvessels and CD204-positive macrophages were also examined. As a result, the mitotic index of cancer cells was significantly lower in ILT and LN-Mic than PT and LN-Mac (p<0.001). Moreover stromal reaction in ILT and LN-Mic was less prominent than in PT and LN-Mac (p<0.001). Immunohistochemical study revealed that EGFR expression level and frequency of geminin positive cells in ILT and LN-Mic were significantly lower than in PT and LN-Mac (p<0.05). The number of stromal cells indicated by staining of CD34, CD204 and smooth muscle actin α in ILT and LN-Mic also was significantly lower than in PT and LN-Mac (p<0.05).

Conclusion:
In lung squamous cell carcinoma, drastic microenvironmental changes (e.g., growth factor receptor expression and proliferative capacity of cancer cells and structural changes in stromal cells) occur during both the process of lymphatic permeation and the progression into macrometastases.

Background:
Background: Non small cell lung cancer (NSCLC) metastasis remains a major cause for patient mortality marking the underlying molecular mechanisms as important therapeutic targets. The progression of cancers from primary tumors to invasive and metastatic stages accounts for the overwhelming majority NSCLC patients deaths. Understanding the molecular events which promote metastasis is thus critical in the clinic. Deregulation of protein synthesis is integral to the malignant phenotype and translational control is emerging as an important factor in tumorigenesis. Indeed, over-expression of translation initiation factors eIF4E and eIF4GI in NSCLC was associated with patients' poor survival. Thus, in this study we aimed to assess the direct role of eIF4E and eIF4GI in NSCLC and their effect on migration and metastasis formation.

Methods:
Methods: eIF4E/ eIF4GI knockdown (KD) in NSCLC cell lines (H1299, H460) was achieved by siRNA. Following transfection the cells were tested for changes in eIF4E/eIF4GIs' targets (SMAD5, NFkB, cMYC, HIF1α), migration (scratch) and pro/ anti Epithelial-Mesenchymal Transition (EMT) markers (N-Cadherin, Slug, ZEB1, E-Cadherin, Claudin, ZO-1, microRNA). Importance of eIF4E and eIF4GI KD to NSCLC phenotype was further corroborated with the inhibitors ribavirin and 4EGI-1. Lastly, we tested for changes in essential microRNA implicated in NSCLC cell migration and EMT.

Results:
Results: Downregulation of eIF4E/eIF4GI significantly decreased their established targets (20-35%↓, 48h, p<0.05) indicating compromised activity. Diminished NSCLC cell lines' migration upon eIF4E/eIF4GI KD was also witnessed (65-82%↓, 48h, p<0.05). Moreover, we demonstrated reduced levels of EMT inducers together with elevated levels of EMT suppressors (40-90%↓, 48h, p<0.05). Finally, we showed that eIF4E/eIF4GI KD affected microRNAs critically involved in migration and EMT processes.

Conclusion:
Conclusions: Our study shows that targeting eIF4E/eIF4GI reduces migration and EMT, both essential for metastasis, thereby underscoring the role of translation initiation in NSCLC metastatic tumor formation. Understanding the molecular events which promote metastasis and improving the means of foretelling their development is a major goal of current clinical research. We suggest that targeting translation initiation in NSCLC with clinically employed drugs that inhibit eIF4E/eIF4GI (Ribavirin/ 4EGI) may afford a valid and effective therapeutic strategy in NSCLC patients and may diminish lung cancer metastatic spread and morbidity and improve the patient's life quality.

Background:
Cigarette smoking not only promotes lung carci­nogenesis, but it has also been demonstrated to promote the progression of lung cancer. Despite nicotine being a major component of cigarette smoke, it is not carcinogenic when acting alone. Instead, it is believed to function as a tumor promoter that stimulates the processes of invasion and metastasis. In the present study we aimed to determine the effect of nicotine on the migratory activity of lung cancer cells.

Methods:
The effect of nicotine on the migration of lung cancer A549 cells was evaluated by a wound healing assay. Hepatocyte growth factor (HGF) was used as a pro‑migratory stimulus. During several of the experiments, specific inhibitors of α7‑nicotine acetylcholine receptor (α7‑nAchR), phosphoinositide kinase‑3 (PI3K) and extracellular signal‑related kinase (ERK)1/2 were included. The phosphorylation levels of Akt and ERK1/2 were examined using a cell‑based protein phosphorylation assay.

Results:
Nicotine did not induce cell migration by itself, but it instead promoted HGF‑induced cell migra­tion (Figure). The effects of nicotine were inhibited by the pretreatment of the cells with the α7‑nAchR inhibitor, methyllycaconitine, and the PI3K inhibitor, LY294002. The mitogen‑activated protein kinase/ERK kinase kinase inhibitor exerted modest, but non‑significant inhibitory activity on the effect of nicotine. Nicotine did not induce Akt phosphorylation by itself, but instead promoted the HGF‑induced phosphorylation of Akt. It was also observed that nicotine had no effect on ERK1/2 phosphorylation.Figure 1



Conclusion:
These results indicate that nicotine, when alone, does not have a pro‑migratory func­tion, but instead enhances responsiveness to the pro‑migratory stimulus emitted by HGF. This study provides an insight into the mechanism of tumor promotion by demonstrating that nicotine and α7‑nAchRs act in synergy with the HGF‑induced PI3K/Akt signaling pathway, increasing the sensitivity of lung cancer cells to HGF, and thereby promoting cell migration, a vital step in invasion and metastasis.

Background:
Non small cell lung cancer (NSCLC) belongs to the most frequently diagnosed cancer entities and is one of the leading causes of cancer related death worldwide. Deregulation of protein synthesis has received considerable attention as a major step in cancer development and progression. Protein synthesis is regulated at multiple stages, including translation of mRNA into proteins. Studies suggest that ribosomal protein synthesis plays a direct role during tumor-initiation. Crucial for this translation process are eukaryotic initiation factors (eIFs), which ensure the correct 80S ribosome assembly. eIFs are linked to the MAPK and the mTOR signalling pathways, which have become major targets in cancer therapy. Mutations or deregulated expression of eIFs influence cell growth and proliferation, and contribute to carcinogenesis. We hypothesized that eIFs represent crossroads for carcinogenesis in lung cancer and might serve as potential biomarker.

Methods:
Expression profiling of paired NSCLC and non-neoplastic lung tissue (NNLT) from 1.000 individuals were studied by immunohistochemistry on tissue micro arrays (TMAs) with antibodies against the eIF subunits 2α; 3C; 3H; 3M; 4E and 6. eIF expression was evaluated with respect to the staining intensity (intensity score 0-3; 0: no staining, 1: weak, 2: moderate and 3: strong) and percentage of positive tumor cells (proportion score; 0-100%). In addition, the protein and mRNA expression levels of eIFs and mTOR pathway members were determined in 25 patients by Western Blot analysis and qRT-PCR. For the statistical analysis α was set to 5%.

Results:
Western Blot analysis of NSCLC revealed a significant up-regulation of mTOR and the eIF subunits p2α, 2α, 1A, 4A, and eIF6 compared to NNLT (p< 0,05). The mRNA levels of NSCLC also displayed a significant upregulation of the eIF subunits 2α, 4A, and eIF6 compared to NNLT. Immunohistochemistry highlighted a stronger staining in neoplastic cells for eIF2α, eIF4E, eIF3H and eIF6.

Conclusion:
Our data indicated that eIFs are significantly upregulated in NSCLC, suggesting an important contribution of eIFs and mTOR signalling to the development and progression of lung carcinomas. A better understanding of the molecular mechanisms in pulmonary carcinogenesis is necessary for the development of novel treatment strategies.

Background:
Hypoxia-inducible factor (HIF) is important for cancer progression, resistance to therapy and development of cancer itself. A family of transcription factors HIF is heterodimer consist of α subunits (HIF-1α, HIF-2α, HIF-3α), which forms an active complex with β subunits also known as the aryl hydrocarbon nuclear translocation (ARNT) then stimulate various target genes, termed as hypoxia response element (HRE). However, a genetic variant of HIF was not fully understood. Previously, we reported that HIF1A polymorphism associated with TP53 status in lung cancer patients, and transcriptional activity of the HIF-1α variants in A549 lung cancer cells was significantly greater than that of the wild type, especially in cells containing a mutant type of p53. We also found that one of HIF2A (EPAS1) polymorphism significantly associated with poorer prognosis of lung cancer patients, and the nucleotide substitution might affect HIF2A expression through transcriptional regulation in vitro.

Methods:
In this study, we tried to clarify a role of genetic variations of HIF3A gene, and started evaluations of six polymorphisms located in HIF3A loci (rs3764609, rs3764610, rs3764611, rs375220, rs3810302, rs3826796) in 83 Japanese lung cancer patients as a pilot study.

Results:
We performed sequence analysis of genomic DNA and success identify HIF3A polymorphisms by direct sequencing. Genotype distributions of each SNP showed good agreements with the Hardy-Weinberg equilibrium. We found the rs3810302 have different genotype distribution compare healthy Japanese data base (HapMap), p=0.011. Then, some loci of HIF3A showed significant associated with clinicopathological of lung cancer patients (stage, cancer differentiation, histology, etc).

Conclusion:
Our preliminary study suggested that some of HIF3A polymorphisms showed significantly important associations with lung cancer clinicopathological. More studies were further required to focus on its relationship.

Background:
The anti-EGFR monoclonal antibody (mAb), necitumumab, has been recently approved in combination with chemotherapy, as 1st-line treatment for advanced lung squamous cell carcinoma (LSCC) patients, but with minimal survival benefit. Evidence continues to accumulate that signal transducer and activator of transcription 3 (STAT3) is a promising molecular target for cancer therapies. STAT3 is activated by tyrosine phosphorylation in response to EGF and interleukin-6 (IL-6). In addition to STAT3, IL-6 activates the Src family kinases, and subsequently YES-associated protein 1 (YAP1). STAT3 and Src-YAP1 activation contributes to EGFR inhibitor resistance and concomitant targeting of EGFR and STAT3-Src may represent an effective treatment strategy for LSCC.

Methods:
RNA was isolated from six LSCC cell lines and the mRNA expression analysis of EGFR, STAT3, Src and YAP1 was performed by TaqMan based qRT-PCR. Cell viability was assessed by MTT (thiazolyl blue) assay after treatment with necitumumab and evodiamine, an alkaloid isolated from the dried, unripe Evodia rutaecarpa (Juss.) Benth fruit that exerts an anticancer effect by inhibiting STAT3 and Src. Western blotting was performed to assess the effect of necitumumab on EGFR downstream signaling pathways.

Results:
We first evaluated the expression of EGFR in our panel of LSCC cell lines. We found that almost all of them homogeneously express high levels of EGFR. We then assessed the effect of necitumumab on EGFR downstream signaling in the SK-MES1 cell line. Treatment of SK-MES1 cells with 25ug/ml of necitumumab for seven days was unable to ablate STAT3, Src or YAP1 mRNA expression. Consistent with this, we found that necitumumab suppressed EGFR, ERK1/2 and AKT phosphorylation but increased STAT3 phosphorylation on the critical tyrosine residue 705 in a time and dose-dependent manner. We examined the growth inhibitory effect of the necitumumab and evodiamine combination. We performed an MTT cell proliferation assay on SK-MES1 cells and we used a constant ratio drug combination method to determine synergy, additivity, or antagonism. The combination of necitumumab and evodiamine resulted in a clear synergism in SK-MES1 cells as measured by the combination index (CI) analysis, with a CI of 0.74. Experiments in the rest of our LSCC cell lines are ongoing.

Conclusion:
Herein we have examined the role of STAT3 and Src-YAP1 in the context of treatment with the FDA-approved EGFR mAb, necitumumab. Our data provide initial evidence that co-activation of STAT3 and Src-YAP1 may limit the cellular response to EGFR inhibition in LSCC.

Background:
Lung cancer ranks as the first most common cancer and the first leading cause of cancer-related death in China and worldwide. Due to the difficulty in early diagnosis and the onset of cancer metastasis, the 5-year survival rate of lung cancer remains extremely low. JAK2 has emerged as pivotal participant in biological processes, often dysregulated in a range of cancers, including lung cancer. Recently we found that JAK2 might play an important role in lung cancer pathogenesis as an oncogene. While our understanding of JAK2 in the onset and progression of lung cancer is still in its infancy, there is no doubt that understanding the activities of JAK2 will certainly secure strong biomarkers and improve treatment options for lung cancer patients.

Methods:
The expression levels of JAK2 mRNA and protein were assayed using the RT-PCR and Western Blot assay respectively. MTT assay, Scratch-wound healing assay, Transwell migration and invasion assay were conducted to study the proliferation, migration and invasion abilities of lung cancer cells independently. The shRNA and overexpression plasmids of JAK2 were conducted.

Results:
JAK2 is up-regulated in lung cancer tissues when compared with their adjacent non-tumor tissues, and was associated with lymph node metastasis. Downregulation of JAK2 inhibits the proliferation, migration and invasion abilities of lung cancer cells. Moreover, overexpression of JAK2 induced the proliferation, migration and invasion abilities of lung cancer cells.

Conclusion:
These findings demonstrate that JAK2, whose expression is up-regulated in lung cancer, may participate in lung cancer progression by regulating cancer cells proliferation, migration and invasion.

Background:
Chemotherapy against nonsmall cell lung cancers is remarkably progressing. Nanomaterials are searched for this therapy. Graphene oxide is also suggested as one of promising therapeutic materials. Graphene is an allotrope of carbon with honeycomb structure. It may show the diverse biologic effects from minimal to highly toxic effect according to the cell types.The goal of this study was to define the differential cell death mechanism of graphene oxide on lung adenocarcinoma cells and macrophages with focusing autophagy.

Methods:
A549 cells and Raw264.7 cells were cultured in Dulbecco's modified eagle's medium with 10 % fetal bovine serum and treated with graphene oxide(GO). GO was treated to the cells in the range of 5 ~ 200 ug/ml for 24 and 48 hours. Cell survival was examined with light microscopy and MTT assay. Protein expression was checked by Western blots for LC3A/B-I, II, NBR1, p62/SQSTM1, mTOR, Bec-1 and PU.1(monocyte/macrophage-specific transcription factor).

Results:
Higher concentrations of graphene oxide induced increasing cellular death with different intensity between A549 and Raw264.7 cells. LC3B-I to II conversion (autophagy) was increased by GO in A549 cells and decreased in Raw264.7 cells. Expression of NBR1 and p62 showed same directional change in both cell types. The mammalian TOR was also decreased in A549 cells. PU.1(monocyte/macrophage-specific transcription factor) was decreased in Raw264.7 cells. Bec-1 and GAPDH was not affected by GO in both cells.

Conclusion:
This study showed the opposite response in autophagy in A549 cells and Raw264.7 cells. Although the precise mechanisms are mandatory to be defined, graphene may be used for selective targeting against lung adenocarcinoma with preserving immune function.

Background:
Abnormal increases in reactive oxygen species (ROS) in cancer cells serves as a target for tumor-selective killing. Also, several experimental and clinical trials studied the effect of the hyperoxia condition by difference of response between normal cells and cancer cells. In this study, the hypothesis tested was that normobaric high oxygen concentration would have anti-cancer effects such as inducing apoptosis on human lung cancer cell line.

Methods:
Human bronchial epithelial cells (Beas-2b) and human alveolar adenocarcinoma cells (A549) were exposed with hyperoxia condition in a time-dependent manner. The changes in the cell morphology, viability and protein expressions such as p53 and ERK were examined after the exposure of hyperoxia (90% O~2~). In addition, to investigate whether hyperoxia condition affects the production of ROS and cell cycle regulation, cells were analyzed by a flow cytometry.

Results:
Exposure to the hyperoxia caused morphologic changes such as atypical nuclei and numerous mitotic figures which inhibited the cell viability in a time-dependent manner in A549 (p <0.01). In addition, the colony formation was suppressed selectively in A549 exposed to hyperoxia. Although not statistically significant, A549 exposed to hyperoxia showed increases in the ROS levels compared with Beas-2b. Also, the hyperoxia condition caused a progression delay in the G2/M cell cycle significantly in A549 (p <0.01). In hyperoxia exposed A549 cells, the phosphorylation of ERK 1/2 (p-ERK 1/2) was reduced while the phosphorylation of p53 was increased.

Conclusion:
This study showed that hyperoxia may have anti-cancer effect by decreasing cell viability and the colony forming ability. ROS generation by hyperoxia may cause to suppress the p-ERK, it related with the activation of p53 and G2/M cell cycle arrest. In conclusion, our data suggests that the anti-cancer effect of hyperoxia may relate to the ROS through oxidative stress mediated ERK signaling and cell arrest.

Background:
Normal lung epithelium cells may act in concert with tumour cells, given that bystander effects may exist between the two. This interaction may lead to inappropriate activation of pro-oncogenic signalling pathways, which may result in high mutational load and tumour heterogeneity. The aim of this project is to evaluate the effects of non-small cell lung cancer (NSCLC) cells on an immortalised normal bronchial epithelial cell line.

Methods:
A normal bronchial epithelial cell line (HBEC4) was exposed to A549 (adenocarcinoma), H460 (large cell carcinoma) and SK-MES-1 (squamous cell carcinoma) NSCLC cell lines in a trans-well co-culture system. Cellular characteristics were examined using a Cytell Cell Imaging System (cell number, viability, apoptosis, cell cycle). The gene expression profile was also determined in terms of inflammatory mediators, stem cell markers (RT-PCR) and miRNA profiling (Nanostring). The proliferative effect of NSCLC cancer exosomes was also examined (BrdU ELISA) on the HBEC4 cell line.

Results:
A number of functional and gene modifications were observed in the HBEC4 cell line after seven days of co-culture. While patterns were similar amongst all NSCLC subtypes, SK-MES-1 elicited the most significant effects in terms of cell number, viability, cell cycle progression and proliferative potential of isolated cancer exosome fraction. Promotion of both inflammatory mediators and stem cell marker expression was evident at the mRNA level. There was no apparent consensus between NSCLC subtypes and miRNA expression, as exposure to each cell line resulted in distinct profiles of miRNAs in HBEC4 cells. Bioinformatic analysis of miRNA target genes, demonstrated that pathways such as p53, MAPK, VEGF, TLR and Wnt were amongst those altered.

Conclusion:
Cancer cells may promote significant genotypic and phenotypic alterations within the normal lung epithelium though multiple mechanisms. These modifications may, in part, contribute to the heterogeneity of lung cancer tumours and influence response to both chemotherapeutics and targeted agents.

Background:
Arginine depletion has shown promising anticancer effects among arginine auxotrophic cancers that are deficient in argininosuccinate synthetase (ASS) and/or ornithine transcarbamylase (OTC). Pegylated arginase (PEG-BCT-100 (rhArg1peg5000)) works as an arginine depletor by converting arginine to ornithine. However, accumulated ornithine can be channeled via ornithine decarboxylase (ODC) to produce polyamines that are known to promote tumor growth. We postulate that ODC inhibition could rescue anticancer effects of BCT-100 in lung adenocarcinoma.

Methods:
A panel of 7 lung adenocarcinoma cell lines (H23, H358, HCC827, H1650, H1975, HCC2935 and HCC4006) was used to study the in vitro effect of BCT-100 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The in vivo effect of BCT-100 was studied using 5 nude mice xenograft models lines (H358, HCC827, H1650, H1975 and HCC4006). Protein expression and arginine concentration were investigated by Western blot and ELISA respectively. TUNEL assay was performed to identify apoptosis.

Results:
BCT-100 reduced in vitro cell viability across different cell lines. However, BCT-100 could only suppress tumor growth in HCC4006 xenograft model, while paradoxical growth stimulation was observed in H358, HCC827, H1650 and H1975 xenograft models. Upon BCT-100 treatment, ODC was induced in two solid tumor xenograft models (H1650 and H1975), while unaltered in cystic tumor xenograft models (H358 and HCC827) and the remaining solid tumor (HCC4006) xenograft model. In both H1650 and H1975 xenografts, combined BCT-100 and α-Difluoromethylornithine (DFMO, an ODC inhibitor) significantly suppressed tumor growth compared with control or single arm treatments with median survival doubled compared with control group. Apoptosis was activated in combination arm in both xenograft models. In HCC4006 xenograft model, the tumor suppression effect of BCT-100 arm and DFMO/BCT-100 arm was similar. Apoptosis was noted in DFMO, BCT-100 and DFMO/BCT arms.

Conclusion:
Inhibition of ODC by DFMO is essential in BCT-100 (pegylated arginase) treatment in lung adenocarcinoma.

Background:
Smoking independent lung cancers are consisted mainly of female patients, but the molecular background of this epidemiological feature other than EGFR mutation is still vague. Several studies have reported the correlation between female hormone related factors and the prognosis of lung cancer, but the results are still inconsistent. We focused on the expression of aromatase, estrogen receptor alpha (ER alpha), and estrogen receptor beta (ER beta) to investigate the carcinogenesis of smoking independent lung cancer.

Methods:
Immunohistochemistry staining (IHC) of aromatase, ER alpha, and ER beta was performed against formalin fixed tissues from 38 never-smoking patients who underwent complete surgical resection between 2012 and 2013. Among them, adequate RNA of the tumor and adjacent normal lung were extracted from deep frozen tissues of 31 patients. Considering the IHC results, quantitative RT-PCR (qRT-PCR) was performed to measure the expression level of aromatase and 3 different exons of ER alpha (exon4-5, exon6, and exon7) which composes the ligand binding motif using the Taqman© method.

Results:
Extra-nuclear expression of ER alpha with IHC showed significant correlation with pathological invasiveness, statistically. qRT-PCR results showed decreased expression of ER alpha exon 7 in invasive tumor tissues, compared with their adjacent normal tissues. This is consistent with previous in vitro results indicating that extra-nuclear ER alpha were exon7 splicing variants. There was no difference of ER alpha exon7 expression between normal and tumor tissues in non-invasive lung cancer tissues. When considering EGFR mutation status, EGFR wild type lung cancers showed lower ER alpha exon7 expressions compared with EGFR mutated lung cancers.

Conclusion:
Extra-nuclear expression of ER alpha, which may represent exon7 splicing variants of ER alpha, correlates with pathological invasiveness in smoking independent lung cancer. It may also have a part in carcinogenesis of EGFR wild type lung cancer.

Background:
The overwhelming majority of deaths due to lung cancer result from metastatic progression of the disease. Cytokines, a group of proteins involved in cell signaling, play an important role in activating the migratory and invasive capabilities of cancer cells, and studies have implicated the stromal-derived factor 1 (SDF-1/CXCL12)-CXCR4 cytokine signaling axis in the progression of several metastatic cancers, including that of the lung. Our previous investigations have shown that survival outcomes of female stage IV non-small cell lung cancer (NSCLC) patients with high CXCR4 levels are significantly worse compared to those of patients with low CXCR4, whereas male patients show no difference in survival. Studies in NSCLC cell lines have observed a link between CXCR4/SDF-1 and estrogen receptor (ER) function, as well as proliferation in response to treatment with estradiol (an estrogen) specifically in female cell lines. These previous results form the rationale for this project, which explores potential sex differences in the motility of NSCLC cell lines in response to cytokine and estrogen stimulation.

Methods:
Western blotting and PCR methodologies were used to assess the downstream activation of CXCR4 and estrogen receptor signaling as a means to confirm their activity in the cell lines studied. The migratory potential of NSCLC cells was measured using wound healing migration assays (scratch tests). Cells were incubated in phenol red-free RPMI 1640 media with or without the reagents of interest (SDF-1, beta-estradiol, estrogen and CXCR4 antagonists, among others) and the migration of cells into the wound was quantified to approximate the metastatic behavior of NSCLC cells in the presence or absence of the aforementioned stimuli.

Results:
All NSCLC cells studied showed high levels of CXCR4, but ER expression varied within our cell line panel, largely by gender of origin. Our preliminary data show a tentative but observable difference in how male and female NSCLC cells respond to both stimulation and inhibition of the CXCR4 axis. In addition, estrogen and SDF-1 co-stimulation induces a greater increase in cell motility of female NSCLC cells.

Conclusion:
The results observed may suggest a possible mechanism, through interactions between CXCR4 and estrogen receptor signalling pathways, to explain the extreme survival differences between male and female stage IV NSCLC patients with high CXCR4 expression.

Background:
Klotho gene was known as one of the anti-aging gene. We previously reported that the expression of the Klotho gene was an important postoperative prognosticator for lung large cell neuroendocrine carcinoma and lung small cell carcinoma. Recently, it has been shown that the Klotho gene suppresses epithelial-mesenchymal transition (EMT). In this study, we examined the association between the expression of Klotho and the regulation of EMT in lung squamous cell carcinoma.

Methods:
We examined the expression of Klotho in patients with lung squamous cell carcinoma, who received surgical resection or photodynamic therapy, by immunohistochemical analysis. In order to elucidate the association between the expression of Klotho and expression of EMT related protein, such as E-cadherin, N-cadherin, Vimentin and Snail, we transfected GFP-Klotho plasmid DNA into human squamous lung cancer cell line SQ5. Twenty four hours later, we sorted GFP-positive cells by flowcytometry using FACSCantoII (BD Biosciences, CA, USA), and then we examined the protein levels by Western blot analysis.

Results:
By immunohistochemical analysis, Klotho expression was observed in not only normal bronchial epithelial cells but also centrally located early lung cancers, which were all carcinoma in situ and treated by PDT. However, in lung cancer patients with invasive and or advanced squamous cell carcinoma who received surgical resection completely, Klotho expression was observed in only 4 patients (13%). In SQ5 cells transiently overexpressing GFP-Klotho, the expression of N-cadherin,which is one of mesenchymal markers, was completely inhibited compared with the SQ5 cells transfected with GFP vector. Overexpression of Klotho affected the regulation of neither other mesenchymal markers such as Vimentin and Snail nor epithelial marker, E-cadherin.

Conclusion:
We conclude that the expression of Klotho was related to the cancer invasive ness and Klotho inhibited the expression of N-cadherin, and regulates the EMT in lung cancer. Klotho may play an important role in cancer treatment and molecular-targeted therapy.

