Virtual Library

Abstract:
Background Cancer is set to become a major cause of morbidity and mortality in coming decade in every region of the world.Methods The mortality, morbidity and treatment variants in Russia were evaluated.Results The incidence of lung cancer morbidity in Russia in 2014 numbered 57 685, mortality – 49 730. Standardized index incidence rate demonstrates improvement of men since year 2009. It was 55,0 on 100 000 population in 2009; 49,15 in 2013 and 49,0 in 2014. It means a 10,9% decrease since 2009 to 2014. It takes stable first place in men. In women the same years showed different values: 7,0; 7,17; 7,3 at 2009, 2013 and 2014 years respectively, that means + 4,3%. It takes 10-12 places of all malignant diseases among women. Among men lung cancer is on the first place (26,6%) in mortality rates. A non-interventional, prospective cohort study included 838 patients, average age 58,7; male – 78,4%; female – 21,6%, smokers – 26,5%; ex-smokers – 24,1%, current smokers – 49,4%. Disease stages at diagnosis were: stage I-II – 36,8%; stage III – 37,8%; stage IV – 25,4%. It was squamous-cell carcinoma – 54,3%; adenocarcinoma – 31%; BAR – 6,4%; LCC – 2,9%, adenosquamous carcinoma – 2,3%, other – 3,1%. Proportion of EGFR positive tumors constitutes 10,1% (85/838) pts. Surgery was performed for 393 pts (46,9%). Radiotherapy was administered to 145 pts (17,8%). 370 pts (44,2%) underwent first-line CT and 96 (11,8%) – second-line. The treatment depends on the morphology type of the tumor. We consider four main types NSCLC (AdenoCa and SCC), LCLC and NETs. NETs group included typical and atypical carcinoids, LCNEC, SCLC. We participated in 53 different multicenter international trials in lung cancer, including 930 patients. Outside of these protocols we analyzed 567 pts with advanced NSCLC: 255 pts with squamous cell cancer (SCC) and 250 pts with non-SCC for 1st line chemotherapy (CT). ORR was 20-25% for platinum-duplets, 1-year survival – 27,5-37,5 month. The 1-year survival showed best results in absolute figures with paclitaxel and platinum compounds in SCC and gemcitabine and platinum compounds in non-SCC, but the value was not statistically significant neither for 1-year nor for median survival. For SCLC new combination with irinotecan and platinum compound showed ORR – 55,1% and stabilization of disease in 24,3% of pts.Conclusions Nowadays the treatment approaches to lung cancer in Russia depends from morphological type of tumors, IGC results and needs further investigations.

Abstract:
Lung cancer is still the leading cause of cancer death in China. The estimated new lung cancer cases and deaths were 733,300 and 610,200 in 2015, respectively. Non-small cell lung cancer (NSCLC) remains the predominant form of the disease in China, with majority of patients being diagnosed at advanced stages. Thus this presentation will focus on advanced stage NSCLC. Current treatment strategy The current treatment algorithm for wild-type non-squamous and squamous NSCLC were shown in Figure 1 and 2, respectively. Figure 1 Figure 1. Treatment algorithm for non-squamous NSCLC (wild-type) Figure 2 Figure 2. Treatment algorithm for advanced squamous NSCLC For patients with activating EGFR mutations, EGFR-TKIs therapy will be used as front-line therapy. Commercial available EGFR-TKIs in China include Gefitinib, Erlotinib and Icotinib. For patients harbouring an ALK rearrangement, crizotinib will also be considered as first-line treatment. When failed from EGFR-TKIs or ALK-Inhibitor therapy, patients will be treated according to clinical model of disease progression. For patients with asymptomatic progression, continuing EGFR-TKIs or ALK-Inhibitor is recommended. For patients with local progression, EGFR-TKIs or ALK-Inhibitor will also be continued with additional local therapy such as whole brain radiation. However, for patients with aggressive progression, EGFR-TKIs or ALK-Inhibitor will be substituted by chemotherapy. Unfortunately, it is difficult to overcome drug resistance according to molecular mechanism because novel agents such as Osimertinib and Alectinib haven’t been approved by Chinese FDA. Challenge and perspective 1. Genetic alterations assays Genetic alterations are frequent in Chinese NSCLC patients. According to PIONEER study (NCT01185314), which is a prospective molecular epidemiology study in newly diagnosed advanced lung adenocarcinoma, the EGFR active mutation rate is 50.2% in Chinese patient population. The incidence of EGFR mutations in patients who never smoked can be as high as 59.6%. ALK rearrangement is also common in this patient population. In a large cross-sectional study enrolled 1160 NSCLC patients, the incidence of ALK rearrangements is 8.1%. Noteworthy, 44% of patients younger than 30 years old harbor ALK rearrangements. However, genetic alterations test rate used to be low in China. According to a large national survey, the EGFR mutation test rate was only 9.6% in 2011. However, as the turnover time shortens, the testing fee decreases, and ctDNA testing becomes available, the EGFR/ALK assays have turned into routine practice in China. Moreover, NGS platforms detecting panels of mutations are commonly used in some leading centers now. 2. Novel agent availability There is severe delay in the approval for novel agents by Chinese FDA. For instance, Bevacizumab was approved by FDA for treatment of NSCLC in 2006, while it was approved by Chinese FDA 9 years later. To improve availability of novel agents, Chinese oncologists are active in participation in international multi-center clinical trials. In addition, more and more innovative drugs have been developed by domestic pharma industry and entered clinical trials (Table 1). Moreover, Chinese FDA makes new policies to encourage innovative drugs and accelerating new drug application.

Agent ID	Classification	Indication	Phase
Avitinib	Mutation selected EGFR-TKI	EGFR T790M Mutation	Phase I
Apatinib	VEGFR-TKI	Nonsquamous NSCLC in 3L	Phase III
Famitinib	VEGFR-TKI	Nonsquamous NSCLC in 3L	Phase III
Theliatinib	EGFR inhibitor	EGFR amplification	Phase I
Volitinib	c-MET inhibitor	c-MET amplification	Phase I
SHR-1210	PD-1 antibody	NSCLC in 2/3L	Phase I

Table 1. Innovative drugs from China in clinical trials 3. Lung cancer prevention The incidence rate of lung cancer remains high in China between 2000 and 2011. Factors that have contributed to this issue include tobacco smoking and air pollution. 50% adult Chinese men were current smokers in 2010. In addition, smoking rates in adolescents and young adults are still rising in China. To reduce tobacco use in China, the government enact a strict smoking control law in Beijing in June 2015. However, the air pollution is still a severe problem and needs to be improved urgently. 4. Economic burden There are several factors which have contributed to the heavy economic burden of lung cancer patients in China. First, the residents’ income is still low in China. In 2015, per capita disposable income (one year) was only $3300. Second, the cost of anti-cancer drugs is very high (Crizotinib/cycle $8500, Gefitinib/cycle $2200, Pemetrexed/cycle $3000, and Bevacizumab/cycle $4500). Moreover, only 20% of whole medical expense can be covered by insurance, and majority of targeted drugs can’t be covered.

Abstract:
Lung cancer is the commonest type of cancer in males and the leading cause of cancer death in both sexes world-wide. It is also the commonest in men in India accounting for 11.3% of all new cancers and also is the most common cause of cancer death (13.7%). In contrast to a decline trend in men in developed countries with a plateau for females, in India, the incidence continues to rise for both males and females. Data from the population based cancer registries developed under the National Cancer Registry Program of the Indian Council for Medical Research(ICMR) indicates that there is wide geographical variability in the incidence of this disease in different parts of the country. The highest age adjusted incidence rates of 45 per 100000 population are seen in the North-East region of India and are similar to areas reporting the highest incidence rates in some parts of the US and Europe. In other areas of India, especially the Western region, the age adjusted incidence rates are as low as 2 per 100000 population. The demographic profile including age, gender, stage, histology and even the molecular epidemiology (prevalence of EGFR mutations and ALK rearrangements) varies considerably in different parts of India. However, the overall incidence is much lower than that compared to many western countries. The demographic profile of lung cancer seen in India needs special mention. In the past, a single-centre large series of 1009 patients presenting to our institute from 1977-86 had shown squamous histology to the commonest (34.3%) followed by adenocarcinoma (25.9%) and small cell lung cancer (SCLC; 20.3%). Subsequent analysis of 250 patients presenting to us three decades later (2007-09), we found that the histological pattern was largely unchanged with squamous still being the commonest (34.8%) followed by adenocarcinoma (26.0%) and SCLC (18.4%). The male-female ratio as well as the current/ex-smoker to never-smoker ratio was also similar between the two cohorts. A possible reason for the lack of change in demographic profile of lung cancer was thought to be related to the fact that ‘bidi’ and NOT cigarette is the most common form of tobacco smoking in India. The ratio of bidi to cigarette smoking in India ranges from 2.5:1 to 7.0:1 in different parts of India and unlike cigarette making, there has been no change in the process of bidi manufacturing which is primarily a cottage industry. The other important aspect related to its association of quantified tobacco smoke exposure. The smoking index (SI; number of combined bidis and cigarettes smoked per day multiplied by number of years smoked) has been developed for this purpose. Patients can be categorized as either never-smokers (SI=0), light to moderate smokers (SI=1-300) and heavy smokers (SI≥301). In a cohort of 520 non-small cell lung cancer (NSCLC) patients, we observed that age, gender, histological type and stage differed significantly between the three groups. Never-smokers had significantly more females (52%), were younger (mean age 54.5 years), lesser squamous histology (28%), more advanced stage (IIIB/IV; 92%), more metastatic disease (67.4%) and more extra-thoracic metastases (42%) while group of heavy smokers had more males (98%), were older (mean age 61.2 years), more squamous histology (58%), lesser advanced stage (81%), lesser metastatic disease (39%) and lesser extra-thoracic metastases (17%). We have identified another risk factor in women to be the exposure to Biomass fuel. Majority of patients (approximately 83% of NSCLC histology at our centre) present with advanced stage(IIIB/IV) at the time of diagnosis and are managed non-surgically. Misdiagnosis as tuberculosis and empirical treatment with anti-tubercular drugs prior to referral to higher centre is one of the important causes for delayed diagnosis of this disease in India. Developing and under-developing countries are often constrained with regards to availability of health care and other resources necessary for appropriate management of the health related requirements of their population and this holds true for lung cancer as well. Some of the challenges in resource constrained settings include: · Large population with high population density · Illiteracy and poor health awareness · Sub-optimal economic and infrastructure inputs for health care · Suboptimal ratios of doctor and nurses for population · Overburdened hospitals and health care facilities · Huge burden of TB that hinders differentiation by the primary physician with lung cancer Important issues in resource constrained settings include choosing the platinum agent as well as the non-platinum agent. Decision on dose intensity may also be influenced by similar factors (efficacy, tolerance, toxicity profile and packaging strengths of marketed drugs). A list of some of the important factors influencing decision are shown below.

Characteristic	Relative importance
Disease related
Age	++
Gender	+
Histology	+++
Molecular profile of tumor	++
Stage	+
Performance Status	+++
Unrelated to disease
Co-morbid illnesses	+
Socio-economic background/financial constraints	+++
Medical reimbursement/insurance issues	+++
Wishes of patient/family members	++
Frequency of hospital visits	++

Dr. D. Behera Senior Professor & Head, Dept. of Pulmonary Medicine, Postgraduate Institute of Medical Education & Research, Chandigarh - 160012 (INDIA) Email: [email protected] Select References and suggested reading 1. Behera D, Balamugesh T. Lung cancer in India. Indian J Chest Dis Allied Sci 2004; 46 : 269-81 2. Behera D. Managing lung cancer in developing countries: difficulties and solutions. Indian J Chest Dis Allied Sci 2006; 48: 243-4 3. Jindal SK, Behera D. Clinical spectrum of primary lung cancer: review of Chandigarh experience of 10 years. Lung India 1990; 8: 94-98 4. Singh N, Aggarwal AN, Gupta D, Behera D, Jindal SK. Unchanging clinico-epidemiological profile of lung cancer in North India over three decades. Cancer Epidemiol 2010; 34: 101-4. 5. Behera D, Balamugesh T. Indoor air pollution as a risk factor for lung cancer in women. J Assoc Physicians India 2005; 53: 190-2. 6. Singh N, Aggarwal AN, Gupta D, Behera D, Jindal SK. Quantified smoking status and non-small cell lung cancer stage at presentation: analysis of a North Indian cohort and a systematic review of literature. J Thorac Dis 2012; 4: 474-84. 7. Singh N, Behera D. Lung cancer epidemiology and clinical profile in North India: Similarities and differences with other geographical regions of India. Indian J Cancer 2013; 50: 291 8. Singh N, Aggarwal AN, Behera D. Management of advanced lung cancer in resource constrained settings : a perspective from India. Expert Rev Anticancer Ther 2012: 12: 1479–95. 9. Maturu VN, Singh N, Bal A, Gupta N, Das A, Behera D. Relationship of epidermal growth factor receptor activating mutations with histologic subtyping according to International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society 2011 adenocarcinoma classification and their impact on overall survival. Lung India 2016; 33: 257-66. 10. Bal A, Singh N, Agarwal P, Das A, Behera D. ALK gene rearranged lung adenocarcinomas: molecular genetics and morphology in cohort of patients from North India. APMIS 2016 Aug 8; DOI: 10.1111/apm.12581 [Epub ahead of print]

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Abstract:
Lung cancer has been the most common cancer in the world for several decades. The number of new cases estimated in 2012 is 1.8 million cases (12.9% of the total), 58% of which occurred in the less developed regions. The disease remains as the most common cancer in men worldwide (1.2 million, 16.7% of the total) with the highest estimated rates in Nothern America (33.8%) and Northern Europe (23.7%), a relatively high rate in Eastern Asia (19.2) and the lowest rates in Western and middle Africa (1.1 and 0.8 respectively). In developing countries, lung cancer is the most common cancer among males and the third most common cancer among females. Lung cancer is the most common cause of death from cancer worldwide estimated to be responsible for nearly one in 5 (1.59 million deaths, 19.4% of the total) (1). Temporal analyses reveal that significant reductions in lung cancer mortality have been observed in developed countries due to increased awareness of the harmful effects of smoking , asbestos and other factors The role of early detection is also evident. (2). In contrast, lung cancer incidence and mortality rates have increased in some low and medium resourced countries (3). The regional differences are mainly due to increased tobacco smoking in the developing countries , smoking waterpipe, cannabis or even passive and secondary smoke and in the mean time there is lack of proper tobacco control. There are also occupational risk factors such as asbestos exposure, dust, fumes, nickel ,silica and insecticides and up till now there are areas that have not banned asbestos or succeeded to control occupational and environmental exposure and incidence of mesothelioma is increasing. (4) Many studies have shown that cases have genetic susceptibility to develop lung cancer specially in North Africa. Another important factor specially the Middle East North Africa is the increase in the elderly population that may be attributed to better infection control and improvement of general health care . As life expectancy continues to increase throughout the African continent, the burden of cancer is likely to increase. Given that an estimated 32,640 new lung cancer cases will be seen in Africa in 2015 ( 5) .We have to remember also that cancer diagnosis rate in Africa is relatively low and patients present usually in an advanced stage so underreporting may be another factor . Accordingly, it is essential to know the magnitude of lung cancer in different regions in Africa by having cancer registry for the countries . So, obstacles to the global fight against lung cancer include lack of registry in some parts of Africa, low public awareness of lung cancer and absence of screening for the high risk cases , overburdened treatment centers and insufficient financial support. The ways to combat all these obstacles start by setting strategies for prevention and earlier detection in the low income countries. Public health awareness of the risk factors that cause lung cancer and the importance of avoiding / stopping smoking and banning asbestos should be clear and this is the role of public health authorities, medical journals and public media. The war against tobacco companies should start and everyone should understand the danger of smoking. This is done also by cooperation of scientific organizations of governmental and non governmental organizations. Also, we should reduce air pollution and regulate the occupational exposure of the employees to avoid the appearance of lung cancer and mesothelioma. As for early detection , screening can help in high risk patients and many authorities and NGOs can help to catch the early cases. In the mean time there should be ways to access modern imaging techniques to detect the cancer and use the minimal requirements for diagnosis and care . Accordingly it is essential to set the treatment guidance protocols to facilitate the management of the patients and to educate and train the doctors that should acquire degree granting programs and get certificates in the oncological field. It is mandatory to to lower the cost of health care to encourage the patients to go for treatment and to get the proper care. There should be special dealing for the economic pressure and avoidance of financial toxicities for the patient. The last point that have to be ameliorated in Africa developing countries is research through International collaboration as studying genetic polymorphism and relation to smoking and changing patient concept about drugs received in clinical trials that use new drugs, proper investigations and lower the cost of treatment and may get better outcome. References 1- Globocan 2012 (IARC): Estimated cancer incidence, mortality and prevalence worldwide, section of cancer surveillance 2- Jemal A, Center MM, DeSantis C, Ward EM (2010) Global patterns of cancer incidence and mortality rates and trends. Cancer Epidemiol Biomarkers Prev 19: 1893-1907. 3- Sankaranarayanan R, Jayant K, Brenner H 2011: An overview of cancer survival in Africa, Asia, the Caribean and central America: the case for investment in cancer health services. IARC Sci Publ: 257-291. 4- Gaafar RM, Eldin NH (2005) Epidemic of mesothelioma in Egypt. Lung Cancer 49: S17-S20. 5- Tao Z, Shi A, Lu C, Song T, Zhang Z, etal. 2014: Breast cancer : Epidemiology and Etiology. Cell Biochem Biophysi

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Abstract:
General considerations Early stage lung cancer - a term in transition Generally early stage lung cancer is understood as stage I and stage II non-small lung cancer. An alternative understanding of early stage lung cancer is resectable disease. However, both definitions are imprecise and subject to development and Expertise. 1. Defining early stage lung cancer as resectable disease depends on regional philosophies and local expertise and therefore is the most unreliable and variable definition. The term resectability focuses on the T factor of the tumour and describes the ability of the surgeon to achieve radical resection. In contrast operability includes any potential regional and systemic spread and focuses more on the N and M descriptors. 2. Defining early stage lung cancer based on mediastinal nodal involvement neglects the fact, that single station N2 (N2a) is associated with the same five-year survival as multistation N1 (N1b). This touches on the term locally advanced disease, which in fact also means different things for different people. For the oncologist locally advanced disease usually means N2 involvement with the consequent call for chemotherapy. For the surgeon locally advanced disease primarily addresses the T factor and is used for T3 or T4 tumours, indicating more extended resections in the absence of N2 disease. In summary, terms like early stage, locally advanced stage or advanced stage should be avoided since they do not properly describe a clinical situation nor are they guiding therapy. If the term early stage lung cancer should be maintained for any reason, there is need for revisions. The five-year survival of stage I and stage II non-small lung cancer is a range of less than 30 to more than 90% and the survival expectedly mainly depends on nodal involvement. The estimated median five-year survival of patients with Screening detected T1N0 NSCLC is a reported 92%. Even nodal negativeT3 tumours are associated with almost 60% five year survival following radical resection. On the other hand involvement of multiple N1 lymph nodes results in a much worse prognosis of about 35%. However, for this presentation the current definition of stage I and stage II non-small lung cancer was used. Preoperative staging Resectability of lung cancer for technical reasons in general, and in early stage lung cancer in particular, very rarely is an issue. Oncological operability has to be defined preoperatively along international guidelines. The European Society of Thoracic Surgeons (ESTS) recently has ublished revised guidelines for preoperative mediastinal lymph nodes staging for non-small cell lung cancer. Only one selected group of patients with tumours of less than 3 cm in diameter (cT1) in the outer third of the lung without signs of nodal involvement at CT scan, PET scan or PET CT (cN0) may directly undergo surgical resection. All other clinical situations require invasive preoperative staging by bronchoscopy plus EBUS/EUS. If the absence of nodal involvement is verified by EBUS/EUS this patient may also directly undergo surgery. In the presence of radiologically suspect mediastinal lymph nodes and negative EBUS/EUS further confirmation is recommended using mediastinoscopy or thoracoscopy. If mediastinal nodal involvement is histologically verified by any means the patient has to undergo multimodality treatment. All clinical findings are to be discussed in an interdisciplinary tumour board for proper therapy planning. [1] Surgical therapy of early stage NSCLC Surgery remains the cornerstone of treatment of early stage non-small lung cancer for patients willing to accept the procedure-related risks. Goal of any surgical intervention for early stage lung cancer is the complete resection of the primary tumour together with regional lymphatic nodes. The standard for any resection with curative intent is defined by anatomical lung resection. In early stage lung cancer the predominant type of resection is lobectomy or bilobectomy, sometimes along with bronchoplastic or angioplastic procedures or extended resections for locally invading T3 tumours. Pneumonectomy particularly in the treatment of early stage lung cancer is rarely used. Gold standard of surgical resection for lung cancer is lobectomy. This standard is based on a prospective multi-institutional randomized trial comparing limited resection with lobectomy for peripheral T1N0 non-small cell lung cancer published in 1995. [2 ]In the absence of more recent prospective randomised trials lobectomy still must be considered the surgical procedure of choice for patients with peripheral T1N0 non-small cell lung cancer. An extensive body of literature mainly composed of retrospective studies supports the use of radical anatomical segmentectomy for peripheral cT1N0M0 non-small lung cancer with less than 2 cm in diameter, certainly for older patients with limited cardiopulmonary function. However, caution should be taken to promote a widespread indication for intentional segmentectomy in young good surgical candidates until the results of the ongoing randomised controlled trials become available.[ 3,4] The role of minimally invasive surgery Minimally invasive anatomical resection for lung cancer carried out by means of video-assisted thoracic surgery (VATS) has been increasingly carried out during the past years. A systematic review and meta-analysis of randomized and nonrandomized trials published in 2009 reported an improved five-year survival and reduced systemic recurrences in patients who received VATS lobectomy. [5 ]A multicentric propensity-matched analysis of more than 1000 patients, of which 700 had undergone VATS lobectomy confirms, that thoracoscopic lobectomy is associated with lower morbidity as compared with thoracotomy. The positive impact of minimally invasive surgery in the treatment of lung cancer particularly applies to the elderly. [6] Regarding long-term survival after video-assisted thoracoscopic lobectomy a meta- showed a survival benefit in the favour of VATS with a difference in survival of 5% at five years. The reason for this observed survival benefit may be attributed to a less pronounced compromise of the immunocompetence after the surgical trauma. [7] The role of mediastinal lymph node dissection The rationale for a formal mediastinal lymph node dissection is multifold. The distribution pattern of mediastinal lymph node metastasis is not predictable and skip metastasis are seen in up to 30% of patients. Even small tumours may present with unexpected N2 disease with an incidence of 6-10%. The operative morbidity is not significantly influenced by a systematic mediastinal lymph node dissection. Recommended standard of mediastinal lymph node dissection is the removal of all mediastinal tissue containing lymph nodes in a systematic Approach within anatomical landmarks. The most recent randomized controlled trial published in 2011 did not find a survival benefit by complete mediastinal lymphadenectomy in patients with early stage lung cancer, but the results should not be generalized to patients staged only radiographically or those with higher stage tumours. The recommendation from this study is that a formal mediastinal en-bloc dissection may still affect survival and certainly optimally stages patients. In the subgroup analysis no difference between VATS and open lobectomy was observed for number of lymph nodes harvested and regarding long-term survival.[8] As minimally invasive surgery along with unilateral mediastinal lymphadenectomy generally prolongs operation times and the requirement of single lung ventilation the advantages for the elderly population has to be questioned and discussed individually. An alternative to thoracoscopic unilateral lymphadenectomy is offered by video-assisted mediastinal lymphadenectomy through the neck (VAMLA). The approach is similar to transcervical mediastinoscopy and allows for a radical bloc dissection of all mediastinal lymph node stations. Besides the benefit of bilateral lung ventilation during this phase of the operation a bilateral mediastinal lymphadenectomy offers improved surgical radicality. Alternatives to surgical resection and the role of primary radiotherapy In patients unfit for surgery SABR is the treatment of choice for peripherally located stage I non-small cell lung cancer. If SABR is not available a hypofractionated radiotherapy is advocated. A systematic Review comparing outcomes of SABR and surgery in patients with severe COPD revealed a higher 30 day mortality following surgery but similar overall survival at one and three years. [9] In a meta-analysis of 19 out of 318 papers with the best evidence addressing a comparison of SABR and surgical wedge resection both methods proved as reasonable alternatives to lobectomy in high risk surgical patients. In this analysis SABR was associated with reduced local recurrence compared to wedge resection and should be considered when wedge resection is planned due to anatomical location and size of the primary tumour in a patient who is high risk for surgery. [10] Although local tumour control may be comparable or even superior to extra-anatomic surgical resection a quite high rate of late radiological changes after stereotactic ablative radiotherapy for early stage lung cancer has to be considered. At one year follow-up the predicted probability of having expected or pronounced radiological changes after SABR were 65 and 22%. These changes included phenomena like mass-like appearance, radiation fibrosis, and rib fractures, which sometimes are difficult to differentiate from tumour recurrence. Summary The ACCP guidelines address the question, who had to be considered a high risk candidate for surgery. With the advent of minimally invasive resection, the criteria to classify a patient as too ill to undergo an anatomic lung resection are being redefined. Surgical resection remains the primary and preferred approach to the treatment of stage I and II NSCLC in patients with good or low surgical risk. Primary radiation therapy remains the primary curative intent approach for patients who refuse surgical resection or are determined by a multidisciplinary team to be inoperable. [11] References 1. Revised ESTS guidelines for preoperative mediastinal lymph node staging for non-small-cell lung cancer. De Leyn P, Dooms C, Kuzdzal J, Lardinois D, Passlick B, Rami-Porta R, Turna A, Van Schil P, Venuta F, Waller D, Weder W, Zielinski M. Eur J Cardiothorac Surg. 2014 May;45(5):787-98 2. Randomized trial of lobectomy versus limited resection for T1 N0 non-small cell lung cancer. Lung Cancer Study Group. Ginsberg RJ; Rubinstein LV. Ann Thorac Surg. 1995; 60(3):615-22; discussion 622-3 3. Tsutani Y, Miyata Y, Nakayama H, et al. Oncologic outcomes of segmentectomy compared with lobectomy for clinical stage IA lung adenocarcinoma: propensity score-matched analysis in a multicenter study. J Thorac Cardiovasc Surg 2013;146:358-64. 4. Zhao X, Qian L, Luo Q, et al. Segmentectomy as a safe and equally effective surgical option under complete video-assisted thoracic surgery for patients of stage I non-small cell lung cancer. J Cardiothorac Surg 2013;8:116, 5. Yan TD, Black D, Bannon PG, McCaughan BC. Systematic review and metaanalysis of randomized and non-randomized Trials on safety and efficacy of videoassisted thoracic surgery lobectomy for early-stage non-small cell lung cancer. J Clin Oncol 2009; 27: 2553–2562 6. Thoracoscopic lobectomy is associated with lower morbidity compared with thoracotomy.Villamizar NR, Darrabie MD, Burfeind WR, Petersen RP, Onaitis MW, Toloza E, Harpole DH, D'Amico TA. J Thorac Cardiovasc Surg. 2009 Aug;138(2):419-25. 7. Long-term survival in video-assisted thoracoscopic lobectomy vs open lobectomy in lung-cancer patients: a meta-analysis. Taioli E, Lee DS, Lesser M, Flores R. Eur J Cardiothorac Surg. 2013 Feb 14. 8. Darling GE, et al. Randomized trial of m diastinal lymph node sampling versus complete lymphadenectomy during pulmonary resection in the patient with N0 or N1 less than hilar) non-small cell carcinoma. J Thorac Cardiovasc Surg 011;141:662-70 9. Early and locally advanced non-small-cell lung cancer (NSCLC): ESMO Clinical Practice Guidelines for diagnosis, Treatment and follow-up. J. Vansteenkiste, D. De Ruysscher, W. E. E. Eberhardt, E. Lim, S. Senan, E. Felip & S. Peters, on behalf of the ESMO Guidelines Working Group 10. Mahmood S, Bilal H, Faivre-Finn C, Shah R. Is stereotactic ablative radiotherapy equivalent to sublobar resection in high-risk surgical patients with stage I non-small-cell lung cancer? Interact Cardiovasc Thorac Surg. 2013 Nov;17(5):845-53. 11. Treatment of stage I and II non-small cell lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines.Howington JA, Blum MG, Chang AC, Balekian AA, Murthy SC. Chest. 2013 May;143(5 Suppl)

Abstract:
Radiotherapy is a curative treatment for early-stage NSCLC. Following hypofractionated radiotherapy in 15 once-daily fractions of 4 Gy to biopsy-proven tumors, a prospective multicenter study reported a 3-year local control rate of 82.7% (95% CI = 69.7% to 90.5%) [Cheung PC, 2014]. In the past decade, stereotactic ablative radiotherapy (SABR or SBRT) has become established as the guideline-recommended standard of care for medically inoperable patients with a peripheral early-stage NSCLC, as 5-year local control rates of 90% have been reported [Louie AV, 2015]. SABR is usually delivered in 3-8 fractions, utilizes small margins for positional uncertainty, 4-dimensional computed tomography (4DCT) for treatment planning, multiple conformal beams or arcs for delivery, and cone-bean CT scans for daily setup. Where facilities for SABR are unavailable, hypofractionated radiotherapy delivered using 4DCT planning remains an acceptable curative treatment. Diagnosis Population studies reveal that a significant proportion of elderly patients, as well as those with severe co-morbidities, do not receive any treatment. Guidelines recommend that a tissue diagnosis be obtained before initiating treatment for early-stage NSCLC, but also permit the use of SABR following review by an expert tumor board, in tumors where the calculated probability of malignancy is high [Vansteenkiste J, 2014; Callister ME, 2015]. However, any decision to proceed to a FDG-PET directed SABR approach in less fit patients must take into account the likelihood of benign disease. Given a high incidence of pulmonary tuberculosis, guidelines for Asia have recommended performing early non-surgical biopsies in Asian patients [Bai C, 2016]. Toxicity Treatment-related grades 3-4 toxicity are uncommon following SABR to peripheral lung tumors, while local control rates are approximately 90% [Louie AV 2015]. Commonly reported toxicities are chest wall pain, rib fractures, and except in patients who have pre-existing interstitial lung disease (ILD), the incidence of high-grade radiation pneumonitis is low. A systematic literature review of SABR in patients with ILD reported a treatment-related mortality in 15% [Chen H, Proc ASTRO 2016]. Follow-up Guidelines recommend 6-monthly CT scans for up to 3 years following SABR, followed by annual scans thereafter. The assessment of radiological changes can be challenging in a sub-group of patients during long-term follow-op, and the so-called high-risk radiological features [HRF] can identify patients in whom a biopsy is warranted [Figure 1, Huang K, 2014]. The HRF’s identified in the literature are an enlarging opacity at primary site, a sequential enlarging opacity, enlarging opacity after 12-months, a bulging margin, loss of linear margin, loss of air bronchogram and cranio-caudal growth [Huang K, 2014]. Initial reports on surgery for local failures following SABR indicate that this salvage procedure can be performed safely [Allibhai Z, 2012; Hamaji M, 2015; Verstegen N, Proc ELCC 2015]. Figure 1 The observed rates for a second primary lung cancer following SABR appear similar to those following surgery [Verstegen N, 2015]. In this situation, a subsequent course of SABR can generally be performed safely. Operable patients The role of SABR in fit patients remains a topic of active debate. Indirect comparisons of outcomes following the two modalities have revealed conflicting results. The role of SABR in surgical patients is currrently being investigted in 3 prospective randomized studies (NCT02468024, NCT02629458, NCT01753414), with a fourth study (VALOR) scheduled to open shortly.

Abstract:
Adjuvant Chemotherapy of Completely Resected NSCLC Glenwood D. Goss Lung cancer remains the leading cause of cancer death world-wide and accounts for approximately 28% of all cancer deaths(1,2). Surgical resection is the cornerstone of therapy for early stage disease, but relapse is high with 30-60% of patients with resected NSCLC still dying of their disease. Despite the results of a 1995 meta-analysis demonstrating a non-significant 5% survival advantage at five years with the addition of adjuvant cisplatin-based chemotherapy, no large randomized studies conclusively demonstrated a benefit following resection until 2003(3). Five large randomized trials were undertaken to determine if adjuvant platinum-based chemotherapy after curative surgery for NSCLC conferred a survival advantage: ALPI; IALT; JBR10; CALGB 9633; and ANITA (4,5,6,7,8). Three of these five trials showed statistically significant improvements in overall survival, ranging from 4% [IALT] to 15% [JBR10] at 5 years, corresponding to an absolute improvement in relapse-free survival from 49% to 61%. Of the two trials that did not demonstrate improved survival, one [ALPI] suffered from poor compliance to the treatment regimen (69%), and the second was a smaller trial (n=344) limited to patients with stage IB disease [CALGB 9633], which was likely underpowered to detect a statistically significant improvement in overall survival. Interestingly, despite being limited to patients with stage IB disease, CALGB 9633 did demonstrate an overall survival hazard ratio comparable to the other adjuvant trials (HR=0.8) that included patients with more advanced disease, despite not achieving statistical significance. Since the publication of original adjuvant chemotherapy trials, a number of meta-analyses have confirmed the benefit of adjuvant platinum-based chemotherapy after surgical resection for NSCLC(9,10). In these meta-analyses, all-stage (IB-IIIA) hazard ratios were in the range of HR=0.86, corresponding to an absolute benefit for chemotherapy on overall survival of 4-5% at 5 years. The benefit, however, was demonstrated to be stage dependent (albeit using older staging criteria versions), with the benefit only reaching statistical significance for stages II and III. While the role of adjuvant chemotherapy in stage I disease is controversial (11), subgroup analyses in a number of trials in high-risk patients with stage IB disease (tumours≥4cm) suggests that there may be an overall survival advantage with adjuvant chemotherapy in this subgroup of patients, comparable to those observed in stage II and III disease [Strauss 2008]. In 2009 the long term follow up of the IALT study (with a median follow up of 7.5 years) was reported. Results showed a beneficial effect of adjuvant chemotherapy on overall survival (hazard ratio [HR], 0.91; 95% CI, 0.81 to 1.02; P = .10) and on disease-free survival (HR, 0.88; 95% CI, 0.78 to 0.98; P = .02). However, there was a significant difference between the results of overall survival before and after 5 years of follow-up (HR, 0.86; 95% CI, 0.76 to 0.97; P = .01 v HR, 1.45; 95% CI, 1.02 to 2.07; P = .04) with P = .006 for interaction. Similar results were observed for disease-free survival. The analysis of non-lung cancer deaths for the whole period showed an HR of 1.34 (95% CI, 0.99 to 1.81; P = .06) suggesting that those patients receiving adjuvant chemotherapy had a higher death rate from non- lung causes after 5 years(12). However these conclusions were not support by the findings of Butts and colleagues reporting on JBR10 with a median follow-up was 9.3 years (range, 5.8 to 13.8). Adjuvant chemotherapy continued to show a benefit (hazard ratio [HR], 0.78; 95% CI, 0.61 to 0.99; P = .04). There was a trend for interaction with disease stage (P = .09; HR for stage II, 0.68; 95% CI, 0.5 to 0.92; P = .01; stage IB, HR, 1.03; 95% CI, 0.7 to 1.52; P = .87). Adjuvant chemotherapy resulted in significantly prolonged disease specific survival (HR, 0.73; 95% CI, 0.55 to 0.97;P = .03). Observation was associated with significantly higher risk of death from lung cancer (P = .02), with no difference in rates of death from other causes or second primary malignancies between the arms. They concluded that prolonged follow-up of patients from the JBR.10 trial continues to show a survival benefit for adjuvant chemotherapy(13). Recently in a post hoc analysis of ECOG 1505, a trial of adjuvant chemotherapy +/- bevacizumab for early stage NSCLC, Wakelee and colleagues had the opportunity to compare four different cisplatin doublet regimens namely, cisplatin with one of vinorelbine, docetaxel, gemcitabine or pemetrexed. Median follow-up time for each chemotherapy doublet was: vinorelbine 54.3 months; docetaxel 60.3 months; gemcitabine 57.0 months; and pemetrexed 40.6 months respectively. The arms were well balanced for the major prognostic factors apart from smoking where the rate was slightly lower in the pemetrexed arm. There was no difference in the median number of cycles between arms. Both in the nonsquamous and squamous subgroups there was no difference in overall survival (nonsquamous logrank p=0.18 and squamous p=0.99) and disease free survival (nonsquamous p=0.54 and p=0.83). The authors concluded that there did not appear to be a difference in outcome between cisplatin doublet regimens(14). Despite the established benefit of adjuvant chemotherapy after curative surgery for NSCLC there is still much to be done with approximately 50 % of patients still dying from disease. Furthermore, not all patients with early stage disease are eligible or willing to undergo chemotherapy following complete surgical resection [Booth 2010]. As such, the long-term prognosis of patients with NSCLC, even among those with early stage disease, remains poor. Therefore it is imperative that we find new and better therapies to improve upon the results of surgical resection and adjuvant chemotherapy. . References: 1. American Cancer Society. Cancer Facts & Figures 2012. Atlanta: American Cancer Society; 2012. 2. Jemal A, Siegel R, Ward E et al. Cancer Statistics 2007. CA Cancer J Clin 2007; 57: 43-66. 3. L. A. Stewart, S. Burdett, J. F. Tierney, J. Pignon on behalf of the NSCLC Collaborative GroupSurgery and adjuvant chemotherapy (CT) compared to surgery alone in non-small cell lung cancer (NSCLC): A meta-analysis using individual patient data (IPD) from randomized clinical trials (RCT). Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings (Post-Meeting Edition).Vol 25, No 18S (June 20 Supplement), 2007: 7552 4. Scagliotti GV, Fossati R, Torri V et al. Randomized study of adjuvant chemotherapy for completely resected stage I, II, or IIIA non-small-cell lung cancer. J Natl Cancer Inst 2003; 95: 1453–61. 5. Arriagada R, Bergman B, Dunant A et al. Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer. N Eng J Med 2004; 350: 351-60. 6. Winton T, Livingston R, Johnson D et al. Vinorelbine plus cisplatin vs observation in resected non-small-cell lung cancer. N Eng J Med 2005; 352: 2589-97. 7. Strauss GM, Herdone JE, Maddaus et al. Adjuvant paclitaxel plus carboplatin compared with observation in stage IB non-small cell lung cancer: CALGB 9633 with the Cancer and Leukemia Group B, Radiation Therapy Oncology Group, and North Central Cancer Treatment Group Study Groups. J Clin Oncol 2008; 26: 5043-51. 8. Douillard JY, Rosell R, De Lena M et al. Adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage IB-IIIA non-small cell lung cancer (Adjuvant Navelbine International Trialist Association [ANITA]): a randomized controlled trial [published erratum appears in Lancet Oncol 2006; 7: 797]. Lancet Oncol 2006; 7: 719-27. 9. Pignon JP, Tribodet GV, Scagliotti G et al. Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE Collaborative Group. J Clin Oncol 2008; 26: 3552-9. 10. NSCLC Meta-analyses Collaborative Group. Adjuvant chemotherapy, with or without postoperative radiotherapy, in operable non-small-cell lung cancer: two meta-analyses of individual patient data. Lancet 2010; 375: 1267-77. 11. Wakelee H, Dubey S, Gandara D et al. Optimal adjuvant therapy for non-small cell lung cancer – how to handle stage I disease. Oncologist 2007; 12: 331-7. 12. Arriagada R, Dunant A, Pignon JP, et al. Long-Term Results of the International Adjuvant Lung Cancer Trial Evaluating Adjuvant Cisplatin-Based Chemotherapy in Resected Lung Cancer JCO January 1, 2010 vol. 28no. 1 35-42 13. Butts C, Ding K, Seymour L,et al. Randomized Phase III Trial of Vinorelbine Plus Cisplatin Compared With Observation in Completely Resected Stage IB and II Non–Small-Cell Lung Cancer: Updated Survival Analysis of JBR-10. Journal of Clinical Oncology, January 1, 2010 vol. (28) 1 29-34. 14. H.A. Wakelee[1], S.E. Dahlberg[2], S.M. Keller te al. E1505: Adjuvant chemotherapy +/bevacizumab for early stage NSCLC: Outcomes based on chemotherapy subsets. ASCO Annual Meeting, 2016 Abstr 8507: E1505 Chemotherapy subsets. 15. Booth CM, Shepherd FA, Peng Y et al. Adoption of adjuvant chemotherapy for NSCLC: a population-based outcome study. J Clin Oncol 2010; 28: 3472-8.