Background:
We are presenting our view of the similarities between the human/cancer cell and a theoretical biological computer. We would like to challenge the actual view on the cancer cell actions as random processes. Our hypothesis is that cancer cell is behaving as a biological computer with programmed actions and that should have an impact on the way we are dealing with cancer.

Methods:
Section not applicable

Results:
We suggest that the cancer cell should be analyzed as a digital system. Normal versus erratic cell function could be compared to normal versus erroneous computer program. In that case, we should try to find the program that has gone awry and modify it to stop the cancer instead of trying to block the peripheral effects of that program which is leading to sub-optimal results. If the cell has a program code, it could not be in the genome that we have decoded. In the digital point of view the genome is only static part representing data sets. What really makes a difference is the program code operating on that data set. In our model, that could be only the “non-coding” DNA. From our point of view, the human cell is biological computer consisting of the input units in the cell membrane, analog/digital converters in the cytoplasm and digital processing unit in the nucleus. The result of that processing is than converted through digital/analog converters (mRNA), activating different processes in the cytoplasm or leading to the synthesis of new molecules. Blocking the effectors pathways can lead to temporary slowing down of the tumor until the program code finds the solution for that obstacle. In our model, the permanent termination can be achieved only by blocking the program code. To do so, we have to find which part of the program code is active in cancer cell and with methods of reverse engineering find the solution to correct/debug/stop that program from execution.

Conclusion:
In our opinion the shift in paradigm of cancer cell from a biological mechanism to a biological computer should be made. Tailoring research based on that premise with the tools used in analyzing the unknown program code and modified to a biological system could lead to better understanding and treatment of cancer.

Background:
Cigarette smoking (CS) is well known to cause lung cancer. In addition to the mechanisms of tumorigenesis of lung cancer with CS, a lot of evidences are currently accumulating that CS induces epithelio-mesenchymal transition (EMT) in tumor cells. The correlation of CS with the malignant properties of lung cancer remained elusive in clinical settings. Here we examined the clinical significance of CS with regard to EMT and malignant progression in human lung adenocarcinoma.

Methods:
Clinical samples were obtained from the 239 cases of resected lung adenocarcinoma which were consecutively operated from January 2001 to December 2007 in our institution. Pathological stage distribution of the cases by TNM classification (WHO, 7th edition) was below: 1A: 118, 1B: 71, 2A: 22, 2B: 4, 3A: 23, 3B: 1. Smoking history was taken from all the patients, then their smoking status were classified into 3 groups according to the Brinkman Index (Non;0, n=109: Light;1~400, n=29: Heavy;401~, n=101). The samples were immunostained against E-cadherin and Vimentin using tissue microarrays of resected specimens to assess the activation level of EMT. Then, we classified into 3 groups: the group ‘N’, E-cadherin(E+) and Vimentin(V-), “null” EMT activation; the group ‘F’, E-/V+, ‘full’ EMT; the group ‘P’, E-/V- or E+/V+, ‘partial’ EMT. The numbers of the group F/ P/ N were 38/ 93/ 108, respectively. Furthermore, DNA samples were extracted from frozen surgical samples and the mutations for the hot-spot exons of EGFR, K-ras, and p53 were detected by SSCP or direct sequencing methods. The differences of survival duration, pathological invasive factors, DNA mutations and EMT activation level were statistically analysed among smoking groups.

Results:
Significant difference was found in 5-year survival rate among 3 smoking groups: Non, 89.1%; Light, 89.5%; Heavy, 61.7%. Smoking status was significantly associated with EMT activation, DNA mutation status, local invasive factors, and lymph-node metastasis. In the tumors harboring either wild-type K-ras or wild-type p53, heavy smoking was associated with EMT activation (p<0.0001, p=0.0002, respectively), whereas no correlation with regard to EGFR staus.

Conclusion:
Smoking amounts had a significant association with EMT activation level and malignant progression of human adenocarcinoma. Heavy smoking was related with EMT activation of the tumors either with wild-type K-ras or p53.

Background:
Emerging evidence has implicated ELF3 involvement in cancer signaling pathways. To determine the biological basis to pursue ELF3 as a novel therapeutic target, we investigated the role of ELF3 in lung adenocarcinoma (LUAD). Using a multi-omics approach in two independent cohorts of LUAD we (a) discover genetic mechanisms driving aberrant expression of this oncogene, (b) identify the protein-protein-interaction (PPI) partners of ELF3, and (c) determine the specific functions of ELF3 in LUAD using model systems.

Methods:
Comprehensive, multi-omic data was collected from the BC Cancer Research Centre (BCCRC), The Cancer Genome Atlas (TCGA), and several mouse models of LUAD tumourigenesis. ELF3 cellular localization was visualized by immunofluorescence. ELF3 knock-down and overexpression was achieved by lentiviral vector delivery for in vitro and in vivo assays. Physical protein-protein interaction (PPI) networks obtained from IID were overlaid onto cancer and non-malignant gene expression data from TCGA and 11 restructured datasets from Gene Expression Omnibus. PPIs were interrogated to investigate malignancy-associated ELF3 interactions. Pathway analysis was performed using pathDIP. Survival analysis was performed using the log-rank method.

Results:
ELF3 was significantly overexpressed in both cohorts, remarkably in >70% of cases (p=1.64E-21). However, mutation of known upstream regulators was not sufficient to explain the frequency of ELF3 overexpression. Instead, the ELF3 locus underwent frequent (>80%) genetic alteration including focal amplification and promoter hypomethylation, which corresponded with increased expression. ELF3 was predominantly localized to the nucleus, consistent with its transcription factor function. Analysis of PPI networks indicated highly LUAD-specific ELF3 interactions whereby loss and gain of interactions lead to reprogramming of LUAD transcriptional networks, including loss of TNFα pathway, and gain of TGFβ pathway, PI3K pathway, and translesion (DNA repair) pathway interactions. Furthermore, EGFR, KRAS, and MYC transgenic models of LUAD tumourigenesis all displayed a marked increase (6 to 8-fold) in ELF3 expression signifying its importance to LUAD of varied genetic backgrounds. In culture, ELF3 regulated proliferation, viability and anchorage-independent growth. In animal models, ELF3 knock-down cells underwent negative clonal selection, suggesting ELF3 expression is beneficial to tumour growth. Clinically, high expression of ELF3 was associated with poor survival regardless of tumour stage.

Conclusion:
Overexpression of ELF3 reprograms protein-protein-interactions in LUAD leading to the activation of cancer-specific pathways, and producing oncogenic phenotypes. Depletion of ELF3 with shRNAs reverses tumour cell growth, suggesting ELF3 is a promising therapeutic target. In addition to ELF3, interruption of cancer-specific PPIs also represents a therapeutically actionable strategy.

Background:
Cell culture models may be crucial to study lung microvascular endothelium and its role in cancer progression and treatment. In addition, a high heterogeneity among endothelial cells from various districts has been demonstrated, with greater difference on the lymphatic circulatory system. Organ-specific endothelial cells are essential to elucidate signalling pathways involved in the pathogenetic mechanisms of neoplastic lung diseases and to provide novel approaches to reach the goal of a true personalized therapy. The aim of the present study was to isolate and characterize lymphatic endothelial cells from neoplastic and healthy human lung.

Methods:
A simple and unexpensive method, requiring minimal equipment and accessories was utilized to harvest, isolate and expand lymphatic endothelial cells from human lung (Lu-LECs). Specifically, samples from lung cancer (T) and spared distal lung (D) of 46 patients undergoing lobectomy or pneumonectomy for NSCLC, were processed. To obtain a pure population of Lu-LEC, a two-step purification tool based on sorting with monoclonal antibody to CD31 and podoplanin coated paramagnetic beads was employed. Immunohistochemical analysis on harvested pulmonary tissues was performed to assess the presence or absence of neoplastic cells and to identify blood and lymphatic vasculature.

Results:
The purity of cultured endothelial cells was ascertained by morphologic criteria including TEM analysis, immunocytochemistry, flow cytometry and functional assays. T and D Lu-LECs were positive for CD31. Moreover, to define the lymphatic phenotype, we examined the specific markers podoplanin, LYVE-1 and Prox-1. No significant immunophenotypic differences between D and T Lu-LEC were detected by FACS. Although T Lu-LEC were larger than D Lu-LEC, morphologic features as cytoplasmic microvescicles, Weibel-Palade Bodies and aggregate of parallel intermediate filaments were equally observed. Cells were characterized in vitro for the ability to express several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation and in the development and progression of cancer. Cultured lymphatic endothelial cells variably expressed VEGFR-2, VEGFR-3, PDGFR-beta, c-met, and IGF-1R according to D or T origin. Moreover, FGFR-1 and EGFR-1 were present in a large fraction of T and D Lu-LEC. Matrigel assay documented that T Lu-LEC more efficiently organized in tubular structures at early time point when compared to D counterpart. Conversely, wound healing assay revealed that D Lu-LEC had a superior migratory ability.

Conclusion:
Primary lines of LECs from the human lung have been consistently obtained and may represent an important tool to study NSLCL microenvironment, lymphoangiogenesis and anti-cancer therapy.

Background:
Chronic obstructive pulmonary disease (COPD) is serious lung disease that is often associated with development of lung cancer. It is well known that both diseases share many common risk factors, most prominently smoking. Much less is known about molecular link between these two pathologies. How to predict which COPD patients will develop lung cancer? Can COPD drugs reduce or increase lung cancer risk?

Methods:
To answer these question we analyzed molecular data from tumour and normal tissue samples obtained from 72 lung cancer patients, comprising methylation, copy number aberrations, gene expression and microRNA expression data acquired from each sample. Various matching spirometric parameters, were used as indicator of severity of the airflow limitation in patients with COPD and were evaluated as potential prognostic indicators with respect to survival. We studied molecular aberrations to identify those that correlate with these parameters or differ between COPD and non-COPD patients. Using data from Broad Institute's Connectivity Map (CMAP), we analyzed gene expression effects of various pharmacological compounds, to identify potential benefits/hazards in administration of various drugs (and their combinations) typically used for treatment of COPD and/or lung cancer, with respect to prognosis of patients with COPD vs. those without COPD.

Results:
We identified group of 619 genes and 20 microRNAs whose expression is significantly associated with patient's COPD status (and severity of the disease). COPD-associated genes significantly enrich pathways related to G2 M phase of the cell cycle, G-protein coupled receptors signalling, Rho GTPases signalling and several cancer-related pathways. We found that subset of these genes constitute prognostic signature that was subsequently validated using independent publicly available dataset (HR = 2.66, p = 0.01, N = 204, GSE31210). We have also shown that alternative signature with similar prognostic power can also be constituted by COPD-associated micoRNAs (HR = 2.07, p = 0.036, N = 189, TCGA LUAD miRNAseq data). By subsequent CMAP analysis we then identified drugs that significantly (p < 0.01) affect expression of the COPD-associated genes in a manner that may improve the patients prognosis, and those that may cause its worsening. First mentioned include fenspiride – drug for obstructive airways disease and urological anti-infective phenazopyridine. Interestingly, we found calcium folinate - frequently used as a detoxifying agent for antineoplastic treatment, including treatment of lung cancer, as a potentially harmfull.

Conclusion:
Genes and microRNAs associated with COPD are significantly associated with prognosis of the lung cancer patients.

Background:
The association between pulmonary inflammation and lung cancer is well established. Smokers with chronic obstructive pulmonary disease (COPD) have higher risk of developing lung cancer than smokers without this condition. However, the molecular events underlying the association between inflammation and cancer in the lung are poorly understood. To better understand this association, an A/J mouse model was recently developed which combines exposure to the tobacco specific lung carcinogen NNK and the pro-inflammatory agent LPS. Using an innovative mass spectrometry based DNA adductomic approach, we plan to measure the DNA damage resulting from these exposures in this model.

Methods:
Both NNK and LPS can induce DNA modifications (DNA adducts), if not eliminated or repaired these adducts can result in miscoding events that can lead to misregulation of normal cellular growth control mechanisms and ultimately may result in cancer formation. Traditionally, the standard LC-MS methodology used for DNA adduct measurement focuses on the investigation of small numbers of anticipated DNA adducts based on a priori assumptions regarding their formation from specific exposures or chemicals. This approach does not account for the complexity of in vivo DNA adduct formation resulting from endogenous sources such as oxidative stress, lipid peroxidation or aberrant metabolism, or as a result of exposure to complex mixtures of chemicals which cannot be completely anticipated or predicted. To address this limitation, we have developed a high resolution/accurate mass data dependent-constant neutral loss-MS[3] methodology for DNA adductomics using ion trap-orbital trap technology to screen for all DNA modifications simultaneously.

Results:
We have successfully tested our method on mixtures of standards and applied it to lung DNA samples collected from mice exposed to NNK and to LPS. Our method allowed for the detection of the expected DNA adducts resulting from NNK as well as a number of endogenous DNA adducts resulting from lipid peroxidation, oxidative stress and aberrant metabolism resulting from the LPS induced inflammatory process.

Conclusion:
These results confirm the ability of our DNA adductomic approach to characterize the DNA damage deriving from these exposures. Our comprehensive DNA adductomic approach contributes to the development of new tools needed to investigate lung carcinogenesis, to elucidate its mechanisms and dissect the molecular pathways involved in inflammation-driven lung cancer, with the ultimate goal of identifying preventive and therapeutic strategies.

Background:
Lung cancer is the most prominent cancer in human with the highest mortality rate among cancer patients in both genders nowadays. Several models of primary lung cancer research are in use, however, no systematic evaluation of optimal models is available. Here, we assess and reappraise the most robust models of primary lung cancer for their suitability of cancer evolution and targetability for new therapeutics.

Methods:
Three models of primary lung cancer were evaluated: (I) Carcinogen (urethane or diethylnitrosamine (DEN)) induced lung cancer model, established via three intraperitoneal (i.p.) injections to BALB/c and C57BL/6 mouse strains. Five and ten months after injections, mice were assessed for tumor incidence. Lewis Lung Carcinoma (LLC) cell line was employed for an orthotopic development of lung tumor in syngeneic mouse. The cell line was injected (II) intravenously (i.v.) or (III) subcutaneously (s.c.) to establish lung tumor models in 14 days. Tumor nodules and tumor necrosis were confirmed by microscopy. Immunohistochemistry (IHC) of markers of proliferation (p-Histone3, inhibitor of differentiation 1 (Id1), and Ki67), immune cells (CD4, CD8, B220, F4/80, and NKp46), vascular structure (CD31), stroma (alpha-actin) were performed for a finer characterization of the tumor.

Results:
Ten months after i.p. injections of carcinogens, we found that the urethane model stably induced tumor nodules (90%: 9/10) when compared to DEN (30%: 3/10). BALB/c strain was significantly more susceptible for the urethane induced tumor development compared to C57BL/6 strain. Injection of LLC cell line via i.v. developed diffuse lung tumor without metastasis to other organs. s.c. injection also stably developed single tumor nodule (~500mg). IHC revealed that all tumors were consistently positive for the proliferation markers, and F4/80+ cells and CD4+ cells infiltrated into tumors significantly more than CD8+, B220+, or NKp46+ cells. Heterogeneous distributions of CD31+ cells and alpha-actin+ cells were observed in overall tumor models.

Conclusion:
The urethane-induced lung tumor is reliable and reproducible with a high rate of development and seems superior to DEN induced tumor, but need a long time period to develop. In contrast, the i.v. and s.c. tumor models are established within short time ranges. The tumors developed by s.c. enable for the analysis of the tumor only without adjacent tissue bias. The involvement and characteristics of immune cells found within tumors were comparable across all models. Injections by i.v. or s.c. of cell line to mouse can be considered as an alternative yet convenient model to develop various different types of lung cancers.

Background:
We have identified a novel serine/threonine protein kinase, MAP3K19, whose expression in normal lung was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. We have also found MAP3K19 to be expressed in multiple, primary NSCLC tumor samples, as well as human lung cancer cell lines, including A549. The kinase is transcriptionally upregulated in cells upon various types of cell stress, including oxidative, endoplasmic reticulum and osmotic stress.

Methods:
Using two different murine xenograft models, we assayed the role of MAP3K19 to inhibit the growth of either primary human NSCLC tumors and A549 cells using small molecule inhibitors.

Results:
The ability of i.v. injected A549 cells to colonize and grow in the lung was significantly reduced in mice that received orally administered, selective MAP3K19 inhibitors. Similar results were observed in a subcutaneous xenograft model, as A549 tumor cell growth was inhibited by both MAP3K19 antagonists and other standard of care kinase inhibitors. These studies also showed an additive anti-proliferative effect when gefitinib or sorafenib and MAP3K19 inhibitors were co-administered. Importantly, xenograft models using primary human NSCLC tumors implanted subcutaneously in immunodeficient mice showed a statistically significant inhibition of tumor growth when the mice were treated with the orally administered MAP3K19 antagonists. IHC analysis of the tumors showed that mice treated with the MAP3K19 inhibitors also had decreased levels of Ki-67, c-myc, p27 and phospho-Bim staining and increased caspase-3 staining.

Conclusion:
These results suggest a molecular mechanism by which MAP3K19 may inhibit tumor cell growth, and further suggest that inhibition of MAP3K19 either by itself or in combination with other therapies may represent a new avenue for the treatment of NSCLC. The clinical development of the MAP3K19 inhibitor is expected to initiate Phase I clinical trials in early 2017.

Background:
Lung cancer stem cells are considered to be responsible for lung cancer progression. However, little is known about how they actually promote lung cancer progression and metastasis.

Methods:
We retrovirally introduced three defined factors (OCT3/4, SOX2, and KLF4) into lung cancer cell line, A549. We evaluated cancer stem cell properties in the A549 cells transduced with the three factors (OSK-A549) in terms of their chemo resistance, and sphere formation ability. We also assessed lung cancer organoid constructing ability by co-culturing with mesenchymal stem cells (MSC) and human umbilical vein epithelial cells (HUVEC).

Results:
OSK-A549 cells formed dome-shaped colonies in 10 to 15 days after transfection. These colonies were picked up for further expansion in DMEM/10%FBS medium, and we named these cells OSK-A549-Colony cells. Induced OSK-A549-colony cells were more resistant to cisplatin than parental A549 cells. Cell cycle analysis revealed that the rate of G0/G1 cells was significantly increased in OSK-A549-colony cells. Sphere forming ability was enhanced in OSK-A549-colony cells. These results suggested that OSK-A549-colony cells acquired the properties of lung cancer stem like cells. Co-culture with MSC and HUVEC showed that A549 and OSK-A549-colony cells could form large spheres equally, however, HE staining of spheres revealed that OSK-A549-colony cells could form much denser spheres than those of parental cells (Figure). As the morphology was similar to real lung cancer tissue, we named this spheres “lung cancer organoids”.Figure 1



Conclusion:
By introducing defined factors, A549 cells acquired lung cancer stem cell like properties, and these cells could form lung cancer organoids by co-culturing with MSC and HUVEC. Analysis of these organoids might enable us to elucidate the molecular mechanism of lung cancer progression and metastasis.

Background:
Hypoxia, a major phenomenon in solid tumors, can promote the metastatic potential of tumor cells which is associated with chemoresistance and poor prognosis. It was reported that various angiogenesis factors including VEGF and HIF, were associated in cancer development and progression by hypoxia. In addition, both epithelial-mesenchymal transition (EMT) and cancer stem cells play an important role in malignant progression in many human tumors. We investigated the effect of hypoxic stress on the angiogenesis, EMT and stemness acquisition in lung cancer.

Methods:
Normal lung cell (BEAS-2B) and lung cancer cell lines (A549, H292, H226 and H460) were incubated in either normoxic or hypoxic (below 1% O~2~) conditions. For transcriptome analysis, mRNA of BEAS-2B and A549 cell lines were analyzed using next-generation sequencing (HiSeq 2500 system). For further validation, angiogenesis markers were analyzed by western blotting. EMT was assessed with western blotting, wound healing assay and Matrigel invasion assay, and stem cell characteristics were assessed with RT-PCR, immunostaining, soft agar colony formation assay, sphere formation assay and in vivo mice tumor model.

Results:
Next-generation sequencing revealed significant changes in the expression of angiogenesis, EMT and stem cell markers after hypoxic stress. Among the angiogenesis markers, VEGF and HIF-2α were increased. EMT markers related in hypoxia showed decrease in E-cadherin and increase in fibronectin, vimentin, N-cadherin, α-SMA, Snail, Slug, ZEB1 and ZEB2. Stem cell markers such as CXCR4, Oct4 and Nanog were increased at least one lung cancer cell line in hypoxic condition compared with in normoxic condition. Functional assays for EMT and stemness acquisition indicated that hypoxic stress increased wound healing, Matrigel invasion, sphere formation and in vivo mice tumor formation.

Conclusion:
These results suggest that hypoxia induces angiogenesis markers expression which is associated with EMT and stemness acquisition in lung cancer.

Background:
Transforming growth factor-β (TGF-β) is known to inhibit cell growth in benign cells but promotes tumor invasion and metastasis by inducing an epithelial-mesenchymal transition (EMT). EMT is a differentiation switch through which epithelial cells differentiate into mesenchymal cells. It occurs in the process of tissue morphogenesis during development, wound repair and cancer progression in adult tissues. EMT is often associated with acquisition of stem-like characteristics. In this study, we investigated whether EMT induced by TGF-β could acquire stem-like characteristics in lung cancer.

Methods:
Human normal epithelial (BEAS-2B) and cancer (A549, H292, H226 and H460) cell lines were incubated with 10 ng/ml of TGF-β for 3 days. Transcriptome and methylation analysis of BEAS-2B and A549 cells treated with TGF-β were performed by using next-generation sequencing (HiSeq 2500 system). Western blotting was performed to analyze the expression of epithelial marker (E-cadherin) and mesenchymal markers (fibronectin, vimentin, N-cadherin and α-SMA). RT-PCR was performed to analyze the expression of variable stem cell markers (CD44, CD133, CXCR4, ABCG2, CD117, ALDH1A1, EpCAM, CD90, Oct4, Nanog, SOX2, SSEA4, and CD166). Wound healing assay, Matrigel invasion assay and sphere formation assay were used to assess functional characteristics of EMT and stemness acquisition.

Results:
Next-generation sequencing revealed significant changes in the expression of stem cell markers, CD44, ALDH1A1 and CD90 in both BEAS-2B and A549 cells. The changes in the expression of EMT and stem cell markers induced by TGF-β were variable according to lung cell lines. Except for H460 cell line, lung cell lines showed at least one or more increased stem cell markers expression with TGF-β. Functional analysis revealed increased wound healing, Matrigel invasion and sphere formation after TGF-β treatment

Conclusion:
TGF-β induced EMT was associated with acquisition of stem-like characteristics. Various expression patterns of stem cell marker were observed according to different lung cancer cell lines.

Background:
As a cancer stem cell marker, CXCR4 has been known to be closely associated with cell survival and stemness acquisition. Previous studies reported that the level of CXCR4 is increased after hypoxic condition in several types of cancer. However, the mechanism of the increased CXCR4 expression has not been well understood. We investigated whether aberrant promoter demethylation could induce CXCR4 activation by using hypoxic stress in lung cancer.

Methods:
Human normal lung cell (BEAS-2B) and lung cancer cell lines (A549, H292, H226 and H460) were incubated under hypoxic condition. Transcriptome and methylation analysis using next-generation sequencing were performed by HiSeq 2500 system. For further validation, CXCR4 expression was analyzed by RT-PCR and western blotting. To determine whether CXCR4 is reactivated, cell lines were treated with a DNA methyltransferase inhibitor (AZA). Hypoxia-induced DNA demethylation was identified by methylation-specific PCR and bisulfite sequencing. Stem cell characteristics were assessed by sphere formation assay and in vivo mice tumor model.

Results:
Next-generation sequencing results revealed that CXCR4 expression was increased after hypoxic condition, whereas CXCR4 methylation was reduced. CXCR4 was activated by either hypoxic condition and treatment with AZA. MSP showed decreased CXCR4 promoter methylation in hypoxic condition compared with normoxic condition. Functional stem cell assay indicated that hypoxic stress increased sphere formation and in vivo mice tumor formation.

Conclusion:
These results suggest that hypoxia induces stem cell characteristics which are related with CXCR4 reactivation by promoter demethylation.

Background:
The degree of histologic cellular differentiation of a lung cancer has been associated with prognosis but is subjectively assessed. We hypothesized that information about tumor differentiation of individual cancers could be derived objectively from cancer gene expression data, and would allow creation of a cancer phylogenetic framework that would correlate with clinical, histologic and molecular characteristics of the cancers, as well as predict prognosis.

Methods:
We utilized mRNA expression data from NSCLC samples to explore the utility of ordering samples by their distance in gene expression from that of stem cells. A differentiation baseline was obtained by including expression data of human embryonic stem cells (hESC) and human mesenchymal stem cells (hMSC) for solid tumors, and of hESC and CD34+ cells for liquid tumors.

Results:
We found that the correlation distance (the degree of similarity) between the gene expression profile of a tumor sample and that of stem cells oriented lung cancers in a clinically coherent fashion. Cancers most similar to stem cells in gene expression are in general undifferentiated, larger, more likely to be node positive and more FDG avid on PET imaging. Most importantly,patients with cancers with gene expression patterns most similar to that of stem cells had poorer overall survival. Figure 1



Conclusion:
A stem cell oriented phylogeny of lung cancers objectively orients cancers by level of differentiation in a clinically coherent fashion. Lung cancers most similar to stem cells in expression are associated with a poorer prognosis after treatment.

Background:
Cancer stem cells (CSC) are a subpopulation of cells in the tumor with capacity for self-renewal and differentiation. Due to these characteristics, CSC are referred to as tumor initiating cells. Several studies suggest that CSC might be responsible for metastasis and resistance to conventional therapies leading to tumor recurrence. A challenge in cancer biology is to discover the biomarkers for specific types of cancer and the development of probes capable of identifying these targets. Thus, the objective of this study is the development of DNA aptamers for selective identification of the molecular signature of lung CSC.

Methods:
A549 lung carcinoma cells were used as target to perform the isolation of aptamers from a random library of DNA through the cell SELEX technique. For negative cycles for removal of DNA molecules binding to common epitopes between different cell types, blood cells were used.