Abstract:
Perspectives of Targeted Therapies and Immunotherapy in Completely Resected NSCLC - Heather Wakelee, USA The use of four cycles of cisplatin-based adjuvant chemotherapy is now the standard of care for patients with resected stage II and IIIA NSCLC and is commonly used for patients with larger (at least 4 cm in size) stage IB tumors. The survival benefit with adjuvant chemotherapy though is limited with meta-analyses revealing a 4-5% absolute survival benefit at 5 years for patients receiving adjuvant cisplatin-based chemotherapy.[1,2]Some recent attempts to improve outcomes with the addition of other agents to cisplatin doublets (or as longer term therapy) have been disappointing. The addition of bevacizumab to chemotherapy in the ECOG-ACRIN E1505 adjuvant trial failed to show a benefit in disease free survival (DFS) or overall survival (OS).[3] The use of the MAGE-A3 vaccine in the MAGRIT trial was similarly negative.[4] With knowledge about molecular drivers of NSCLC and targeted treatment options in advanced disease, multiple studies are either completed or underway to study molecularly targeted agents in earlier stages of lung cancer, particularly with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). In metastatic NSCLC the EGFR TKIs produce superior response and progression free survival (PFS) compared with platinum doublet chemotherapy in treatment naïve patients with tumors with activating EGFR mutations (EGFRmut).[5,6]Similar outcomes with significant response and PFS improvements with the anaplastic lymphoma kinase (ALK) inhibitor crizotinib compared to chemotherapy have been reported in patients with tumors harboring translocations of ALK.[7] Encouraging data from retrospective and non-randomized trials looking at adjuvant EGFR TKI use led to randomized trials. Earlier trials that did not select based on EGFRmut status were negative, but more recent trials have been more encouraging. The phase III RADIANT trial selected patients with resected early stage NSCLC for EGFR expression by IHC/FISH, but not by EGFR mutation status, and randomized them to adjuvant erlotinib or placebo. [8] The primary end point was DFS in the full data set, with secondary analyses focused on patients with tumors harboring del19 or L858R EGFR mutations. No differences were found in DFS or OS based on treatment arm for the nearly 1000 patients who were enrolled. In the EGFRmut subset (N=161) DFS did favor erlotinib (HR 0.61, 95% CI = 0.384-0.981, p = 0.0391), but this was not considered statistically significant, as the primary endpoint of the trial was negative. The overall survival results, while still immature, were not in favor of the erlotinib arm, even in the EGFRmut subset. The conclusion from this study is that adjuvant EGFR TKI therapy requires further investigation and should not be considered a standard treatment option at this time. Multiple ongoing trials are exploring adjuvant EGFR TKI (and adjuvant ALK TKI) therapy for resected early stage NSCLC patients with tumors harboring the appropriate molecular marker.(Table 1) The ongoing trials are looking not only at whether or not an OS benefit can be obtained with adjuvant molecularly targeted therapy but also duration of therapy and the potential to use EGFR TKIs instead of chemotherapy in selected patients. The largest United States study is the NCI National Clinical Trials Network (NCTN) ALCHEMIST trial. The study is open to patients with resected early stage (IB-IIIA) NSCLC who are screened for EGFR activating mutations and ALK translocations. Patients with tumors harboring EGFR mutations or ALK translocations enter the appropriate sub-study and, after completion of all planned adjuvant chemotherapy or radiation therapy, are randomized to targeted TKI therapy or placebo for 2 years. Both sub-studies will enroll approximately 400 patients (410 EGFR; 378 ALK) and are powered for an OS endpoint. Patients without actionable mutations can enroll on the ANVIL sub-study looking at adjuvant nivolumab, a PD-1 targeted agent.(Table 1) Globally most targeted therapy adjuvant trials are being conducted in Asia, particularly China and Japan. ADJUVANT (C-TONG 1104) trial in China and IMPACT WJOG6410L in Japan are phase III trials for patients with resected stage II-IIIA EGFRmut NSCLC comparing gefitinib to cisplatin/vinorelbine using DFS as the primary endpoint.(Table 1) Other trials outlined in Table 1 are exploring variations on this theme using gefitinib or icotinib and either after or instead of adjuvant chemotherapy. The PD-1 inhibitors nivolumab and pembrolizumab are approved for the second line treatment of advanced stage NSCLC and will likely be utilized in first-line in the near future.[9-11] Based on their promise in advanced stage NSCLC, multiple trials with PD-1 and PD-L1 agents are ongoing. Most studies are for patients who have completed adjuvant chemotherapy (though some allow chemotherapy naïve patients) and they predominantly randomize patients to approximately 1 year of PD-1 or PD-L1 inhibitor therapy. Most include testing for PD-L1 expression, but do not exclude patients with low tumor levels of PD-L1. Many are placebo controlled.(Table 1) Chemotherapy has helped improve outcomes but continued investigations with novel approaches will be necessary to continue to improve cure rates for patients with resected early stage NSCLC. The use of molecularly targeted agents for patients with tumors containing EGFRmut or ALK translocations are promising with validation studies ongoing and the hope of immunotherapy is being investigated as well in multiple global trials. Table 1. Ongoing Phase III Targeted and Immunotherapy Adjuvant Trials

Trial	Description	Primary Endpoint(s)
C-TONG 1104 NCT01405079	*gefitinib vs. cisplatin/vinorelbine	3-year DFS
GASTO1002 NCT01996098	*Chemo then icotinib vs obs	5-year DFS
BD-IC-IV-59 NCT02125240	*Chemo then icotinib vs. placebo	2-year DFS
WJOG6401L IMPACT	*Gefitinib vs. cisplatin/vinorelbine	5-year DFS
ALCHEMIST A081105/E4512	*Erlotinib vs. placebo: ALK^ crizotinib vs placebo	OS
ALCHEMIST/ANVIL	&EGFR/ALK wildtype; US NCI NCTN, Nivolumab vs obs	OS/DFS
Impower010	Restricted to PD-L1+ Global, Atezolizumab vs. placebo	DFS
MEDI4736	&Global, MEDI4736 vs placebo	DFS
Keynote-091	&ETOP/EORTC, Pembrolizumab vs placebo	DFS

All EGFR studies include stage II-IIIA All PD-1/PD-L1 studies open to IB (4cm) – IIIA after adjuvant chemotherapy N: Number of estimated enrollment DFS: disease-free survival; OS: overall survival *EGFR deletion 19 or exon 21 L858R mutation only ALK^ : Positive for ALK translocation by FISH &- regardless of PD-L1 status US NCI NCTN: United States National Cancer Institute, National Clinical Trials Network References: 1. Pignon JP, Tribodet H, Scagliotti GV, et al: Lung Adjuvant Cisplatin Evaluation: A Pooled Analysis by the LACE Collaborative Group. J Clin Oncol, 2008 2. Group NM-aC, Arriagada R, Auperin A, et al: Adjuvant chemotherapy, with or without postoperative radiotherapy, in operable non-small-cell lung cancer: two meta-analyses of individual patient data. Lancet 375:1267-77, 2010 3. Wakelee HA, Dahlberg SE, Keller SM, et al: Randomized phase III trial of adjuvant chemotherapy with or without bevacizumab in resected non-small cell lung cancer (NSCLC): Results of E1505. Journal of Thoracic Oncology Proceedings WCLC 2015:Abstr: Plen04.03, 2015 4. Vansteenkiste JF, Cho BC, Vanakesa T, et al: Efficacy of the MAGE-A3 cancer immunotherapeutic as adjuvant therapy in patients with resected MAGE-A3-positive non-small-cell lung cancer (MAGRIT): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol 17:822-835, 2016 5. Mok TS, Wu YL, Thongprasert S, et al: Gefitinib or Carboplatin-Paclitaxel in Pulmonary Adenocarcinoma. N Engl J Med, 2009 6. Sequist LV, Yang JC, Yamamoto N, et al: Phase III Study of Afatinib or Cisplatin Plus Pemetrexed in Patients With Metastatic Lung Adenocarcinoma With EGFR Mutations. J Clin Oncol, 2013 7. Solomon BJ, Mok T, Kim DW, et al: First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N Engl J Med 371:2167-77, 2014 8. Kelly K, Altorki NK, Eberhardt WE, et al: Adjuvant Erlotinib Versus Placebo in Patients With Stage IB-IIIA Non-Small-Cell Lung Cancer (RADIANT): A Randomized, Double-Blind, Phase III Trial. J Clin Oncol 33:4007-14, 2015 9. Brahmer J, Reckamp KL, Baas P, et al: Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer. N Engl J Med 373:123-35, 2015 10. Borghaei H, Paz-Ares L, Horn L, et al: Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med 373:1627-39, 2015 11. Herbst RS, Baas P, Kim DW, et al: Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet 387:1540-50, 2016

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Abstract:
Abstract – IASLC – 2016 Vienna, Austria Chronicity is a word that over the past few years has been utilized when talking about lung cancer treatments. From the developments of the TKIs in the early 2000’s to the approval of immunotherapy for lung cancer in 2015, we are seeing that progression free survival and in some instances overall survival continues to be on the rise. So what does this mean for the medical oncology community? Patients can be on therapies longer than a year and sometimes for several years. We now as providers face the challenge of becoming experts in the management of long-term toxicity of these agents. Side effects of the targeted and immunotherapy drugs are not as predictable as their chemotherapy predecessors, and we are now dealing onset times ranging from days to years after beginning therapy. Immunotherapy drugs are the newest treatment craze and rightfully so, as we have seen documented 12 month overall survival in both non-squamous and squamous cell carcinoma for some of these agents and even up to 24 months for some patients. Although this has brought optimism to both providers and patients alike, it has also brought forth a multitude of side effects that remind us that we are still novices in this field and necessitate the collaboration with our non-oncologic colleagues as some of these side effects can be life-long. This lecture will review the mechanism of action of the immunotherapy agents as well as review those that are currently available for NSCLC with review of the data leading to their approval and the current and potential future challenges that lie ahead.

Abstract:
With the emergence of immunotherapy in lung cancer, patients now have access to treatments that have the potential to improve prognosis. Patients diagnosed with advanced non-small cell lung cancer (NSCLC) (either squamous or non-squamous) have previously had limited treatment options. With the emergence of new drugs, particularly in the immune-oncology setting, this is now changing. Recent clinical trial evidence demonstrates that compared with docetaxel, patients who received Nivolumab or Pembrolizamab had better overall survival and also significantly fewer Grade 3-4 adverse events (AEs). However the nursing experience of caring for lung cancer patients on an immunotherapy remains quite limited. Up to recent times immunotherapy drugs were limited to the clinical trial setting or early patient access schemes. Often patients on clinical trials are managed and monitored by research nurses which can further limit the experience for Lung Cancer Clinical Nurse Specialists caring for patients on immunotherapy. The two main clinical trials for immunotherapy in the UK were CHECKMATE (Nivolumab) and KEYTRUDA (Pembrolizamab). The aim of this presentation is to look at two patient case studies and review their experience of taking an immunotherapy. The presentation will focus on how immunotherapy has impacted on their lung cancer and also on their life. As part of the patient case studies there will be a focus on the nursing role in supporting and caring for patients on immunotherapy in a safe and effective manner. The presentation will examine the main adverse event profiles of immunotherapy and how these differ to chemotherapy. The presentation will open with a brief synopsis of the mode of action of immunotherapy which is a 2 minute film. The main focus of the presentation however, will be on the patient experience and the nurses’ role. Currently in the UK there is a scarcity of information available to oncology nurses on the nursing care of patients on immune-oncology treatments. However, there are many transferable skills which can be utilised when caring and supporting patients and their carers who either about to commence on immune-oncology. According to Reiger (2003) oncology nurses have a better opportunity than any other member of the healthcare team to develop the required rapport for effective educational pre-treatment consultations with both the patient and the carer. With regards to lung cancer clinical nurse specialists this rapport is often developed from the point of meeting the patient and carer at diagnosis and then supporting them through first line treatment and beyond to follow up. Often oncology nurses will build a rapport with patients when they attend to undergo treatment and are often a point of contact for patients to report side effects to. Including the patient’s carer in pre-treatment consultations and at key points in the patient pathway is very important. Often patients will be receive a lot of information about their diagnosis and proposed treatment plans and this can be very overwhelming (Malton 2002). Having the carer present means that they can ask questions about treatment side effects, how long the intended benefit of the treatment is and how the patient will be monitored during treatment. The pre-treatment consultation should be undertaken separately to the consent to treatment appointment to allow both the patient and the carer to digest information and write down any questions about the treatment they may have. In the current setting patient information for Nivolumab is under design meaning there may be limited information available to give the patient. There are patient alert cards which patients can keep in their wallets which detail the main side effects of Nivolumab. However, it is also important the nurse undertaking the pre-treatment consultation has a good understanding of Nivolumab, how it works and the potential side effects to monitor for so that they can counsel the patient and their carer accordingly and be able to answer any questions they may have. A clear and concise approach should be used in the pre-treatment consultation with key communication skills of planning, preparation and delivering the message, listening and questioning skills should also be implemented (Wiffen 2007). References. Malton S (2012) How to counsel cancer patients about their oral chemotherapy. Clinical Pharmacist 4: 171 Wiffen P, Mitchell M, Snelling M, Stoner N (2007) Oxford Handbook of Clinical Pharmacy. 1st edn. Oxford University Press, Oxford Rieger P, Yarbro C (2003) Role of the oncology nurse. In: Cancer Medicine. 6th edn. Kufe DW, Pollock RE, Weichselbaum RR, eds. BC Decker, Hamilton

Abstract:
Where Are We With TKI Toxicities? (Extended Abstract) Thoracic oncology nursing is now entering over 10 years of experience with managing tyrosine kinase inhibitor (TKI) toxicities, most notably, epidermal growth factor receptor (EGFR) inhibitor associated rash. The three approved EGFR inhibitors used to treat non-small cell lung cancer (NSCLC) are afatinib, erlotinib, and gefitinib. Most recently, the drug Osimertinib, for EGFR mutation resistance known as T790M, has now been approved for use. Grading of the EGFRI rash has been difficult due to its inconsistency in comparison to non-EGFRI rashes in the general medical community and other oncologic rashes. Consensus guidelines for the management of EGFR inhibitor associated rash have been produced and disseminated in the oncology community.[1,2] There is a correlation between EGFRI rash and overall survival in NSCLC patients, making it imperative to keep patients with rash on the EGFRI therapy.[3,4] It remains a challenge for oncology nurses to understand and manage this sometimes severe rash (see figure 1). Figure 1. Other cutaneous toxicities such as scalp rash, paronychia, hypertrichosis- namely trichomegaly (see figure 2), fissuring, pruritis, and xerosis have all been reported. The Multinational Association for the Supportive Care in Cancer (MASCC) has produced recommendations for these toxicities as well.[2] While they are often a minor annoyance, they can sometimes also become severe and cause dose reductions and a significant impact on activities of daily living (ADL’s). Identification, prevention, and management are important tasks for oncology nurses to master to allow patients to remain on therapy. Figure 2. Other classes of TKI toxicities include the Anaplastic Leukemia Kinase (ALK) inhibitors, where there are currently 3 drugs approved for use, alectinib, ceritinib, and crizotinib. Each of the ALK inhibitors carries different yet important toxicities, ranging from nausea/vomiting, diarrhea, edema, bradycardia, pneumonitis, myalgias with elevated CPK levels, and hepatotoxicity. Several other TKI’s are in development for potential use in lung cancer, such as HER2 inhibitors, BRAF inhibitors, and drugs targeting pathways dealing with RET, MET and KRAS (see table 1).[5-7]

BRAF mutations	4% NSCLC Most common is V600E Drugs in trials: dabrafenib, vemurafenib, dasatinib,
RET rearrangements	1-2% NSCLC Highly associated with young, never-smokers Drugs in trials: vandetanib, cabozantinib, sunitinib, ponatinib
MET amplification	Drugs in trials: crizotinib, tivantinib, onartuzumab, MET inhibitors
HER2 mutations	Drugs in trials: trastuzumab, afatinib, dacomitinib, neratinib
KRAS mutations	25-30% NSCLC, most common mutation MEK, PI3K, FAK inhibitors

It is important for thoracic oncology nurses to have a firm understanding of these drugs and their toxicities. Management strategies must be tailored to the patient’s symptoms and side effects, as well as to the specific drug and dosage. References: 1. Burtness B, Anadkat M, Basti S, et al (2009). NCCN task force report: management of dermatologic and other toxicities associated with EGFR inhibition in patients with cancer. JNCCN, 7(suppl 1):S5-S21. Available at http://www.nccn.org/JNCCN/PDF/2009_Derm_Tox_TF.pdf 2. Lacouture ME, Anadkat MJ, Bensadoun RJ, et al (2011). Clinical practice guidelines for the prevention and treatment of EGFR inhibitor-associated dermatologic toxicities. Support Care Cancer, 19(8):1079-1095. DOI:10.1007/s00520-011-1197-6 3. Lee SM, Khan I, Upadhayay S, et al (2012). First-line erlotinib in patients with advanced non-small-cell lung cancer unsuitable for chemotherapy (TOPICAL): a double-blind, placebo-controlled, phase 3 trial. Lancet Oncol, 13(11):1161-1170. DOI:10.1016/S1470=2045(12)70412-6 4. Liu H, Wu Y, Lv T, et al (2013). Skin rash could predict the response to EGFR tyrosine kinase inhibitor and the prognosis for patients with non-small cell lung cancer: a systematic review and meta-analysis. PLOS One. DOI:10.1371/journal.pone.0055128 5. Cardarella S, Ogino A, Nishino M, et al. (2013). Clinical, pathologic, and biologic features associated with BRAF mutations in non-small cell lung cancer. Clin Cancer Res. 2013; 19:4532-4540. 6. Tsuta K, Khono T, Yoshida A, et al. (2014). RET-rearranged non-small-cell lung carcinoma: a clinicopathological and molecular analysis. Br J Cancer. 110:1571-1578. 7. Awad MM, Oxnard GR, Jackman DM, et al. (2016). MET Exon 14 mutations in Non-small-cell lung cancer are associated with advanced age and stage dependent MET genomic amplification and c-MET overexpression. J Clin Oncol. 34(7):721-30.

Background:
Current methods for circulating tumor cell (CTC) detection are mostly include an enrichment step and the subsequent immunostaining-based identification of CTCs by epithelial and leukocytes markers. These methods are limited by loss and damage of CTCs during the enrichment and fail to determine the malignancy and drug targets of putative CTCs.

Methods:
We describe an enrichment-free, metabolic-based assay for rapid detection of tumor cells in the pleural effusion and peripheral blood samples. All nucleated cells are plated on microwell chips that contain 200,000 addressable microwells. These cells are labeled with a fluorescent anti-CD45 antibody (leukocyte marker), a fluorescent glucose analog (2-NBDG) and a dead cell marker (EthD-1). The microwell chips are imaged by a computerized high-speed fluorescent microscope in three colors and the bright filed. A computation algorithm analyzes the images and identify candidate tumor cells that are viable, CD45 negative, and exhibit high glucose uptake (EthD-1[-]/CD45[-]/2-NBDG[high]). A micromanipultor is then utilized to retrieve single tumor cells based on recorded addresses for single-cell sequencing.

Results:
EthD-1[-]/CD45[-]/2-NBDG[>100] cells are identified as candidate tumor cells. Single-cell sequencing based on a small panel of driver oncogenes (EGFR, KRAS, PIK3CA) shows that >60% of candidate tumor cells are true tumor cells harboring mutations in the panel. Single-cell whole exome sequencing results show all candidate tumor cells have high mutation frequency in dirver oncogenece and tumor suppressors from Qiagen's Human Lung Cancer Panel. Meanwhile, CD45[-]/EthD-1[-]/2-NBDG[>100] tumor cells show heterogenieity in cytokeratin (CK) expression, and only ~40% of these tumor cells are found CK positive. Figure 1



Conclusion:
We have developed a simple and functional-based method to rapidly identify tumor cells with high glucose uptake in the clinical liquid samples without enrichment. These tumor cells are addressable, enabling single-cell manipulation and sequencing. Clinical feasibility of this assay has been established by testing samples from a cohort of patients.

Background:
Chronic inflammation plays an important role in lung carcinogenesis and in chronic obstructive pulmonary disease (COPD), and is accompanied with alterations in specific serum-proteins. Both COPD and lung cancer are associated with smoking behavior, and 40-70% of lung cancer patients have COPD. The aim of the study is to compare levels of specific serum markers related to inflammation, extracellular matrix remodeling (ECM) and endothelial cell activation in patients with lung cancer and COPD.

Methods:
Blood samples were collected from 208 lung cancer patients with stage I-IIIA disease before surgery in addition to blood samples from 47 COPD patients, stage I-IV (4 patients in stage I, 16 in II, 19 in III and 8 in IV). Six of COPD-patients used oral steroids, 28 used inhaled corticosteroids. Serum levels of various markers were measured by enzyme immunoassays.

Results:
Of 17 proteins (table 1), 9 were significantly elevated in the COPD group compared to lung cancer group including proteins associated with lung cancer in other studies as OPG, PTX3, ePCR, GDF15 and endostatin. Only 3 proteins, CRP, vWF og GDF15 reflecting systemic inflammation and endothelial cell activation, were more abundant in serum from lung cancer patients, and one of these (CRP) significantly so.

Table 1. Serum proteins measured in our study.

Protein short name	Protein full name
OPG	Osteoprotegrin
ePCR	Endothelial cell protein C receptor
vWF	Von Willebrand factor
PTX3	Pentraxin 3
Axl	Tyrosine-protein kinase receptor
CXCL16	C-X-C motif chemokine ligand 16
DLL1	Delta-like protein 1
Cats	Cathepsin S (Chloramphenicol acetyl transferase)
GDF15	Growth differentiation factor-15
Endostatin
CD147	Cluster of differentiation 147 (Basigin. EMMPRIN)
sTNFR1	Tumor necrosis factor receptor 1
CRP	C-reactive protein
Alcam (CD166)	Activated leukocyte cell adhesion molecule
PARC	p53-associated parkin-like cytoplasmic protein
sCD163	Cluster of differentiation 163
Gal3BP	Galectin-3-binding protein

Conclusion:
Chronic inflammation plays an important role in both diseases: lung cancer and COPD. However, it seems that inflammation as determined by these selected markers is more pronounced in patients with COPD as most of the biomarkers levels were significantly higher in these patients than lung cancer group.

Background:
Colorectal cancer (CRC) is one of the most common malignancy and the most frequent cause of cancer-related death in Western countries. In patients with CRC, the presence of liver or lung metastases (LMs) seriously affects survival, and the early diagnosis and resection of LMs significantly improves the outcome. Unfortunately, the sensitivity of imaging studies in detecting LMs is low, because the onset of solitary pulmonary nodules is common during follow-up, the most part of them are not malignant. The aim of this study was to evaluate the accuracy of serum carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-7 and cyrokeratin-19 fragment (CYFRA 21-1) as predictive markers of LMs from CRC.

Methods:
We retrospectively reviewed the medical charts of 21 patients with a history of CRC who developed histologically confirmed solitary or multiple PMs. There were 13 (61.9%) men and 8 (38.1%) women, with an overall median age of 65 years (range 31-82 years). Controls were 24 age-matched patients with CRC in whom the presence of PMs was excluded using 18F-FDG PET. The receiver operating characteristic (ROC) curve was used to obtain the optimal threshold value (cutoff point) for each TM.

Results:
The optimal cutoff was set at 5 ng/mL, 7.5 ng/mL, 250 pg/mL, and 2.8 ng/mL for CEA, VEGF, MMP-7, and CYFRA 21-1, respectively. The sensibility, specificity, positive (PPV) and negative (NPV) predictive value, and accuracy are reported in the Table. The logistic regression analysis excluded CYFRA 21-1 from the model, and thus we calculated the results also considering the combination of CEA+VEGF+MMP-7. The area under the ROC curve (AUC) was 0.712 (95% CI: 0.432-0.802).

RESULTS	CEA	VEGF	MMP-7	CYFRA 21-1	CEA+VEGF+MMP-7
Sensitivity	71.4% (52.1-90.7)	80.9% (64.2-97.7)	85.7% (70.5-99.9)	84.2% (67.8-99.9)	90.5% (77.9-99.9)
Specificity	91.7% (80.6-99.9)	95.8% (87.8-99.9)	95.6% (87.8.99.9)	91.7% (80.6-99.9)	99.6% (98.7-99.9)
Positive predictive value	88.2% (72.9-99.9)	94.4% (83.9.99.9)	94.7% (84.7-99.9)	88.9% (74.4-99.9)	95.0% (85.4-99.9)
Negative predictive value	78.6 % (63.4-93.8)	85.2% (71.8-98.6)	88.5% (76.2-99.9)	88.0% (75.3-99.9)	99.1% (97.9-99.9)
Likelihood ratio positive	28.57	19.43	20.57	10.11	212.62
Likelihood ratio negative	0.31	0.20	0.15	0.17	0.10
False positive rate (α)	8.33%	4.17%	4.17%	8.33%	0.43%
False negative rate (β)	28.57%	19.05%	19.05%	15.79%	9.52%
Clinical accuracy	82.2%	88.9%	91.1%	84.4%	93.3%

Conclusion:
The periodic assay of CEA+VEGF+MMP-7 together may help to suspect the presence of LMs, suggesting the need to anticipate further evaluations.

Background:
Effective discrimination between lung cancer and benign tumours is a common clinical problem in the differential diagnosis of solitary pulmonary nodules. While most solitary pulmonary nodules are benign, around 20% of cases represent an early stage lung cancer. The presence of cell-free DNA (cfDNA) in plasma of lung cancer patients demonstrates promising diagnostic implications and could be considered as an auxiliary tool in the differential diagnosis of solitary pulmonary nodules by evaluating cancer-specific biomarkers, such as hypermethylated tumor DNA fragments. We developed a simultaneous methylation profiling of 21 distinct tumor suppressor genes (TSGs) in plasma cfDNA using MS-MLPA assay.

Methods:
The methylation profiling of 21 TSGs in plasma cfDNA of 32 resectable NSCLC (I-IIIa) patients and 8 subjects with benign lung nodules (hamartoma, fibrosis, granuloma) was performed using optimized MS-MLPA assay.

Results:
25/32 (78%) NSCLC and 8/8 (100%) benign-nodule cfDNA samples presented at least one TSG methylation, however the number of hypermethylated TSGs was much higher in NSCLC group. APC (frequency 18% samples), MLH1 (18%), ATM (13.6%), DAPK1 (13.6%), HIC 1 (13.6%), and RARβ (9%) were the most frequently methylated genes in NSCLC, while TIMP3 (75%), MLH1 (25%) and TP73 (37.5%) – in benign patients.

Conclusion:
The optimized MS-MLPA assay allowed simultaneous detection of multiple methylated TSGs in plasma cfDNA. The MS-MLPA showed good performance in samples with diverse cfDNA concentrations suggesting that methylation detection rate depends on the methylated DNA content in a sample. The study is on-going. The groups are to be extended and other benign lung pathologies evaluated.

Background:
Much has been studied regarding the prognostic role of circulating tumor cells (CTCs) in NSCLC. CTM (defined as clusters of 3 or more cancer cells detected in peripheral blood) relationship to prognosis was previously published for small cell lung cancer (SCLC), demonstrating worst prognosis for the presence of CTM. No relevant data, however, was published for CTM in NSCLC. The objective of this study is to define the presence of CTM as a prognostic factor for survival in NSCLC and its molecular characteristics.

Methods:
It was performed a retrospective evaluation of 31 metastatic NSCLC patients positive for CTC, which were previously enrolled for CTC research in a single institution. CTC and CTM were detected by ISET (Isolation by Size of Epithelial/Trophoblastic Tumor Cells, Rarecells, France®). Included patients had metastatic disease treated in multiple lines of cytotoxic treatment. Analysis included frequencies, demographic characteristics and survival variables, including Progression Free Survival (PFS) and Overall Survival (OS), with PFS as primary endpoint. The PFS and OS were calculated based on the date of first CTC collection and first progression after collection (PFS) or death (OS). Molecular characterization was performed by immunocytochemistry for Transforming Growth Factor beta receptor (TGFßR) and Matrix Metalloproteinase 2 (MMP2).

Results:
The primary endpoint was not met. Presence of CTM did not have statistically significant influence on PFS or OS in our population. Eight patients were positive (CTM+) and 23 were negative (CTM-) for CTM. Median follow-up was 13.3 months (m). Median age was 65.5 years in CTM- and 69.6 years in CTM+ patients. Remaining demographic variables were balanced between groups. All patients had progressive disease and 9 were still alive at the time of analysis. Median PFS (mPFS) was 6.9m for CTM- and 4,5m for CTM+, with p=0.59. Median OS (mOS) was paradoxically greater in CTM+, without statistical significance (27.3m for CTM- and 31.6m for CTM+; p=0.83). Molecular characterization did not have any prognostic impact on both CTM- and CTM+ groups. No patient was positive for TGFßR and 2 CTM+ patients were positive for MMP2 (10/31 patients with isolated CTCs positive for MMP2).

Conclusion:
This retrospective analysis showed no impact on survival for the presence of CTM in NSCLC, which was opposite to findings of positive prognostic value of CTM for SCLC where CTM was a negative factor for survival. Molecular characterization also did not show differences between groups. The findings warrant further evaluation in dedicated research.

Background:
Circulating tumor cells (CTCs) are tumor cells shed from primary tumor and circulate in the peripheral blood. CTCs, as a surrogate of distant metastasis, can be potentially useful in diagnosis and monitoring therapeutic effects in malignant tumors. Among a variety of systems for detection of CTCs, the “Cellsearch” is the only approved system for clinical use. However, EpCAM-negative tumor cells, such as those originating from non-epithelial cells and those undergoing epithelial-mesenchymal transition (EMT) cannot be captured with the “CellSearch” that is an EpCAM-based isolation system. Therefore, we have developed a novel polymeric microfluidic device (“Universal” CTC-chip) that can capture CTCs with or without EpCAM expression (AACR 2015). In the present study, we examined CTCs-detection performance of the CTC-chip in patients with thoracic malignant tumors (lung cancer [LC] as an “EpCAM-positive” tumor and malignant pleural mesothelioma [MPM] as an “EpCAM-negative” tumor) in comparison with that of the CellSearch.

Methods:
Peripheral blood sampled from each patient was divided and subjected to quantitative evaluation of CTCs with the CTC-chip as well as with the “CellSearch”. The CTC-chip, coated with an anti-EpCAM antibody, was used to capture CTCs in the blood samples (n=19) from lung cancer patients. To capture CTCs in the samples (n=11) from MPM patients, the CTC-chip was coated with an antibody against podoplanin that is expressed on the mesothelioma. After immuno-staining for cytokeratin and CD45 on the chip, a captured cell containing Hoechst-positive nucleus and cytokeratin-positive/ CD45-negative cytoplasm was judged as a CTC. The CTC-count for each sample was represented as the number per 7.5mL of the blood.

Results:
The median CTC-count detected with the CTC-chip in LC was 50 (range, 0-270), which was significantly higher than that (the median CTC-count, 0; range, 0-47) with the CellSearch (p<0.01). In the peripheral blood sampled from MPM patients, CTC was detected in only one patient using the CellSearch, but was detected in all 11 patients with the median CTC-count of 144 (range 0-470).

Conclusion:
The“universal” CTC-chip achieved higher performance in detection of CTCs of thoracic malignant tumors as compared with the CellSearch.

Background:
Promoter DNA hypermethylation is a well characterized epigenetic event and has been linked with early stages of lung carcinogenesis through inactivation of tumor suppressor genes. In this study, we studied the methylation status of APC, DAPK, and GSTP1 genes in tissue biopsy and serum of lung cancer patients and cancer-free controls.

Methods:
In this prospective study, 160 primary lung cancer patients and 70 cancer-free controls undergoing bronchoscopy for benign disease were recruited. DNA was isolated from tissue biopsy and serum of all the subjects and methylation-specific PCR of APC, DAPK, and GSTP1 was carried out after bisulfite conversion. Association of DNA methylation with various clinico-pathological parameters and survival was determined in lung cancer patients.

Results:
The methylation rates of APC, DAPK, and GSTP1 in tissue biopsy were 83.1%, 83.1%, and 78.1% for lung cancer patients and 72.9%, 70%, and 70% for cancer-free controls. The methylation rates of APC, DAPK, and GSTP1 in serum were 52.5%, 30.6%, and 65.6% for lung cancer patients and 14.3%, 18.6%, and 30% for cancer-free controls. In lung cancer patients, all three genes were methylated at significantly higher frequency in tissue biopsy than matched serum samples. No significant correlation was observed between methylation of any of three genes with clinico-pathological parameters, including survival.

Conclusion:
Present study did not demonstrating any evidence suggesting the role of promoter DNA methylation of APC, DAPK, and GSTP1 in lung carcinogenesis. However, follow-up of cancer-free controls, who were positive for DNA methylation, is required to confirm their role in early stages of lung carcinogenesis.

Background:
Tissue availability is a crucial point in NSCLC. The introduction of Liquid Biopsies allows to determine circulant biomarkers, specifically using free DNA. To simultaneously analyze multiple patients sample at high sensitivity, Next Generation Sequencing (NGS) can be narrowed to target a limited number of actionable genes. Here we prospectically applied a lab-developed narrowed gene panel (SiRe) to produce a DNA library covering 568 actionable mutations in six gene (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα).

Methods:
This daily clinical practice study was performed on cfDNA obtained from Non Small Cell Lung Cancer blood samples (serum and plasma) prospectically collected either prior to treatment administration in patients without tissue availability (n = 46) or after a progressive disease (n = 19) from a first line gefitinib (n = 14) or afatinib (n = 5) therapy.

Results:
SiRe detected an activating EGFR mutation in 4/46 (8.9%) cases and in T790M in 9/19 (47.4%) at the time of tumor progression. Using tissue data as gold standard, the SiRe panel showed a sensibility of 90.5% and specificity of 100%.

Conclusion:
The SiRe panel is an effective tool enabling the implementation of NGS for cfDNA mutational profiling in molecular pathology practice.

Background:
Next generation sequencing (NGS) has been increasingly used in oncology practice but proven practically difficult when serial tumor specimens are needed. The objectives of this study were to determine feasibility and explore clinical utility of serial NGS analyses of circulating tumor DNA (ctDNA) in patients (pts) with advanced solid tumors undergoing treatment.

Methods:
ctDNA digital NGS was performed by a CLIA-certified lab (70-gene panel with mutant allele fraction (MAF) quantification). ctDNA results were retrospectively analyzed and decreases/increases/stability of molecular tumor load (MTL) defined here as MAFs of truncal driver mutations were correlated with clinical and radiographic response to treatment (response, progression, or stable disease, respectively).

Results:
From Jan 2015 to July 2016, 38 consecutive pts with advanced lung tumors (84% LUAD, 5% LUSC, 5% SCLC, 5% NOS) receiving treatment (Table) had serial ctDNA analyses (median 2, range 2-7). ctDNA alterations were detected at least once in 37 (97.4%) pts. Changes in MTL correlated with or predicted all (95% CI, 82.0-99.8%) radiological and/or clinical responses except for the patient with no genomic alteration detected. MTL results clarified response status when radiographic responses were difficult to assess in 9 (28%) of pts with either complex pleural disease (n=6), pneumonitis during PD-1 inhibitor therapy (2). Two MTL change patterns were observed: 1) clonal changes while receiving targeted therapy, including EGFR (12), ALK (3), MET (2), ERBB2 (2); 2) global changes to PD-1 inhibitors, chemotherapy or radiation. Representative tumor response maps will be presented. Table. Summary of tumor types and cancer treatment.

Cancer Type	Targeted Therapy	Immunotherapy	Chemotherapy	Radiation	TOTAL
LUAD	14	8	7	3	32
LUSC	1	1	0	0	2
SCLC	0	0	2	0	2
NOS	1	0	1	0	2
All	16	9	10	3	38

Conclusion:
Serial liquid biopsies and ctDNA digital NGS are feasible and clinically useful in monitoring MTL and genomic alterations during cancer treatment, especially in situations when radiographic responses are equivocal. Prospective evaluation of impact on clinical decision making is warranted.

Background:
PFTK1, a novel cyclin-dependent kinases, plays pivotal roles in cell proliferation, differentiation and cell cycle regulation. It has been reported that cell motility and invasiveness could be enhanced by PFTK1 in kinds of tumors. However, the function of PFTK1 in NSCLC metastasis is not clear. The aim of this study was to explore the potential role of PFTK1 in NSCLC metastasis.

Methods:
Expression of protein PFTK1 was assessed by immunohistochemistry staining in tissue microarrays, containing paired tumor tissue and adjacent NSCLC tissue from 119 cases of human lung cancer. PFTK1 was knocked down by shRNA interference method both in human H1299 and 95C cells. Then we applied H1299 and 95C cells that PFTK1 expression was inhibited into the next study. The effect of PFTK1 on cell migration and invasion was explored by cell wound healing assay and transwell assay. Western blot was used to detect whether PFTK1 influences the expression of EMT related proteins β-catenin, vimentin and ZEB1. Cytoskeleton preotein F-actin was observed using cell immunofluorescence test.

Results:
Immunohistochemistry staining of 119 NSCLC patients showed that a high level of PFTK1 expression was correlated with lymph node metastasis and T stage(P<0.05). And detailed analysis indicated that the high expression of PFTK1 was associated with poor prognosis for NSCLC patients (P<0.05). In addition, suppression of PFTK1 inhibited cell migration and invasion in H1299 and 95C cells. Inhibition of PFTK1 decreased the expression of β-catenin, vimentin, as well as ZEB1. Cytoskeletal protein F-actin was also decreased by the down-regulation of PFTK1.

Conclusion:
We reported for the first time that PFTK1 was overexpressed in samples of NSCLC. The high expression of PFTK1 was associated with lymph node metastasis, T stage and poor prognosis for NSCLC patients. Furthermore, our data indicated that PFTK1 promoted cells migration and invasion by regulating the expression of cytoskeletal protein F-actin and modulating EMT events. Therefore, our findings suggest that PFTK1 would be a potential target to development of therapies for NSCLC.

Background:
Aberrant expression of miRNAs has been found in human cancers and has not only been used as diagnostic, prognostic, and predictive biomarkers, but also as potential therapeutic target. In this study, we compared the expression profile of miRNAs in the serum of NSCLC patients with that of non-malignant respiratory disease patients and healthy controls.

Methods:
For this prospective pilot study, a total of 10 subjects were recruited, 2 each of lung adenocarcinoma (ADC), squamous cell carcinoma (SQC), pulmonary tuberculosis (TB), chronic obstructive pulmonary disease (COPD), and healthy controls, from Outpatient Department of Pulmonary Medicine and Sleep Disorders, AIIMS, New Delhi. Approximately 5 ml. of peripheral blood was collected; serum was separated and total RNA was isolated using miRNeasy serum/plasma kit (QIAGEN). Small RNAs were purified from total RNA; libraries of 18 to 50 nt small RNAs were prepared with the TruSeq RNA Library Prep Kit (Illumina), and mature miRNAs were profiled using illumina TruSeq Sequencing Chemistry on illumina HiSeq 2000 next generation sequencing (NGS) platform. Quality check was performed and high quality raw data was mapped on to miRBase database. The expression profile of miRNAs in each subject was analyzed using miRNAkey software and fold change was performed to identify differentially expressed miRNAs in NSCLC as compared to controls.

Results:
Using NGS, we were able to detect 1074, 1013, 299, 268, and 907 known miRNAs in the serum of lung ADC, SQC, TB, COPD and healthy controls, respectively. A number of miRNAs, such as let-7, miR-10b-5p, miR-15a-5p, miR-23b-5p, miR-92a-3p, miR-148b-3p, miR-185-3p, miR-192-5p, miR-320a, miR-329-3p, miR-342-3p, miR-375, miR-449a, miR-486-5p, miR-497-5p, miR-584-3p, miR-1908-5p, and miR-3195 were found to be downregulated, while miR-10a-5p, miR-148a-3p, and miR-197-3p were found to be upregulated in NSCLC as compared to non-malignant respiratory disease controls and healthy controls (>2 fold change; p<0.05). Further, few miRs, including miR-93-3p, miR-130b-5p, miR-196b-5p, miR-337-3p, miR-378f, miR-382-5p, miR-424-3p, and miR-1271-3p were found to be specifically expressed in NSCLC.

Conclusion:
A number of miRNAs were found to be dysregulated in the serum of NSCLC patients than controls. The present study is being continued in a larger set of subjects to validate the findings & also the expression patterns of target genes of differentially expressed miRNAs is being analyzed by real-time reverse-transcriptase PCR to find their relevance in lung carcinogenesis. This may prove to be useful for developing non-invasive diagnostic and prognostic tools as well as tools to monitor therapeutic efficacy in NSCLC.

Background:
Lung cancer is the leading cause of cancer death worldwide. 25 -30% of lung cancers are histologically squamous cell carcinomas (SCC). Despite recent advances in immunotherapy for lung SCC, traditional cytotoxic chemotherapies currently remain the mainstay of treatment. However, over the course of treatment, patients with lung SCC inevitably acquire chemotherapy resistance. This results in poor overall survival of advanced stage lung SCC of only 9 to 11 months. Repetitive exposure of lung cancer cell lines to chemotherapeutic drugs enables investigation of molecular mechanisms of acquired chemotherapy resistance in vitro. We are studying the role of miRNAs in this process. MiRNAs are small non-coding nucleic acids that regulate gene expression. They are involved in numerous cellular pathways, including therapy resistance. MiRNA serve as biomarkers and have recently become therapeutic targets or therapeutics themselves.

Methods:
We induced chemotherapy resistance in lung SCC cell lines LUDLU-1, Calu-1, SK-MES-1 in vitro by repetitive drug treatment over a period of 6 – 12 months. Agents used to develop resistance included Cisplatin, Gemcitabine, Paclitaxel and Vinorelbine. Cell viability after 3 days of chemotherapy treatment was measured by MTT assay and drug dose causing a 50% growth inhibition (IC~50~) was calculated. Total RNA including miRNA was extracted. Expression of 754 miRNA was measured by TaqMan OpenArray Human MicroRNA array.

Results:
After 15 -25 cycles of chemotherapy lung SCC resistant cells showed a statistically significant increase in IC~50~ values: Cisplatin up to 12.4 (n-fold); Gemcitabine 40.2 – absolute resistance (n-fold); Paclitaxel 30.9 – 110.1 (n-fold); Vinorelbine 4.8 -19.3 (n-fold). Resistance was stable and passed on to daughter cells. MiRNA expression of resistant cells was compared to parental, drug sensitive cells and is illustrated by heatmaps and volcano plots. Analysis of expression patterns revealed upregulation and downregulation of specific miRNAs in drug resistant cells. We are currently investigating the function of these dysregulated miRNAs in promoting chemotherapy resistance. Further, we are testing if certain miRNA are suitable targets to improve chemotherapy response.

Conclusion:
We identified changes of miRNA expression patterns after induction of chemotherapy resistance with various drugs used for lung SCC treatment. These findings may lead to development of new predictive biomarkers and to new miRNA-based drugs.

Background:
Chemotherapy to non-small cell lung cancer (NSCLC) remains a big limitation; chemo-resistance which has been reported regulating by one of cancer stem cells (CSC) marker cluster determinant (CD) 44 expression in NSCLC. Here, we demonstrated that the importance of CD44 in chemo-resistance of NSCLC, subsequently, develop and evaluate the hyaluronan (HA)-liposome as a drug delivery system for overcoming chemoresistance by efficiently delivery CD44 targeting siRNA to NSCLC cells.

Methods:
First, the relationship between expression of CD44 and sensitivity of the chemotherapy was evaluated in NSCLC cells (H1299, H1703, H1793, H1435, H2087, H358, H522, H460) using flow cytometry (FACS) and MTT assay. The expression of CD44 was confirmed as inversely proportion to the sensitivity of the chemotherapy in these NSCLC cells. Furthermore, in order to confirm that correlation between CD44 expression and chemo-resistance of NSCLC, we generated and characterized cisplatin resistant cell lines, and indicated that CD44 expression on resistant cells significantly increased compared to wild type cells.

Results:
Figure 1 Chemo-sensitivity of resistant cells are directly associated with expression of CD44 by knockdown of CD44 expression using siRNA. For overcoming drug-resistance in lung cancer, we developed a HA-liposome drug delivery system which can specifically target to CD44, effectively delivered CD44 siRNA to CD44 overexpressed resistant NSCLC cells. And, the HA-liposome (CD44 siRNA) successfully inhibited the expression of CD44 on resistant cells and improved the sensitivity to cisplatin.



Conclusion:
We demonstrated the corelation of chemoresistance and expression of CD44 in NSCLC, and successfully developed HA-liposome drug delivery system for significantly inhibit the expression of CD44. This study supported future investigation HA-liposome (CD44 siRNA) as possible chemotherapy carrier for targeting CD44, to assess for inhibiting chemoresistance using various drug delivery in NSCLC.

Background:
Immunoglobulin (CD79A) binding protein 1 (IGBP1) binds to PP2Ac and exerts an anti-apoptotic effect. We have already reported that IGBP1 overexpression occurs during the course of malignant progression of lung adenocarcinoma (Sakashita S et al., Pathol Int. 2011). However, the molecular mechanism of IGBP1 overexpression is still unclear. A few reports have documented mutation, hypomethylation, or amplification of IGBP1, but only one study has suggested that down-regulation of miR-34b leads to high expression of IGBP1 (L-P Chen et al. Oncogene. 2011). In this study, we have detected miR-3941 as another functional microRNA that influences the expression status of IGBP1.