Results:
CSC from A549 were expanded in vitro as tumorspheres and stemness marker expression profiles analyzed by flow cytometry. Flow cytometry comparing the cells labeled with the initial library or with the selected aptamers, showed in the latter a large increase in the labeled population. This highly fluorescence-tagged aptamer-labeled cell population was also positive for CD90, described as a marker for cancer stem cells. This double-labeled population was isolated by cell sorting with the aptamers being purified from the cells, sequenced and grouped into families based on homology between sequences. Eight aptamers were identified, whose affinity and specificity are currently being analyzed.

Conclusion:
The cell line A549 has a population of cells with stem cell characteristics. Furthermore, the selected aptamers are able to identify a subpopulation of cells, which express stem cell markers. Financial support: FAPESP; CNPq (Brazil)

Background:
Cancer stem cells (CSCs) are a small subset of tumor cells that exhibit stem cell-like properties and contribute in treatment failure. CSCs can be distinguished from other cancer cells on the basis of specific markers. Although the clinical impact of these markers is unclear, they may have a prognostic or predictive value in NSCLC.

Methods:
To clarify the expression and prognostic significance of several CSC markers in non-small cell lung cancer, we retrospectively analyzed 368 patients with adenocarcinoma (n = 226) or squamous cell carcinoma (n = 142). We correlated the expression of six CSC markers – CD133, CD44, aldehyde dehydrogenase 1 (ALDH1), sex determining region Y-box 2 (SOX2), octamer binding transcription factor 4 (OCT4), and Nanog – with clinicopathologic and molecular variables and survival outcomes.

Results:
In adenocarcinoma, CD133, ALDH1 and CD44 expression was associated with low pathologic stage and absence of lymphovascular invasion, while Nanog expression correlated with high histologic grade, lymphatic invasion and increased expression of Snail-1, a transcription factor associated with epithelial-mesenchymal transition. CSC marker expression was also associated with histologic subtypes in adenocarcinoma. Multivariate analysis showed that high Nanog expression was an independent factor associated with a poor prognosis in adenocarcinoma.

Conclusion:
In conclusion, we have shown that CSC markers may be prognostic factors in NSCLC, and high Nanog expression is an independent prognostic factor for poor survival that may be associated with EMT features in ADC patients. In addition, the clinicopathologic implication of CSC markers in lung ADC differed from those in tumors arising from other organs. Thus, the impact of CSC marker expression should be considered in a tumor/organ specific manner.

Background:
The cancer stemness niche that could promote and induce the reprogramming of cancer stem cells (CSCs), is considered as one of the key factors in cancer metastasis, tumor recurrence and drug resistance. The behaviour of this specific tumorous microenvironment may correlate with poor prognosis of the disease. However, the contribution of the cancer stemness niche on regulating the differentiation or de-differentiation of CSCs remains unclear.

Methods:
To investigate the mystery of the niche and to discover the genetic and epigenetic machineries along the reprogramming process; here, using the lung CSC/stromal cells co-culture model, integrating with both genome-wide transcriptome and DNA methylome, the gene expression and DNA methylation patterns were analyzed in lung CSCs and the differentiated clones.

Results:
We found that the stromal cells-incubated lung CSCs (StriCLS1) were significantly characterized as the CSC enriched population; with highly expressed stemness makers, nanog, oct3/4, sox2, and klf4, and showed relatively higher percentages of side population, ALDH activity and tumorigeniety than differentiated cells in both in vitro tumor sphere forming ability and in vivo xenograft model (only 10 StriCLS1 cells could generate tumor formation in NSG mice). We found that these stemness characteristics diminished as removing the feeder stroma cells and could be restored after re-co-culture with the tumorous stroma cells. Although the whole exon-seq data showed the comparability of StriCLS1 and differentiated CLS1 CSCs on the DNA sequences; the gene expression and DNA methylation patterns revealed significantly changes. The results showed that stemness and its reprogramming were related to paracrine/autocrine networks (IGFBPs, WNTs/Hedgehog, HGF/Met, LIF/LIFR), metabolism shift (PDKs and LDHs), and other drug-resistance or stress-response signalings (ABCG2 and AKTs), indicating that these key factors were regulated via the niche which may be affected by the DNA methylation patterning.

Conclusion:
We conclude that the cancer stemness reprogramming is a well-regulatory process via the paracrine/autocrine connective networks in the tumor microenvironment. This process may contribute to cancer progression, and assist the tumor growth and evolution under different stresses. This study provides new insight into the importance of crosstalk between CSCs and the cancerous microenvironment that could be targeted as potential genetic/epigenetic signaling regulators for anticancer therapy.

Background:
X-linked inhibitor of apoptosis protein (XIAP) and second mitochondrial-derived activator of caspase (Smac) are two important prognostic biomarkers for cancer. They are negatively correlated in many types of cancers. However, their relationship is still unknown in lung cancer.

Methods:
RT-PCR and Western blot were performed to explore the correlation between Smac and XIAP at the level of mRNA and protein in NSCLC patients. Full-length XIAP, Smac and mature Smac were generated by PCR and cloned into pcDNA3.0-Flag/Myc. The location of mature Smac and full-length Smac was detected by immunofluorescence. MTT assay and Flow cytometry were detected the cell viability and apoptosis of transfected cells. Caspase-3 activity was masured by Caspase-3 activity assay. Co-immunoprecipitation assay was done to reveal the direct relation between XIAP and Smac. Nude mouse xenograft experiment further proved the relation and the function of Smac and XIAP in vivo.

Results:
In this study, we found that there was a negative correlation between Smac and XIAP at the level of protein but not mRNA in NSCLC patients. However, XIAP overexpression had no effect on degrading endogenous Smac in lung cancer cell lines. Therefore, we constructed plasmids with full length of Smac (fSmac) and mature Smac (mSmac) which located in cytoplasm instead of original mitochondrial location, and confirmed by immunofluorescence. Subsequently, we found that mSmac rather than fSmac was degraded by XIAP and inhibited cell viability. CHX chase assay and ubiquitin assay were performed to illustrate XIAP degraded mSmac through ubiquitin pathway. Overexpression of XIAP partially reverted apoptotic induction and cell viability inhibition by mSmac, which was due to inhibiting caspases-3 activation. In nude mouse xenograft experiments, mSmac inhibited Ki67 expression and slowed down lung cancer growth, while XIAP partially reversed the effect of mSmac by degrading it.

Conclusion:
In conclusion, XIAP inhibits mature Smac-induced apoptosis by degrading it through ubiquitination in NSCLC.

Background:
SASH1 (SAM and SH3 domain-containing protein 1) is a recently identified gene with tumour suppressor properties and has a role in induction of apoptosis. Previous work has shown that 90 % of lung cancer cell lines have a decrease in SASH1 mRNA levels (Zeller et al., 2003), however little characterisation of SASH1 function in lung cancer has been undertaken.

Methods:
We evaluated SASH1 expression in transformed normal and malignant non-small cell lung cancer cell lines. We also utilised cell based assays to study the effects of altered SASH1 levels on cell survival and proliferation. Identification of a novel SASH1 targeting drug was performed through connectivity mapping.

Results:
SASH1 protein expression was down regulated in two of the five lung cancer cell lines compared to immortalized normal bronchial epithelial cells. Prognoscan assessment identified decreased SASH1 mRNA expression reduced patient survival. The depletion of SASH1 in lung cells resulted in a significant increase in cellular proliferation in cancer lung cells. Connectivity mapping predicted the drug Chloropyramine would lead to an increase in SASH1 expression. We demonstrated that Chloropyramine upregulates SASH1 in malignant cell lines. In keeping with this we have demonstrated the Chloropyramine inhibited lung cancer proliferation in vitro. We also explored the role of SASH1 in apoptosis. Following ultraviolet light exposure SASH1 is cleaved by Caspase-3. The C-terminal fragment of SASH1 then translocates from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild type SASH1 or cleaved SASH1 amino acids 231-1247 leads to an increase in apoptosis, however loss of the SASH1 cleavage site and/or nuclear translocation prevents this initiation of apoptosis. Mechanistically SASH1 cleavage is required for the translocation of the transcription factor NF-κB to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a co-dependence between SASH1 and NF-κB for this process.

Conclusion:
We have shown that SASH1 contributes to apoptosis via a NF-κB-dependent mechanism. Agents that upregulate SASH1, such as chloropyramine or SASH1 gene therapy, are potential novel approaches to the management of NSCLC in the future.

Background:
Ensartinib (X-396) is a novel, potent anaplastic lymphoma kinase (ALK) small molecule tyrosine kinase inhibitor (TKI) with additional activity against MET, ABL, Axl, EPHA2, LTK, ROS1 and SLK. We report data on the ALK TKI-naïve and crizotinib (C)-resistant NSCLC pts treated with ensartinib. Clinical trial information: NCT01625234

Methods:
In this multicenter expansion study, pts with ALK+ NSCLC were treated with ensartinib 225 mg daily on a 28-day schedule. Pts had measurable disease, ECOG PS 0-1, and adequate organ function. Untreated brain metastases (CNS) and leptomeningeal disease were allowed. Next Generation Sequencing (NGS) was performed on plasma samples collected at baseline and on study and compared with central tissue results (FISH/IHC). All pts were assessed for response to therapy using RECIST 1.1 and for adverse events (AEs) using CTCAE version 4.03.

Results:
80 pts (51% female) have been enrolled. Median age 54 (20-79) years, 64% ECOG PS 1. Of 40 ALK+ NSCLC pts evaluable for response; partial response (PR) was achieved in 23 pts (58%) and stable disease (SD) in 8 pts (20%). In the C-naïve pts (n = 8), PRs were observed in 7 pts (88%). In the 22 pts with prior C but no other ALK TKI, 14 pts (64%) achieved PR and 6 (27%) SD. In the 10 pts who had received two or more prior ALK TKIs, there was 2 PR, 2 SD (40% DCR). CNS responses (50% PR) have been observed in both C-naïve and C-resistant pts. Plasma and tissue genotyping were available on 27 pts (26 ALK+ and 1 ALK-). ALK was detected in plasma in 16 pts, all of whom had a response to therapy. 2 pts with PD were tissue +ve and plasma -ve. 9 plasma samples were unevaluable. Serial sequencing demonstrated a decrease in ALK in pts responding and an increase at the time of progression. The most common drug-related AEs (≥ 20% of pts) included rash (53%), nausea (32%), vomiting (26%), fatigue (23%), and pruritus (21%). Most AEs were Grade (G) 1-2. The G3 treatment-related AEs were rash (8 pts), fatigue (2 pts), pruritus (2 pts), edema (2 pts), decreased appetite (1 pt), nausea (1pt), and vomiting (1pt).

Conclusion:
Ensartinib is well-tolerated with response in both C-naïve and C-resistant ALK+ NSCLC pts, as well as pts with CNS disease. Plasma sequencing appears to be promising to select pts for therapy and monitor for response and development of acquired resistance.

Background:
Pulmonary sarcomatoid carcinoma (PSC) is a poorly differentiated subtype of non-small cell lung cancer (NSCLC). Compared with other subtypes of NSCLC, PSC has higher aggressive courses and far worse survival. The incidence of anaplastic lymphoma kinase (ALK) rearrangement is controversial, and clinical benefit from anti-ALK treatment in PSC remains unknown.This study aimed to reveal the reliable frequency and the clinical-pathologic characteristics of pulmonary sarcomatoid carcinoma (PSC) with anaplastic lymphoma kinase (ALK) rearrangement, and to provide insight into the translatability of anti-ALK treatment in this treatment-refractory disease.

Methods:
Immunohistochemistry (IHC) staining using a Ventana anti-ALK (D5F3) rabbit monoclonal antibody was performed in 141 PSC specimens collected from multiple medical centers. IHC-positive cases were then confirmed using ALK fluorescent in situ hybridization (FISH). The incidence rates and clinical-pathologic characteristics of ALK-rearranged PSC were then analyzed. Response to the ALK inhibitor crizotinib in a patient with ALK-rearranged PSC was evaluated according to the response evaluation criteria for solid tumors (RECIST) version 1.1.

Results:
A total of 5 of 141 (3.5%) of PSCs showed ALK rearrangement-positive by IHC and then were confirmed by FISH. Two were carcinosarcomas and the other three were pulmonary pleomorphic carcinoma (PPC). Strong positive ALK rearrangement was observed in both the epithelioid and sarcomatoid components. ALK rearrangement was mutually exclusive with mutations in EGFR and KRAS. The median age of ALK-positive patients was younger than that of ALK-negative patients. PSCs in never-smokers was more likely to harbor ALK rearrangement than those in former or current smokers (P <0.05). A 40-year-old woman diagnosed with ALK-rearranged PPC experienced a partial response (-32%) to the ALK inhibitor crizotinib.

Conclusion:
The incidence rates of ALK rearrangement in PSC in the Chinese population are similar to those of other subtypes of NSCLC. PSCs in younger never-smokers are more often to harbor ALK rearrangement. ALK inhibitors may serve as an effective treatment for ALK-rearranged PSC.

Background:
Anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) rearrangements represent two of the most frequency fusion targets in lung adenocarcinoma. The aim of this study was to investigate the clinicopathological characteristics, coexistence and treatment of ALK and ROS1- rearrangement patients in lung adenocarcinoma without EGFR mutations.

Methods:
Patients who harbored EGFR wild-type gene were screened for ALK and ROS1 gene in four Hospitals in China. ALK and ROS1 rearrangements were detected using RT-PCR. Progression free survival curves were plotted using the Kaplane-Meier method.

Results:
Seven hundred and thirty-two patients enrolled in current study.Of the 732 patients, the median age was 59 years (range: 28–81). ALK and ROS1 rearrangements were detected in 89 (12.2%) and 32 (4.4%) patients,respectively. One patient was identified with ALK/ROS1 coexistence. Both of ALK and ROS1 positive were predominantly found in younger and non-smokers. More patients with ALK/ROS1 rearrangements were associated with TTF1 expression，napsin A expression and solid predominant adenocarcinoma subtype(Figure 1 ). Figure 1Figure 2





Conclusion:
ALK and ROS1 rearrangements frequency was enriched in EGFR wild-type patients and ALK/ROS1 coexistence was rare. The ALK and ROS1 arrangements were associated with age, smoking status, TTF1 expression, napsin A expression and solid predominant adnocarcinoma subtype.

Background:
ALK (Anaplastic Lymphoma Kinase) translocation is a rare oncogenic driver in NSCLC (Non Small Cell Lung Cancer) (3 to 5%) and few data are published on the management of these patients outside patients included in clinical trial. objective:to investigate clinical characteristics and management of these patients in real world setting.

Methods:
inclusion of patients with a diagnosis of NSCLC harboring ALK translocations between January 2012 and December 2014, collection of demographic and clinical characteristics, risk factors, Progression free survival (PFS), Overall Survival (OS), mode of progression and therapeutic management

Results:
132 patients recruited in 31 centers: 67(51%) men; age: 60.1 ± 14,5 years; PS 0/1 at diagnosis: 89%; non smokers:79%, adenocarcinoma: 93%; Stage at diagnosis 4/3/2-1:74%/19%/7%: co-mutations EGFR n=2, BRAF n=2, KRAS = 1, HER2 = 1. Outcomes of stage IV (n=97): first line treatment: chemotherapy: 75%, Best supportive care (BSC): 1 %, anti ALK: 24 %; response rate, disease control rate (DCR) and PFS to first line treatment: 42 %,64% and 7.5 months (CI 5.9 ;9.5), second line treatment (n=60): chemotherapy: 25%, anti BRAF 72%, BSC 3 %; response rate, DCR and PFS to second line treatment: 43.4%,70% and 4,7 months (CI 4.0; 8.1); 2 years-OS: 56.7% (CI 45.5 ;70.4), mediane OS was not reach.

Conclusion:
In this real world analysis, the majority of NSCLC patients with ALK translocation were non smokers,adenocarcinoma and appears to have a better survival to NSCLC pts without oncogenic driver. Clinical trial information: Supported by an academic grant from Lilly, Astra Zeneca, boehringer ingelheim

Background:
The purpose of the study was to explore the association between the percentage of ALK+ cells in fluorescent in situ hybridization (FISH) and the clinical efficacy of ALK inhibitor.

Methods:
A total of 73 patients (pts) with ALK rearrangement who were identified the percentages of ALK+ cells in FISH and were treated with ALK inhibitor, were enrolled at three participating institutions. Retrospectively, we evaluated progression-free survival (PFS) by log-rank test and Kaplan–Meier method.

Results:
Median % ALK+ cells in FISH was 54% (range 15-100%). Fifty (68.5％) pts used crizotinib (CRZ) and 23 (31.5%) pts used alectinib (ALC) as the first ALK inhibitor. Among 57 pts who were evaluated by immunohistochemical (IHC) staining, 10 had no detectable expression of the ALK protein and the percentage of ALK+ cells was all within 15-29%. The PFS of pts with 15-29% ALK+ cells was shorter than that with 30% or more ALK+ cells (CRZ group: 1.4 months [95% CI: 0.2-4.2] in 15-19% ALK+ cells, 3.25 months [0.47-Not Estimated (NE)] in 20-29%, 6.77 months [4.27-12.6] in 30-100%, p < .001. ALC group: 6.4 months [3.03-16.8] in 20-29%, 23.1 months [7.8-NE] in 30-100%, p = 0.547). Moreover, among pts with 15-29% ALK+ cells, median PFS of pts with IHC- was significantly shorter than that with IHC+ (CRZ group: 1.3 vs 5.6 months, p = 0.009. ALC group: 0.87 vs 40.3 months, p = 0.004).

Conclusion:
The case in 15-29% ALK+ cells and IHC- could not obtain the benefit of the ALK inhibitor. However, if the case is IHC+, the long PFS could be expected despite the percentage of ALK+ cells in FISH.

Background:
Although several tyrosine kinase inhibitors have potent antitumor activity against ALK-rearranged non-small cell lung cancers (NSCLC), resistance to these small molecules emerges through a number of mechanisms. Preclinical evidence suggests that ALK-positive NSCLCs can also be successfully targeted immunologically using vaccine-based approaches. Immunologic responses against the ALK protein have been reported in ALK-positive anaplastic large cell lymphoma, and we sought to determine whether ALK could be recognized by the immune systems of patients with ALK-positive NSCLC.

Methods:
Serum was collected from 32 ALK-positive and 29 ALK-negative NSCLC patients over the course of routine clinical care who had consented to an institutional review board approved translational research protocol. We developed a novel enzyme-linked immunosorbent assay (ELISA) to detect autoantibodies against the ALK cytoplasmic domain in patients with ALK-rearranged NSCLC, and the specificity of these autoantibodies was validated using Western blot analysis. Short peptides spanning the length of the ALK cytoplasmic domain were synthesized to more narrowly define the precise immunogenic peptide sequences.

Results:
Among 32 ALK-positive NSCLC patients, very high ALK autoantibody titers were detected in the serum of 3 patients (9%), and ALK autoantibodies were not detected in any of the 29 patients with ALK-negative NSCLC. These autoantibodies specifically recognized only the ALK cytoplasmic domain and not the ALK extracellular domain. Epitope mapping demonstrated that the autoantibodies from each of the 3 patients with high autoantibody titers recognized distinct ALK peptide sequnces within the ALK cytoplasmic domain.

Conclusion:
ALK is capable of being recognized by the immune systems in some patients with ALK-positive NSCLC. Further investigation is needed to determine whether the presence of anti-ALK antibodies impacts prognosis in NSCLC. The naturally immunogenic properties of ALK in NSCLC may be able to be exploited using therapeutic ALK vaccines in patients.

Background:
Crizotinib is recommended as first-line therapy for non-small cell lung cancer (NSCLC) patients with ALK rearrangements. Following the approval of second-generation ALK inhibitors for patients who progress on or are intolerant to crizotinib, this study describes how physicians monitor for progression, diagnose progression, and alter treatments following progression on crizotinib therapy.

Methods:
A panel of US oncologists was surveyed regarding their monitoring practices and criteria for diagnosing progression on crizotinib. From March to June 2016, the oncologists provided data retrospectively from the medical charts of their adult patients diagnosed with locally-advanced or metastatic ALK+ NSCLC who progressed on crizotinib following the US approval of the first second-generation ALK inhibitor, ceritinib, in April 2014. Time to clinician-defined progression and treatment changes following progression were assessed using the medical chart data.

Results:
28 oncologists responded to the survey. Data was abstracted on 74 ALK+ NSCLC patients who progressed on crizotinib. 49% of the patients were male; 50% were never smokers. 81% of patients received crizotinib in first line; the median age at initiation was 61 years. Most physicians (71%) reported monitoring for radiographic progression every 3-4 months. In terms of course of action when new lesions are detected, physician response varied: most physicians (75%) prefer to add local therapy and resume crizotinib following a symptomatic isolated lesion, while, following multiple symptomatic lesions, 96% and 64% of physicians prefer to switch to a new therapy depending upon whether the lesions were systemic or isolated to the brain, respectively. Among the study sample, progression on crizotinib, as defined by physicians, was detected after a median of 10.4 months. 86% of patients discontinued crizotinib within 30 days of diagnosis of the physician-defined progression. Among all patients who discontinued, 77% switched to ceritinib, 14% to chemotherapy, and 1% to alectinib; the remaining 7% did not receive additional systemic antineoplastic therapy.

Conclusion:
The findings from this physician survey and retrospective chart review of ALK+ NSCLC crizotinib-treated patients suggest that physician response to the development of new lesions varies depending upon the location and extent of the lesions. Once physicians considered their patients to have progressed, most immediately switched their patients to ceritinib, a second-generation ALK inhibitor.

Background:
The identification of EML4-ALK translocations in 3%-5% of NSCLC resulted in the approval of numerous targeted therapies of these lung cancers. Consequently, the availability of new ALK inhibitors forced the development of diagnostic assays, although their validation is limited by the restricted number of available specimens. As the significance of the current standard test (tissue-based FISH) is progressively questioned, development of improved accompanying diagnostics for the predictive biomarker is of high medical need. Overcoming discrepancies of FISH such as tissue sample quality and inter-observer variability, we validated a liquid biopsy test for analysis of EML4-ALK fusion transcripts. For the first time, the availability of a defined cohort of NSCLC patients allowed the determination and clinical interpretation of EML4-ALK in exosomal RNA derived from plasma specimen.

Methods:
We developed and validated an exosome-based biopsy test for EML4-ALK to be run in a CLIA laboratory. The assay enables the discriminative detection of the three major fusion variants v1, v2 and v3(a,b,c). In detail, exosomal RNA is isolated from low-volume plasma (≤ 2ml) of NSCLC patients and subjected to highly sensitive and specific RT-qPCR. During analysis, each sample is confirmed by defined test controls and process tracking. The final diagnostic results of all clinical specimen are correlated with clinical response data.

Results:
Section is not applicable. Cohort analysis is ongoing. Therefore, we will submit our results as late-breaking abstract.

Conclusion:
Section not applicable. We will determine the variant-specific expression of EML4-ALK in plasma samples of a clinically defined, medium-sized NSCLC cohort. Based on specimen data, we are able to test for correlation of the biomarker derived from exosomes with respective tissue results and clinical response of a patient. Complete results and conclusions will be based on the analysis of all clinical samples. As the respective cohort is currently in the status of sample collection and testing for EML4-ALK (ongoing until October 2016), the final data will be submitted as late-breaking abstract.

Background:
ALK and ROS1 kinase inhibitors have achieved tremendous success in the treatment of lung cancer patients. However, the emergence of drug resistance limits their long term clinical applications. The mechanisms of resistance often include gene amplification, acquired mutations, bypass signaling, and epithelial-mesenchymal transition (EMT). The bypass and EMT-based resistances constitute the majority of the resistant patient population, especially after multiple kinase inhibitor treatment. None of the current ALK or ROS1 inhibitors can overcome bypass or EMT-based resistance when applied as a single agent therapy. SRC kinase has been identified to contribute broadly to cancer treatment resistance via participation in signaling pathways required for DNA synthesis, control of receptor turnover, actin cytoskeleton rearrangement, migration, adhesion, invasion, motility, and survival. SRC/FAK signaling plays important roles in regulating antitumor immunity, cancer stem-like properties, and EMT. Here we deployed a polypharmacology approach to combatting multiple resistance mechanisms spontaneously.

Methods:
Recombined enzyme assays, enginnered cell lines and H2228 cells were used to evaluate TPX-0005 in in vitro and in vivo models.

Results:
TPX-0005 is a potent ALK/ROS1/TRK inhibitor with a rigid three-dimensional macrocyclic structure and a much smaller size (MW <370) than current ALK/ROS1/TRK inhibitors. The compact structure allows TPX-0005 efficiently target the center of ATP binding site and be able to circumvent the steric interference from clinical resistant mutations. Therefore, TPX-0005 potently inhibited both wild type and mutant ALK/ROS1/TRK fusion proteins including gatekeeper and solvent front mutations at low nanomolar concentration. In addition to its primary targets, TPX-0005 is also a potent SRC/FAK inhibitor. H2228 lung cancer cell line, endogenously expressing EML4-ALKv3 protein, is refractory to crizotinib and ceritinib in cell proliferation assay (IC~50~ ~1 μM). The upregulation of multiple RTKs including EGFR and IGFR, as well as cancer stem cell marker CD44 in H2228 cells is believed to confer the primary resistance to selective ALK inhibitors. Inhibition of SRC/FAK kinases will modulate RTK expression and cancer stem-like properties to restore the sensitivity to ALK inhibitor. TPX-0005 inhibited the phosphorylation of EML4-ALK (IC~50~ 13 nM), SRC and FAK (IC~50~s 70-80 nM), along with other downstream signaling targets in H2228 cells, leading to dose-dependent down-regulation of EGFR and CD44 expression levels. As a result, TPX-0005 overcame the primary resistance and effectively inhibited cell proliferation (IC~50~ ~0.1 μM) and cell migration of H2228 cells.

Conclusion:
TPX-0005 exerts unprecedented polypharmacology profile for combatting multiple resistance mechanisms including acquired mutations, bypass signaling, cancer stemness, and metastasis, that warrants further clinical investigation.