Methods:
We performed microRNA array analysis using total RNA extracted from fresh specimens of invasive lung adenocarcinoma (IGBP1+) and minimally invasive adenocarcinoma (IGBP1-). We compared the results of microRNA array with microRNAs listed in TargetScan (a microRNA database) that would potentially bind to IGBP1. Using reverse transcription-quantitative PCR (RT-qPCR), we analyzed the expression levels of candidate microRNAs in frozen specimens of lung adenocarcinoma. We also validated these microRNAs by checking IGBP1 expression and cell proliferation after they had been transfected into lung adenocarcinoma cell lines (A549, PC-9) and confirmed the direct effect of the microRNAs by luciferase reporter assay.

Results:
Using microRNA array and TargetScan, we selected 6 microRNAs (miR-34b, miR-138, miR-374a, miR-374b, miR-1909, miR-3941). RT-qPCR analysis showed that these microRNAs were down-regulated in invasive adenocarcinoma (IGPB1+) relative to adjacent normal lung tissue (IGBP1-) (Fig1A). We transfected these microRNAs into lung adenocarcinoma cell lines, and all of the microRNAs suppressed IGBP1 expression. Among these microRNAs, miR-34b and miR-3941 depressed luciferase activity by targeting 3’UTR-IGBP1 in the luciferase vector (Fig1B). Figure 1



Conclusion:
We have found that miR-3941 targets IGBP1 in addition to miR-34b. Down-regulation of both microRNAs can lead to high expression of IGBP1, and this is thought to be associated with progression of lung adenocarcinoma.

Background:
Across multiple cancer histologies, many significantly down-regulated genes reside within chromosomal regions with increased number of copies, and vice versa. These “paradoxical genes” have been usually ignored as a noise, but could be a consequence of epigenetic regulatory mechanisms, including microRNA-mediated control of mRNA transcription.

Methods:
To identify paradoxical genes in lung adenocarcinoma (LUAD) we curated and analyzed gene expression and copy number aberrations across 1,064 LUAD samples, including newly-generated aCGH data from 65 samples. We then analyzed 9 LUAD microRNA expression studies to compile a list of consistently deregulated microRNAs. Finally, using microRNA:gene networks from mirDIP we examined possible association between microRNAs and paradoxical genes.

Results:
We identified 85 genes whose differential expression consistently contrasts the aberrations of their copy numbers. 70 genes were validated using TCGA-LUAD data. We showed that paradoxical expression of these genes is associated with 19 microRNAs, whose significant deregulation in LUAD has been consistently reported. Importantly, these genes form a clinically significant prognostic signature.Figure 1Figure 2





Conclusion:
Paradoxical gene expression, caused by microRNA deregulation, is preserved across patient cohorts, and forms a prognostic LUAD signature.

Background:
There are two main types of lung cancer: non small cell lung cancer (NSCLC) which represents 80-85% cases of lung cancer and small cell lung cancer (SCLC) which is about 10-15% cases of lung cancer. The 5-year survival rate for patients with lung cancer vary depending on the stage of the cancer when it is diagnosed. Unfortunately, most of patients with lung cancer are diagnosed on later stage of disease (stage III and IV). In our research we try to find marker among miRNA that can predict occurring of lung cancer on the earlier stage.

Methods:
Isolation of miRNA from plasma was performed by miRCURY RNA Isolation Kit – Biofluids (Exiqon) from NSCLC patients and controls. Synthesis of cDNA and qPCR were carried out using miRCURY LNA[TM] Universal RT microRNA PCR with LNA[TM] enhanced PCR Primers (Exiqon). Statistical calculations were executed on 11 samples as a Pre-eliminary data.

Results:
Results are shown in the following table.Figure 1



Conclusion:
We assed level of 5 different miRNAs circulating in the blood of NSCLC patients using qPCR. Our initial results show that different miRNA can be used to stratify patients and miRNA. Expression of hsa-miR-451a is decreasing in NSCLC versus negative control. Interestingly up-regulated hsa-miR-660-5p was recently described as a prognostic marker in breast cancer but our result preliminary results showed constant decrease in hsa-miR-660-5p expression in all patients’ groups vs controls. The examination on the bigger cohort of patients is necessary to receive a more statistically significant and conclusive data.

Background:
We previously reported the identification of diagnostic miRNA signatures in plasma samples of lung cancer patients detected by low dose computed tomography (LDCT) screening. Circulating miRNAs are released into the bloodstream by different mechanisms such as passive leakage from damaged cells or active secretion through extracellular-vesicles or protein complexes

Methods:
To evaluate the potential origin and the release of the 24 miRNAs of the diagnostic signature we analyzed their expression by real-time or digital PCR in both cells and conditioned medium (CM) from cancer cell and different cell types of the lung microenvironment. Lung tissues and cell-blocks were analyzed by miRNAs in situ hybridization (ISH). Modulation of miRNAs after in vitro treatments known to induce changes associated with cancer progression, in different cell types was assessed and correlated to changes observed in circulating miRNAs signatures.

Results:
24-miRNAs analysis showed higher abundance in specific cellular components such as mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells or mir-451 and 142-3p in hematopoietic cells. Generally, tumor cells showed lower levels of miRNAs compared to bronchial epithelial cells. MiRNAs specific localization in lung tissue was confirmed by ISH. We observed that mir-451 is specifically expressed in lung interstitial alveolar walls while mir-126 by endothelial cells outside the tumor bulk; miR-145 is characteristic of fibroblast and muscle cells and miR-142-3p of hematopoietic cells, fibroblast and muscle whereas mir-21 is over-expressed in the tumor. The analysis of miRNAs in CM showed that miRNAs secretion is correlated with cellular expression for most cell types (Pearson correlation range: 0.41-0.80). Interestingly, platelets and granulocytes were the components that mostly secreted miRNAs. In vitro experiments showed that endothelial cells under hypoxic condition up-regulate mir-126 and that mir-145 was up-regulated and secreted in lung cancer-associated compared to normal fibroblasts. Interestingly, during conversion of T lymphocytes into T regulatory cells up-regulation of mir-15b, mir-19b and mir-320 was observed whereas mir-15b and mir-197 were up-regulated in the conversion of macrophages into M2 phenotype. Modulation of miRNAs in immune and stromal cells was consistent with up-regulation of the same miRNAs observed in plasma samples.

Conclusion:
Our findings on the origin of circulating miRNAs support the conclusion that plasma miRNAs are heterogeneous and secreted by different cellular components of lung microenvironment rather than by tumor cells. In particular, we demonstrated that a pro-tumorigenic and immunosuppressive microenvironment contributes to the de-regulation of miRNAs observed in plasma of lung cancer patients.

Background:
Lung cancer is the leading cause of cancer related mortality and approximately 80% is represented by non-small cell lung cancer (NSCLC). In the last decade, age of patients at diagnosis has decreased, with an incidence of approximately 13.4% in patients under 50 years. Previous studies have hypotesized that lung cancer in young patients could represent a separated clinicalpathological entity, however it is still controversial whether younger patients have a different outcome compared with their older counterparts. MicroRNAs (miRNAs) have recently been defined to play a key role in cancer pathogenesis and their aberrant expression has been suggested as a potential biomarker of prognosis in lung adenocarcinoma. To understand the molecular features of young and old adenocarcinoma patients, we investigated the expression levels of a panel of miRNAs selected from recent literature.

Methods:
Thirty-five lung ADC from patients under 50 years old were enrolled as the younger group and thirty-five ADC older than 50 years were collected as the older group. After miRNA isolation from formalin-fixed and paraffin-embedded tumor tissues, the expression levels of 30 miRNAs were analyzed by NanoString technology and compared between the two groups. Survival data were used to assess the prognostic impact of miRNAs. The software miRgator v3.0 was used to predict the putative pathways targeted by miRNAs.

Results:
The analysis revealed that 7 miRNAs (miR-25-3p, miR-29c-3p, miR-33a-5p, miR-144-3p, miR-153-3p, miR-342-5p and miR-485-3p) were differently expressed in the two groups (Mann-Whitney U test, p<0.05). All these miRNAs showed higher expression levels in young compared to old patients, and their predicted targets included EGFR, MET, VEGF-A, TP53 and PDGFRa. miR-144-3p had an opposite influence on overall survival, since its upregulation was associated with a better outcome in young patients (p= 0.01) and a worse prognosis in the old group (p= 0.03).

Conclusion:
Our study provides new insights about the role of miRNAs in lung adenocarcinoma occurring in young patients. We observed that lung cancer in young and old patients may be influenced by different regulatory mechanisms since we found 7 miRNAs as downregulated in the older group, probably due to distinct age-related genetic and epigenetic alterations. Moreover, one of the deregulated miRNAs showed a different prognostic impact in the two groups thus confirming that young and old patients deserve a specific clinical approach. Further validations are needed to better define if an age-based genomic signature could be used as prognostic marker in lung cancer.

Background:
Squamous cell carcinoma (Sq) is second major histological subtype of lung cancer. Unlike in the case of adenocarcinoma (Ad), Sq has only few molecular target drug. MicroRNA (miR) is a major part of post-transcriptional regulators functioning as tumor suppressor genes or oncogenes. MiR will regulate target molecules related to carcinogenesis and malignancy in Sq.

Methods:
Using The Cancer Genome Atlas dataset including copy number variation, RNA sequence, miR sequence, clinicopathological feature from 484 lung cancer cases, the correlation between genomic copy number and expression of miR was analyzed. 245 samples of Sq and 239 samples of Ad were included. The raw counts of each mature miR fragments with different precursor were merged and calculated from miR-seq isoform files by R project (http://www.r-project.org/) Segmented copy number variation datasets were processed with R package CNTools of Bioconductor project. Independent two-group Mann-Whitney U test was used to compare different expression between Sq and Ad. MiR expression according to copy number variation was analyzed using Pearson correlation coefficient r-score. To identify the miR target sites of mRNAs, targetscan-Perl scripts were used (http://www.targetscan.org/).

Results:
From 1,001 mature miR fragments, 34 miRs were identified as the candidates especially for Sq distinguished from Ad. Furthermore, four miRs were up-regulated in amplified regions and independently associated with poor prognosis in Sq. Moreover, those who had the tumor with high expression in three of four miR simultaneously showed worst prognosis. To explore miR-mRNA network, we also predicted the target genes for each miR. From 734 common target genes, three showed positive correlation with the expression of three miRs.

Conclusion:
Three miRs up-regulated in Sq were associated with poor prognosis through the regulation of three common target genes.

Background:
Lung cancer is one of the leading causes of cancer-related death in the world. Metastasis is the main cause of death. There is still lack of ideal predictive and prognostic biomarkers. Human lncRNA plays important structural and functional roles in many biology progresses. Increasing studies have demonstrated that the abnormal expression of lncRNA is correlated to cancer progress in variant types of cancer, and lncRNA is considered as a potential valuable biomarker for diagnosis, treatment and prognosis of cancer.

Methods:
Here, we investigated the lncRNA expression profile in lung adenocarcinoma with lymph node metastasis and without lymph node metastasis (n=5 vs 5) by microarray assay. Three lncRNAs were selected for further verification by qRT-PCR in a training cohort including 118 paired lung cancer and the adjacent normal tissues and a test cohort including 60 paired tissues. In addition，we analyzed the correlation of the lncRNAs with clinicopathological features and survival.

Results:
We observed 245 significantly differentially expressed lncRNAs between lung adenocarcinoma with lymph node metastasis and without lymph node metastasis. Gene ontology analysis showed that the lncRNAs may be involved in several important biological progresses. ROC curves revealed that a 3-lncRNA signature distinguished not only lung cancer with lymph node metastasis from lung cancer without lymph node metastasis but also lung cancer from normal tissue. Moreover, the 3-lncRNA signature showed prognostic value by survival analysis. Multivariable Cox regression analysis showed that the signature was an independent prognostic factor for lung cancer patients. In another independent test cohort including 60 paired lung cancer and normal tissues, we validated the diagnostic and prognostic values of the 3-lncRNA signature.

Conclusion:
Our results identified a new 3-lncRNA signature for the diagnosis and prognosis of lung cancer, suggesting the potential clinical utility of lncRNA biomarkers in lung cancer.

Background:
Early-stage lung cancer patients have a five-year survival rate greater than 70%, however this benefit is not exploited due to late diagnosis. Moreover, for the early-stage patients eligible to surgical intervention the long-term survival is also reduced by the high risk of relapse following surgery. The identification of circulating biomarkers is an attractive and less invasive way to improve the management of lung cancer patients. MicroRNAs (miRNAs) post-transcriptionally modify gene expression and are thus involved in cancer through controlling different cellular processes. Dysregulation of their expression contributes to lung cancer progression both in tissue samples and in the blood stream (plasma/serum). The aim of our study is to assess a miRNA profile from serum patients to identify circulating biomarkers useful to predict surgery outcome in the early-stage NSCLC patients

Methods:
16 early-stage NSCLC patients were enrolled. Serum samples before (pre) and after (post) surgery together with surgical tumor tissue were collected from each patient. Extracted RNA was enriched to construct library. Raw sequencing reads were aligned to hg19 human genome and miRNAs were annotated using miRBase v21. After reads normalization, differentially expressed miRNAs were identified through edgeR package. p‑value < 0.01 was considered as statistically significant.

Results:
miRNA deep sequencing analysis on 16 NSCLC patients: surgical tissue, pre-surgical and post-surgical serum samples lead to detect a total of 2500 miRNAs. MiRNA expression profile data were explained by the Venn diagram (figure1) Figure 1 MiR-125b-5p resulted significantly down-regulated in serum samples (pre and post-operative) compared to tumor tissue, while it increased in serum of patients after tumor removal. Therefore miR-125b-5p expression could be influenced by tumor resection because of its involvement in different tumorigenic processes.



Conclusion:
miRNA deep sequencing revealed circulating biomarkers potentially involved in lung tumor progression after surgery. MiRNAs could be useful to follow disease recurrence and to improve survival rate of early-stage patients.

Background:
PIWI-interacting RNAs (piRNAs) are small (24-32 nucleotides) non-coding RNAs. Their functions, widely conserved across species, are associated to epigenetic control of gene expression and maintenance of genomic stability by the repression of mobile elements. In humans, >23,000 piRNAs are known, showing tissue-specific expression patterns. While the aberrant expression of individual piRNAs has been identified in some cancer types, the role of piRNA co-expression networks in the development of lung tumors and their utility as molecular markers remains unexplored. By analyzing over 7000 piRNA transcriptomes from human tumors and non-malignant tissues, we have identified lung cancer (LC) specific expression networks associated with clinically-relevant tumor features and patient prognosis.

Methods:
We developed a custom small-RNA sequence analysis pipeline to generate >7,000 human piRNA transcriptomes. piRNA expression baseline was deduced from 6,378 piRNA transcriptomes (non-malignant/tumors) from 11 organ sites. In lungs, we analyzed 1,082 tumors and 209 non-malignant samples from two cohorts: BC Cancer Agency (BCCA) and The Cancer Genome Atlas (TCGA). Network analysis was performed using the weighted gene co-expression network analysis (WCGNA). We evaluated tumour aggressiveness by considering correlation to several clinical parameters, including stage, number of mutations, nodal/distant metastasis, and overall/disease-free survival. piRNA survival signatures were identified using a Cox Proportional Hazard model.

Results:
A subset of piRNA showed robust expression in somatic tissues. Expressed piRNAs display organ-specific patterns and mainly map to coding transcripts, suggesting a role in regulation of gene expression. In lungs, 204 piRNAs were consistently expressed in both LC cohorts. Tumor piRNA expression profiles are markedly different from their non-malignant counterparts (133 piRNAs were differentially expressed). The patterns differ between the adenocarcinoma and squamous cell carcinoma, and were influenced by smoking status. Network-based analysis identified piRNA expression changes in two modules of piRNAs are associated with aggressiveness tumor features, such as increased number of mutations, tumor size and nodal metastasis. Finally, combined expression of piRNAs define signatures associated with patient overall and recurrence free survival.

Conclusion:
We provide evidence of somatic, tissue-specific human piRNA expression. In lungs, aberrant expression patterns are associated with well-established etiological factors of cancer and seem to contribute to lung cancer subtype-specific biology. We discover that specific piRNA-based expression patterns characterize aggressive lung tumors and also exhibit prognostic value. The unique expression patterns of piRNAs offer an opportunity to better understand lung cancer-specific biology as well as develop novel prognostic markers for clinical application.

Background:
Deregulation of small RNAs at the imprinted DLK1-DIO3 locus has been linked to lung adenocarcinoma (LUAD) patient outcome. While the contribution of microRNAs (miRNAs) is established, the role of Piwi-interacting RNAs (piRNAs), small RNAs involved in epigenetic regulation of gene transcription, is unexplored. We quantified expression of piRNAs and miRNAs mapping to this locus in two independent cohorts of LUAD and assessed the ability of a combined miRNA/piRNA signature to improve patient outcome stratification.

Methods:
Expression levels (RPKM) for miRNA/piRNA were determined from small RNA sequencing experiments from two cohorts (TCGA, n=154, 5-year follow up; BCCA, n=77, 8-year follow up). Associations with patient overall survival (OS) and recurrence free survival (RFS) were calculated by inputting miRNA and piRNA expression combinations into a Cox proportional hazard model. Risk scores were calculated by multiplying the expression value for each gene by its hazard coefficient, and summed per sample. Risk scores were ranked and divided into tertiles for log-rank survival analysis. DNA-level piRNA targets were predicted using MiRanda based on sequence complementarity in the region 3.5kb upstream of the transcription-start site of all human transcripts from ENSEMBL. Transcript-level miRNA targets were predicted using the miRDIP algorithm, which integrates 13 miRNA target prediction algorithms and six miRNA prediction databases.

Results:
Only 7 out of 138 piRNAs mapping to the locus were expressed. A combined miRNA/piRNA signature improved both OS and RFS predictions compared to signatures of miRNAs or piRNAs alone. In TCGA, log-rank analysis of risk groups indicated only the miRNA/piRNA signature significantly stratified patients (OS p=0.0038, RFS p=0.0229) into low, intermediate, and high risk groups compared to separated miRNA or piRNA signatures. Similarly, in the BCCA dataset, only the combined miRNA/piRNA signature significantly stratified high, intermediate, and low risk groups (p=0.0019). Target prediction of piRNAs and miRNAs from the signature indicated that 34 genes may be regulated at both the DNA (piRNA) and mRNA (miRNA) level.

Conclusion:
We find the combination of miRNA and piRNA expression derived from the DLK1-DIO3 locus produces a superior stratification of patient outcome than either metric alone. While the contribution of miRNAs to patient risk stratification is established, the improved model performance derived from the addition of piRNAs adds another layer of gene regulation at the DNA-level. Model performance is optimal when these two small RNA species are considered simultaneously; suggesting their coordinated biological effects as a result of deregulation at this locus in LUAD.

Background:
Improved understanding of the molecular mechanisms driving lung cancer progression can lead to novel therapeutic strategies to improve the currently poor patient treatment outcome. Deregulation of microRNA (miRNA) expression in malignant cells activates molecular pathways that drive tumor progression such as epithelial-mesenchymal transition (EMT). We identify miRNA paralogs, miR-106a and miR-106b, to be elevated in metastatic lung adenocarcinoma (LUAD). We assess whether these two highly similar miRNAs share the same functions in vitro, and measure how their elevated expression increases invasiveness or induces EMT in LUAD tumor.

Methods:
MiRNA expression was obtained from small RNA sequencing data derived from clinical primary LUAD specimens and paired non-malignant tissues (60 localized, 27 with lymph node invasion). Non-invasive, epithelial LUAD cell lines with low endogenous miR-106a/b levels were transfected and co-transfected with overexpression vectors for miR-106a and miR-106b. Invasiveness of experimentally-modulated tumor cells was assessed in vitro by Boyden chamber assay and in vivo using a zebrafish model, and expression of EMT markers was determined by Western Blot. Predicted miRNA targets were identified using mirDIP portal. To identify putative genetic mechanisms of mir-106a/b overexpression, DNA copy number, methylation, and Gene Set Enrichment Analysis (GSEA) were performed. Clinical associations were computed in an independent cohort of TCGA LUAD samples.

Results:
Both miR-106 paralogs were significantly overexpressed in LUAD samples with lymph node invasion. However, increased expression of miR-106b alone or together with miR-106a, but not miR-106a alone, enhanced metastatic phenotypes, and correlated with increased mesenchymal and decreased epithelial marker expression. Predicted targets include EP300, a transcriptional activator of E-cadherin, and members of the TGFβ signaling pathway. Copy number and methylation status did not correlate with miRNA expression; however, GSEA analysis revealed enrichment of E2F transcription factor targets in LUAD with high expression of either miR-106 paralogs. Furthermore, expression of miR-106 paralogs was significantly positively correlated with E2F1 and E2F2, suggesting that upstream regulation by E2F is a potential mechanism. Interestingly, miR-106a and miR-106b expression was associated with poor survival and advanced stage when stratified by mesenchymal marker vimentin.

Conclusion:
Although both miR-106a and miR-106b are overexpressed in metastatic LUAD, the strongest prognostic association was found in LUAD with a mesenchymal expression signature and high expression of both miRNAs. Our cell models suggest that miR-106b may play a direct role in EMT, with miR-106a influencing tumor progression via alternative mechanisms. Inhibition of one or both of these miRNAs may provide a strategy for treating advanced stage disease.