Background:
In advanced lung cancer patients the gold standard for detecting ALK gene rearrangements is fluorescence in situ hybridization (FISH), and ALK protein expression can be also evaluated by immunohistochemistry (IHC). A single analysis performed alone may not detect all the ALK-positive cases and some patients with discordant FISH and IHC respond to tyrosine kinase inhibitors (TKIs). In this study we evaluated ALK aberrant expression in lung cancer patients by a reverse-transcription (RT)-PCR, to investigate its clinical utility and its concordance with FISH and IHC.

Methods:
ALK aberrant expression was retrospectively investigated on RNA from formalin-fixed paraffin-embedded tissue (FFPEt) of 24 advanced lung adenocarcinoma patients, previously evaluated by FISH and IHC. We used a one-step Scorpion RT-PCR that allowed in a single reaction either the mRNA reverse transcription and the cDNA amplification for ALK kinase-domain, normally not expressed, and a control gene, to assess RNA quality.

Results:
Results are reported in Table 1.Figure 1



Conclusion:
Despite the instability of mRNA from FFPEt, only 2 samples resulted inadequate for RT-PCR. RT-PCR was in disagreement with both FISH and IHC in one case, which is likely to be a RT-PCR false positive. RT-PCR did not detect ALK aberrant expression in a FISH positive case, which was negative also by IHC; unfortunately, this patient died after a cycle of pemetrexed therapy, before undergoing a second line TKI treatment. The presence of ALK rearrangements does not necessarily imply increased protein levels, because of the complex transcriptional and post-transcriptional regulations, so further analysis at RNA levels may clarify discrepancy between FISH and IHC allowing a better stratification of patients who could benefit from TKIs. Therefore, according to our results the RT-PCR evaluating ALK aberrant expression regardless of the fusion partners should be considered for introduction into routine ALK testing in lung cancer.

Background:
Gene fusion of EML4-ALK and gene mutation in ALK kinase domain are important determinants of the response of ltargeted therapy in lung cancer. In this study, Droplet Digital PCR (ddPCR) were used to assess the ALK gene status of blood-based nucleic acid, to develop a non-invasive assessment for treatment and progresses of lung cancer.

Methods:
Three FFPE sanples and 17 peripheral blood samples were collected from 11 patients with lung cancer, 5 of which were detected 2 – 5 times follwing their treatments. ddPCR technology were used to identify rearrangement of EML4-ALK in RNA from the peripheral blood samples and FFPE samlples, and mutations in ALK kinase domain from 12 of the 17 peripheral blood. Correlation of responses to ALK inhibitors and progress in tumor based on ALK gene status were analysed.

Results:
Rearrangement of EML4-ALK were detected in all the 3 (100%) FFPE samples, and 2 of 4 (50%) blood samples by ddPCR in initial. Gene mutations including L1152R, C1156Y, F1174L, L1196M, D1203N and G1269A in ALK kinase domain were detected in 10 of 12 (88.3%) blood samples after ALK inhibitors treatment. L1152R and D1203N were detected more frequent (29.6% for both), and other 4 mutations were detected a frequency of 3.7%. For most patients detected more than 1 time, the abundance of EML4-ALK, L1152R and D1203N were found decreased after ALK inhibitors treatment (Fig. 1). LDK378 were used after resistance to crizotinb developed in patients BMS, ZQ and HSR. C1156Y, which interferes drug binding, were found in patients BMS and ZQ who showed poor response to LDK378 treatment ; whereas L1196M, which enhances drug binding, was found in patient HSR who showed good response to LDK378 .Figure 1



Conclusion:
ALK gene status in peripheral blood detecting by ddPCR could be a viable approach for non-invasive analysis of tumour genotype in real time.

Background:
The phase 3 study PROFILE 1014 showed a superior outcome of crizotinib over pemetrexed-cisplatin/carboplatin (PCC) in ALK+ NSCLC.[1] PROFILE 1029 is an ongoing phase 3 study of similar design (NCT01639001) conducted in an East Asian population in China, Hong Kong, Malaysia, Taiwan, and Thailand. Here, we present findings on patient-reported symptoms and QoL.

Methods:
Patients with previously untreated, ALK+ advanced NSCLC were randomized 1:1 (stratification: ECOG PS 0 or 1 vs 2) to crizotinib 250 mg PO BID or pemetrexed 500 mg/m[2] with cisplatin 75 mg/m[2] or carboplatin to an AUC of 5–6 mg·min/mL, IV Q3W for ≤6 cycles. The EORTC QLQ-C30 and lung cancer-specific module (QLQ-LC13) questionnaires were completed at baseline, days 1, 7, and 15 of Cycle 1, day 1 of each subsequent cycle, and at end-of-treatment or withdrawal. Mixed model analyses were used to evaluate changes from baseline and between treatment arms.

Results:
Patients, who received crizotinib (n=103) had significantly longer median time to deterioration (TTD) for chest pain, dyspnea, or cough compared to PCC (n=98) (2.8 mo [95% CI: 1.4, 6.9] vs 0.3 mo [95% CI: 0.3, 0.5], respectively; HR=0.432 [95% CI: 0.307, 0.610]; P<0.0001). Crizotinib treatment, compared to PCC, was associated with a significantly greater change from baseline in global QoL and physical, role, cognitive, and social functioning (Table). QLQ-C30 results demonstrated that crizotinib-treated patients experienced either improvement or reduced worsening of most symptoms compared to PCC-treated patients. QLQ-LC13 data showed improvement or reduced worsening across all symptoms for crizotinib, relative to PCC. Figure 1



Conclusion:
Crizotinib treatment significantly delayed TTD of lung cancer symptoms (chest pain, cough, dyspnea). Crizotinib was associated with significantly greater improvements from baseline in key patient-reported lung cancer symptoms and global QoL, compared with PCC. Reference: 1. Solomon BJ, et al. J Clin Oncol. 2016 [Epub ahead of print].

Background:
Brigatinib, an investigational next-generation ALK inhibitor, has yielded promising activity in crizotinib-treated ALK+ NSCLC patients in a phase 1/2 trial (NCT01449461). As responses and adverse events (AEs) varied with starting dose, two brigatinib regimens are under evaluation in ALTA (NCT02094573).

Methods:
Patients with crizotinib-refractory advanced ALK+ NSCLC were randomized 1:1 to receive brigatinib at 90 mg qd (arm A) or 180 mg qd with a 7-day lead-in at 90 mg (arm B) and stratified by presence of brain metastases at baseline and best response to prior crizotinib. Primary endpoint was investigator-assessed confirmed ORR per RECIST v1.1.

Results:
222 patients were enrolled (arm A, n=112/arm B, n=110). Median age (A/B) was 51/57 years, 55%/58% were female, 74%/74% previously received chemotherapy, and 71%/67% had brain metastases. As of February 29, 2016, 64/112 (57%) patients in arm A and 76/110 (69%) patients in arm B were receiving brigatinib; median follow-up was 7.8/8.3 months. The Table shows investigator-assessed endpoints by arm and subgroup for select baseline characteristics. Independent review committee–assessed endpoints (A/B, n=112/n=110; as of May 16, 2016): confirmed ORR 48%/53%, median PFS 9.2/15.6 months. Any-grade treatment-emergent AEs (≥25% overall frequency; A/B, n=109/n=110 treated): nausea (33%/40%), diarrhea (19%/38%), headache (28%/27%), cough (18%/34%); grade ≥3 events (excluding neoplasm progression; ≥3% frequency): hypertension (6%/6%), increased blood CPK (3%/9%), pneumonia (3%/5%), increased lipase (4%/3%). A subset of pulmonary AEs with early onset (median onset: Day 2) occurred in 14/219 (6%) treated patients (3%, grade ≥3); 7/14 patients were successfully retreated. No such events occurred after escalation to 180 mg in arm B.

Conclusion:
In each arm, brigatinib yielded substantial responses and prolonged PFS, with an acceptable safety profile. 180 mg with 90 mg lead-in was not associated with increased early pulmonary events and showed a consistent improvement in efficacy, compared with 90 mg, particularly with respect to PFS.

Investigator-Assessed Endpoints by Arm and Subgroup

	Confirmed ORR, n/N(%)			Median PFS, months
Arm	A	B	A+B	A	B	A+B
All patients	50/112(45)	59/110(54)	109/222(49)	9.2	12.9	11.1
Prior chemotherapy
Yes	35/83(42)	44/81(54)	79/164(48)	8.8	12.9	11.8
No	15/29(52)	15/29(52)	30/58(52)	9.2	8.1	9.2
Race
Asian	18/39(46)	18/30(60)	36/69(52)	8.8	11.1	11.1
Non-Asian	32/73(44)	41/80(51)	73/153(48)	9.2	12.9	11.8
Brain metastases at baseline
Yes	31/80(39)	43/74(58)	74/154(48)	9.2	11.8	11.1
No	19/32(59)	16/36(44)	35/68(51)	7.4	15.6	15.6
Best response to prior crizotinib
Partial+complete	36/71(51)	47/73(64)	83/144(58)	11.1	15.6	15.6
Other	14/41(34)	12/37(32)	26/78(33)	7.4	12.9	9.2
ORR=objective response rate PFS=progression-free survival

Background:
Patients with advanced NSCLC typically experience symptoms compromising their quality of life (QoL), making this an important therapeutic goal. PROFILE 1029 (NCT01639001) is an ongoing open-label, Phase 3 study in East Asian patients with previously untreated, ALK+ advanced NSCLC in China, Hong Kong, Malaysia, Taiwan, and Thailand. Patient-reported health status outcomes are presented.

Methods:
Patients were randomized 1:1 (stratification: ECOG PS 0 or 1, 2) to crizotinib 250 mg PO BID or pemetrexed 500 mg/m[2] IV with either cisplatin 75 mg/m[2] or carboplatin (PCC) to achieve an AUC of 5–6 mg·min/mL, IV q3w for ≤6 cycles. Health status assessed using the EuroQoL 5D (EQ-5D) was a secondary endpoint of the study. The EQ-5D, consisting of a visual analogue scale (VAS) score ranging from 0 (worst imaginable health state) to 100 (best imaginable health state) and a descriptive measure (no, some or extreme) of problems in 5 dimensions: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression, was to be completed at baseline and on day 1 of each cycle until treatment termination or withdrawal. The data were analyzed using a mixed model analysis.

Results:
The proportion of patients completing at least one question on the EQ-5D questionnaire ranged from 97.6% to 100% for crizotinib-treated patients over 42 cycles of treatment and from 98.0% to 100% for PCC-treated patients over a maximum of 6 cycles of treatment. The estimated overall change from baseline in the EQ-5D VAS was 3.4209 (95% confidence interval [CI]:1.20, 5.64) for crizotinib and ‑0.4927 (95% CI: -2.75, 1.77) for PCC. The difference between crizotinib and PCC was 3.9136 (95% CI: 0.85, 6.98; P<0.05). The estimated overall change from baseline in the EQ-5D utility score was 0.0502 (95% CI: 0.02, 0.08) for crizotinib and 0.0077 (95% CI: -0.02, 0.04) for PCC. The difference in utility score changes for crizotinib versus PCC was 0.0425 (95% CI: 0.00, 0.08; P<0.05). End of treatment descriptive results showed no deterioration from baseline across most EQ-5D dimensions.

Conclusion:
A statistically significantly (p-value <0.05) greater improvement from baseline in general health status, as assessed by the EQ-5D VAS and utility scores, was observed for crizotinib-treated patients compared with PCC-treated patients.

Background:
In the open-label, phase 1 ASCEND-1 study (NCT01283516), ceritinib demonstrated durable whole body and intracranial responses in patients with ALK-rearranged (ALK+) non-small cell lung cancer (NSCLC) (Kim et al. Lancet Oncol 2016). Median progression-free survival (PFS) was promising in ALK inhibitor (ALKi)-naïve patients (18.4 months), most of whom had received one or more lines of chemotherapy. Here, efficacy and safety of ceritinib are summarized in a subset of treatment-naïve patients enrolled in ASCEND-1.

Methods:
Patients with ALK+ NSCLC enrolled worldwide received ceritinib 750 mg/day (fasted). Efficacy and safety were evaluated in a subset of patients who had not received any prior systemic antineoplastic therapy. Data cut-off was 16 November 2015.

Results:
Overall, 246 patients with ALK+ NSCLC (83 ALKi-naïve and 163 ALKi-pretreated) received ≥1 dose of ceritinib, of whom 16 had not received any prior systemic antineoplastic therapy. Among the 16 treatment-naïve patients, three (18.8%) had baseline brain metastases, five (31.3%) an ECOG performance status of 0, and all had stage IV disease. Median time from primary site diagnosis to ceritinib initiation (range) was 1.8 months (1.0–35.9). At data cut-off, median duration of exposure (range) was 18.5 months (0.9–35.7) and median duration of follow-up (range) was 29.6 months (4.7–39.1). In these 16 treatment-naïve patients, per investigator assessment, the overall response rate was 68.8% (95% confidence interval [CI]: 41.3, 89.0) and the disease control rate was 87.5% (95% CI: 61.7, 98.4). Median duration of response was 21.1 months (95% CI: 5.5, 31.1). Median investigator-assessed PFS was 19.3 months (95% CI: 4.2, 26.3), and median overall survival was 39.1 months (95% CI: 19.1, 39.1). Among three patients with baseline brain metastases, one had brain metastases selected as target lesion and achieved a partial intracranial response. The most frequently reported any-grade adverse events (AEs), regardless of study drug relationship, were diarrhea (93.8%), nausea (81.3%), ALT or AST increase (each 81.3%), and vomiting (62.5%). AEs requiring intervention were predominantly managed with dose reduction/interruption. Overall, 13 patients (81.3%) discontinued treatment, due to disease progression (n=6), consent withdrawal (n=3), AEs (n=2), administrative problems or death (each n=1).

Conclusion:
Ceritinib demonstrated durable efficacy in treatment-naïve patients with ALK+ NSCLC. Safety was consistent with the overall ASCEND-1 study population. An ongoing, prospective, phase 3 study (ASCEND-4) in which patients are randomized to receive either ceritinib or chemotherapy will provide further evidence for the clinical benefit of ceritinib in previously untreated patients with ALK+ NSCLC.

Background:
Alectinib is an FDA-approved ALK TKI, for treatment of patients with ALK+ metastatic NSCLC who have progressed on, or are intolerant to, crizotinib. Systemic and CNS efficacy was demonstrated in two single-arm, phase II studies (NP28673 [NCT01801111] and NP28761 [NCT01871805]). We report the pooled systemic efficacy and safety analysis of alectinib from 2016 cut-offs 22 January, NP28761 and 1 February, NP28673.

Methods:
Patients were ≥18 years, had locally advanced or metastatic ALK+ NSCLC [FDA-approved FISH test] and had progressed on, or were intolerant to, crizotinib. Patients received oral alectinib 600mg twice daily until disease progression, death or withdrawal. The pooled analysis assessed objective response rate (ORR) by an independent review committee (IRC) using RECIST v1.1 (primary endpoint in both studies); disease control rate (DCR); duration of response (DOR); progression-free survival (PFS); overall survival (OS); and safety.

Results:
The pooled dataset included 225 patients, (n=138 NP28673; n=87 NP28761). Median age was 53 years, 60% of patients had baseline CNS metastases and 77% had received prior chemotherapy. The response-evaluable (RE) population by IRC included 189 patients (84%). Median follow-up was 18.8 months (0.6–29.7). In the RE population (n=189) ORR by IRC was 51.3% (95% CI 44.0–58.6; all partial responses), a DCR of 78.8% (95% CI 72.3–84.4), with a median DOR of 14.9 months (95% CI 11.1–20.4) after 58% of events. In patients with prior chemotherapy (n=148), IRC ORR was 49.3% (95% CI 41.0–57.7); DCR: 79.1% (95% CI 71.6–85.3); median DOR: 14.9 months (95% CI 11.0–21.9) after 59% of events. In patients who were chemotherapy-naïve (n=41), IRC ORR was 58.5% (95% CI 42.1–73.7); DCR: 78.0% (95% CI 62.4–89.4); median DOR: 11.2 months (95% CI 8.0–NE) after 54% of events. In the total pooled population (n=225) median PFS by IRC was 8.3 months (95% CI 7.0–11.3) after 69% of events and median OS was 26.0 months (95% CI 21.4–NE) after 43% of events. Grade ≥3 adverse events (AEs) occurred in 40% of patients and the most common were dyspnoea (4%), elevated levels of blood creatine phosphokinase (4%) and alanine aminotransferase (3%). The mean dose intensity was 94.6%. Fourteen patients withdrew due to AEs; 20.9% had AEs leading to dose interruptions/modification.

Conclusion:
This pooled analysis confirmed alectinib has robust systemic efficacy with a durable response in this population and in patients with or without prior chemotherapy. Alectinib had an acceptable safety profile.

Background:
Comparing the efficacy of ALK inhibitors in post-crizotinib therapy of ALK+ NSCLC is hampered by the lack of comparator ALK inhibitor treatment arms in pivotal studies. An indirect naive comparison was undertaken to explore study results for the investigational ALK inhibitor brigatinib and the currently available agents alectinib and ceritinib following progression on crizotinib. Baseline characteristics were examined to determine if the distribution of prognostic factors differed across studies, and outcomes were compared.

Methods:
Patient characteristics and study outcomes (objective response rate [ORR], progression-free survival [PFS], duration of response [DOR], and adverse events [AEs]) for alectinib, brigatinib, and ceritinib were extracted from pivotal study publications identified in a systematic literature review, alectinib prescribing information, and brigatinib data in post-crizotinib settings. Outcomes were compared over the longest follow-ups reported.

Results:
All pivotal studies were multicenter and open label; populations were similar in median age, sex ratio, and baseline disease stage. Slight imbalances among studies exist in Eastern Cooperative Oncology Group/World Health Organization performance status and central nervous system metastases at baseline. ORR was numerically higher in the brigatinib phase 1/2 study for subjects receiving 180 mg once daily with 7-day lead-in at 90 mg versus other studies (Table). Median PFS and DOR were also higher in brigatinib studies versus alectinib and ceritinib studies; PFS 95% confidence intervals did not overlap between brigatinib and ceritinib studies. Rates of discontinuation due to AEs were similar across studies, but AE-related dose reductions were most frequent in ceritinib studies.Figure 1



Conclusion:
Pivotal trials for ALK inhibitors share many similarities, making an indirect comparison possible. Naive comparison suggests brigatinib may have a favorable efficacy profile compared with currently available therapies, while ceritinib may require dose reduction more frequently to manage AEs. Further analyses are needed to determine the magnitude and direction of potential bias.

Background:
Previous retrospective studies suggested that lung cancer patients with anaplastic lymphoma kinase (ALK) gene rearrangements are associated with sensitivity to pemetrexed chemotherapy. To determine the efficacy of pemetrexed based chemotherapy compared with non-pemetrexed based chemotherapy, we retrospectively evaluated clinical outcome in ALK positive non-small cell lung cancer (NSCLC) patients.

Methods:
We identified 126 patients with advanced, ALK-positive NSCLC who received 1st line cytotoxic chemotherapy from Seoul National University Hospital and Seoul National University Bundang Hospital. We compared response rate, progression-free survival, and overall survival according to chemotherapy regimens. We also analyzed the intra-cranial time to progression and proportion of ALK-positive cells as a predictive factor of pemetrexed efficacy.

Results:
Forty eight patients received pemetrexed based chemotherapy and Seventy eight patients received non-pemetrexed based chemotherapy as first line systemic treatment. One hundred eighteen patients received platinum double combination chemotherapy. The pemetrexed based chemotherapy group shows superior overall response rate (44.7% versus 14.3%, p<0.001) and disease control rate (85.1% versus 62.3%, p=0.008). Pemetrexed based chemotherapy group had longer progression free survival (6.6 months versus 3.8 months, p<0.001). Exposure to pemetrexed and exposure to second generation ALK inhibitor were independent prognostic factors of overall survival (p=0.016 and p=0.011, respectively). Intra-cranial time to progression (TTP) was similar among treatment group (32.7 months versus 35.7 months, p= 0.733). Proportion of ALK positive cells was not statistically significant predictive factor of survival in pemetrexed based chemotherapy.

Conclusion:
Pemetrexed based regimen may prolong progression free survival compared with other regimens in ALK positive NSCLC in the first line setting. Exposure to pemetrexed is associated with improved survival compared with that of premetrexed-naive controls in ALK positive NSCLC.

Background:
It is estimated that 3-5% of non-small cell lung cancers (NSCLC) are ALK-positive. Crizotinib was the first approved ALK inhibitor for metastatic NSCLC, demonstrating efficacy in clinical trial settings. However, there is less data on the utilization and patient outcomes associated with crizotinib in real-world clinical practice. Objectives of this study consisted of describing demographic and disease characteristics, treatment patterns, and outcomes in US community practice.

Methods:
This was a retrospective, observational study of adult crizotinib-treated ALK-positive patients with metastatic NSCLC who received treatment between 9/1/2011 and 10/31/2014, with follow up through 12/31/15. Data were obtained via programmatic queries of the US Oncology Network/McKesson Specialty Health electronic health record database, supplemented with chart abstraction. Patients in clinical trials or diagnosed with other primary cancers were excluded. Descriptive statistics were calculated overall and by line of therapy (LOT), performance status, and brain metastases. Overall survival (OS) and time to treatment failure (TTF) were estimated from crizotinib initiation using the Kaplan Meier (KM) method.

Results:
We identified 70,300 NSCLC patients, of which n=199 ALK-positive crizotinib treated patients met eligibility criteria during the study period. Crizotinib was prescribed as first line (1L) in n= 123 (61.8%) and second line or greater (≥2L) in n= 76 (38.2%). The majority (88.9%) had confirmed adenocarcinoma histology and 32.1% had brain metastases at initial diagnosis. Median age at crizotinib initiation was 60.0 years (range 27.1-88.2); 54.8% were never smokers, 33.7% were former smokers and 77.4% had an ECOG performance status of 0 or 1. Treatment of 250 mg twice daily was most commonly prescribed (88.4%) with dose unchanged from prior dose in 79.4% of patients. Patients remained on crizotinib for a median duration of 8.5 months (range 0.23-48.3). The primary discontinuation reason was progression (n=91, 58.7 %) however only 3.2% of patients were identified as discontinuing crizotinib as a result of treatment-related toxicity. With a median follow-up time of 16.3 months (range 2.2-46.6), median OS from crizotinib initiation was 33.8 months (95% CI=24.3-38.8) in the overall population with 1 and 2-year survival rates of 79.0% and 61.3%. OS was similar across LOT (p= 0.9093) and by brain metastases (p=0.2775). Median TTF was 9.5 months in the overall population and was similar by LOT (p=0.6808) and brain metastases (p= 0.1603).

Conclusion:
Outcome endpoints were similar between groups, although potentially limited by small sample size. Results from this study were consistent with findings from clinical trials.

Background:
Crizotinib, as the standard treatment for use in first-line treatment of anaplastic lymphoma kinase (ALK)-positive advanced non-small-cell lung cancer (NSCLC), showed superiority over platinum-based chemotherapy in advanced ALK-positive lung adenocarcinoma.Undoubtedly, the resistance to crizotinib is a current bottleneck which limits its clinical application. However, there are few reports about clinical failure to crizotinib, especially the correlation between the failure patterns of crizotinib and survival benefit.

Methods:
Totally,171 ALK-positive NSCLC patients treated with crizotinib were reviewed at the Guangdong General Hospital in China from October 2010 to July 2016.The status of ALK rearrangement was assessed by Lysis ALK Break Apart fluorescence in situ hybridization,reverse transcription polymerase chain reaction, or Ventana ALK immunohistochemistry.Chi-square test and Kapla-Meier survival curve were used to analyze the results statistically.

Results:
Among enrolled patients,47.5%(81/171) gained secondary resistance,10.5%(18/171) had primary resistance and 4 patients stopped taking crizotinib because of the occurrence of unacceptable toxicities including anasarca,ventricular tachycardia and hepatic insufficiency. Moreover,49 patients had no progression,in which 2 patients had taken crizotinib more than 5 years uninterruptedly.In the patients with secondary resistance (n=81),46 were male and 63 were never smokers.Brain metastases occurred in 27.1%(22/81) at the baseline,half of which(11/22) still had brain progression after the treatment of crizotinib.On the contrary,21 patients without brain metastases at the baseline were evaluated at disease progression because of brain metastases.We classified patients with secondary resistance into several categories according to the failure patterns of crizotinib, such as dramatic,gradual and local progression.There were 47(58.0%), 2(2.5%) and 32(39.5%) patients for dramatic, gradual and local progression respectively.The patients with dramatic progression had an inferior progression-free survival with crizotinib to those with gradual and local progression (9.8 vs 11.9 months),which did not achieve statistical significance.The post progression survival(PPS) of dramatic progression group is 10.4 months.The PPS of other group is 20.5 months comparatively.Patients with dramatic progression showed shorter overall survival when compared with other patients (26.7 vs 41.0 months, P=0.042).

Conclusion:
Dramatic progression was prevalent in ALK-positive lung adenocarcinoma beyond failure to crizotinib, and predicted poor overall survival.

Background:
Although most patients with ALK-positive non‒small-cell lung cancer (NSCLC) who benefit from treatment with crizotinib ultimately develop progressive disease (PD), continuing crizotinb beyond the initial PD (CBPD) in these patients may be beneficial. In this retrospective study, we investigated whether Chinese patients with advanced ALK-positive NSCLC benefit from CBPD, including patients with CNS progression and those who had received local therapy, and whether any factors are predictive of a longer post-initial progression-free survival time (PFS2).

Methods:
Data on 33 patients with ALK-positive NSCLC who had achieved disease control with initial crizotinib therapy were studied. The impact of continued crizotinib therapy (defined as >3 weeks of crizotinib treatment after the first documentation of PD) on the patients’ PFS2 time was assessed after adjusting for potential confounding factors.

Results:
With initial crizotinib therapy, the objective response rate (ORR) and median PFS time (PFS1) in the 33 patients studied were 63.6% and 8.6 months, respectively(Figure 1). The brain was the most commonly involved disease progression site (n = 20; 60.6%); 14 patients (42.4%) received local therapy before and after crizotinib. With continued crizotinib therapy after the first documentation of PD, the median PFS2 time for all 33 patients was 16 weeks(Figure 1), and in those with CNS progression but systemic disease control it was 30 weeks. Patients who received local therapy after disease progression had a significantly longer PFS2 time compared with those who did not (p = 0.039). Multivariable Cox regression analysis showed that the PFS1 time with initial crizotinib treatment and local therapy were independent predictors of PFS2. Figure 1



Conclusion:
This study provides further evidence of the benefit of continuing crizotinib therapy in Chinese patients with progressive ALK-positive NSCLC. Patients with a longer PFS1 and those who received local brain therapy had better survival with continued crizotinib therapy.

Background:
Ceritinib (Zykadia) is a recently approved second-line agent for anaplastic lymphoma kinase (ALK+) non-small cell lung cancer (NSCLC). The current study sought to describe healthcare providers’ (HCPs) decisions to treat with ceritinib and to describe patient-reported side effects, perceived effectiveness and attitudes toward ceritinib.

Methods:
One-on-one telephone interviews were conducted with HCPs caring for patients treated with ceritinib using a semi-structured interview guide designed to explore treatment decision-making, adverse events (AEs) and their management. Patients with current or past experience of ceritinib completed semi-structured telephone interviews designed to capture their experience. A thematic analysis of interview transcripts was conducted using qualitative analysis software, MaxQDA.

Results:
Study participants comprised 10 HCPs (6 oncologists, 4 nurses) and 18 patients (9 female) aged 34-78 years (mean=51.0; SD=11.3). HCPs reported relying on two main factors when deciding to switch patients to ceritinib or to next-line treatment after ceritinib: evidence of sufficient clear-cut progression and poor tolerance to treatment. Four HCPs reported considering clinical trials or other newly approved drugs instead of ceritinib. Patients and HCPs concurred that the most frequently reported side effects of ceritinib include diarrhea (n=15 patients; n=9 HCPs), nausea (n=13; n=10), vomiting (n=12; n=6), and abdominal pain (n=10; n=7). Dose reduction, antiemetic and anti-diarrheal medications, and home remedies (e.g. ginger ale, crackers) were reported as being effective at managing these side effects prophylactically or once they occurred. Taking ceritinib with food was reported by 5 patients and 4 HCPs, and helped to improve nausea, vomiting or abdominal pain. Patients reported that ceritinib was effective in achieving or maintaining symptom control for cough (n=12 of 12 patients symptomatic at diagnosis) and shortness of breath (n=9 of 11 patients symptomatic at diagnosis). Of 14 patients with lung tumors at start of ceritinib, 13 reported positive tumor response during treatment. Three of 7 patients with brain metastases achieved reduction or no evidence of disease with ceritinib in combination with other interventions (e.g., radiation). Patients were asked what they liked about ceritinib: tumor response and symptom control, an extension of life, or improvement in quality of life were key themes. Patient-reported dislikes included side effects and number of pills. Of 14 patients asked specifically, all stated the benefits of ceritinib outweigh its side effects.

Conclusion:
Patients perceived ceritinib as an effective treatment for ALK+NSCLC. AEs were reported to be manageable and patients were willing to manage these in order to experience the treatment benefits.

Background:
Ceritinib is the first second-generation ALK inhibitor approved in the US to treat ALK+ non-small cell lung cancer (NSCLC) patients who progressed on or were intolerant to crizotinib. This study provides the first real-world description of the characteristics, treatment patterns, and early outcomes of ALK+ NSCLC patients who received ceritinib in clinical practice.

Methods:
From March to June 2016, 23 US oncologists provided data retrospectively from the medical charts of their adult patients diagnosed with locally-advanced or metastatic ALK+ NSCLC who received ceritinib following crizotinib therapy. Clinical characteristics, treatment patterns, and early outcomes on ceritinib were assessed. Best response on ceritinib was evaluated using RECIST criteria.

Results:
Participating oncologists reviewed charts of 58 ALK+ NSCLC patients treated with ceritinib. 41% of the patients were male, 52% were never smokers, and median age at ceritinib initiation was 63 years. Patients started ceritinib following a median of 10.6 months on crizotinib; 21% of patients had prior chemotherapy experience. At ceritinib initiation, many patients had multiple distant metastases, most commonly in the liver (60%), bone (53%), and brain (38%). While 71% initiated ceritinib at 750mg once daily, 19% received 600mg once daily, and 10% received 450mg once daily. 17% of patients were instructed to take ceritinib with food; 50% were instructed to fast. Median follow-up after ceritinib initiation was 3.8 months. Although follow-up was short, most patients achieved either a complete (8%) or partial (61%) response on ceritinib, regardless of metastatic site present at initiation (Table). Among the 21 patients who discontinued ceritinib, 6 received alectinib, 2 chemotherapy, 2 immunotherapy, and 11 received no further antineoplastic therapy.

Conclusion:
These early findings of ceritinib use in clinical practice suggest that ceritinib is effective at treating crizotinib-experienced ALK+ NSCLC patients, regardless of the location of metastatic sites. Future studies with longer follow-up are warranted. Figure 1

Background:
Lung cancer with ovarian metastasis or adnexal metastasis harboring anaplastic lymphoma kinase gene (ALK) translocation is rare. Crizotinib, a novel anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor, has already shown an impressive single-agent activity in ALK-positive lung cancer.

Methods:
Our case is the first report of crizotinib effective for ALK-positive adenocarcinoma with adnexal metastasis. A 33-year-old woman was diagnosed with adnexal metastasis from non-small cell lung cancer (NSCLC). Histological examination of the tumors showed lung adenocarcinoma. The right lung biopsy tissue and left adnexal mass biopsy tissue were both revealed the presence of an ALK rearrangement by Ventana (D5F3) ALK immunohistochemistry (IHC) assay (Ventana Medical Systems, Roche, Inc).

Results:
The patient experienced a remarkable tumor response to crizotinib treatment.Figure 1



Conclusion:
Although the adnexal location is an uncommon metastasis site from lung cancer, oncologists should be aware of the possibility of such metastasis for female patients with ALK rearrangement NSCLC. Considering this remarkable response, we conclude that the presence of adnexal metastasis in NSCLC patients with ALK rearrangement should be attentive.

Background:
Here, we present the patient-reported outcomes (PROs) of ceritinib versus chemotherapy as first-line treatment for advanced ALK+ NSCLC.

Methods:
Untreated, ALK+, advanced, nonsquamous NSCLC patients (N=376) were randomized (1:1) to ceritinib 750 mg/day (n=189) or chemotherapy (n=187; [pemetrexed 500 mg/m[2 ]plus cisplatin 75 mg/m[2] or carboplatin AUC 5-6] for 4 cycles followed by maintenance pemetrexed). PROs were assessed using EORTC quality-of-life questionnaire (QLQ-C30), the lung cancer module (QLQ-LC13), Lung Cancer Symptom Scale (LCSS), and EQ-5D.

Results:
Median treatment exposure was 66.4 weeks for ceritinib and 26.9 weeks for chemotherapy. PRO compliance was high, ≥80% at most timepoints. Ceritinib significantly prolonged time to deterioration of lung cancer-specific symptoms (pain, dyspnea, and cough) versus chemotherapy in both LCSS and QLQ-LC13 instruments (composite endpoints for LCSS, HR=0.61 [0.41, 0.90]; and QLQ-LC13, HR=0.48 [0.34, 0.69]). Time to deterioration in LC13 questionnaire was significantly longer with ceritinib versus chemotherapy (23.6 [20.7, NE] vs 12.6 [8.9, 14.9] months) (Table). In the QLQ-C30 instrument, 4 of 5 functional domains and 6 of 9 symptom scales improved with ceritinib (P< 0.05); 2 scales related to gastrointestinal symptoms indicated deterioration for ceritinib. In agreement with most other scales showing symptom improvement, ceritinib demonstrated significant improvements in Global Health Status/QoL in the same instrument (QLQ-C30, P<0.001) as well as for EQ-5D-5L index (P<0.001) and EQ-5D-5L VAS (P<0.05 from cycle 13 until 49). Figure 1



Conclusion:
Untreated ALK+ NSCLC patients experienced significantly greater improvements in lung cancer-specific symptoms on treatment with ceritinib. General health status was significantly improved with ceritinib versus chemotherapy. Overall, PRO results from all 4 instruments independently showed improvements highlighting the consistency and robustness of these findings.

Background:
In Slovakia, since October 2012, crizotinib has been available for the treatment of adults with previously treated ALK-positive advanced NSCLC, based on the therapeutic indication approved by the European Medicines Agency. The purpose of this study was to assess the results achieved with crizotinib in the treatment of NSCLC in clinical practice in Slovakia, and to compare them with the results from the key clinical trial PROFILE 1007.

Methods:
In the multicentre retrospective study, approved by the Ethical Committee of the Specialised Hospital of St Zoerardus Zobor, the data of 34 ALK-positive patients from 8 centres were reviewed. Data regarding ALK testing and results were obtained from the central laboratory database (Comenius University Jessenius Medical Faculty and Martin's Biopsy Centre). Data regarding patients were obtained from the databases of participating institutions and patient files. Fluorescence in situ hybridisation (FISH) with break-apart probes was used for the confirmation of ALK rearrangement in all cases. Response to treatment was evaluated using RECIST criteria v. 1.1. Statistical analyses were performed using MedCalc[®] software. PFS and OS were estimated using the Kaplan–Meier method.

Results:
Between October 2012 and December 2015, 34 ALK-positive patients with locally advanced or metastatic NSCLC were treated with crizotinib, 31 of them after the first-line chemotherapy. Characteristics of patients: median age, years (range): 58 (23-77), ECOG/WHO PS: 0, 1, 2, 3 in 1, 23, 6, and 4 patients, respectively. Histology: adenocarcinoma in 33 cases, NSCLC, NOS in one. Patients with locally advanced disease: 2, with metastatic disease: 32. Median PFS was 18 months (95%CI: 13 - 22), median OS (number of events: 13, 38,24%): 32 months (95%CI: 18 - 32), response rates: CR + PR: 3 + 23, 76,5% (95%CI 50-100%), SD: 7, 21,5%, PD: 3, 8,8%, not stated: 1. There was a signifcant improvement in PS within 2 month, mean difference: - 0,62, p = 0,0025. Grade 3/4 toxicities occurred in 15/2 patients. Crizotinib was permanently discontinued due to AEs in 2 patients only. PFS and OS in our study were numerically better in comparison with PROFILE 1007. On the other hand, common grade 3 toxicities occured also more often.

Conclusion:
Conclusion: Our study provides real-world evidence of the efficacy of crizotinib in patients with ALK-positive NSCLC, treated outside of clinical trials. 

Background:
Patients with non-small cell lung cancer (NSCLC) harboring ALK rearrangements have been shown to exhibit a good response to ALK-inhibitor treatment. However, serious adverse events are observed in some patients. Therefore, it is important to precisely evaluate severity of adverse events and treat properly.

Methods:
We performed a retrospective study of the efficacy and safety of ALK inhibitors in ALK-positive lung cancer patients. Between September 2013 and April 2016, 9 patients receiving ALK inhibitors in our department were analyzed. All patients gave informed consent for the use of their clinical data.

Results:
The median age was 67 years (range, 54-74 years), and the histological type of cancer was adenocarcinoma in all cases. One patient had stage IIIB, four patients had stage IV and four patients had postoperative recurrence. Seven cases were fluorescence in situ hybridization (FISH) positive / immunohistochemistry (IHC) positive, and two cases were FISH positive / IHC negative. Crizotinib and alectinib were administered orally to seven and eight patients, respectively. In evaluable cases, the disease control rate was more than 80% both crizotinib and alectinib. The median progression-free survival was longer in alectinib treatment than in crizotinib treatment (133 days (range, 8-635 days) vs 51 days (range, 3-452 days)). Among 7 patients received crizotinib, the adverse events included grade 4 increased ALT and AST levels in 1, grade 3 pneumonitis in 2, grade 2 edema in 1, and grade 2 increased creatinine level in 1. Among 8 patients received alectinib, the adverse events were included grade 2 pneumonitis in 2 and grade 2 skin disorder (drug eruption) in 1. Severe adverse events such as pneumonitis and liver dysfunction were observed within 40 days, and in these cases, treatment could not be continued. Visual disorder, gastrointestinal symptoms such as nausea, vomiting and constipation were more frequent in crizotinib treatment, but these symptoms were reduced by switching from crizotinib to alectinib. Drug discontinuation rate because of adverse events was higher in crizotinib treatment than in alectinib treatment (71% vs 50%). No treatment-related death occurred.

Conclusion:
Although ALK inhibitors have therapeutic effect against ALK-positive NSCLCs, severe adverse events occur in some patients. Based on these adverse events as well as the efficacy in each agent, alectinib treatment may be recommended in first line setting for ALK-positive NSCLC patients.

Background:
Approximately 3–7% of lung tumors harbor Anaplastic Lymphoma Kinase (ALK) fusions. The aim of the current study was to characterize the population of patients with ALK positive non small cell lung cancer (NSCLC) treated at our Institution.

Methods:
Retrospective analysis of 26 ALK positive NSCLC, diagnosed between December/2008 and February/2016. Eligible patients had lung adenocarcinoma harboring ALK translocation according to fluorescent in situ hybridization. Best response was assessed using RECIST (version 1.1).

Results:
Twenty six patient cases are reported, diagnosed between 01/12/2008 and 29/02/2016. Median age was 56 years, 60.7% of patients were women, and 71.4% were never-smokers. Twenty-four (92.3%) were adenocarcinomas. All patients were EGFR negative. Twenty (76.9%) were stage IV. Fifteen patients (57.7%) were treated in first line with palliative chemotherapy (CT), thirteen of them with platinum/pemetrexed. Twelve patients (46.1%) were treated with crizotinib, two in first line, nine in second line and one in third line; one patient was treated with ceritinib in fourth line. As major adverse events there were eight cases (30.7%) of venous thromboembolism, including five (19.2%) pulmonary embolisms. ALK directed therapy, namely crizotinib, was safe and well-tolerated. Most of the patients (91.7%) were treated with the standard dose of 250mg twice per day. One patient needed dose reduction due to hepatotoxicity (G3, CTCAE.V4). The most frequent treatment-related adverse events were emesis (G1) vision disorders (G1), and increased AST/ALT (G3). Three patients treated with CT had grade 3 toxicity (pneumonia with respiratory failure, anemia, peripheral neuropathy). Median follow-up of study population was 13.5 months. In patients treated with crizotinib objective response rate (ORR = complete response + partial response) was 50% and clinical benefit (CB = complete response + partial response + stable disease) was 75%. In patients treated with CT ORR was 6.7% and CB was 73%. Seven (26.9%) patients died during the study period. Median overall survival has not been reached.

Conclusion:
ALK directed therapy provided increased benefit and lower toxicity compared to CT. During the study period, there were several treatment guidelines updates impacting the patient’s management. Presented results are consistent with the published literature.

Background:
ROS1 gene-rearrangement in non-small cell lung cancer (NSCLC) patients has recently been identified as a driver gene and benefited from crizotinib treatment. However, no data is available for ROS1-positivity NSCLC about chemotherapeutic options and prognostic data. We investigated pemetrexed-based treatment efficacy in ROS1 translocation NSCLC patients and determined the expression of thymidylate synthetase (TS) to provide a rationale for the efficacy results.

Methods:
We determined the ROS1 status of 1750 patients with lung adenocarcinoma. Patients’ clinical and therapeutic profile were assessed. In positive cases, thymidylate synthetase (TS) mRNA level was performed by RT-PCR. For comparison, we evaluated the TS mRNA status and pemetrexed-based treatment efficacy from 170 NSCLC patients with anaplastic lymphoma kinase (ALK) translocation (n=46), EGFR mutation (n=50), KRAS mutation (n=32) and wild-type of EGFR/ALK/ROS1/KRAS (n = 42).

Results:
Thirty-four ROS1 translocation patients were identified at two institutions. Among the 34 patients, twelve with advanced stage or recurrence were treated with pemetrexed-based first-line chemotherapy. The median progression-free survivals of pemetrexed-based first-line chemotherapy in ROS1 translocation, ALK translocation, EGFR mutation, KRAS mutation and EGFR/ALK/ROS1/KRAS wild-type patients were 6.8, 6.7, 5.2, 4.2 and 4.5 months, respectively (P=0.003). The TS mRNA level was lower in patients with ROS1-positive than ROS1-negative patients (264±469×10[-4] vs. 469 ± 615×10[-4] , P=0.03), but similar with ALK-positive patients (264±469×10-4 vs. 317±524×10[-4], P=0.64).

Conclusion:
Patients diagnosed with ROS1 translocation lung adenocarcinoma may benefit from pemetrexed-based chemotherapy. TS mRNA level enables the selection of therapeutic options for ROS1 translocation patients.

Background:
Chromosomal rearrangements involving the c-ros oncogene 1 (ROS1) have been described as a subset of non-small cell lung cancer (NSCLC). Recently Crizotinib has exhibited marked therapeutic efficacy in the treatment of the ROS1 fusion NSCLC. However, resistance often occurs and repeated biopsy is necessary for tumor genotyping and underlying resistant mechanism. Circulating tumor DNA (ctDNA) represents a promising way to assess tumor genetic profile non-invasively. This study aims to assess whether liquid biopsies accurately screen disease diagnosis and reflect the response to Crizotinib treatment through analysis of ctDNA for ROS1 fusions in patients with lung adenocarcinoma, and to elucidate the underlying mechanisms of ROS1 targeted drug resistance.

Methods:
Twelve plasma samples were collected from a cohort of 4 patients with ROS1 fusion advanced stage lung adenocarcinoma, confirmed by fluorescent in situ hybridization (FISH) in tissue. A prospective-retrospective analysis on ctDNA was further performed from archived plasma samples using our ctDNA panel with concurrent CT or MRI imaging at the baseline and 8-week intervals during responsive Crizotinib treatment, and at progressive disease.

Results:
All patients showed detectable levels of ROS1 fusion in ctDNA at baseline. Upon treatment with Crizotinib, response rate is inversely correlated with levels of ROS1 fusion. One patient with progressive disease, patient 1, exhibited a detectable CD74-ROS1 fusion with 13.5% concentration at baseline; it was undetectable at partial response and re-elevated to 8.2% accompanied by an acquired G2032R mutation when disease progressed.

Conclusion:
Our ctDNA panel could be applied clinically to detect ROS1 fusion from plasma for accurate screening and convenient monitoring where detection correlates with disease status, and could distinguish mutations associated with Crizotinib induced resistance in patients with NSCLC, thus facilitating personalized cancer therapy.

Background:
ROS1 gene rearrangement refers to rare genetic aberrations, occurs in 1-2% of patients with NSCLC. of ROS1 rearrangement patient This subgroup of patients is high sensitive to crizotinib therapy.

Methods:
Clinical case Patient, male, 87 y.o., observed in our clinic since July, 2013. Diagnosis: Lung adenocarcinoma , metastases in the both lungs, pleura, supra- and subclavicular lymph nodes, bones, Т3N3М1b, stage IV. EGFR «-», ALK «-». Concomitant diseases: Ischemic heart disease. The heart rhythm disorder (ventricular and supraventricular extrasystole). Treatment performed: 1st line:-Alimta + carboplatin х6 cycles (July - November 2013). Partial response. Disease progression in September 2014. Chemotherapy was proceeded according to the previous regimen. After 4 cycles in February 2015 the further progression of disease was observed. Biopsy of axillar lymph node was performed. Molecular testing found ROS1 gene rearrangement. On 02.04.2015 crisotinib was started with dose reduction 250 mg/day. The next day bradycardia (cardiac rate 39 beats per minute) developed, QTc was prolonged to 558 msec (initially 470 msec). Crizotinib administration was stopped. Development of this adverse event as a rule is the cause of withdrawl of crizotinib. However, high probability of targeted therapy response and absence of alternative in choosing of antineoplastic therapy made us use all the opportunities for correction of adverse events. 10.04.2015 implantation of dual chamber pacemaker was performed and crizotinib 250 mg/day administration was proceeded. The patient’s condition was satisfactory, there were no other adverse events and dose of crizotinib was increased to the standard on 21.04.2015.

Results:
In 7 weeks after beginning of targeted therapy (28.05.2015) PET-CT was performed - local areas of abnormal uptake of FDG were not detected. The patient continues crizotinib at the current time – last PET-CT was done 04.07.2016. Complete response remains. The duration of response is 15 months. Figure 1



Conclusion:
Therefore, we pronounced the early and durable response.

Background:
In Mexico, 8,600 new cases of lung cancer are annually diagnosed, with 22 daily deaths.[1] Nintedanib is a multikinase inhibitor, acts as a potent oral anti-angiogenic blocking the vascular endothelial growth factor (VEGFR 1-3), the fibroblast growth factor receptor (FGFR 1-3) and the platelet-derived growth factor receptor (PGFR α y β). The LUME-Lung 1 trial showed an improvement with docetaxel + nintedanib in overall survival of patients with lung adenocarcinoma accomplished disease control in 60.2%.[4 ]

Methods:
We present a descriptive trial, with a clinical data collection of patients with advanced lung adenocarcinoma who progressed to a platinum-based, first-line treatment (+ bevacizumab), included in the compassionate-use program of nintedanib, carried out between March 2014 and September 2015 in 38 medical centers in Mexico. Treatment consisted in IV dose of docetaxel 75 mg/m[2 ]every 21 days, combined with oral nintedanib 200 mg/BID, administered from 2-21 days until maximum toxicity or disease progression. Primary Objective: Describe patients and tumors characteristics, including previous therapies. Secondary Objectives: Estimate the time under nintedanib treatment, response rate (measured by RECISTv1.1 criteria) and evaluate safety in daily clinical practice (by means of CTACAEv4 criteria). The SPSSv statistics software was used.

Results:
Ninety-nine patients were included. The median of age was 59 years old, 53% were male, 51.5% were smokers, 50% had ECOG 1, 96% stage IIIB and IV, 16% had controlled brain metastases, and 85% had access to a EGFR mutational test (11% out of them positive). First-line, platinum-based chemotherapy was given to 99% of the patients, 7% were administered first-line bevacizumab, 40% receive maintenance treatment, 50% had had partial response as the best response to first-line treatment. The objective response rate was 53%, stable disease 26.5%, disease progression 16.3%, non-evaluable 4% and disease control rate of 79.6%. The combination had an adequate tolerance, mostly toxicities grades 1-2. Toxicities grade 3-4 were mainly fatigue (14%), diarrhea (13%), hiporexia (7%), neutropenia (7%), nausea (6%), vomiting (1%). Nintedanib dose was reduced in 27 patients (27%). The median duration of the treatment with nintedanib was 6.7 months.

Conclusion:
Overestimated responses may be related to the retrospective desing of the study and due to that were valued by investigator wich could influence the results. The Safety of nintedanib in real-life patients was demostrated and not very different from the results in the LUME-Lung 1 trial. Gastrointestinal toxicity were the most frequent side effects, mostly toxicities grades 1-2.

Background:
While the financial aspects of the burden on caregivers for patients with advanced Non-Small Cell Lung Cancer (aNSCLC) have been estimated, limited published information exists on the humanistic burden incurred by these caregivers.

Methods:
Data were taken from a multi-center, cross-sectional study of aNSCLC patients and their caregivers conducted in France, Germany and Italy. The study consisted of three components: medical chart review, patient questionnaire and caregiver questionnaire. Overall, 683 consulting patients and 277 accompanying informal caregivers were recruited via treating physicians. The impact on health related quality of life was measured using the EuroQoL-5D (EQ-5D-3L) while caregiver burden was quantified using the Zarit Burden Interview (ZBI), which consists of 22 items, each rated 0-4. ZBI scores were grouped into: little/no burden (0-20), mild/moderate (21-40), moderate/severe (41-60) and severe burden (61-88). Scores of 24+ were assumed to identify caregivers at risk of depression. Analysis, conducted on 277 matched patient and caregiver forms, was stratified by country and by patients’ line of therapy. Statistical significance was assessed using Mann-Whitney U tests.

Results:
Caregivers’ mean (SD) age was 55.2 (13.0) years; 78.6% were female and 62.3% were the patient’s partner/spouse. Patients’ mean (SD) age was 66.2 (9.7); 73.6% were male and 91.0% had Stage IV NSCLC. Over two-thirds (70.4%) of patients were receiving 1[st] line advanced therapy, while 29.6% were receiving later lines of therapy. The mean (SD) EQ-5D-3L index for caregivers was 0.87 (0.19). Differences in EQ-5D-3L were observed between carers of 1[st] line patients and later line patients (0.89 v 0.83 p=0.003). The mean ZBI score for caregivers was 32.1 (15.6); A quarter (24.0%) of caregivers had little/no burden, 44.6% mild/moderate, 28.8% moderate/severe and 2.6% severe burden; 69.7% of caregivers were identified as at risk of depression. Differences in ZBI were observed between carers of 1[st] line patients and later line patients (30.9 v 34.9 p=0.099).

Conclusion:
Comparing these results with other published ZBI data, the burden suffered by aNSCLC patient caregivers appears to be higher than other conditions studied in Europe, namely , Parkinson’s disease (25.8) another study conducted across advanced cancer (18.5). Caregivers for aNSCLC patients suffer significant humanistic burden in addition to the overall burden faced by patients and is likely to result in additional costs. When assessing the impact of a treatment, the potential to improve the impact on caregivers should also be included.

Background:
BRAF is found mutated in 2-3% of stage IV NSCLC. BRAF inhibitors have been reported to have antitumor activity. A nationwide access to vemurafenib for cancer patients with tumors presenting with BRAF mutations was launched by the French National Cancer Institute (INCa) providing free access to tumor molecular diagnosis. The AcSé-Vemurafenib study is the 2[nd] exploratory multi-tumor 2-stage design phase II trial of AcSé program. We report the preliminary results of the NSCLC cohort in this nationwide program.

Methods:
BRAF mutational status was assessed on INCa molecular genetic platforms by either direct sequencing or NGS. Patients with BRAF mutation (including BRAF V600E and others less common mutations), progressing after at least one standard treatment (including a platinum-based doublet, unless pts were considered as unfit for chemotherapy) were proposed to receive vemurafenib 960 mg BID. Responses were centrally assessed using RECIST v1.1 every 8 weeks.