Background:
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide; however, science has not yet been able to substantially improve the prognosis of lung cancer patients. Accumulating evidence suggests that microRNAs (miRNAs) are key players in the regulation of tumor development and metastasis. Expression of six miRNAs previously shown to play roles in tumor development (miR-146b, miR-128b, miR-21, miR-221, miR-34a, and Let-7a) in other tumor types

Methods:
Expression of six miRNAs previously shown to play roles in tumor development (miR-146b, miR-128b, miR-21, miR-221, miR-34a, and Let-7a) in other tumor types was examined using real-time RT-PCR in 78 specimens of NSCLC.

Results:
The results revealed that patients with low expression of miR-146b had significant shorter median and mean survival time than those with high miR-146b expression (33.00 and 30.44 months versus 42.0 and 36.90 months, respectively; log-rank test P=0.048), thus low miR-146b expression level was associated with poor prognosis in NSCLC patients. Univariate Cox hazard regression analysis demonstrated that miR-146b expression levels tended to be a significant prognostic indicator of NSCLC (adjusted hazard ratio=0.482, 95% CI: 1.409- 29.593, P=0.016). Multivariate Cox proportional hazard regression analysis showed that miR-146b expression levels were an independent prognostic factor for NSCLC patients (hazard ratio=0.259, 95% CI: 0.083-0.809, P=0.020). Furthermore, the effects of miR-146b on NSCLC cell growth and invasion in vitro were investigated. Our findings demonstrate that ectopic expression of miR-146b suppresses proliferation and colony formation ability of lung cancer H1299 cells in vitro. In addition, miR-146b induced G0/G1 phase arrest in H1299 cells and over-expression of miR-146b inhibited lung cancer cell migration and invasion in vitro.

Conclusion:
Our findings demonstrate that miR-146b functions as a suppressor miRNA and prognosis predictor in NSCLC.

Background:
Sub-clones within a cancer diverge due to ongoing accumulation of mutations. We sought to characterize the intratumor heterogeneity and phylogenetic relationships among different histological patterns present in lung adenocarcinomas based on mass spectrometric analysis of tumor subclones.

Methods:
MALDI-TOF mass spectrometry was used to generate proteomics data from different histological regions of 35 resected lung tumors, as well as from 3 basal cell and 3 mesenchymal cell samples. A total of 1985 different histological regions were analyzed from the 35 resected tumors along with the 3 samples each of airway basal cells and mesenchymal stem cells. For each of the 1991 samples, a spectral profile was generated with expression data from 217 peptide mass peaks to allow comparison of the proteomics profiles from the different histological regions from each cancer to the basal and mesenchymal stem cell profiles. Weighted protein co-expression networks were analyzed by using WGCNA package in R. Global and histologic specific networks were generated through using a power adjacency function which defines the similarity between any pairs of proteins The network modules were decided by using average linkage hierarchical clustering and a dynamic tree-cut algorithm. Networks of the different histologies and normal were compared and visualized by heat map methods.

Results:
The clinically more aggressive histologies ( micropapillary/solid) clustered with stem cells and away from normal alveolar tissue (Fig 1) and had severe loss in peptide connectivities (Fig 3). Applying t-SNE dimensionality reduction method showed that subclones from one specimen cluster differently from each other suggesting underlying heterogeneity, with more heterogenous tumors being associated with worse prognosis (Fig 2). Figure 1



Conclusion:
Construction of a phylogenetic tree of lung ACA subclones oriented to stem cells demonstrated that the degree of disruption of a subclone correlated with the degree of similarity of the subclone to stem cells, and with prognosis.

Background:
Randomised trials have shown treatment with programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) inhibitors can provide a survival benefit to patients with advanced stage non-small cell lung cancer (NSCLC). PD-L1 expression, determined by immunohistochemistry (IHC) using different protocols, antibodies and thresholds for positivity for different inhibitors, has been reported to be potentially predictive of clinical outcome. The objective of this study was to compare the staining patterns of prominent PD-L1 IHC assays in clinically relevant NSCLC samples.

Methods:
Consecutive full sections of 20 NSCLC samples, comprising five each of resection, core biopsy, cytology, and pleural fluid samples, underwent IHC with the following anti-PD-L1 antibodies/autostainers: 22C3/Link 48, 28-8/Bondmax, SP142/Bondmax, SP263/Benchmark XT, E1L3N/Benchmark XT according to publicly-available protocols. PD-L1 expression were scored manually by pathologists according to the percentage of tumour cells (%TC) stained on a continuous scale.

Results:
Using published tumour cell percentage thresholds for 22C3, 28-8, SP142 and SP263 of ≥50%, ≥1%, ≥5%, and ≥25%, the frequency of PD-L1 positive cases were 10%, 15%, 70%, and 15% of cases respectively. When a ≥1% threshold was applied, the corresponding frequencies were 70%, 15%, 95%, 65% respectively, and 55% for E1L3N. Using published thresholds, cases were positive according to 1, 2, 3, 4 and 5 antibodies in 15%, 25%, 25%, 0% and 5% of cases respectively. Sorting of cases according to increasing %TC staining revealed a similar order of cases between antibodies, albeit with differences in %TC quanta and occasional exceptions to the order. Spearman rho analysis indicated %TC staining significantly (p<0.05) correlated between most antibody pairs, except 28-8 and 22C3, 28-8 and SP142, and 28-8 and E1L3N. Unsupervised hierarchical clustering revealed two subgroups, comprising of SP142/SP263 and 22C3/28-8/E1L3N.

Conclusion:
The classification of cases as PD-L1 positive can vary significantly according to the antibody and protocol used. Differences were more likely due to protocol dependent staining intensities and nominated thresholds for positivity, rather than differences in antibody affinity for different epitopes.

Background:
Glucose transporter type 1 (GLUT1) and carbonic anhydrase IX (CAIX) represent glucose metabolism and tissue hypoxia, respectively. The purpose of this study is to measure the GLUT1 and CAIX expression and volume-based 18F-fluorodeoxyglucose positron emission tomography (FDG PET) parameters in non-small cell lung cancer (NSCLC) patients and examined the correlations between above parameters and their prognostic significance.

Methods:
We evaluated GLUT1 and CAIX expression by immunohistochemical methods and volume-based FDG PET parameters in 269 NSCLC patients treated with surgical resection (158 adenocarcinoma, 93 squamous cell carcinoma and 18 other type carcinoma). Metabolic tumor volume (MTV) and total lesion glycolysis (TLG) of NSCLC were measured using pretreatment 18F-FDG PET/CT. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off values of MTV or TLG. Correlations between GLUT1, CAIX, MTV, TLG, and clinicopathological factors were assessed, and prognostic significance was determined.

Results:
The GLUT1 expression was identified in 99% of squamous cell carcinoma and 50% of adenocarcinoma. In patients with adenocarcinoma, GLUT1 expression showed positive correlation with MTV or TLG (P = 0.012 and P = 0.037, respectively). In patients with squamous cell carcinoma, correlation analyses between GLUT1, MTV and TLG were not performed because most cases of squamous cell carcinoma showed GLUT1 positivity. There was no correlation between CAIX expression, MTV and TLG in squamous cell carcinoma and adenocarcinoma. In cases with adenocarcinoma, GLUT1-positive patients had lower overall survival (OS) rates and lower recur-free survival (RFS) rates than GLUT1-negative patients (P < 0.001 and P = 0.004, respectively). CAIX expression was not significantly associated with either OS or RFS in squamous cell carcinoma and adenocarcinoma. Patients with high MTV or TLG had significantly lower OS rates and lower RFS rates than patients with low MTV or TLG in adenocarcinoma. High GLUT1, TLG and MTV were significantly associated with adverse clinicopathologic variables. When patients with adenocarcinoma were stratified into two groups (High GLUT1 and TLG vs. the other 3 groups), high GLUT1 and TLG was an independent prognostic marker for OS in multivariate analysis (P = 0.003).

Conclusion:
Our results show that high GLUT1 expression levels were significantly associated with MTV or TLG in patients with adenocarcinoma. High GLUT1 and TLG was an independent prognostic marker for OS. High GLUT1 and TLG can be used to identify a subgroup of patients with adenocarcinoma at high risk of recurrence or progression who may benefit from aggressive chemotherapy or radiotherapy.

Background:
Compared with non-smoking counterparts, smoking-associated lung cancers is characterized by a high frequency of somatic mutations, including mutations of DNA repair genes, a high burden of neoantigens, and increased immunogenicity. B7-H3 (also known as CD276) belongs to a family of immune modulators that includes PD-1 and PD-L1 (also known as B7-H1 or CD274). Considering the better response to PD-L1 inhibitors in smokers’ lung cancer, we examined the prognostic interaction of tumor B7-H3 expression with smoking history in lung adenocarcinoma patients.

Methods:
Using tissue microarrays of consecutive 270 cases of lung adenocarcinoma, we evaluated tumor B7-H3 expression by immunohistochemistry. We examined the prognostic association of B7-H3 expression in strata of smoking history, using Cox proportional hazards regression analysis and the log-rank test. Additionally, we used logistic regression analysis to examine the correlations between B7-H3 expression levels and clinicopathological/molecular features of lung adenocarcinoma.

Results:
The association of B7-H3 expression with survival significantly indeed differed by smoking history (Pinteraction=0.014); B7-H3 high-expression was associated with inferior survival in moderate/heavy-smoking patients (smoking index [SI]≥400) (hazard ratio [HR]=3.07, 95% confidence interval [CI]=1.74-5.49, P=0.0001) (log-rank test: P<0.0001), but not in non/light-smoking patients (SI<400) (HR=2.24, 95% CI=0.63-1.96, P=0.64) (log-rank: P=0.64). High B7-H3 expression was associated with smoking patients (univariable odds ratio [OR]=2.63, 95 % CI=1.51-4.65, P=0.0005) and independently associated with EGFR wild-type status (multivariable OR=2.80, 95% CI=1.38-5.84, P=0.0042).

Conclusion:
We demonstrated that the prognostic association of B7-H3 expression indeed differed according to smoking history. The current study also showed significant association of high B7-H3 expression with EGFR wild-type and smoking patients, indicating the potential effectiveness of anti-B7-H3 therapy for EGFR wild-type or smokers’ lung adenocarcinoma.

Background:
Stathmin 1 is a cytosolic phosphoprotein that plays a crucial role in the control of cellular division and proliferation by regulating microtubule dynamics. In addition, Stathmin1 is associated with tumor growth and progression. Our study aimed to determine differences in overall (OS) and disease free survival (DFS) in patients with non-small lung cancer (NSCLC) stratified by STMN1 tumor expression.

Methods:
Using inmmunohistochemistry, Stathmin1 expression was determined in resection specimens from 423 NSCLC patients. The relationship between Stathmin1 protein expression and overall and disease free survivalwas assessed using Kaplan Meier survival curves and compared using log-rank statistics. Cox proportional hazards regression determined the hazard for death stratified by Stathmin1, adjusting for clnicopathological characteristics.

Results:
The STMN1 protein was expressed in 58% of NSCLC cases, 54% of Adenocarcinoma, 63% of Squamous cell carcinoma, and 100% of large cell neuroendocrine carcinoma (LCNEC) and its expression was significantly associated with advanced T and N factors, advanced pathological stages, positive lymphatic permeation, positive vascular invasion, and poor or mediate differentiation. STMN1 was a negative prognostic factor for OS (hazard ratio [HR], 2.212; 95% confidence interval [CI]: 1.544-3.169; p< 0.001) and DFS (HR, 2.685; 95% CI: 1.897-3.798; p< 0.001). Multivariable analysis confirmed that STMN1 protein expression was an independent predictor of an unfavorable OS (HR, 1.480; 95% CI, 1.006 to 2.176; p = 0.046) and DFS (HR, 1.605; 95% CI, 1.105 to 2.329; p = 0.013) in all NSCLC patients, and of an unfavorable DFS (HR: 2.128, 95% CI, 1.293 to 3.503; p = 0.003) in Stage I NSCLC patients.

Conclusion:
STMN1 expression was an independent prognostic factor for NSCLC, even when restricted to early stage patients.

Background:
In the absence of a targetable mutation, cisplatin based chemotherapy is the backbone of NSCLC treatment. However, a diverse patient population combined with complex tumour heterogeneity is hampering its’ clinical utility. Although intrinsic and acquired resistance to cisplatin is common, the mechanisms have not yet been fully elucidated. However, some studies have suggested that inflammatory pathways may play a key role in chemo-resistance. The aim of this project is to increase our understanding of inflammatory mediated cisplatin resistance in NSCLC.

Methods:
A number of isogenic cell line models of NSCLC (adenocarcinoma, squamous cell carcinoma, large cell carcinoma) cisplatin resistance were utilised to assess the role of inflammation in chemo-resistance. These included a sensitive parental cell line (PT) and a matched resistant subtype (CisR). The cell lines were screened for NFKB and a number of inflammatory mediators including chemokines and TLRs at the mRNA (RT-PCR/qPCR) and protein level (Western Blot/ELISA). A specific NFKB inhibitor, DHMEQ, and recombinant chemokines were employed to further characterise inflammatory pathways in PT and CisR cells in terms of cisplatin sensitivity, proliferation (BrdU ELISA), cellular viability (Cytell Cell Imaging System) and DNA damage response (Comet). An in vivo study was also completed using DHMEQ alone and in combination with cisplatin.

Results:
A number of NFKB targets and responsive pathways are deregulated in CisR cells compared with their matched sensitive PT cell line. Amongst others, CCL2 and CCL5 were altered across all NSCLC subtypes. Preliminary data suggests that DHMEQ enhances cisplatin sensitivity in both PT and CisR cells, conversely recombinant chemokines elicit a protective effect. Additionally, DHMEQ treatment resulted in opposite affects on CCL2 and CCL5 mRNA levels in the PT and CisR cell lines. This may reflect an alternative pathway hierarchy within the cells. Further characterisation is ongoing assessing chemokine specific inhibitors. Although, in vivo data suggests a trend of decreased tumour growth in the DHMEQ cohorts compared with vehicle control, the data was not significant. However, tumour samples appeared more necrotic with DHMEQ and are currently being characterised using IHC for necrosis and proliferation.

Conclusion:
Targeting chemokines downstream of NFKB may provide a means to overcome inflammatory mediated acquired and intrinsic NSCLC chemo-resistance. Given the increased significance of immuno-oncology agents to harness the body’s own immune system in the fight against cancer, these agents may also prove fruitful in re-sensitising patients to chemotherapy.

Background:
Ribosome is a subcellular organelle, which serves as the site of biological protein synthesis. Ribosomal protein L29 (RPL29) is one of the proteins composing ribosome, and it expresses in cell surface as well as in cytoplasm, especially showing its high expression in cancer cells.

Methods:
We retrospectively reviewed 92 patients who underwent surgical resection for non-small cell lung cancer between June 2010 and January 2012. Preoperative serum anti-RPL29 levels were measured by the indirect enzyme-linked immunosorbent assay. The cut-off value was represented by the median of anti-RPL29 levels.

Results:
The patients consisted of 60 males and 32 females, and their average age was 68.7 years (range: 44-87 years). Adenocarcinoma was the most common subtype (n = 69), which was followed by squamous cell carcinoma (n = 13), adenosquamous cell carcinoma (n = 4), pleomorphic carcinoma (n =4), and large cell carcinoma (n = 2). Postoperative pathological stage consisted of stage IA (n = 28), IB (n = 28), IIA (n = 11), IIB (n = 2), and IIIA (n = 23). EGFR activating mutations were found in 35 patients (32 adenocarcinomas, 2 adenosquamous cell carcinomas, and 1 pleomorphic carcinoma). The median of anti-RPL29 levels in 92 cases was 0.351. Three-year and five-year overall survival rate was 62.7% and 56.6%, respectively, in the patients whose serum anti-RPL29 level was less than the median, and 90.0% and 83.7%, respectively, in the patients with the median or more of anti-RPL29 levels (P = 0.005). In the multivariate Cox proportional hazard model, anti-RPL29 level being the median or more and pathological stage IA were identified as independent prognostic factors (P = 0.013 and P = 0.017, respectively).

Conclusion:
Serum anti-RPL29 levels may be a novel prognostic marker for non-small cell lung cancer.

Background:
radiation-induced lung toxicity (RILT) are observed today in patients who have undergone thoracic irradiation for the treatment of lung malignancy. Radiation-induced damage to normal lung parenchyma remains the dose-limiting factor in chest radiotherapy. However, the radiative dosage is not effective for most of patients because of avoiding RILT. Therefore, novel diagnosis methods reveal individual potential RILT are required. For now, increasing evidence illustrates that exosomes in circulating fluids provide a promising way as biomarkers for noninvasive disease diagnosis. Exosomes are 30–150 nm particles which are released from cells into the extracellular environment and thousands of proteins have been identified in plasma exosomes. Whether exosomal proteomics analysis could benefit lung cancer patients with appropriate radiative dosage and prevent RILT remains to be studied.

Methods:
Plasma samples were collected from RILT patients with grade I and II, and no RILT individuals matched with age, gender and blood collection time after 10 to 30Gy radiation within 6 months. Plasma exosomes were accessed by 110,000×g ultracentrifugation and visualized by NS300 equipment. The raw data of exosomal proteomics profiles of RILT patients and no-RILT individuals were generated by LC-MS and its expression were verified by western blot.

Results:
In the present study, we revealed 17 exosomal protein participated in wounding response and two of them were correlated with RILT clinic stage. A2M (Alpha-2-macroglobulin) was decreased in RILT patients and FGB (Fibrinogen beta chain) was increased in RILT patients. Furthermore, A2M was decreasing from no radiative damage patients to that of RILT grade I to II, and the FGB expression in exosomes showed positive correlation with RILT from low to high level. The patients with low FGB expression in plasma exosomes could tolerate higher radiative dosage until the FGB was upregulated in plasma.

Conclusion:
LC-MS is an efficient method for exosomal proteomics analysis and we reveal two stable targets A2M and FGB, which could indicate the potential of patients suffering RILT after radiotherapy. The two novel targets could serve as promising diagnosis biomarkers for avoiding RILT.

Background:
Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor with a short survival time arising from the mesothelial cells of the pleura. MPM is mainly associated with asbestos exposure and a strong inflammatory reaction. The common treatment of MPM combines macroscopic complete resection and adjuvant or neoadjuvant chemotherapy, respectively. Soluble mesothelin and osteopontin are current available biomarker for malignant mesothelioma with moderate sensitivity and specificity. Glycodelin is an immune system modulator well described during pregnancy. It is involved in invasion of the trophoblast and in regulation of the immunotolerance between the maternal immune system and the fetus.

Methods:
With a commercial ELISA, we measured the glycodelin serum concentrations of patients with MPM. In addition, we analyzed the glycodelin gene expression using quantitative PCR and stained glycodelin in formalin-fixed paraffin embedded tissue slides.

Results:
We found high glycodelin concentrations in the serum of patients with MPM compared to benign lung diseases. Patients with high glycodelin serum concentrations exhibited a worse overall survival. Moreover, glycodelin serum levels correlated with tumor response to treatment. A comparison of soluble mesothelin-related proteins (SMRP) and glycodelin in the serum of a large patient cohort demonstrated that the detection of both soluble factors can increase the reliable diagnostic of MPM. Glycodelin mRNA and protein was highly expressed in MPM tumors compared to normal lung tissue.

Conclusion:
In this study, we first described the expression of glycodelin in MPM. Altogether, glycodelin seems to be a new potential serum biomarker for the aggressive malignant pleural mesothelioma.

Background:
The adoption of next-generation sequencing (NGS) may help to identify single nucleotide variants (SNVs), small insertions–deletions (indels), and larger structural variations including chromosomal rearrangements. Many molecular alterations have protein-level associations that can be questioned using immunohistochemistry (IHC). The goal of our work was investigated molecular patterns of predictive biomarkers and new genes involved as potential therapeutic targets with an emphasis on protein IHC and their translational promise.

Methods:
We studied 212 formalin fixed and paraffin embedded tissues: 8 high-grade and 20 low-grade neuroendocrine carcinomas(NEC), 102 adenocarcinomas(ADC), 65 squamous cell carcinomas (SCC) and 17 large cell carcinomas(LCC), placed in tissue microarrays(TMAs). EGFR,P53,KRAS,ALK,ERBB2,PTEN,BRAF,VEGF,CD24 and CD44 were examined using IHC and Aperio system. DNA extracted(QIAmp) from a subset was used to analyze several variants including EGFR, ERBB2, PIK3CA, MMP2, SNAI, VGFA, VIM, ZEB1, AXL, CD44, CD276, and CDH1, using the TruSeq Custom Amplicon assay on the Illumina MiSeq System, resulting data set for 80 LC were analyzed using the Variant-Studio software and correlated them with the clinocopathological data.

Results:
The median age of the patients was 64 yrs (minimum-maximum, 24-88yrs). The population included 98(44.5%) women; 32(14,5%) never smokers and 100(45,5%) former smokers; only 2(1%) Asians. Our image analysis showed that the median IHC protein expressions were similar between low and high-grade NEC, but those were different compared to others (P<0.05). Indeed, EGFR, EBB2, P53 and BRAF IHC expression were significantly lower in NEC group compared to other subtypes (P<0.05). Overall, LCC have lower protein expression than ADC and SCC, specially P53 and VEFG. We detected several drivers mutation including EGFR 22%(19/80), ERBB2 2%(5/80), immune regulated genes CD276 6.8%(17/80) and CTLA 15.4%(39/80). We observed also EMT gene mutations as CD44 30.7%(78/80), MMP2 2.8%(7/80), VGFA 2%(5/80), CDH1 2.4%(6/80), SNAI 2.8%(7/80), VIM 1.2%(3/80), and ZEB1 4%(12/80). Interestingly, a significant higher AXL and CD44 gene expression was found in ADC and SCC specimens compared to NEC(P=0.001 and P=0.04,respectively) Similar to the protein expression in overall low gene expressions was also observed in LCC compared to others.