Results:
From Oct. 13, 2014 to June 15, 2016, 65 patients were enrolled including 55 NSCLC harboring BRAF V600E and 10 pts with other activating mutations (2 G466, 3 G469, 1 G596, 3 K601 and 1 N581). 55 patients received vemurafenib and had at least one post-baseline assessment. Median age: 67 years (range 40–84), 51% females and 100% non-squamous histology. Median number of prior chemotherapy lines: 1 (0 –5). Most frequent grade ≥3 adverse events (AEs) were skin (18% of patients) and gastrointestinal toxicities (16%). Among the 39 BRAF V600E NSCLC patients evaluable for the best overall response (BOR) with a minimum follow-up of 4 months, 15 PR, 8 SD, 10 PD, 5 deaths before assessment and 1 missing were observed. The objective response rate was 38.5% [95% CI:23.4-55.4], and the disease control rate 59% [42.1-74.4]. Median duration of response was 5.1 months [1.8-9.2]. Progression-free survival (PFS) at 4 months was 48.2% [31.8-62.8]. No response was reported among the 7 evaluable patients with other BRAF mutations with 5 PD, 1 death before assessment and 1 missing as BOR ; PFS at 4 months was 14.3% [0.7-46.5]. 18 patients were still on treatment at the cut-off date, 47 have stopped vemurafenib (25 PD, 15 AEs, 1 death, 1 doctor’s decision, 5 patient’s decisions).

Conclusion:
Vemurafenib provided response rate and DCR in BRAF V600E pretreated NSCLC but was not found efficient in NSCLC with other BRAF mutations. These results underline the interest of integrating BRAF V600E in biomarkers routine screening.

Background:
Airway stenting is undoubtedly the mainstay procedure for treating patients with malignant airway stenosis to prevent a variety of airway symptoms. Suffocation death is the most painful ending for those patients. The impact of airway stent treatment to avoid this tragic event was investigated.

Methods:
Between 2000 and 2014, 57 patients underwent airway stenting in our department for malignant airway stenosis. They included 25 lung cancer cases, 15 esophageal cancer cases and 7 thyroid cancer cases. The location of the stenosis was the carina for 31 cases, the right or left main bronchus in 12, and the trachea 14. Either Dumon silicon (n=50) or self-expandable metallic stents (n=7) were used. The effect of airway stenting to prevent suffocation death, and the factors for predicting the prognosis were analyzed.

Results:
There were no cases of in-hospital death. An improvement in airway symptoms was achieved in 54 patients (94.7%) and the median survival after stenting was 3.7 months. At death, only 8 (14%) of those patients died due to direct airway symptoms, including respiration difficulty, even when their general condition was good (Suffocation death group). Conversely, the other 49 patients mostly died due to systemic cancer spread, but all 49 cases had no pain associated with airway symptoms. Therefore, suffocation death appears to have been avoided in those 49 (85.9%) patients (Non-suffocation death group). In a univariate analysis, “Stent migration”, “Tracheal stenosis”, and “Thyroid cancer” were potentially significant factors regarding suffocation death. In a multivariate analysis, “Stenosis at mid trachea” was found to be an independent predictive factor for suffocation death (p = 0.02).

Conclusion:
Suffocation death can be effectively prevented by the use of airway stenting treatment. “Stenosis at mid trachea” is the most problematic factor when attempting to obtain some benefit from stenting and this may be due to the difficulty of achieving accurate stent (mainly straight silicon stent) fixation in such lesions.

Background:
The anaplastic lymphoma kinase (ALK) inhibitor ceritinib is approved at 750 mg fasted for the treatment of patients with ALK-rearranged (ALK+) metastatic non-small cell lung cancer (NSCLC) pretreated with crizotinib. The pharmacokinetic (PK) part of this study (Part 1) compares PK exposure of ceritinib following food consumption versus a fasted state in advanced ALK+ NSCLC patients.

Methods:
Part 1 of this prospective, open-label, multicenter, randomized, 3-arm, phase 1 study (ASCEND-8; NCT02299505) is investigating PK and safety of ceritinib in advanced ALK+ NSCLC patients, treatment-naïve or pretreated with multiple lines of chemotherapy and/or crizotinib. Here, we compare steady-state PK of ceritinib 450 or 600 mg taken with a low‑fat meal versus ceritinib 750 mg fasted (primary endpoint) and report preliminary safety outcomes from Part 1. Part 2 continues to randomize treatment-naïve patients and will assess safety and efficacy.

Results:
As of June 16, 2016 (data cut-off), 137 patients were randomized in a 1:1:1 ratio to each treatment arm; 135 patients received one dose (safety set) and 97 patients had evaluable steady-state PK data. Disease characteristics were comparable between arms. Median follow-up duration was 4.14 months (range, 0.1–13.9). Relative to 750 mg fasted, the 450 mg fed arm demonstrated comparable steady-state PK, while the 600 mg fed arm showed ~25% higher steady-state PK (Table). Preliminary safety data suggests overall frequency of AEs and types of AEs were comparable between arms. However, incidences of gastrointestinal (GI)-related AEs (diarrhea, nausea or vomiting) were lowest, with no grade 3/4 GI AEs reported, in the 450 mg fed arm.Figure 1



Conclusion:
Steady-state PK was comparable in advanced ALK+ NSCLC patients taking ceritinib 450 mg with a low-fat meal versus 750 mg fasted. This study continues to enroll treatment-naïve patients into Part 2 to assess efficacy across the three treatment arms and assess longer safety follow-up.

Background:
Young patients with lung cancer represent a distinct subset of patients with this disease. Studies show that younger patients are more likely to be women and non-smokers. They are diagnosed at a later stage; the histologic type is more likely to be adenocarcinoma and more driver mutations such as in the EGFR gene are found. Prognosis and survival of the younger patients has mostly been shown to be better in the younger population.

Methods:
Retrospective data was collected in a single tertiary hospital between 1/2010 and 12/2015. Patients were divided into 2 age groups: patients who were diagnosed aged younger than 50 years and patients aged older than 60 years. We analyzed demographic characteristics, disease course and survival.

Results:
The younger cohort included 77 patients, with a median age of 45 years. The older group included 107 patients, median age of 68 years. Both groups had similar female to male ratio and had similar ratio of smokers, although the younger had significantly lower median pack years (40 vs. 60, P<0.001). Adenocarcinoma was the most common histopathology in both age groups (64% vs. 71%) and a larger proportion of small cell lung cancer histology was found in the younger cohort (12% vs. 3%, P<0.001). EGFR mutations were more common in the young cohort (18% vs. 13%, P=0.06), as well as ALK translocations (9% vs. 1%, P<0.001) and accordingly, they were treated by more targeted therapies (25% vs. 16%, P=0.015). Although young patients had more brain metastasis (38% vs. 21%, P=0.002), their median survival was not significantly different than the older cohort (24.8 vs.18.2 months p=0.5). yet after performing sub-stratification it was found that patients under 40 years had better median survival (70.8 months P=0.05). Among patients with a driver mutation, median survival was better for younger patients (31.9 vs. 17.0 months, P=0.003).

Conclusion:
Young patients with lung cancer have better median survival; their tumors harbor a higher rate of driver mutations and they have a higher percentage of brain involvement.

Background:
PF-06747775 (PF-7775) is a highly potent, selective third generation irreversible EGFR-TKI, effective against EGFR-TKI sensitizing and resistance (T790M) mutations in NSCLC cell lines; IC50s between 3-12 nM and 26X greater selectivity toward mutant vs. wild-type (WT)EGFR. This is the first report from an ongoing phase I, first in human multicenter study (NCT02349633) of PF-7775 in patients with metastatic EGFRm+ NSCLC.

Methods:
EGFRm+ NSCLC pts, with acquired resistance to EGFR-TKIs enrolled into dose escalation cohorts of PF-7775, orally once daily, beginning at 25 mg. Stable brain metastases were allowed. All pts were assessed for pharmacokinetics (PK), response to therapy, and adverse events (AEs). Prospective central T790M testing was optional for dose escalation cohorts, but is required in subsequent expansion cohorts. Plasma samples were collected from all patients for ctDNA analysis of EGFR mutations.

Results:
Dose escalation is complete. 26 patients enrolled in 7 dose levels (25-600 mg): 58% female, mean age 63.5 years, Asian/Caucasian 61%/34%, 14/25 T790M+. Dosing reached 600 mg and then was expanded at a lower dose for better long term tolerability. RECIST responses were observed at all dose levels. BOR is PR 11(42.3%; 5 T790M+), stable disease 6(23.1%; 4 T790M+), PD 2(7.7%: 1 T790M+), symptomatic deterioration 1(3.8%; 1 T790M+), and indeterminate 6(23.1%; 3 T790M+). The AE profile is very favorable as predicted from the large WT margin. No DLTs were observed. Grade 3 AEs were noted at > 150 mg (diarrhea {n=4, 15.4%} and skin toxicities {n=8, 30.8%}). Figure 1. Best Change from Baseline in Tumor Size (%) Figure 1 PK were generally dose-proportional at doses of 25-600 mg, with a median apparent t~1/2~ of 6 h (range 4-30).



Conclusion:
PF-7775 has demonstrated early signals of clinical activity and is well tolerated in EGFRm+ NSCLC pts with acquired resistance to EGFR-TKIs.

Background:
Although superior clinical benefits of first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in the treatment of advanced non-small-cell lung cancer (NSCLC) had been reported with different sensitivity. The sensitivity of second-line TKIs in NSCLC patients with different EFGR mutations was unknown. the purpose of this study is to investigate the clinical outcome of NSCLC patients with and without brain and/or bone metastases in different EGFR tumor genotypes receiving EGFR-TKIs as a second-line treatment.

Methods:
The treatment outcomes of 166 NSCLC patients harboring either the exon 19 deletion or the L858R point mutation of EGFR treated by second-line TKIs were retrospectively reviewed.

Results:
The disease control rate (DCR) and objective response rate (ORR) of enrolled NSCLC patients were 77.7% and 11.4%, respectively. The exon 19 deletion group had a significantly longer median progression-free survival (PFS) (6.7 vs. 4.5 months, P=0.002) and overall survival (OS) (13.7 vs. 11.7 months, P=0.02) compared with the L858R mutation group for NSCLC patients, as well for patients with brain metastasis [PFS: (6.7 vs. 3.9 months, p<0.001), OS: (13.7 vs. 7.9 months, p=0.006)]. The median PFS and OS for NSCLC patients with bone metastasis were 4.8 months (95% Cl, 4.0-5.6 months) and 12.9 months (95% Cl, 8.7-17.1 months), respectively. No significant difference was observed for patients with bone metastasis for different mutations. EGFR genotype and ECOG PS were independent predictors of PFS for both NSCLC patients with and without brain metastasis. Never smoking (P=0.001), exon 19 deletion (P=0.03), EGOC PS (0-1) (P<0.001) and no brain metastasis (p=0.01) were correlated with longer OS for all NSCLC patients. For patients with brain metastasis, age at disease progression (p=0.009), genotype (p=0.02) and EGOC PS (p<0.001) were independent predictors of OS. For patients with bone metastasis, EGOC PS (p<0.001) was an independent predictor for both PFS and OS. Female (P=0.01), never smoking (P=0.005), number of bone metastases (P=0.03) and EGOC PS (0-1) (P=0.002) were related to a longer PFS. EGOC PS (0-1) (P<0.001) was associated with a longer OS. Rash, fatigue, and anorexia were the three most frequent side effects observed No significant side effects difference between exon 19 and 21 mutation groups were observed.

Conclusion:
NSCLC patients harboring exon 19 deletion achieved better PFS and OS than those with L858R mutation, indicating that EGFR mutations are significant prognostic factors for advanced NSCLC patients with and without brain metastasis receiving second-line EGFR-TKIs treatment.

Background:
LUX-Lung 8 compared second-line afatinib (40 mg/day; n=398) and erlotinib (150 mg/day; n=397) in patients with stage IIIB/IV squamous cell carcinoma (SCC) of the lung. PFS (median 2.6 vs 1.9 months, HR=0.81 [95% CI, 0.69–0.96], p=0.010) and OS (median 7.9 vs 6.8 months, HR=0.81 [0.69–0.95], p=0.008) were both significantly improved with afatinib versus erlotinib. Here we report exploratory molecular (n=245) and immunohistochemical (n=288) analyses of tumour samples to assess the frequency of short variants (SVs) and copy number alterations (CNAs) in cancer-related genes and whether these tumour genomic alterations, or EGFR expression levels, have clinical utility as prognostic/predictive biomarkers in patients with SCC of the lung. We also assessed the predictive utility of the prospectively validated VeriStrat®, a serum protein test (n=675).

Methods:
Archived tumour samples were retrospectively analysed using next-generation sequencing (FoundationOne™). Tumour EGFR expression was assessed by immunohistochemistry; EGFR positivity was defined as staining in ≥10% of cells. Pretreatment serum samples were assigned as VeriStrat-Good or VeriStrat-Poor according to a mass spectrometry signature. Cox regression analysis was used to correlate OS/PFS with genomic alterations (individual or grouped into gene families e.g. ErbB family), EGFR expression levels and VeriStrat status.

Results:
The frequency of ErbB family alterations was low (SVs: EGFR 6.5%, HER2 4.9%, HER3 6.1%, HER4 5.7%; CNAs: EGFR 6.9%, HER2 3.7%). No individual genetic alterations, or grouped ErbB family aberrations, were prognostic of OS/PFS. Treatment benefit from afatinib versus erlotinib was consistent in all molecular subgroups. Most tumours were EGFR-positive by immunohistochemistry (afatinib: 82%; erlotinib: 86%). EGFR expression was not predictive of OS or PFS benefit (EGFR-positive PFS: HR=0.76 [0.57‒1.02]; OS: HR=0.84 [0.63‒1.12]; EGFR-negative PFS: HR=0.87 [0.45‒1.68]; OS: HR=0.77 [0.40‒1.51]). In afatinib-treated patients, both PFS (HR=0.56 [0.43‒0.72], p<0.0001) and OS (HR=0.40 [0.31‒0.51], p<0.0001) were improved in the VeriStrat-Good versus the VeriStrat-Poor group. VeriStrat-Good patients had significantly longer OS and PFS when treated with afatinib versus erlotinib (median OS: 11.5 vs 8.9 months, HR=0.79 [0.63‒0.98]; PFS: HR=0.73 [0.59‒0.92]). In VeriStrat-Poor patients there was no significant difference in OS between afatinib and erlotinib (HR=0.90 [0.70‒1.16]). However, there was no significant interaction between treatment arms and VeriStrat classification.

Conclusion:
Despite comprehensive, multifaceted analysis, no biomarkers were identified that predicted the benefit with afatinib over erlotinib in patients with SCC of the lung. Afatinib is a treatment option in this setting irrespective of patients’ tumour genetics or EGFR expression levels. However, patient outcome strongly depends on VeriStrat status.

Background:
Tyrosine kinase domain gene mutations of the epidermal growth factor receptor gene (EGFR) have proven to be clinically significant in nonsmall-cell lung cancer (NSCLC), particularly in adenocarcinoma. However, EGFR mutations in other type of lung cancer such as squamous cell lung cancer is uncommon.

Methods:
This is a preliminary study of which EGFR mutations were investigated using mutation-specific High Resolution Melting (HRM) polymerase chain reaction (PCR) from cytologic samples of squamous cell lung cancer. Cytological samples were obtained from bronchoscopy or transthoracal needle biopsy. Trained lung pathologist determined the cytological type of the tumor as squamous cell lung cancer. Immunohistological evaluation were not done since the cytological sample were limited.

Results:
Twenty subjects with confirmed squamous cell lung cancer were enrolled between June 2014 -December 2014 in Pulmonary Refferal Hospital/ Persahabatan Hospital Jakarta Indonesia, of which 18/20 (percents) were male, age between 50-75 years old. Eighty percents subjects has stage IV/metastatis with Performance Status of 2-3 at the time of diagnosis. EGFR mutations were detected in 4 of 20 subjects (20%) . Two subjects harbour exon 19 deletion, and 2 subjects harbour L858R mutation.

Conclusion:
These results suggest that EGFR mutations are found in 20% cytologically confirmed squamous cell lung cancer in this group.

Background:
Insulin-like growth factor (IGF) signaling is implicated in acquired resistance to EGFR TKIs in NSCLC. BI 836845 is an IGF ligand-neutralizing antibody that binds to IGF-1 and IGF-2, and inhibits their growth-promoting activities. This Phase Ib trial evaluates BI 836845 in combination with afatinib in patients with NSCLC progressing following prior treatment with EGFR TKIs or platinum-based chemotherapy (NCT02191891).

Methods:
The trial consists of two sequential parts: a dose confirmation part (Part A, reported here) and an expansion part (Part B). In Part A, eligible patients were aged ≥18 years with advanced and/or metastatic NSCLC progressing on EGFR TKIs (patients with EGFR mutations) or platinum-based chemotherapy. Patients receiving prior afatinib therapy below the assigned dose level or <30mg/day, or with progression on an insufficient dose of EGFR TKI prior to the study, were excluded. Part A used a 3+3 dose-escalation design with a starting dose of BI 836845 1,000mg/week (1-hour intravenous infusion) plus oral afatinib 30mg/day, in 4-week cycles. Primary endpoints were the maximum tolerated dose (MTD) of BI 836845 in combination with afatinib, and the occurrence of dose-limiting toxicities (DLTs) during Cycle 1.

Results:
At data cut-off (18 April 2016), 16 patients were treated (BI 836845 1,000mg/afatinib 30mg [n=4]; BI 836845 1,000mg/afatinib 40mg [n=12]). Median age (range) was 60 (48–77) years. Fourteen (88%) patients had activating EGFR mutations. Nine (56%) patients discontinued treatment, mostly due to progressive disease (one patient discontinued BI 836845 for other reasons); seven patients remain on treatment. During Cycle 1, 0/3 patients (afatinib 30mg) and 0/12 patients (afatinib 40mg) had a DLT (one patient [afatinib 30mg] was replaced during Cycle 1 due to a non-DLT adverse event [AE]). Therefore, the MTD and recommended Phase II dose (RP2D) was determined to be BI 836845 1,000mg/week in combination with afatinib 40mg/day. All patients experienced at least one drug-related AE; the most common were diarrhea (n=12; 75%), paronychia (n=11; 69%) and rash (n=10; 63%). Drug-related AEs were mostly grade 1/2 (one patient [afatinib 30mg] had grade 3 stomatitis). No drug-related AEs led to discontinuation and no dose reductions were required for BI 836845 or afatinib.

Conclusion:
The MTD and RP2D of BI 836845 was determined as 1000mg/week in combination with afatinib 40mg/day in patients who have failed prior EGFR TKIs or chemotherapy. This combination demonstrated a clinically manageable safety profile, consisting of AEs commonly associated with afatinib. The expansion part (Part B) is ongoing.

Background:
Patients with non-small-cell lung cancer (NSCLC) carrying specific mutations at epidermal growth factor receptor (EGFR) gene are usually sensitive to treatments with tyrosine kinase inhibitors (TKIs). However, not all EGFR-mutated patients respond equally to TKI treatments, and approximately 20-30% show primary resistance. Although the mechanisms responsible for acquired resistance are known, those responsible for primary resistance are not completely understood. In this study we aimed to assess the role of TP53 mutations in a cohort of advanced EGFR-mutated NSCLC patients receiving first-line TKIs. We analyzed TP53 gene status in relation to outcome in terms of overall response rate, disease control rate (DCR), response duration, progression free survival (PFS) and overall survival (OS).

Methods:
We retrospectively analyzed 136 patients with advanced EGFR-mutated NSCLC treated with first-line TKIs from January 2012 to April 2015. Exons 5-8 of TP53 gene were amplified by PCR and sequenced by direct sequencing on 123 patients. DCR was defined as the sum of complete response, partial response and stable disease. The survival endpoints examined were PFS and OS. PFS was defined as the time from start of first-line treatment to disease progression or death, whichever occurred first. OS was defined as the time from start of first-line treatment to death.

Results:
TP53 mutations were observed in 37 (30.1%) patients:10 (27.0%), 6 (16.2%), 9 (24.3%) and 12 (32.4%) in exons 5, 6, 7 and 8, respectively. DCR was 70% in TP53-mutated patients compared to 88% in TP53-wt patients (relative risk, RR: 3.17 [95% CI 1.21-8.48], p=0.019). In particular, a 42% DCR was observed in patients with TP53 exon 8 mutation compared to 87% in exon 8 wt patients (RR 9.6 [2.71-36.63], p<0.001). Shorter median PFS and OS were observed in patients with TP53 exon 8 mutations compared to other patients (4.2 months vs 12.5 months [p=0.058] and 16.2 months vs 32.3 months [p=0.114], respectively); these differences became significant in the subgroup of patients with EGFR exon 19 deletion (4.2 months vs 16.8 months [p<0.001] and 7.6 months vs not reached [p=0.006], respectively), hazard ratio (HR) 6.99 (95% CI 2.34-20.87, p<0.001) and HR 4.75 (95% CI 1.38-16.29, p=0.013), respectively.

Conclusion:
TP53 mutations, in particular exon 8 mutations and those defined as nondisruptive, reduce responsiveness to TKI treatment and induce a worse prognosis in EGFR-mutated NSCLC patients, especially in those carrying exon 19 deletions.

Background:
This study has been designed to evaluate the differential effect of EGFR mutation status (exon 19 versus 21) on PFS and OS in treatment naïve advanced EGFR Mutation positive adenocarcinoma lung treated with Gefitinib as first line agent

Methods:
This was a post hoc analysis of EGFR mutated (exon 19 and 21) advanced-stage (Stage IIIB or IV), chemotherapy-naive NSCLC patients treated with gefitinib as first line in a phase 3 randomized study. Patients were treated with Gefitinib 250 mg daily. Patients underwent axial imaging for response assessment on D42, D84, D126 and subsequently every 2 months till progression. Responding or stable patients were treated until progression or unacceptable toxicity. SPSS was used for statistical analysis. Kaplan Meier method was used for survival estimation and log rank test for comparison. Cox proportion hazard model was used for multivariate analysis.

Results:
141 patients were eligible for analysis of which 78 were males and 63 were females. 127 patients (90.1%) were ECOG 0-1 while 14 patients (9.1%) were ECOG >1. Exon 21 mutation was present in 65 patients (46.1%) and exon 19 mutation in 76 patients (53.9%). 133 of 141 patients were evaluable for response. Response rate of patients having exon 19 mutation was 72.9% (51 patients, n=70) while it was 55.6% in patients having exon 21 mutation (35 patients, n=63) {p=0.046}. Median PFS in exon 19 mutated patients was 9.3 months (95% CI 6.832-11.768) compared to 7.8 months (95% CI 5.543-10.047) in exon 21 mutated patients (p=0.699). The median OS in exon 19 mutated patients was 19.8 months (95 % CI 16.8-22.7) and 16.5 months (95% CI 10.9-22.1) in exon 21 mutated patients (p=0.215).Only female gender had a positive impact on both PFS and OS on multivariate analysis.

Results:
141 patients were eligible for analysis of which 78 were males and 63 were females. 127 patients (90.1%) were ECOG 0-1 while 14 patients (9.1%) were ECOG >1. Exon 21 mutation was present in 65 patients (46.1%) and exon 19 mutation in 76 patients (53.9%). 133 of 141 patients were evaluable for response. Response rate of patients having exon 19 mutation was 72.9% (51 patients, n=70) while it was 55.6% in patients having exon 21 mutation (35 patients, n=63) {p=0.046}. Median PFS in exon 19 mutated patients was 9.3 months (95% CI 6.832-11.768) compared to 7.8 months (95% CI 5.543-10.047) in exon 21 mutated patients (p=0.699). The median OS in exon 19 mutated patients was 19.8 months (95 % CI 16.8-22.7) and 16.5 months (95% CI 10.9-22.1) in exon 21 mutated patients (p=0.215).Only female gender had a positive impact on both PFS and OS on multivariate analysis.

Background:
The identification of activating epidermal growth factor receptor (EGFR) mutations is essential for deciding therapy of non-small cell lung cancer (NSCLC) patients. Circulating cell-free tumor DNA (cftDNA) holds promise as a non-invasive methodology for tumor monitoring in solid malignancies. Among advanced NSCLC patients with an acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs), about 50% carry T790M mutation, but its frequency in EGFR-TKI-naıve patients and dynamic change during therapy remains unclear. We hypothesized that EGFRmutation analysis detection in cftDNA for NSCLC may be feasible for monitoring treatment response to EGFR-TKIs and also predict drug resistance.

Methods:
EGFR sensitive mutations and T790M were analyzed using digital PCR (d-PCR) (Quant studio 3D, life technologies) in longitudinally (at baseline, at 4, 8, 20, 60, 120, 180, 270, 360 days) collected plasma samples (n=50) from 8 tissue-confirmed EGFR-mutant NSCLC patients treated with an EGFR-TKI (Gefitinib N = 4; Erlotinib N = 1; Afatinib N = 3). DNA extracted from plasma of 8 healty blood donors were used to detect the specificity of EGFR mutant assay. Tumor assessment was performed according to RECIST criteria 1.1 every two months.

Results:
The sensitivity of d-PCR in plasma versus tissue was 71.4%. No EGFR mutation was present in the 8 control cases (specificity of 100%). Of four patients who developed progression disease (PD), in the samples of progression, T790M was detected in 75% of cases. The frequency of T790M in pre-TKI plasma samples was of 37.5%. EGFR sensitive mutations decreased at PD while T790M mutation increased in 75% of patients. Patients with concomitant pre-TKI EGFR 19 deletion and T790M showed a PD before of 12 months compared to those with L858R. T790M was frequently detected when new lesions were developed. Four patients had T790M level decreased to undetectable level with longer PFS than those with detectable T790M in blood.

Conclusion:
Our results indicated that d-PCR was a highly sensitive and useful method for detecting the T790M mutation. Moreover, dynamically monitoring T790M change might help determining EGFR-TKI resistance. We thank Italian Association for Cancer Research (AIRC) for supporting the study.

Background:
VeriStrat is a proteomic test validated to be prognostic and predictive of overall survival (OS) to EGFR Tyrosine Kinase Inhibitors (TKIs) in EGFR wild type patients[1]. Serum Amyloid A1 (SAA1) and its two truncated forms are responsible for 4 of the 8 peaks overexpressed in Veristrat (VS) Poor classified patients. The aim of the study was to explore if the microenvironment, with its complex network mediated by inflammation and angiogenesis could represent the base of the aggressive disease behavior of VS Poor subjects. Moreover, the activity of the immune escape axis PD-1/PD-L1 was explored.

Methods:
Plasma biomarkers analyses were retrospectively performed on plasma baseline samples of 244 patients enrolled in the PROSE trial[1], while exploratory tissue analysis was performed on 37 available histological specimens. Circulating levels of HGF, VEGF, FGF, Cromogranin A (CgA) and its pro- and anti-angiogenic fragments (fragment 1-373 and 1-76, respectively) were determined by an ELISA assay. PD-L1 expression both on tumor cells and inflammatory elements of the tumor microenvironment, and the tissue expression (T) of HGF and its receptor c-MET were measured by immunohistochemistry.

Results:
CgA, HGF, VEGF, and FGF plasma levels were statistically significantly higher in VS Poor subjects. High plasma HGF levels were associated with lower PFS (3.4 versus 2.0 months, HR 1.67; 95% CI 1.25-2.23; p<0.001) and OS (11.2 versus 6.4 months, HR 1.64; 95% CI 1.12-2.23 p=0.002). High PD-L1- tumor expression was associated with worse PFS (5.9 versus 1.9 months, HR 2.28; 95% C.I. 1.14-4.57; p<0.020) and a trend for lower OS (14.6 versus 6.7 months, HR 1.47; 95% CI 0.85-2.53; p=0.165), but not significantly associated with VS status (p=0.656). At the multivariate analysis, CgA, HGF and VEGF were independently associated with VS Poor status. When clinical variables were also included (histology and PS), multivariate analysis evidenced VEGF as the only independent biomarker associated with the VS Poor classification (p=0.0013). Plasma HGF levels (HR 2.083; 95% C.I. 1.306-3.321; p=0.0021) and tumor PD-L1 expression (HR 2.579; 95% CI 1.036-6.421; p=0.0417) remained independent prognostic biomarkers for shorter PFS.

Conclusion:
Inflammation and angiogenesis appear to be associated with the complex processes at the base of the Veristrat signature. Plasma HGF levels and tumor tissue PD-L1 are prognostic in terms of a worse PFS, but VeriStrat remains the only highly reproducible clinically relevant biomarker associated with OS. [1]V Gregorc et al, The Lancet Oncology, p713, 15(7),2014.

Background:
Third-generation tyrosine kinase inhibitors (3rd-TKIs) have proven effective in patients with EGFR T790M who progress on prior EGFR TKI therapy. Median progression-free survival (PFS) on a 3rd-TKI was 9-10 months for T790M+ patients compared to 2-4 months for T790M- patients. PFS is similar regardless of the specimen used to assess T790M (tissue, plasma, or urine). Using simulation analytics, the primary study aim was to assess the cost effectiveness of a urine-testing strategy (UTS) versus a tissue-testing strategy (TTS) for T790M detection in patients with EGFR-positive lung adenocarcinoma and progression on prior TKI therapy.

Methods:
Analytics followed International Society for Pharmacoeconomics and Outcomes Research (ISPOR) and Society for Medical Decision Making (SMDM) guidelines for Good Modeling Practices, and Consolidated Health Economic Evaluation Reporting Standards (CHEERS) for reporting findings. Outcomes and economic implications were assessed from the perspective of a third-party US payer, stratified by government versus commercial fee rates. Endpoints were PFS, overall survival (OS), direct medical resources used (biopsies, chemotherapy, post-progression) and related costs. Data sources were published reports of randomized drug trials and current data, which includes accuracy results of tissue versus urine testing (Trovagene, San Diego, CA), Medicare fee schedules, and available adjustments for fees in commercial markets. A state-transition analysis and Markov model tracked patients from stable disease, progression, and to death. Full univariate and multivariate sensitivity analyses were performed to assess the robustness of findings and factors that most influenced outcomes and costs.

Results:
Median PFS after treatment with 3rd-TKI was 3.4 months if tumor testing is T790M- versus 9.7 months if T790M+. Because urine testing can be used in patients for whom biopsy cannot be performed or when tissue testing reveals indeterminate results, PFS and OS were slightly increased using the UTS. UTS resulted in avoidance of a biopsy procedure, potential complications, and tissue-based molecular testing in approximately 48% of patients, leading to a 2- to 10-fold total cost savings relative to the unit cost for a urine test. Within the robust variations in input parameters, the cost of a biopsy procedure/complications and tissue-based molecular testing were the most influential factors.

Conclusion:
UTS is a dominant scenario to TTS by saving costs and improving patient experience (e.g., PFS/OS, and reduction in biopsy related complications). This result is based on LEVEL I evidence from a large, randomized trial that showed PFS is similar among patients regardless of urine versus tissue testing for T790M mutation status.

Background:
This study aimed to assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations including L858R, E19-dels, T790M, and G719X from circulating tumor DNA (ctDNA) in patients with non-small cell lung cancer (NSCLC).

Methods:
Plasma samples were collected from 20 patients with NSCLC including detailed clinical information along with data regarding treatment response. ctDNA was extracted from 10 mL plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Extracted ctDNA was analyzed using two real time-amplification refractory mutation system-quantitative PCR platforms (cobas® EGFR Mutation Test: cobas; and AmoyDx® EGFR 29 Mutations Detection Kit: ADx), one digital platform (Droplet Digital[TM] PCR, ddPCR: Bio-rad), and one next-generation sequencing platform (firefly NGS: Accuragen).

Results:
If a positive result was obtained from any one of the four platforms, the sample was categorized as positive. We identified 15 EGFR mutations in 20 patients with NSCLC using the four platforms, for which 7, 11, 10, and 12 mutations were detected by ADx, cobas, ddPCR, and firefly NGS, respectively. Among the 15 EGFR mutations, six and seven EGFR alterations demonstrated an allele frequency of more or less than 1% (group A or B, respectively), and two exhibited unknown allele frequency. In group A, 5, 5, 5, and 6 EGFR mutations were detected by ADx ,cobas, ddPCR, and firefly NGS, respectively. The positive coincidence rate of any two platforms ranged from 66.7% to 100% and the kappa value varied from 0.787 to 1.000 in group A. In group B, 1, 5, 5, and 6 EGFR mutations were detected and the positive coincidence rate of any two platforms ranged from 16.7% to 100% and the kappa value varied from 0.270 to 1.000. The output of cobas, ddPCR, and firefly NGS were highly correlated, whereas ADx displayed weak concordance with these three platforms in group B. In addition, we identified 75 wild-type loci when EGFR alleles identified as negative by one or more platforms were considered as negative. ADx, cobas, ddPCR, and firefly NGS uncovered 73, 69, 70, and 68 EGFR wild-type loci, respectively. The concordance and negative coincidence rates between any two platforms were over 90%.

Conclusion:
The detection rate and concordance were probably affected by the abundance of EGFR mutations and the sensitivity of different platforms. Three platforms, including cobas, ddPCR, and firefly NGS, exhibited higher positive coincidence and detection rates when the allele frequency was lower than 1%.

Background:
Circulating tumor DNA (ctDNA) are short DNA fragments released into the systemic circulation by rapid cell turnover, and excreted into the urine. Urinary ctDNA-based detection of oncogenic mutations is a non-invasive modality that can help in clinical decision-making, especially when tissue biopsies are not available. When conventional imaging modalities are inconclusive, quantitative assessment of systemic ctDNA burden has the potential to assess response to therapy. In this case series we assessed EGFR mutation status at baseline and at intervals following administration of tyrosine kinase inhibitor (TKI) therapy to determine whether EGFR systemic mutation load correlated with disease burden and therapeutic response.

Methods:
Four patients on anti-EGFR (TKI) were prospectively monitored for quantitative assessment of systemic mutant allele burden of activating and resistance EGFR mutations (Exon 19 deletions, L858R and T790M) in urine. EGFR mutations were quantitatively interrogated by short footprint mutation enrichment PCR followed by next-generation sequencing assays. Systemic mutant allele burden was compared to assessment of tumor burden computed by standard imaging modalities.

Results:
Patients 1, 2, and 3 were originally diagnosed with EGFR-positive NSCLC. Targeted molecular testing of systemic urine ctDNA revealed high EGFR mutation burden and the presence of the T790M resistance mutation at the time of progression on TKI therapy (>550 copies/10[5] genome equivalents (GEq)). Interestingly, the extent of radiographic progression in patient 3 was not completely clear, and urinary T790M along with clinical assessment of pain helped determine progression prior to obtaining pleural effusion results. After initiation of a 3[rd] generation TKI (patient 1: ASP8273, patients 2 and 3: osimertinib), all patients experienced an appreciable decrease in the EGFR mutation burden, which was consistent with clinical improvement prior to radiographic imaging. Patient 4 presented with multiple lung nodules at diagnosis and a high systemic L858R mutant allele burden (>550 copies/100,000 GEq). Two months after initiation of first-line TKI, the main lesion and lymph nodes slightly improved, but the lung nodules progressed. The high systemic L858R burden persisted at the same level as pre-therapy.

Conclusion:
Urinary ctDNA-based quantitative assessment of systemic EGFR mutant allele burden is a non-invasive molecular diagnostic testing modality that correlates with tumor burden and response to therapy.

Background:
The lung specific GPA score is an index, commonly used for patients with non-small cell lung carcinoma (NSCLC) and brain metastasis (BM) in order to predict overall survival (OS) with the help of four easy-to-use items: age at the diagnosis of the brain metastasis; Karnofsky Performance Status (KPS), presence of extra cranial metastasis (ECM) and number of brain metastasis (Sperduto PW et al. J Clin Oncol 2012; 30:419‑25). However, this tool might not be appropriate to patients harboring EGFR activating mutations, which are known to have better prognosis than those with no activating mutations. The goal of our study was to determine if the Lung specific GPA score is adapted to population with these mutations.

Methods:
We retrospectively analyzed 108 Caucasians patients diagnosed with NSCLC between 2000 and 2014. Clinical features, systemic treatments (chemotherapy or EGFR tyrosine kinase inhibitors) and local brain treatment (Whole Brain Radiotherapy (WBRT) or surgery or Stereotactic Radiosurgery (SRS) were examined. OS were compared to the expected OS according to the lung specific GPA score as described by Sperduto.

Results:
Ninety-eight patients (91%) had EGFR activating mutations, whereas 10 (9%) were undetermined. 69% of them were nonsmokers. 77/108 patients (71.3%) were woman. 53/108 patients (49%) had BM: 30 patients had synchronous BM whereas 23 patients had metachronous BM. 20/53 patients (38%) were treated with WBRT. BM is still a burden for NSCLC patients harbouring EGFR activating mutations, (median OS 42 vs 25.5 months, p = 0.0052). OS from BM diagnosis was significantly superior in patients with EGFR activating mutations, compared to the expected OS in accordance with Sperduto’s GPA score (1): group 0-1 (11.5 months IC 95% [8.5-25.7] vs 3.02 months IC 95% [2.63-3.84]); group 1.5-2 (28 months IC 95% [24.3-NR] vs 5.49 months IC 95% [4.83-6.40]) (p = 0.026). Among EGFR activating mutations, OS were also significantly longer in group 1.5-2 than in group 0-1 (28 vs 11.5 months) (p = 0.026)

Conclusion:
Currently, Lung specific GPA is not accurate enough to estimate survival time for EGFR mutant patients. However, this tool is still reliable to distinguish different prognosis for patient with EGFR activating mutations, according to their GPA score. In conclusion, EGFR status modifies OS of patients and should be integrated in the items of the GPA score. These results should be validated in further prospective studies.

Background:
The emergence of T790M mutation (T790M) represents the main mechanism of acquired resistance (AR) to 1[st] and 2[nd] generation tyrosine kinase inhibitors (TKIs) in EGFR mutant patients (p). Recently, 3[rd] generation inhibitors (T790Mi) have demonstrated activity in EGFR mutant (mu) patients with AR to TKIs harboring T790M. Serum and plasma have been used as an alternative to tissue to detect both sensitizing EGFRmu and T790M. We evaluated if (1) T790M could be monitored along T790Mi therapy in p with baseline T790M in serum, (2) T790M loss could be correlated to clinical and radiographic response, and (3) T790M disappears soon in rapid responders.

Methods:
10 p out of a total of 15 T790M+p treated with T790Mi were selected according the baseline T790M+ in serum. Baseline characteristics, data on changes in T790M in serum; and radiographic and symptom changes along T790Mi therapy were collected. T790M in serum was detected using a PNA-locked nucleic PCR clamp-based technique. T790M was evaluated at baseline and at certain times after T790Mi initiation.

Results:
80% of the p were female and never smoker; 100% were adenocarcinoma, Caucasian, del19, and were treated with previous TKI, with a median (m) time to treatment failure of 11.25 months (mo) [range (r)1-19 mo]. P received 2 previous treatments (r1-6), 40% had a rebiopsy for T790M evaluation, had 3 metastatic sites (r1-6), and had a PS 1 in 70% of the cases. 5 p were evaluable for response with 2 SD and 3 PR as best response (BR) in the 1[st ]evaluation. 7 out of 9 p evaluable for clinical response, experienced an improvement in baseline symptoms as soon as 3 weeks (w) after starting T790Mi, only 1 p experienced an increase in pain, but not related to bone M1. T790M was lost in 80% of the p and it was not detected in serum at 3 or 6 w after the T790Mi initiation in 2 out of 4 and 4 out of 7 evaluable p, respectively.

Conclusion:
T790M detection can be lost early along T790Mi treatment. The decrease in symptom burden is seen in p with loss of T790M. PR and SD represent the BR in p with loss of T790M. The loss of T790M in the serum may be a marker of symptomatic and radiographic response to T790Mi. Future evaluation would demonstrate if the reappearance of T790M mutation in serum could be a marker of resistance to T790Mi.

Background:
First generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as gefitinib (GEF) or erlotinib (ERL) are effective for patients harboring EGFR mutations positive non-small cell lung cancer (NSCLC). To know the impact of metastatic status on patients treated with EGFR-TKIs is important. The objective of this study is to know the impact of metastatic status including brain metastasis, bone metastasis, liver metastasis and pleural effusion on the prognosis of patients treated with first generation EGFR-TKIs.

Methods:
The pathology files of National Hospital Organization Kinki-chuo Chest Medical Center from 2009 to 2014 were retrospectively reviewed and 533 patients were EGFR mutation positive NSCLC. Patients with stage IA to IIIA and small cell lung cancer were excluded. From 299 stage IIIB, IV and relapsed NSCLC patients, 178 patients treated with GEF or ERL as first line treatment were enrolled for the study. Metastatic status, progression free survival (PFS), overall survival (OS) and response rate (RR) were investigated. We also evaluated the relationship between the number of metastatic organs and prognosis.

Results:
Fifty-one patients were male and median age at the time of first line treatment was 72 years (range 39-91). Number of patients with adenocarcinoma was 168 and 156 patients were treated with GEF. In log-rank test, PFS and OS were significantly worse in patients with brain metastasis (8.0m vs 13.2m, p= 0.008; 20.2m vs 38.0m, p< 0.001 respectively), bone metastasis (8.8m vs 15.4m, p< 0.001; 24.0m vs 32.1m, p= 0.020 respectively), liver metastasis (6.7m vs 12.5m, p< 0.001; 13.4m vs 32.1m, p< 0.001 respectively) and pleural effusion (10.8m vs 12.2m, p= 0.033; 21.9m vs 34.9m, p= 0.004 respectively) at the time of first line treatment compared with patients without each metastasis. In cox proportional hazards models multivariate analysis, bone metastasis had correlated with worse PFS (HR 2.110, 95%CI 1.437-3.093, p< 0.001) and moreover, brain metastasis had correlated with worse OS (HR 2.414, 95%CI 1.464-3.951, p< 0.001). There were no relationships between metastatic status and RR. Furthermore, the much number of metastasized organs (no metastasis, 1 metastasis and 2 or more metastasis) was significantly related to worse PFS (19.3m vs 12.5m vs 6.6m, p< 0.001) and OS (46.1m vs 29.9m vs 15.1m, p< 0.001).

Conclusion:
Bone metastasis had correlated with worse PFS and brain metastasis had correlated with worse OS in patients treated with first generation EGFR-TKIs. Moreover, the much number of metastasized organs was related to worse PFS and OS.

Background:
Primary resistance to EGFR-TKIs was generally defined as disease progression in less than 3 months without any evidence of objective response. Although possible mechanisms have been investigated in several preclinical and retrospective studies, little is known about the molecular backgrounds of primary resistance.

Methods:
Random Sample of Cases was used to screen TKI-sensitive patients to match with the primary resistant patients on the basis of clinical characteristics (smoking history, EGFR mutations etc.). DNA was extracted from the tumor and their matched normal material. The paired-end whole exome sequencing (WES) of DNA was performed on the Illumina HiSeq 2500 sequencing platform.

Results:
Totally, five patients exhibiting primary resistance to EGFR-TKIs were enrolled and each was randomly matched with one patient possessing TKI sensitivity (Table1). The mean depth of the WES was 100x. The mean number of nonsynonymous SNV per sample was 195 (range 97 to 348) in TKI-resistant group versus 84 (range 60 to 101) in TKI-sensitive group (P=0.059). Consistent with the initial result of Sanger sequencing, all 10 patients were found with EGFR sensitizing mutations (exon 19 deletions or L858R point mutation in exon 21), but no T790M mutation; the resistance group present with a lower EGFR mutant allele frequency than the sensitive group. Next generation sequencing of TKI-resistant specimens detected KRAS amplification (CN~tumor ~/ CN~normal ~= 2.6) in one of five patients, and MET amplification (CN~tumor ~/ CN~normal ~= 2.3) in another one. The recurrent mutation genes included FAT4, RBM10, TANC2, ACAN, PPFIA2, UBR4, XIRP2 and PRAMEF1. Figure 1



Conclusion:
Next-generation sequencing offers more complete genomic analysis to understand the mechanism of differential responses to EGFR-TKIs, which can lead to more precision therapy. KRAS amplification appears to be a newly mechanism underlying primary resistance to EGFR-TKIs in patients harboring TKI-sensitive EGFR mutations. However, it needs to be validated in a larger cohort.

Background:
Bcl-2- like protein 11 (BIM) is a key protein in promoting apoptosis. BIM deletion polymorphism has been proposed as the intrinsic EGFR-TKI resistance and to predict poor response to EGFR-TKI treatment. However, there were conflict results of BIM deletion as the predictive biomarker in previous studies and EGFR-TKI reimbursement is a problem in most low and middle income countries. This study evaluated sequence of EGFR-TKI therapy and role of BIM deletion to maximize the cost-effectiveness of treatment in Thai population.

Methods:
Advanced EGFR-positive NSCLC patients were identified from database between 9/2012 and 12/2014. Retrospective review of 185 medical records was performed. Only 139 patients received EGFR-TKI. Archive tissues were available 129 samples. RT-PCR amplification designed to detect BIM deletion (2903 bp) in intron 2. The correlations of BIM deletion, sequence of EGFR-TKI, and survivals were analyzed.

Results:
Prevalence of BIM deletion was 26/129 (20.2%). Median follow-up time was 17.4 months. BIM deletion patients had trend of shorter both PFS and OS compared to wild-type. L858R patients who received first-line EGFR-TKI had significant longer PFS and higher RR compared to whom received later-line EGFR-TKI (12.6 vs 6.3 mos, P=0.03), (78% vs 49%), respectively. OS in EGFR-positive NSCLC was significantly longer in patients whom received EGFR-TKI compare to chemotherapy alone (28.9 vs 7.4 mos, HR= 0.25 [0.16-0.40], P<0.001). The absolute OS difference between patients whom received EGFR-TKI compared to chemotherapy alone was significantly longer in BIM wild-type group (28.9-7.4=21.5 months) compared to BIM deletion group (25.8-17.9=7.9 months). Figure 1



Conclusion:
BIM deletion polymorphism could be one of the predictive biomarker to maximize the benefit of EGFR-TKI treatment. Furthermore, L858R patients have longer survival if received EGFR-TKI as the first-line treatment. These results could help the low and middle income countries to maximize the cost-effectiveness and to solve reimbursement problem of EGFR-TKI therapy.

Background:
Epidermal growth factor receptor (EGFR) mutations are predictive marker of EGFR-tyrosine kinase inhibitor (TKI) therapy. We compared the sensitivity of EGFR mutation detection techniques between matched tumor tissue and peripheral blood sample in patients with lung adenocarcinoma.

Methods:
We collected the paired samples from plasma and paraffin-embedded tumor tissue in 202 patients before EGFR-TKIs. DNA extraction was performed using the QIAamp MinElute virus spin kit and EGFR mutation analysis was done by two detection methods. One is the PNAClamp[TM] which is the PNA-based PCR clamping that selectively amplifies only the mutated target DNA sequence as minor portion in mixture with the major wild type DNA sequences. The other is the PANAMutyper[TM] EGFR kit, which use PNA clamping-assisted fluorescence melting curve analysis to perform mutation detection and genotyping. The degree of agreement was evaluated by Cohen's kappa value.

Results:
The EGFR mutation rates by PANAMutyper[TM]R and PNAClamp[TM] were 51.0% vs. 47.0% in tissue, and 22.2% vs. 11.4% in plasma sample, respectively. Overall concordance rates and the degree of agreement between tissue and plasma samples was better in PANAMutyper[TM]R (69.3%, k=0.393, p<0.001) than PNAClamp[TM] (62.3%, k=0.211, p<0.001). Sensitivity of plasma EGFR mutations was higher (41.7% vs. 22.1%, p<0.001) and false negative rate was lower in PANAMutyper[TM]R test (58.3% vs. 77.9%, p<0.001). Response to EGFR-TKI was correlated with plasma EGFR mutation by PANAMutyper[TM]R (p=0.025) unlike PNAClamp[TM ](p=0.829).

Conclusion:
The sensitivity and concordance rate of PANAMutyper[TM]R test were better than standard PNAClamp[TM] test. So this technique can be useful to detect EGFR mutation in circulating cell-free DNA sample.

Background:
Several studies have shown that overexpression of thyroid transcription factor (TTF-1) may be associated with the presence of epidermal growth factor receptor (EGFR) gene mutations and predict survival in patients with non-small cell lung cancer (NSCLC). Nevertheless, the potential significance of TTF-1 immunostaining as a predictor of response to EGFR tyrosine kinase inhibitors (TKIs) has received limited research attention thus far. The aim of this study was to further explore the potential association between TTF-1 immunohistochemical expression and response to EGFR TKIs in patients with lung adenocarcinoma.

Methods:
The medical records of 129 patients with stage IV lung adenocarcinoma, treated at the Oncology Unit of “Sotiria” Athens General Hospital between January 2011 and December 2014, were retrospectively reviewed. All patients had received treatment with EGFR TKIs (erlotinib or gefitinib) and had a known TTF-1 immunohistochemical expression status in formalin-fixed, paraffin-embedded tumour tissue. Demographic and clinicopathological features (age, gender, smoking status and performance status), TTF-1 immunohistochemistry and EGFR mutation status results were correlated to each other as well as with the response rate (RR) to EGFR TKIs, using univariate and multivariate regression analysis.

Results:
Median age of our study population was 68 years (range 26-88 years), while the majority were male (79/129 cases, 61.2%). Patients with EGFR mutant tumors had significantly higher response rates to EGFR TKIs compared to patients with wild-type EGFR tumors (p=0.001). TTF-1 positive staining was weakly associated with the presence of EGFR mutations, without reaching the level of statistical significance (p=0.053). Most importantly, TTF-1 positive staining was not significantly correlated with RR to EGFR TKIs, when gender, age, smoking status, EGFR mutation status and PS were included in the multivariate model.

Conclusion:
The present results failed to demonstrate any independent association between TTF-1 overexpression and the RR to EGFR TKIs in patients with advanced-stage lung adenocarcinoma. Prospective data from larger cohorts of patients are needed to clarify the exact predictive value of TTF-1 immunostaining in this setting.

Background:
EGFR-mutations are associated with adenocarcinoma, female, never/light smoking and East-Asian ethnicity. Several clinical studies have shown that EGFR-TKIs are superior to platin-based chemotherapy in EGFRmut[+] NSCLC patients; however there is insufficient literature that examines differences in survival based on East-Asian ethnicity, independent of the EGFR mutation type, gender, smoking history and disease recurrence in an all EGFRmut[+ ]NSCLC cohort.

Methods:
A retrospective study of EGFRmut[+] NSCLC patients treated with EGFR-TKIs was conducted at the Tom Baker Cancer Centre in Calgary, Canada between March 2010 and December 2014. Ethnicity was defined by place of birth. Comparison of smoking history was made using Pearson χ[2] statistic. Survival analyses according to gender, smoking history and EGFR mutation type were estimated using the Kaplan-Meier method, with comparisons made between groups by the log-rank test. All statistical analyses were conducted using STATA. Statistical significance was assumed for a two-tailed p value < 0.05.

Results:
A total of 138 patients were identified (61 [44.2%] Asians; 77 [55.8%] non-Asians). Of these, 92 died, eight were lost to follow up and 38 were alive as of June 1, 2016. In the Asian subgroup, the median age was 64.4 years, 67% were female, and 71% were never/non-smokers. Meanwhile, in the non-Asian subgroup, the median age was 66.5 years, 61% were female and only 39% were never/non-smokers. The median PFS for Asians vs non-Asians was 8.7m and 8.2m, while the median OS was 25.6m and 16.1m, respectively. The log rank test demonstrated a statistically significant difference in survival between Asians vs non-Asians (p= 0.026). KM curves showed that: (i) smoking was an important demographic feature as non-Asians with a smoking history had the worst median survival (17.5m); Pearson χ[2] showed that the overall relationship between ethnicity and smoking history was significant, p < 0.001; (ii) Asian-females/males had longer survival (23.4m; 21.5m, respectively) than non-Asian females/males (19.7m; 14.5m, respectively) and (iii) regardless of EGFR-mutation type, Asians (exon 19 deletions: 25.9m, exon 21 (L858R): 20.4m) outlived non-Asians (exon 19 deletions: 19.6m and exon 21: 14.9m).