Conclusion:
We detected different patterns of protein and gene alteration in LC with predominant low expression in NEC. Furthermore, the high expression of EMT genes as AXL and CD44 observed among ADC and SCC can be a evidence that those genes might be a distinctive RTK in these tumor than in NEC tumor suggesting that targeting these genes will be benefit as anti-cancer treatment.

Background:
Lung cancer (LC) is the leading cancer-related cause of death worldwide with a 5-year survival rate of only 8%. Smoking is the main risk factor for lung malignancies, but not every smoker develops LC. DNA methylation alterations in tumor tissue lead to genome instability and thus can contribute to malignant cell transformation. Smoking-associated blood DNA methylation changes have been shown to indicate LC risk. We aimed to investigate differential blood DNA methylation in healthy smokers for the identification of novel oncogenes.

Methods:
We performed Illumina 450K epigenome-wide DNA methylation arrays for 66 smoking prediagnostic lung cancer case-control pairs. The blood DNA samples for this nested case-control design came from the European Prospective Investigation into Cancer and Nutrition (EPIC) Heidelberg cohort. Differentially methylated candidate CpGs were then tested in lung tumor versus adjacent normal tissue for differential methylation in multiple patient sample sets. Additionally, we tested whether differential methylation leads to differential gene expression in lung tumor versus adjacent normal tissue. The top candidate CpG proximal gene was evaluated in functional LC cell based assays to investigate the consequences of its elevated expression in LC.

Results:
The top differentially methylated candidate CpGs from EPIC were also found differentially methylated in lung tumor versus adjacent normal tissue. The two top hypermethylated CpGs were located within differentially methylated regions (DMRs) and the proximal or associated genes were differentially expressed in lung tumors. The top DMR revealed the regulation of gene expression by DNA methylation of a downstream ubiquitin E3 ligase. Deregulated expression of that gene was associated with LC cell proliferation, migration and glucose uptake in vitro. The gene was found to be involved in the activation of AKT by mTORC2. When the gene was knocked down, apoptotic genes which are suppressed by activated AKT in LC cells were re-expressed. Expression of a cell cycle promoting regulator stimulated by activated AKT was found repressed in LC knock down cells.

Conclusion:
We identified differential DNA methylation in blood from healthy smokers who later developed LC. The top differentially methylated CpGs were also differentially methylated in lung tumor versus adjacent normal tissue samples. The top DMR lead to expression of a proximal ubiquitin E3 ligase. We showed, that this gene is crucial for the mTORC2-mediated activation of AKT. This ubiquitin E3 ligase has not previously been associated with cancer but our findings identify it as a novel epigenetically deregulated LC oncogene.

Background:
Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease associated with an up to 10-fold increased risk of lung cancer (LC). COPD and LC share common etiologies including genetic susceptibilities and risk factors, such as smoking. This study systematically characterizes the molecular overlap between COPD and LC.

Methods:
Small airway gene expression data was obtained from subjects with spirometry measures (n=267) (GSE37147). Genome-wide, multi-omics data for lung adenocarcinoma (LUAD) tumor and non-malignant lung tissues from two cohorts (TCGA, n=515; BCCA, n=90) was analyzed. Weighted correlation network analysis (WGCNA) was applied to identify clusters (modules) of highly correlated genes across airway expression profiles. Combined module expression (eigengene scores) were used to: 1) identify modules negatively associated with FEV~1~ and 2) calculate module preservation in lung tumors. Signaling network, pathway and gene ontology analyses were performed using IID, pathDIP, ClueGo and PARADIGM. Known and predicted protein-protein physical interactions (PPIs) were obtained from IID. Network analysis and visualization was performed in NAViGaTOR.

Results:
A module of 31 genes significantly co-expressed across small airways was negatively associated with FEV~1~ and preserved in LUAD tumors. Genes in this module were enriched in functions associated with cell cycle progression, and known and/or predicted to physically interact in the protein complex critical to mediating G2/M progression. The forkhead transcription factor FOXM1 network was the most highly perturbed entity across 515 LUAD tumors. FOXM1 is an essential mitotic protein, known to regulate expression of genes involved in cell cycle progression, as well as stress response to ROS and DNA damage, angiogenesis and metastasis. COPD-related airway mRNA changes and genes highly altered at the DNA and mRNA level in LUAD tumors directly converge on the FOXM1 regulated mitotic complex proteins and/or FOXM1 transcription factor network.

Conclusion:
FOXM1 is overexpressed in multiple cancer types where it is correlated with poor prognosis and oncogenic transformation of epithelia through induction of genomic instability. The convergence of COPD and LUAD changes on this network may underlie increased LC risk in COPD patients, warranting further exploration as a target for COPD treatment and/or LC prevention or treatment.

Background:
Non-diagnostic bronchoscopic or image guided biopsies of small, solitary pulmonary nodules are a common and frustrating clinical conundrum which can cause thoracic oncologists and patients to opt for radical therapy without a pathological diagnosis. As a result, some patients with benign conditions have unnecessarily undergone radical treatment. This study sought to determine if metabolomics profiling of plasma could be used to distinguish early stage NSCLC cases from cancer-free controls. Furthermore, we sought to determine if phenotypic subtypes of NSCLC could be distinguished from one another using metabolomics profiling.

Methods:
Frozen preoperative plasma samples from a cohort of 48 early stage NSCLC patients (24 Adenocarcinomas, 24 Squamous Cell Carcinomas) and 24 cancer-free controls obtained prior to surgical resection were randomly selected from a provincial lung cancer biorepository for metabolomics analysis. Plasma samples were uniformly thawed, extracted, and analyzed in triplicate by blinded personnel using ultra high performance liquid chromatography/quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). After data mining, metabolomics profiles were quantified and normalized using Mass Profiler Professional (Agilent Technologies, CA, USA) and individual metabolites were identified using the Metlin Database. Partial least square discrimination (PLSD) was used as a prediction model to identify metabolomics profiles which effectively clustered NSCLC cases from Controls and Adenocarcinomas from Squamous Cell Carcinomas.

Results:
More than 17,500 entities were detected using the UHPLC-QTOF-MS technique of which 250 potential metabolomics biomarkers were present in all 72 samples. The PLSD analysis using all detected entities effectively distinguished NSCLC cases from controls with 97% overall accuracy. Several endogenous metabolites were statistically significantly affected in Adenocarcinoma and Squamous Cell Carcinomas cases compared to control samples. Some of the identified compounds include biliverdin IX, serotonin, PE(15:0/20:2) and 3-ketosphingasine. In addition, 3-acetamidopropanal, 9,10-dihydroxy-hexadecanoic acid and anandamide (20:5, n-3) were found in high concentrations in Adenocarcinoma cases compared to Squamous Cell Carcinomas.

Conclusion:
Differences in the metabolomics profiles were apparent and demonstrated the preliminary feasibility of distinguishing early stage NSCLC cases from controls and adenocarcinomas from squamous cell carcinomas using metabolomics techniques. Validation of this methodology on a larger, prospectively accrued, cohort is planned in order to optimize model fit and to assess the diagnostic and NSCLC subtype discriminatory performance in the clinical setting.

Background:
We have investigated the correlation between claudin (CLDN) protein expression and clinicopathological parameters as well as survival in histological subtypes of non-small cell lung cancer.

Methods:
137 pathologic stage I primary bronchial cancers including 49 adenocarcinomas of non-lepidic variants (ADC), 46 adenocarcinomas of lepidic variants (L-ADC), and 42 squamous cell carcinomas (SCC) were examined. Immunohistochemistry (IHC) using antibodies against CLDN1,-2,-3,-4,-7 proteins as well as semiquantitative estimation (IHC scores 0-5) were performed on archived surgical resection specimens.

Results:
Claudin IHC scores of L-ADC differed significantly from ADC (CLDN1: p=0.009, CLDN2: p=0.005, CLDN3: p=0.004, CLDN4: p=0.001, CLDN7: p<0.001, respectively) and SCC (CLDN1: p<0.001, CLDN3: p<0.001, CLDN7: p<0.001, respectively). Highly significant CLDN3-CLDN4 parallel expression could be demonstrated in ADC and L-ADC (p<0.001 in both), which was not observed in SCC (p=0.131). ADC and SCC showed no correlation with smoking, whereas in case of L-ADC heavier smoking correlated with higher CLDN3 expression (p=0.020). Regarding claudin expression and survival, in SCC significant correlation could be demonstrated between CLDN1 IHC positivity and better survival (p=0.038). In NSCLC as a whole, high CLDN2 expression proved to be a better prognostic factor when compared with cases where CLDN2 IHC score was 0-1 vs. 2-5 (p=0.009), however, when analyzed separately, none of the histological subgroups showed correlation between CLDN2 expression and overall survival.

Conclusion:
Our results demonstrated significant claudin expression differences not only between the SCC–ADC and SCC–L-ADC but also between the L-ADC and ADC histological subgroups, which strongly underlines that L-ADC represents a distinct entity within the ADC group. CLDN1 overexpression is a good prognostic factor in NSCLC, but only in the SCC subgroup.

Background:
CXC group chemokine receptors and ligands are well known for their role in immune response, regulation of angiogenesis, tumour development and growth. Understanding of lung cancer pathogenesis requires comprehensive analysis of cell interaction in tumour microenvironment formed by malignant cells, stromal cells and immune cell infiltrate. CXC chemokine receptor 3 (CXCR3) and ELR motif negative CXC chemokine ligands 4, 9, 10 and 11 (CXCL4, CXCL9, CXCL10 and CXCL11) form an axis which is part of complex tumorigenesis process.

Methods:
The study recruited 38 patients with NSCLC ranging from stage IA to IIA undergoing anatomical pulmonary resection between January 2011 and January 2012. Patients were followed to assess relapse rate and survival. CXCR3 expression and tumour infiltrating immune cells (neutrophils, T helper cells - CD4, cytotoxic T lymphocytes - CD8, B cells - CD20, macrophages - CD68 and plasma cells - CD138) were evaluated in resected tumour specimens by immunohistochemistry. For CXCR3 basic annotation parameters included an evaluation of staining intensity (negative, weak, moderate or strong) and fraction of stained cells (rare, <25%, 25-75% or >75%). Blood samples from peripheral vein and from pulmonary vein draining tumour vascular bed were collected at the time of surgery. Levels of CXCL4, CXCL9, CXCL10 and CXCL11 were measured using ELISA and CXCL gradient was calculated. Pearson test was used to assess statistical relationship between CXCL levels, CXCR expression and immune cell infiltrate.

Results:
Majority of tumour specimens despite heterogeneity showed strong CXCR3 expression which was equally intense in tumour cells and stroma. Correlation between tumoral and stromal expression was very strong (r = 0.86, p < 0.001). Tumoral expression of CXCR3 correlated with total number of tumour infiltrating immune cells (r = -0.58, p < 0.01), number of T helper cells (r = -0.5, p = 0.01) and T cytotoxic cells (r = -0.4, p < 0.05). Stromal CXCR3 expression had similar correlation with aforementioned parameters but also included correlation with number of B cells in infiltrate (r = -0.45, p = 0.01). CXCL10 and CXCL11 gradients correlated with stromal CXCR3 expression (r = 0.42, p < 0.05 and r = -0.42, p < 0.05). Moderate statistically significant correlation was found between CXCL4 and CXCL10 gradients and relapse (r=0.39, r=0.35, p<0.05).

Conclusion:
CXCR3 and ELR motif negative CXC chemokine axis plays role in lung cancer pathogenesis and needs further evaluation for better understanding of tumour immunology.

Background:
Tumor heterogeneity is a major impediment to targeted treatment response in a variety of cancers, including lung cancer, the commonest cause of cancer death. However, the extent of heterogeneity at the genomic and proteomic level along with its effects on treatment response may be patient-specific.

Methods:
We undertook comprehensive whole genome, exome or targeted sequencing, together with mass spectrometry-based proteomics analyses on twelve sequentially procured lung and lymph node metastatic sites and normal blood from an African American never-smoker lung adenocarcinoma patient who had survived with metastatic disease for over seven years while being treated with single or combination ERBB2-directed therapies.

Results:
Surprisingly, only 1% of somatic variants were common between the two sites, as revealed by WGS. Interestingly, one novel somatic translocation, PLAG1-ACTA2 was identified in both sites resulting in overexpression of ACTA2 that may have been the driver of early metastasis in this patient. The likely predominant driver of proliferation, ERBB2, was focally amplified along with CDK12, greater in the lung compared to the lymph nodes. However, an ERBB2 L869R mutation was specific to the lymph node. We also discovered a novel CDK12 G879V mutation that was specific to the lung. Isogenic MCF10A cells expressing ERBB2 L869R were more proliferative than those expressing wild type ERBB2. Cells expressing ERBB2 L869R that developed lapatinib resistance showed a mesenchymal phenotype, increased migration, and produced significantly more lung metastases than lapatinib-sensitive ERBB2 wild-type cells in a tail-vein injection assay, implicating this mutation in repeated progression of lymph node metastases. The CDK12 mutation is expected to have resulted in a non-functional kinase, lower expression of DNA damage response genes, greater instability of the lung tumor genome, and increased sensitivity to chemotherapy. Accordingly, there was no metastatic sites evident at autopsy in the lung, suggesting the lung metastatic sites were essentially cured. We further sought to correlate the genomic heterogeneity with alterations in the proteome and phosphoproteome using high-resolution mass spectrometry. For this purpose, we first assembled patient-specific database including all somatic variants, as revealed by WGS, from the lung and lymph node to interrogate the mass spectrometry data. Several aspects of the genomic heterogeneity were evident at the protein-level. These include the identification of the mutant CDK12 G879V peptide and higher expression of ERBB2 in the lung.

Conclusion:
The integrated proteo-genomics analyses reveal unprecedented tumor heterogeneity in a patient with lung adenocarcinoma. However, similarities in key tumor driver pathways remain.

Background:
The differential diagnosis of tuberculous and malignant pleural effusion (PE) is extremely difficult and continues to pose clinical challenges. we aimed to evaluate the utility of pleural fluid Interferon gamma (IFN- γ), Adenosine deaminase (ADA), Tumar necrosis factor-alpha (TNF-a) levels with T cells subsets in differential diagnosis of malignant (MPE) and tuberculous pleural effusions (TPE).

Methods:
Forty patients with pleural effusion (20 tuberculous and 20 malignant) were included in the study. The percentages of CD3 ‏lymphocyte, CD4‏ ‏lymphocyte and Treg (CD4‏ CD25‏‏) T cells in pleural effusion from patients with tuberculous and malignant pleurisy were determined by flow cytometry. The concentrations of TNF-a, IFN-γ and ADA were simultaneously determined in pleural fluids by enzyme linked immunosorbent assay and colorimetric method.

Results:
The ADA activity, TNF-a and IFN-g concentration were significantly higher in tuberculous than MPE (84.22±41.47 vs. 23.19±17.93 U/l : P<0.0001, 122.45±47.69 vs. 35.03±31.88 pg/ml : P < 0.0001 and 2.26±1.62 vs. 0.3±0.20 IU/ml : P<0.0001 respectively ). T-cells subsets (CD3‏ T-cells, CD4 T-cells and T reg cells) were significantly higher in TPE than MPE (76.46% vs. 65.29%; P 0.004, 51.21% vs. 43.50%; P 0.044 and 14.60% vs. 12.43%; P 0.032 respectively). CD3 plus CD4 as well as CD3 plus CD4 plus T reg combinations were all 100% specific for discriminating TPE from MPE. TNF-α plus IFN-γ, TNF-α plus ADA, as well as TNF-α plus IFN-γ plus ADA, were 100% specific for discriminating TPE from MPE. Furthermore, the specificity of combined-diagnostic value of TNF-α, IFN-γ, ADA with T cells subsets was > 95 %.

Conclusion:
The combinations of pleural fluid IFN- γ, ADA, TNF-a levels and T cells subsets could effectively address the challenge of distinguishing tuberculous pleural effusion from malignant pleural effusion.

Background:
PD-L1 agents have shown clinical efficacy. Recent reports have demonstrated the predictive value of PD-L1 immunohistochemistry (IHC) assessment from immune (IC) and tumor cells (TC) in non-small cell lung carcinoma (NSCLC). While assessing percent staining of TC is a task familiar to pathologists, assessment of IC is novel and possibly challenging. As noted in other studies, digital pathology (DP) with image analysis (IA) has the potential to reduce inter-reader (IR) variation in specific situations. However, the impact of DP IA on IR variation in the setting of PD-L1 IHC scoring is unknown as are the effects of different IA approaches (field-of-view [FOV] vs whole tumor [WT]).

Methods:
A cohort of 60 NSCLC formalin-fixed paraffin-embedded tissue samples was stained with PD-L1 IHC (SP142). Three pathologists underwent training for IHC-based manual assay and DP IA interpretation. Three scorers independently and blindly scored each case using digital read (DR, no IA but digital assessment on computer monitor), FOV IA, and WT IA. Data was analyzed using pair-wise overall percent agreement rates (OPA) derived from assay threshold categorical bins.

Results:
For IC scoring, WT IA significantly improved IR agreement and reproducibility rates as compared to DR and FOV-based approaches (table 1). TC WT IA also showed similar improvements.

	TC-DR (%)	TC-FOV (%)	TC-WTA (%)	IC-DR (%)	IC-FOV (%)	IC-WTA (%)
R1-R2	83.1 (71.5-90.5)	89.8 (79.5-95.3)	98.3 (91.0-99.7)	89.1 (77.0-95.3)	91.5 (81.6-96.3)	100.0 (93.9-100.0)
R2-R3	94.5 (85.1-98.1)	87.3 (76.0-93.7)	100.0 (93.5-100.0)	76.7 (62.3-86.8)	88.4 (75.5-94.9)	100.0 (93.5-100.0)
R1-R3	82.1 (70.2-90.0)	85.7 (74.3-92.6)	98.2 (90.6-99.7)	83.9 (72.2-91.3)	95.5 (84.9-98.7)	100.0 (93.6-100.0)

Table 1: NSCLC IR OPA rates. PD-L1 IHC scoring threshold 1% (TC1/IC1), R = Reader (95% confidence interval)

Conclusion:
Compared to DR and FOV IA, WT IA significantly improved pathologists’ PD-L1 IR rates for TC and IC scoring in NSCLC samples. Further studies regarding accuracy and reproducibility are being performed in a larger cohort.

Background:
Nivolumab is the first checkpoint inhibitor approved for the treatment of Squamous (Sq) and non-Squamous (non-Sq) metastatic NSCLC in second-line setting, showing a survival benefit in randomized phase III trials. The aim of this study is to investigate the potential role of baseline peripheral blood cells in relation to clinical outcomes following nivolumab treatment.

Methods:
From November 2015 to to June 2016, we evaluated 45 patients with Sq (n = 10) and non-Sq (n = 35) NSCLC, previously treated with first-line platinum-based chemotherapy, that received nivolumab 3 mg/kg IV on day 1 of each 2 week treatment cycle. Clinical characteristics (T-Stage, lymph nodes involvement, M-Stage) were assessed. Total numbers of leukocytes, myeloid-derived suppressor cells (MDSCs), including both monocytic (Mo-MDSC) and granulocytic (Gr-MDSC) type, regulatory T cells (T-regs), and serum lactate dehydrogenase (LDH) were assessed. Endpoints were correlation with objective response (OR), progression-free survival (PFS, categorized as ≤ 3 or > 3 months) and overall survival (OS). Tumor response was assessed using RECIST criteria, version 1.1, at week 9 and every 6 weeks thereafter until disease progression. Statistical methods were based on univariate analyses (Wilcoxon rank test).

Results:
The median PFS of the overall study population was 3 months. Data about Gr-MDSCs (identified by flow cytometry as Lin-CD15+CD14-CD11b+HLA-DR[low/-]), Mo-MDSCs (Lin-CD14-CD11b+HLA-DR[low/-]) and absolute eosinophil counts (AEC) were available in 37/45 patients (82%) of treated patients. Baseline absolute numbers of Gr-MDSCs, Mo-MDSCs and AEC were greater in patients with good prognosis (PFS > 3 months) and better outcomes. In particular among patients with shorter PFS, the median numbers of Gr-MDSCs, Mo-MDSCs and AEC were significantly lower than those detected in patients with longer PFS (4 vs 13 cell/µl, p=0.01; 4 vs 21 cell/µl, p=0.06; 55 vs 155 cell/µl; p=0.02, respectively). No correlation was observed between T-regs, LDH, absolute neutrophil, monocyte, lymphocyte counts and clinical outcomes.

Conclusion:
A baseline blood signature characterized by low levels of Gr-MDSCs, Mo-MDSCs and AEC is associated with poor outcome (median PFS ≤ of 3 months) in 67.6% of patients treated with nivolumab. In contrast, patients (32.4%) with high levels of these three biomarkers showed a median PFS significant longer than 3 months. Overall survival analysis is ongoing.

Background:
Nintedanib, an oral triple-angiokinase inhibitor, has received regulatory approval in combination with docetaxel based on the demonstrated efficacy as a 2[nd] line treatment for NSCLC patients. Two recently approved immune checkpoint PD1 antagonists have shaken up the established lines of NSCLC therapy. In order to explore the combination potential of Nintedanib with PD1 antagonists, we performed in vivo combination experiments in two syngeneic murine tumor models.