Conclusion:
The demographics from our study are consistent with findings from IPASS and EURTAC. Our current survival estimates suggest that even in a cohort of EGFRmut[+]NSCLC treated with an EGFR-TKI, there seems to be a survival benefit for Asian-ethnicity, even after stratifying by gender, smoking history and EGFR-mutation type. *This study was supported by AstraZeneca.

Background:
T790M is an acquired mutation in patients (pts) with advanced non-small cell lung cancer (aNSCLC) treated with an EGFR TKI. The tumours of approximately 60% pts treated with an EGFR TKI harbour the T790M mutation upon disease progression. With the launch of treatments targeting T790M in EGFR aNSCLC it is important to identify pts with T790M who will benefit from such therapy. However not all pts are able to undergo tissue biopsy for testing and the advent of ctDNA testing via plasma sample may increase the number of pts with access to a T790M test. This analysis assesses outcomes and costs of different diagnostic testing strategies for T790M in aNSCLC post-EGFR TKI.

Methods:
Estimates of T790M mutation prevalence and test performance were applied to a hypothetical cohort of 100 pts. Outcomes were number of true positive / true negative (TP / TN) samples and associated testing costs. The following testing strategies were assessed: i) tissue test only ii) plasma followed by tissue for pts who tested negative using plasma or, iii) both tissue and plasma tests concurrently. Total costs were also estimated for the sample procedure (biopsy or plasma extraction), laboratory testing, and biopsy adverse events (5% rate).

Results:
In sequence or concurrent testing (strategies ii & iii) produced the highest number of TP results (n=57) followed by tissue test alone (n=53). Tissue & plasma concurrently (iii) was associated with the highest total costs followed by tissue test only (i) and the lowest cost plasma followed by tissue for pts who tested negative (ii).

Conclusion:
To maximise identification of T790M, and hence the most pts who would benefit from targeted therapy, access to both plasma and tissue testing is advantageous. Plasma followed by tissue for pts who tested negative may also be a cost minimising option for healthcare systems compared to tissue testing alone.

Background:
: Early detection of resistance conferring epidermal growth factor receptor (EGFR) T790M mutation is of clinical significance in previously EGFR positive NSCLC patients.

Methods:
Plasma from 40 advanced NSCLC patients with known L858R or Del 19 745_750 Mutations were tested for cell free DNA on Droplet Digital PCR. This included biopsy confirmed treatment naive cases as well as those who developed clinical resistance to tyrosine kinase inhibitors. The later cases were examined for previous known and new T790M mutations using highly sensitive Droplet Digital PCR. Where ever feasible the results were compared to the secondary biopsy findings, duration of development of new T790M mutation and its serial plasma quantification results.

Results:
20 Treatment naive biopsy positive DEL19 /L858R cases were tested for cell free DNA. All cases were positive with values ranging from .04% to 24%. 19 cases were checked for primary and secondary mutations on progression of disease. 12 cases showed secondary mutation along with primary mutation. The values of T790M mutation ranged from 0.04% to 4.5%. We also had 3 cases with biopsy and cell free correlation of secondary mutation. None of them correlated.

Conclusion:
Droplet digital PCR is a robust platform to detect the primary driver EGFR mutations and can be used as a substitute / addendum to biopsy for initiating early treatment. It also appears to be too sensitive for detection of secondary T790M mutations. However response to treatment in patients with minimal cell free T790M values in plasma may need to be further investigated for clinical utilization of this platform.

Background:
(Applied for Late-Breaking Abstract Status) IASLC guidelines recommend EGFR mutation testing should be performed at diagnosis of advanced NSCLC to guide treatment decisions. In 2015 an international survey concluded that not all patients were tested or received test results before treatment initiation. This varied between countries and across regions. The aim of a follow-up survey in 2016 was to assess testing rates and HCP treatment choice in advanced NSCLC to identify improvements and changes compared to 2015.

Methods:
Online survey of 707 oncologists in 11 countries (Canada, China, France, Germany, Italy, Japan, South Korea, Spain, Taiwan, UK, USA) between July - August 2016. China was newly added in 2016. For better comparison with 2015 results, China was excluded from the primary results focus

Results:
Globally*, physicians requested EGFR mutation testing prior to first-line therapy of stage IIIb/IV NSCLC in 80% of patients. However, 18% of ordered tests were not received prior to deciding first-line therapy; an improvement on 2015 with 23%. Excluding histology, the main reasons for not testing prior to first-line therapy were insufficient tissue, poor performance status and long turnaround time. While turnaround time significantly reduced year-on-year (2016: 21% vs. 2015: 30%), globally* 24% of patients receive test results after more than 10 business days. Globally* 79% (2015: 80%) of patients with mutations (M+) were treated with tyrosine kinase inhibitors (TKIs), with large country variations on treatment preference (minimum 68% Germany; maximum 98% Taiwan). Prolonging of survival/extending life (54%*) was deemed the most important therapy goal in first-line treatment. 74%* of physicians stated that a clinically relevant increase in overall survival was the most important treatment attribute when choosing a first-line therapy, closely followed by an increase in progression-free survival (68%) and strong improvement of health related quality of life (66%). Perceived differences between TKIs were reported by 49% of physicians. Further detail will be presented at the congress. *Global figures excluding China

Conclusion:
While year-on-year improvements in EGFR testing rates, and availability of test results prior to first-line therapy are seen, a large proportion of EGFR M+ NSCLC patients are still not receiving targeted treatment with TKIs based on mutation status. Incomplete implementation of guidelines is still observed. The main barriers to testing, including receiving results in time, must be addressed if treatment equality for all eligible patients can be achieved. Physician education and closer guideline concordance are key steps to further improve outcomes.

Background:
NSCLC is a heterogeneous and dynamic disease where testing for key mutations is essential. With the emergence of clonal resistance, obtaining serial biopsies to assist in the real-time treatment decision-making has proven challenging. Molecular assessments of circulating tumor (ct)DNA has been previously shown feasible utilizing blood specimens. Here we additionally investigated the utility of serial urine ctDNA analysis in NSCLC.

Methods:
This is a prospective observational study of patients with non-squamous, tissue-confirmed EGFR, KRAS or ALK mutant NSCLC preparing to receive a systemic treatment regimen. Urine and blood specimens were collected at baseline, on treatment and at progression for ctDNA analyses. The primary endpoints were correlation between ctDNA and tumor-based molecular results, and measurable change in ctDNA with response by RECIST v1.1. Blood and urine samples were sent to Trovagene for DNA extraction and mutation enrichment NGS.

Results:
Of the 34 patients enrolled thus far, interim blinded analysis of EGFR activating mutations (L858R, exon 19 deletions) was conducted in 20 patients with EGFR-positive tumors. Of 20 patients, 17 (85%) had detectable concordant EGFR mutation in pre-treatment urine and/or plasma. Of 11 patients with matched serial ctDNA samples, detectable EGFR mutation signal was observed in pre-treatment urine and/or plasma of 9 patients. These 9 patients received first to sixth line treatment with single EGFR TKI (n=3), combination TKIs (n=3), chemotherapy (n=1), immune checkpoint inhibitors alone (n=1) or in combination with TKI (n=1). In 9 of 9 patients, changes in ctDNA levels from baseline to cycle 2 day 1 on therapy correlated with best response to treatment: a 100% decrease in urine and plasma EGFR mutation levels was observed in 6 of 6 patients with partial response (PR, n=3) or stable disease (SD, n=3), while less than a 90% decrease or an increase in urine and plasma EGFR levels was observed in patients with progressive disease (PD, n=3).

Conclusion:
Mutation enrichment NGS testing by urine and plasma ctDNA correctly identified EGFR activating mutations in 85% of patients. Monitoring EGFR levels in urine/plasma enabled accurate assessment of response in advance of radiographic evaluation and regardless of therapy type in 100% of patients, with cut-point of a 90% decrease in EGFR load discriminating between patients with disease control (PR or SD) and patients with progressive disease. With continued enrollment, our study aims to further investigate clinical utility of urine and plasma ctDNA for early detection of resistance and discontinuation of inefficient therapy.

Background:
First-line treatment with afatinib prolongs overall survival in patients with metastatic non-small-cell lung cancer (NSCLC) harboring EGFR exon 19 deletion mutations. Conversely, somatic KRAS mutations are negative predictors for benefit from EGFR-targeting agents. Rapid availability of these biomarker results is mandatory to prevent delayed or inferior treatments. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is well-established for lung cancer diagnosis and staging. Next generation sequencing (NGS) via targeted resequencing allows simultaneous interrogation for multiple mutations, but has its limitations based on the amount of tumor tissue required and assay times. RT-PCR using Light-Cycler technology (LC-RTPCR) is a rapid and sensitive assay to detect somatic mutations in various tissues from NSCLC patients. The study’s aim was to analyze if LC-RTPCR is feasible for rapid EGFRdelEx19 and KRAS Exon 2 mutation detection in EBUS-TBNA samples and to compare results with results obtained via standard NGS mutation analyses.

Methods:
48 surplus EBUS-TBNA samples (38 lymph nodes, 10 primary tumor) from 47 patients with pulmonary adenocarcinoma were analyzed. Two samples were collected per lymph node. One was used for routine cytology, the other was freshly frozen (ff). DNA from ff-biopsies was extracted using Genomic DNA buffer set (QIAgen, Germany). Mutation analysis by LC-RTPCR was conducted as previously described. NGS was performed on MiSeq (Illumina) via targeted resequencing using a customized multiplex-PCR panel covering 36 exons from 11 genes. Mutations were annotated with a minimum frequency of 2%. Processing time was approximately 4 days for NGS and 2 hours for LC-RTPCR analyses.

Results:
NGS of EBUS-TBNA samples was technically feasible for both markers in 22 (46%) samples, for EGFR testing in 32 (67%) samples, and for KRAS in 23 (48%) samples. EGFRdelEx19 mutations were detected in four (8.3% of intention-to-screen), and KRAS Exon 2 mutations in 15 cases (31%) cases. LC-RTPCR was technically feasible in all cases. All mutations detected by NGS were also detected by LC-RTPCR. LC-RTPCR detected three additional KRAS Exon 2 mutations and three additional EGFRdelEx19 mutations. NGS detected additional mutations in 4 cases (2 EGFR Exon 21, 1 KRAS Codon 61, 1 PIK3CA).

Conclusion:
LC-RTPCR is a rapid, highly sensitive method to detect mutations of immediate therapeutic relevance, such as EGFRdelEx19 and KRAS Exon 2 mutations, in limited EBUS-TBNA specimens from metastatic NSCLC patients. It is of value as rapid and sensitive initial assay in a two-step diagnostic process for first-line treatment decision, incorporating broader biomarker panels as second step.

Background:
The most common event responsible for resistance of epidermal growth factor receptor (EGFR)-tyrosine kinase Inhibitor (TKI) is acquisition of the T790M mutation, which occurs in approximately 50% of patients who initially respond to EGFR-TKI. Recently, third-generation EGFR-TKIs have been shown to exert a remarkable effect against T790M resistance mutation-positive non-small cell lung cancer (NSCLC). T790M is an important predictive marker for third-generation EGFR-TKIs; therefore, determining the clinicopathologic characteristics of T790M-harboring NSCLC showing relapse after EGFR-TKI therapy is of high clinical relevance. we evaluated whether T790M mutation is related to clinicopathologic or prognostic factors in patients with relapse of EGFR-mutant NSCLC after treatment with EGFR-TKIs.

Methods:
We retrospectively reviewed T790M status and clinical characteristics of advanced-stage or recurrent NSCLC patients who had received EGFR-TKI treatment and rebiopsy at Kurume University Hospital between March 2005 and December 2015.

Results:
we identified 193 patients with advanced or recurrent EGFR-mutant NSCLC who developed resistance to initial EGFR-TKI treatment. Of these patients, 105 (54%) underwent re-biopsy. Adequate histological specimens were available for 73 of these patients, who were enrolled in the study; 38 (52%) of them had T790M mutation and 35 (48%) did not. T790M mutation was present more frequently in patients with exon 19 deletion mutation (63%, 26 of 41) than in those with L858R mutation (38%, 12 of 32) (p=0.035) and in patients who received EGFR-TKI treatment for at least 10 months in total (71%, 32 of 45) than in those who did not (21%, 6 of 28) (p<0.001). The median PFS after initial EGFR-TKI treatment was longer in the T790M mutation-positive group (13.6 months, 95% CI: 9.2-15.8) than in the negative group (7 months, 95% CI: 3.7-8.5) (p=0.037). The median total duration of EGFR-TKI treatment in patients with T790M mutation was 15.3 months, which was significantly longer than that (8.1 months) in patients without T790M mutation (p<0.001). Multivariate analyze revealed that The type of EGFR mutation (exon 19 deletion mutation versus L858R point mutation, p=0.011, OR=0.21, 95% CI=0.05-0.71) and total duration of EGFR-TKI treatment (>10 months versus <10 months, p<0.001, OR=0.09, 95% CI=0.02-0.28) were significantly associated with the presence of T790M mutation.

Conclusion:
The patients with EGFR exon 19 deletion mutation and long-term treatment with EGFR-TKI exposure demonstrated a high prevalence of T790M mutation. These data are potentially important for clinical decision making in the NSCLC patients with EGFR mutation.

Background:
Lung adenocarcinoma patients harbouring sensitizing EGFR mutations can benefit from treatment with tyrosine kinase inhibitors (TKI). Whenever tumour tissue is inadequate or unavailable, detection of EGFR mutations in circulating cell-free tumour (ct) DNA from plasma is crucial to predict and monitor response to therapy. In this study we compared EGFR status between tumour tissue and plasma, using real-time PCR. Moreover, we evaluated the adequacy of ctDNA for a multi-target mass spectrometry (MS) analysis.

Methods:
EGFR mutations were investigated in paired plasma and tumour tissues from a prospective series of 105 lung adenocarcinoma patients: 79 had no prior TKI treatment and 26 underwent re-biopsy for TKI-acquired resistance. Molecular analyses were performed on tissue by a routine MS test (evaluation of 307 hot-spots in 10 genes including EGFR) and on ctDNA by a validated Scorpion/LNA real-time PCR (evaluation of 30 EGFR mutations). In the 26 postTKI patients ctDNA analysis was performed also by MS.

Results:
1-Plasma versus tissue by real-time PCR: overall sensitivity, specificity and concordance were 64.8%, 95.4% and 82%. In preTKI patients, 17 harboured EGFR sensitizing mutations on tissue,10 detected also in plasma (sensitivity 58.8 %, specificity 100%, concordance 91%). All 26 postTKI patients preserved EGFR sensitizing mutations. Regarding the detection of EGFR T790M resistance mutation, sensitivity, specificity and concordance were 63.6%, 80% and 73%. Specifically, 11 patients were T790M-positive (42%): 7 on both specimens, 4 only on tissue and 3 only on ctDNA. 2-Plasma versus tissue by MS: sensitivity, specificity and concordance for T790M were 50%, 93.3% and 76%. Particularly, 6 patients had a T790M-positive ctDNA: 5 concordant and 1 discordant with tissue; 4 T790M-positive cases on tissue were undetected in plasma; 1 sample was not evaluable.

Conclusion:
The real-time PCR on ctDNA showed a sensitivity consistent with literature and a high specificity, mostly in preTKI group. Concordance rates, influenced by biological and methodological factors, were lower in postTKI group. Indeed, some T790M mutations were detected only on ctDNA, which is expected to effectively mirror tumour heterogeneity better than bioptic samples, thus giving a global view of tumour. Finally, we demonstrated the adequacy of ctDNA for MS, in terms of quantity and quality. The use of a multi-target analysis on ctDNA might improve tumour characterization and response monitoring, evaluating important oncogenes other than EGFR, like PIK3CA, KRAS and BRAF. However, further studies are needed to better explore MS applicability on ctDNA.

Background:
Three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have led to multi-fold improvements in progression free survival of advanced stage non-small cell lung cancer (NSCLC) patients carrying EGFR kinase domain mutations. However, cure is not yet achievable with any EGFR TKI monotherapy, as patients will eventually progress due to acquired resistance. In vitro evidence suggests that minor populations of epigenetically modified drug tolerant cells (DTCs) may be one important mechanism for tumor cells surviving the TKI. We hypothesize that characterizing the genomic and epigenomic alterations observed in DTCs in vivo and comparing them to the bulk tumour will delineate a number of mechanisms of tolerance exhibited by DTCs.

Methods:
DTCs were induced via chronic erlotinib treatment of a lung adenocarcinoma primary derived xenograft (PDX) harbouring an erlotinib sensitive exon 19 deletion. Molecular profiles of DTCs are compared to untreated controls via immunohistochemistry (IHC) and gene expression array. We are now undertaking exome-sequencing, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), methylated DNA immunoprecipitation and sequencing (MeDIP-seq).

Results:
When compared to untreated tumours, DTCs exhibit decreased apoptosis (CC3 IHC) and proliferation (Ki67 IHC). DTCs maintained strong signaling via the EGFR pathway (pERK, pAKT, pS6). DTCs exhibited 2437 significantly differentially expressed genes (DEGs; >1.5-fold change and adjusted p-value <0.05) including multiple cancer stem cell markers (ALDH1A1, ALDH1A3, CD44). DEGs also were involved in vesicle-mediated transport (including lysosomes, exosomes and endosomes), autophagy, stress/unfolded protein response, cytoskeleton organization, chromatin organization, ion pumps and transporters, cell adhesion, WNT, NOTCH, PI3K and MAPK pathways. DTCs remained resistant to three cycles of cisplatin/vinorelbine either alone or when combined with erlotinib. Genomic and epigenomic profiling are on-going and results will be presented.

Conclusion:
DTCs may be a major impediment to cure by single-agent EGFR targeted therapies. Understanding the mechanisms and developing strategies to overcome DTCs may give insights on therapeutic strategy to further improve the survival of EGFR-mutated NSCLC patients.

Background:
Primary EGFR dual mutations comprising T790M and exon 19 or 21 mutation (SM, sensitizing mutations) are rare when common diagnostic methods are used. There are limited data about the treatment results with EGFR-TKIs in this setting. Purpose of this study was to find out the prevalence of primary dual EGFR mutations T790M and SM in the Caucasian population of NSCLC patients in Slovakia, and to evaluate treatment results with EGFR-TKIs in these patients.

Methods:
Retrospective multicentre study. The databases of the molecular/genetic diagnostic centres were searched for patients with dual EGFR mutations T790M and SM. Data regarding patients were obtained from the databases of participating institutions and patient files. Descriptive statistics was used for data analysis.

Results:
Altogether 3883 patients were tested for EGFR mutation from 2010 through 2015. Allele specific PCR was used in majority, high resolution melting analysis, Sanger sequencing and mutant-enriched PCR were also used. Double mutations T790M and SM were found in only six cases, i.e. the observed period prevalence was 0.15%. Patients’ characteristics and the treatment results are in the Table. PS was improved after two months of treatment in patients with initial PS over 1, and remained unchanged in those with PS 1. There were no unexpected AEs. Figure 1



Conclusion:
The prevalence of the dual EGFR mutations T790M and SM is very low in Caucasian population in Slovakia when common testing methods are used. Treatment results seen in this study suggest good effectiveness of the first or second generation EGFR-TKIs even in NSCLC with primary dual T790M and SM mutations. The quantitative analysis of these mutations using the blood sample is available at present. It might be useful in decision making about the use of the first – second or the third generation EGFR-TKI, based on the prevailing mutation.

Background:
Our department is among several centers in the Czech Republic which comprehensively diagnose and treat patients with lung cancer, including mutational analysis of EGFR and subsequent personalised treatment.

Methods:
We present the results of mutational analysis of EGFR gene, and effects of treatment in patients with NSCLC with mutations in this gene who were in treatment in our department in the years 2010 to 2015. We processed the data to get a five year long, single institution experience.

Results:
In 786 examined patients, 65% were male. Average age of was 65.1 years, median 66 years. In these 786 patients, 1039 analyses of EGFR mutation status were done. 10% (79 patients) were EGFR positive. Within the group of EGFR-positive patients 65% were female. Average age was 65.6 years, median age 66 years. 62% (46 patients) with positive EGFR mutation were non-smokers, 33% (25 patients) former smokers and 5% (4 patients) smokers. 92% of patients had adenocarcinomas. The most frequent mutations were in exon 19 and 21. 65 % (51 patients) of the EGFR positive group were treated with TKI, in some of the lines of treatment. TKI was mostly used in the first line of treatment. 35% (28 EGFR positive patients) were not treated with TKI because of PS 3 or 4 (11 patients), prior radical surgery (9 patients) or radical radiotherapy (1 patient). 7 patients left for other center. Median PFS in the group of 23 patients, who were treated with TKI in first line, was 8.0 months (average PFS 8.9 months). The average OS of 31 patients who were treated in the years 2010 to 2015, including lines with chemotherapy, was 18.3 months (median 17, the longest survival 56 and the shortest 2 months). At the beginning of April 2016, 20 patients were still in treatment. The longest survival in this group was 61 months. Three patients with mutation T790M, occurring simultaneously with deletion in exon 19 showed surprisingly good results. One patient was still alive at the beginning of April 2016, and OS of the remaining two patients was 43 and 56 months.

Conclusion:
The majority of EGFR positive patients were treated with TKI, mostly in the first line. The rest of the patients either did not need TKI therapy, or TKI was not indicated because of their overall poor condition. Our experience is similar to the results of larger multicenter studies.

Background:
Patients with lung adenocarcinoma harboring somatic activating mutations in the epidermal growth factor receptor (EGFR) gene have benefited significantly from EGFR tyrosine kinase inhibitors (TKIs). However, a vast majority would eventually develop resistance to such TKIs, resulting in disease progression. T790M, a secondary mutation in exon 20 of the EGFR gene, has been identified as the major mechanism responsible for acquired EGFR-TKI resistance, accounting for roughly 50% of resistance. However, the association of T790M status and clinical outcome remains elusive. Here, we conducted a retrospective study examining the association between T790M status and prognosis on clinical samples obtained from 43patietns with EGFR-mutant lung adenocarcinoma and later acquired resistance to EGFR-TKIs.

Methods:
We performed capture-based targeted ultra-deep sequencing on either tumor biopsies or blood samples of 43 lung adenocarcinoma patients, who harbored EGFR mutations, and subsequently developed resistance to EGFR TKIs. We used BurningRock Biotech’s panels, consisting of critical exons and introns, covering multiple classes of somatic mutations, including single nucleotide variation (SNVs), rearrangements, copy number variations (CNVs) and insertions and deletions (INDELs) to detect genetic alterations both qualitatively and quantitatively.

Results:
We investigated the mutation spectrum after the development of resistance to EGFR-TKIs. Our analysis revealed 64% (18/28) of patients, harboring exon 19 deletions at baseline, acquired T790M at the time of progression. In contrast, only 33% (5/15) of patients, harboring exon 21 L858R substitutions at baseline, acquired T790M, indicating T790M mutation is more commonly found in patients with exon 19 deletions (p=0.052). Next, we associated T790M status with cumulative EGFR-TKI exposure time, and found patients had longer TKI exposure are more likely to acquire T790M mutation. The median TKI exposure time for patients who eventually acquired T790M was 19.20m ±1.86. In contrast, the median exposure time for patients who never acquired T790M 12.20m±1.20m (P =0.017). Furthermore, we investigated the presence of T790M mutation with progression free survival (PFS). Our data revealed that 68% (15/22) of patients with PFS≥12 months acquired T790M mutation after TKI exposure. In contrast, among patients with PFS< 12 months, only 38% of them acquired T790M mutation (p=0.048).

Conclusion:
Patients who eventually acquired T790M mutation have longer cumulative TKI exposure and are associated with longer PFS before the emergence of drug resistance. In addition, we also discovered that patients harboring exon 19 deletions are more likely to develop T790M mutation. Therefore, T790M status can be used as a potential biomarker for prognosis.

Background:
Patients with advanced EGFR-mutated non-small-cell lung cancer (NSCLC) who developed the T790M resistance mutation during treatment with EGFR tyrosine kinase inhibitors (TKIs) benefit from treatment with third-generation EGFR TKIs such as osimertinib. Treatment with osimertinib requires the confirmation of the presence of the T790M mutation by re-biopsy of the tumor or by analysis of cell-free plasma DNA from blood samples (liquid biopsy). The purpose of our study was to compare T790M mutation copy numbers in cell-free plasma DNA with response to osimertinib.

Methods:
From April 2015 to June 2016, we included 44 patients with advanced T790M-positive NSCLC who received osimertinib after previous disease progression with an EFGR TKI and in whom response to osimertinib was evaluable. T790M mutation status was assessed by droplet digital PCR in cell-free plasma DNA. The threshold for T790M positivity was >1 copy/mL.

Results:
The T790M mutation status was assessed in all patients by liquid biopsy and in 18 patients also by re-biopsy of the tumor. All 44 patients were T790M-positive in the liquid biopsy. Two out of 18 (11%) patients had a T790M-negative re-biopsy. Thirty-seven patients (86%) showed a response to treatment with osimertinib: 13 (29.5%) complete responses (CR), 24 (54.5%) partial responses (PR), one (2%) stable disease (SD), and six (14%) progressive disease (PD) (Table 1). We observed no statistically significant association between response to osimertinib and T790M copy numbers (p=0.54; Table 1). The median T790M copy numbers across response categories were: CR 25 copies/mL (range 1.7-38092 copies/mL), PR 14 copies/mL (range 1.6-7282 copies/mL), SD+PD 6 copies/mL (range 1.8-475 copies/mL).

Table 1	Response
Copies/mL	CR	PR	SD	PD
<10	5 (39%)	11 (46%)	0 (0%)	4 (67%)
≥10	8 (62%)	13 (54%)	1 (100%)	2 (33%)

Conclusion:
Patients benefited from osimertinib treatment independent of T790M copy numbers in the blood samples. Although limited by low numbers, we observed a trend towards better response to osimertinib in patients with ≥10 T790M copies/mL.