Methods:
The murine tumor cell lines CT-26 and 4T1 were injected subcutaneously into female BALB/c mice. Established tumors around of 50-100mm³ were randomized into the different treatment groups and treated with vehicle, RMP1-14 (murine anti PD-1, 10mg/kg, i.p., q3or4d), Nintedanib (50mg/kg, p.o., qd) and RMP1-14 plus Nintedanib. Anti tumor efficacy was determined after 3 weeks of treatment. In a separate pharmacodynamic approach larger tumors of~300mm³ were treated for 10 days followed by FACS analyses of the dissociated tumors for various myeloid cell subsets including monocytes/macrophages and granulocytes (Markers: CD11c, CD11b, Ly6C, Ly6G, PD-L1, F4/80), T cells/activation (Markers: CD3, CD4, CD8, CD25, FoxP3, IFN-gamma, PD-1) and NK cells (Markers: CD335/Nkp46).

Results:
Single agent treatment of CT-26 subcutaneous tumors with RMP1-14 and Nintedanib resulted in anti-tumor effect with T/C values of 45% and 63%, respectively. The combination treatment group after 24 days showed a T/C value of 34%. In the RMP1-14 refractory tumor model 4T1 neither anti-PD1 treatment nor nintedanib showed benefit (T/C=88% and 82%). The combination treatment after 26 days resulted in a T/C value of 38%. The particular immune cell infiltrate composition and activation state in the different treatment groups will be reported.

Conclusion:
The combination of angiogenic and immune checkpoint inhibition is an attractive opportunity to improve overall response rates and efficacy based on the dual roles of angiogenic factors in blood vessel formation and immune regulation. In the CT-26 model improved additive efficacy could be demonstrated by combining Nintedanib with anti PD-1. More interestingly, the addition of Nintedanib in the anti PD-1 refractory model 4T1 showed a synergistic combinatorial anti-tumor effect. These data fit well with the hypothesis that interfering with tumor angiogenesis in combination with immune checkpoint inhibition will result in additive and synergistic effects by positively regulating immune cell function and infiltration.

Background:
B7-H3 (CD276) is a type I transmembrane protein that belongs to the B7 immunoregulatory family including PD-L1 (B7-H1) and is upregulated in multiple malignancies including Non-Small Cell Lung Cancer (NSCLC). Clinical activity of monoclonal B7-H3 blocking antibodies such as Enoblituzumab are under investigation. In this study we measured the levels of B7-H3 protein in NSCLC and studied its association with major tumor infiltrating lymphocyte (TIL) subsets, levels of PD-L1, B7-H4 and clinico-pathological characteristics in three independent NSCLC cohorts.

Methods:
We used automated quantitative immunofluorescence (QIF) to assess the levels of B7-H3 (clone D9M2L, CST), PD-L1 (clone SP142, Spring), B7-H4 (Clone D1M8I, CST) CD3, CD8 and CD20 in 634 NSCLC cases from 3 retrospective cohorts represented in tissue microarray format. The targets were selectively measured in the tumor and stromal compartments using co-localization with cytokeratin. Associations between the marker levels, major clinic-pathological variables and survival were analyzed.

Results:
Expression of B7-H3 protein was found in 80.4% (510/634) of the cases and the levels were higher in the tumor than in the stromal compartment. High B7-H3 protein expression level (top 10 percentile) was associated with poor survival in two out of three of the cohorts (p <0.05). Elevated B7-H3 was consistently associated with smoking history across the 3 cohorts, but not with sex, age, clinical stage and histology. Co-expression of B7-H3 and PD-L1 was found in 17.6% of the cases (112/634) and with B7-H4 in 10% (63/634). B7-H4 and PD-L1 were simultaneously detected only in 1.8% of NSCLCs (12/634). The expression of B7-H3 was not associated with the levels of CD3, CD8 and CD20 positive TILs.

Conclusion:
B7-H3 protein is expressed in the majority of NSCLCs and is associated with smoking history. High B7-H3 protein levels may have a prognostic effect in lung carcinomas. Elevated levels of B7-H3 are not associated with lymphocyte infiltration. Co-expression of B7-H3 with PD-L1 and B7-H4 is relatively low, suggesting a non-redundant biological role of these targets and possibilities for combination therapies using monoclonal antibodies.

Background:
PD-L1 expression in NSCLC correlates with increased response to pembrolizumab, supporting its role as a predictive biomarker but the reproducibility of pathologists’ scoring of PD-L1 requires further investigation. The primary objective of the DREAM study was to assess the reproducibility of scoring PD-L1 staining by evaluating intra- and inter-observer reproducibility for the assessment of PD-L1 expression in NSCLC. The secondary objective was to assess the impact of training on reproducibility.

Methods:
The study was a blinded, pathologist reproducibility study of scoring PD-L1 expression in NSCLC cases stained with PD-L1 22C3 pharmDx™ kit using the Dako Automated Link 48 Platform. Two pathologists previously trained and certified by Dako scored 789 specimens to form the gold standard. From these specimens 60 were randomly selected to evaluate a 1% cut-point and 60 for a 50% cut-point. Both sample sets were designed to include 50% positive/negative specimens and 20-30 close to each cut-point. Ten pathologists were randomly assigned to two subgroups. Subgroup 1 analyzed all samples on two consecutive days. Subgroup 2 performed the same assessments, except they received a one hour training session prior to the second assessment.

Results:
The overall percent agreement (OPA) for the analysis of the intra-observer reproducibility was 89.7% (95% CI: 85.7; 92.6) for the 1% cut-point sample set and 91.3% (95% CI: 87.6; 94.0) for the 50% cut-point. The OPAs for inter-observer reproducibility of all ten pathologists were 84.2% (95% CI: 82.8; 85.5) and 81.9% (95% CI: 80.4; 83.3) for the 1% and 50% cut-point sample sets, respectively. There was substantial agreement at both the 1% cut-point (prevalence-adjusted bias-adjusted kappa 0.68 (95% CI: 0.65; 0.71)) and the 50% cut-point (prevalence-adjusted bias-adjusted kappa 0.64 (95% CI: 0.61; 0.67)). Training was found to have no or very little impact on the inter- or intra-observer reproducibility in subgroup 2. The OPAs for the inter-observer reproducibility assessments were 82.0% and 82.3% for the first and second assessments of the 1% cut-point sample set, respectively, and 78.3% and 81.7% for the first and second assessments of the 50% cut-point sample set, respectively. The exploratory analyses showed that the sensitivity and specificity of the pathologists assessments, compared with the gold standard assessment, were 84.3% and 91.3%, respectively, for the 1% cut-point and 56.3% and 94.0%, respectively, for the 50% cut-point.

Conclusion:
There is high intra-observer reproducibility and substantial inter-observer agreement in pathologists’ assessment of PD-L1 expression in NSCLC at 1% and 50% cut-points.

Background:
PD-L1 tests have been approved on histological specimens only. More than 1/3 of NSCLC patients are diagnosed on cytology alone. The hypothesis of this study is that cytological cell block material is as good as histological material for PD-L1 analysis.

Methods:
86 paired samples of malignancies from the lung (NSCLC = 72, other primary malignancies and metastases = 14), where cytological cell block and histological material were available from the same lesion within 6 weeks were stained for PD-L1 on serial sections with PD-L1 IHC 28-8 pharmDx (28-8pharmDx) and PD-L1 IHC 22C3 pharmDx (22C3pharmDx). The partial or complete membrane staining on malignant cells regardless of intensity was assessed as >1%, >5%, >10% and hereafter in 10% increments. Number of malignant cells (< or > 100) and staining heterogeneity were recorded. The pathologist was blinded to previous evaluations of pair members. Overall (OA) and Average Positive (APA) and Negative (ANA) agreement were calculated as well as Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) with histology as the non-reference standard. CIs were bootstrapped.

Results:
Agreement statistics for PD-L1 positivity with different cutoffs in cytology and histology specimens, % (95% CI)

22C3pharmDx
	≥ 1% positive	≥ 50% positive
OA	85 (77–92)	94 (88–99)
APA	83 (73–91)	82 (61–96)
ANA	86 (78–93)	97 (93–99)
PPA	80 (67–92)	100
NPA	89 (79–98)	93 (87–99)
28-8pharmDx
	≥ 1% positive	≥ 5% positive	≥ 10 % positive
OA	87 (80–94)	95 (91–99)	90 (83–95)
APA	86 (77–94)	94 (86–99)	84 (71–93)
ANA	88 (80–94)	96 (92–99)	92 (87–97)
PPA	81 (69–92)	91 (79–100)	79 (63–93)
NPA	93 (85–100)	98 (94–100)	95 (88–100)

There was high agreement between the cytological and histological scores, applying to both pharmDx kits and all cutoffs (Table), not changing by exclusion of cytological cell blocks containing <100 cells nor by exclusion of non-NSCLC. Pearson r[2 ]were 0.87-0.89. Paired samples with PD-L1 staining heterogeneity on histological material seemed to have lower agreement than samples with homogenous staining

Conclusion:
High correlation between staining on cytological cell block material and histological specimens was observed using PD-L1 IHC 28-8pharmDx and PD-L1 IHC 22C3pharmDx, suggesting that cytological material is as good as histological material for PD-L1 IHC analysis. Intra-tumor heterogeneity may contribute to disagreement and more research on the influence of staining heterogeneity is warranted.

Background:
PD-L1 tests as predictive biomarkers for anti-PD-1 immunotherapy for NSCLC have been developed independently and use different assays and cutoffs for positivity. A more flexible PD-L1 testing would allow for efficient use of sparse tissue and pathology resources. The aim of this study was to evaluate the agreement between PD-L1 IHC 28-8 pharmDx (28-8 pharmDx) and PD-L1 IHC 22C3pharmDx (22C3pharmDx) assays in patients with malignancy in the lung. The comparison was made on cytological cell blocks as well as on histological material.

Methods:
Paired samples of malignancies from the lung (NSCLC = 73, other primary malignancies and metastases = 14) where cytological cell block material (N=86) and histologic material (N=87) were available from the same lesion within 6 weeks were stained for PD-L1 on serial sections with 28-8pharmDx and 22C3pharmDx. The partial or complete membrane staining on malignant cells regardless of intensity was assessed as >1%, >5%, >10% and hereafter in 10% increments. The pathologist was blinded to previous evaluations of pair members. Overall Agreement (OA) and Average Positive (APA) and Negative Agreement (ANA) were calculated

Results:
Agreement statistics for 28-8 pharmDx and 22C3 pharmDx with different cutoffs for PD-L1 positivity in cytology and histology specimens, % (95% CI)

PD-L1 IHC 22C3 pharmDx and PD-L1 IHC 28-8 pharmDx on cytological cell blocks
	≥ 1%	≥ 5%	≥ 10 %	≥ 50%
OA	94 (90–99)	98 (94–100)	97 (92–100)	93 (87-98)
APA	93 (87-97)	97 (91-100)	94 (85-100)	80 (61-94)
ANA	95 (90-99)	98 (95-100)	98 (94-100)	96 (92-99)
PD-L1 IHC 22C3 pharmDx and PD-L1 IHC 28-8 pharmDx on histological material
	≥ 1%	≥ 5%	≥ 10 %	≥ 50%
OA	97 (92-100)	99 (97-100)	95 (91-99)	93 (86-97)
APA	96 (92-100)	98 (95-100)	93 (84-98)	80 (62-94)
ANA	97 (92-100)	99 (97-100)	97 (93-99)	96 (92-99)

High OA, APA and ANA were found for agreement between 22C3pharmDx and 28-8pharmDx on cytological clot material, as well as on histological tissue for all cutoffs. The APA at cutoff 50% positivity was the lowest (80%); the CIs were however wide due to small number of positive samples at this cutoff. Pearson r[2 ]were 0.96-0.97

Conclusion:
High correlation between PD-L1 IHC 22C3 pharmDx and PD-L1 IHC 28-8 pharmDx was observed on both cytological and histological specimens, suggesting that these assays may be used interchangeably in testing of lung tumors for PD-L1 expression. However, more data is needed to alter current guidelines.

Background:
Lung adenocarcinoma (AD) is a common variant lung cancer, accounting for about 70% of non-small cell lung cancer. PD1/PD-L1 is a promising immune therapy target which has achieved promising results in the late stage NSCLC patients in the ongoing clinical trials. Because of different accompanying diagnostic antibodies employed in different clinical trials and limited data regarding PD-L1 expression in the small number of patients enrolled in clinical trials, there is an urgent need to examine the expression of PD-L1 in lung cancer samples in order to enrich patients who will benefit from the immune targeted therapy. Our goal was to detect PD-L1 expression and analyze its expression with the clinicopathological characteristics.

Methods:
Protein expression of PD-L1 was examined by immunohistochemistry method using the VENTANA PD-L1 (SP263) rabbit monoclonal antibody. Messenger RNA level of PD-L1 was evaluated by in situ hybridization method. Tissue microarrays (TMAs) containing formalin fixed paraffin embedded (FFPE) lung adenocarcinoma cases were used in this study.

Results:
This study included 133 cases of lung AD totally. PD-L1 expression rate in lung ASC was 16.5% at the mRNA level and 13.5% at the protein level, which the kappa coefficient of the two examination methods was 0.824 (P=0.000, highly correlated). PD-L1 expression in lung AD was found to be highly expressed in female patients and smokers (P=0.019 and 0.002), while no association was identified between PD-L1 expression and age, tumor size, clinical stage, positive pleural invasion, histological type (lepidic or non-lepidic type), lymph node metastasis or therapy methods. Overexpression of PD-L1 was a significantly indicator of shorter recurrence free survival (RFS) time and overall survival duration (P=0.000 and 0.000). Multivariate analysis revealed that PD-L1 expression was an independent risk factor for poor RFS and overall survival (P=0.004 and 0.006).

Conclusion:
PD-L1 overexpression is more frequently observed in smokers in lung adenocarcinoma. PD-L1 expression is an indicator of worse prognosis in surgically resected lung adenocarcinoma patients.

Background:
Myeloid-derived suppressor cells (MDSCs) are implicated in immune suppression of cancer and chronic inflammation. Although PD-1/PD-L1 checkpoint inhibitors show clinical efficacy as immunotherapy for lung cancer, the mechanisms by which lung cancers are refractory to immunotherapy remain to be fully understood. MDCSs are thought to contribute to T-cell exhaustion and have been proposed to play a role in tumor progression. In human lung cancer, the relationship between MDSCs and clinico-pathological factors, and the relevance of MDSCs to its diagnosis and treatment is yet to be elucidated.

Methods:
Tissue specimens were obtained from 30 surgically treated patients with stage I to IV NSCLC. Correlation between immunohistochemical staining for CD33, CD14, CD11b, CD8 and PD-L1 (SP263), and clinico-pathological factors were ebaluated. For PD-L1, membranous staining of any intensity of in more than 25% of tumor cells was considered positive. Histologic subtypes were compared with the degree of MDSCs expression.

Results:
CD33- and CD11b- positive immune cells were more highly expressed in tumor tissue than normal tissue. PD-L1 expression was detected in 23% of tumors, and it correlated with CD8-positive cells. Expression of MDSCs was more prominent in invasive adenocarcinoma than adenocarcinoma in situ, and as well as in tumor tissue compared with normal lung tissues adjacent to the tumor. Expression of MDSCs within the tumor microenvironment correlated with the stage of disease progression and subgroup of histological subtypes.

Conclusion:
These findings suggest that MDSCs expression within the microenvironment of lung cancer tissue is associated with progression of human lung adenocarcinoma.

Background:
Expression of programmed death ligand 1 (PD-L1) is rendered a possible biomarker for immunotherapy targeting programmed death protein 1 (PD1) and PD-L1. Durable clinical responses have been demonstrated in patients with squamous cell carcinoma (SqCC) of the lung. Tumor infiltrating lymphocytes (TILs) are also regarded important players in immunomodulating therapies. Still, there is much uncertainty regarding the expression of the markers and associated tumor characteristics. We aimed to assess the prognostic impact and heterogeneity of PD-L1 expression across two antibodies, in conjunction with TILs, in SqCC of the lung.

Methods:
PD-L1 expression was investigated on a tissue microarray (TMA) of pulmonary SqCC using immunohistochemistry and two different clones (SP142, E1L3N). The cohort comprised all consecutive patients with primary resected pulmonary SqCC, without previous SqCC of other sites, diagnosed 2000-2013 at the Institute of Pathology, University of Bern. Epithelial expression of PD-L1 in primary tumors (n=372) and mediastinal lymph node (LN) metastases (n=27) was evaluated across 8 TMA cores per tumor using the following increment intervals: 0%; 1%; 5%; 10%; 20%; 30%, 50%. Intratumoral and intraepithelial infitrates of CD8+ T lymphocytes were determined. Results were compared with clinic-pathologic parameters including tumor related survival.

Results:
The staining results correlated significantly between the two antibodies (r=0.822; p<0.001) with the best congruence for tumors with ≤1% or ≥30% positivity. These tumors had also very homogenous staining results across all TMA cores, indicating low levels of intratumoral heterogeneity, in contrast to tumors with 1-30% PD-L1 positivity (p<0.001 both antibodies). Stainings correlated significantly between primary tumors and LN metastases (p<0.001 both antibodies). High PD-L1 expression correlated with intratumoral and intraepithelial CD8+ lymphocyte counts in primaries (p<0.001 both antibodies). PD-L1 expression was not significantly associated with pT or pN staging. High PD-L1 expression was associated with shorter tumor related survival. Cut-offs with the best prognostic discrimination were 30% positivity for SP142 and 50% for E1LN3 in univariate analysis (p=0.001/p=0.008). PD-L1 expression, pT category and age of the patients, but not CD8+ TILs were independent prognostic factors in multivariate analysis (HR=2.265, p=0.013 for SP142; HR=2.323, p=0.014 for E1LN3).

Conclusion:
PD-L1 expression was an independent predictor of shorter tumor related survival in pulmonary SqCC across all stages. The homogeneity of PD-L1 expression in “no” or “high” staining tumors across different cores and primary versus LN metastases indicates a valid assessment on small tissue samples and LN-metastases.

Background:
Small Cell Lung Cancer (SCLC) is a highly aggressive tumor. Alough most of SCLC cases are sensitive to chemotherapy, the prognosis of SCLC is still dismal. Programmed death-ligand 1 (PD-L1), which is expressed on many cancer cells, has been reported as a predictive biomarker for immune checkpoint inhibitors treatment in advanced non-small-cell lung cancer. However, expression of PD-L1 in SCLC has not been fully studied.

Methods:
Totally 72 surgical specimens of SCLC ( 42 primary, 30 metastasis) were involved in this study. Only 5 cases had received neoadjuvant chemotherapy before surgery while 67 cases did not. Rabbit anti-PD-L1 monoclonal antibody (Spring Bio, SP142,1:100) was used on tissue microarray to detect the expression of PD-L1 on these cases.

Results:
PD-L1 was strongly expressed on the membrane of tumor cells of 8 cases (11.1%) and in the immune stroma of 35 cases (48.6%). Another 29 cases (40.3%) were totally negative for PD-L1 staining. Expression of PD-L1 was significantly associated with tumor sites (primary, P=0.017) and had a trend of low lymphonode metastasis(P=0.057). There was no significant correlation between PD-L1 expression and other clinical-pathological parameters (age, gender, T staging, nodal status, TNM staging, vascular invasion and treatment).

Conclusion:
PD-L1 was expressed in more than half of SCLC cases and correlated with tumor sites. These data may provide useful information for future research or clinical trial on immune checkpoint therapy for SCLC.

Background:
Programmed death ligand 1 (PD-L1) has become one of the most studied biomarkers in early, and advanced non-small cell lung cancers (NSCLC). Conflicting results have been reported in the literature on the value of PD-L1 in predicting survival in surgically resected lung cancers. Our aim was to evaluate mRNA PD-L1 expression and survival based on the data available from the Cancer Genome Atlas (TCGA), and Cancer Cell Line Encyclopedia (CCLE).

Methods:
To determine the expression profile and clinical correlations of PD-L1 in lung cancers we used publicly available lung adenocarcinoma and squamous cell carcinoma (SCC) data from TCGA, and small cell and NSCLC line data from the CCLE. We performed Kaplan-Meier and correlation analyses to show how PD-L1 expression correlates with overall survival and other clinical variables. Lung cancer and normal tissue expression comparisons were also performed using normal tissue expressions from the Genotype-Tissue Expression (GTEx) project.

Results:
Results: PD-L1 mRNA expression from RNA sequencing was available for 517 lung adenocarcinoma and 501 lung SCC samples in the TCGA. The CCLE database contained PD-L1 expression for 12 small cell and 75 NSCLC cell lines. Lung cancers demonstrated a higher PD-L1 expression than most other cancers and normal tissues, and we found that PD-L1 expression was significantly higher in SCC than in adenocarcinoma (p<0.001). Furthermore, PD-L1 showed a significantly higher expression level in pathologic stage II SCC compared to stages I, III, and IV (p<0.05, p<0.05, p<0.001, respectively). Interestingly, stage IV was associated with a lower PD-L1 expression compared to stages I, II, and III (p<0.001). We did not identify similar expression associations with pathologic stage in adenocarcinoma. However, we found that current and also reformed smokers for less than 15 years had a higher PD-L1 expression in adenocarcinoma (p<0.05), but not in SCC. PD-L1 expression was not significantly associated with a survival difference in any stage or histology subgroups. Using the CCLE data we found that NSCLC cell lines show various expression of PD-L1 that is significantly higher compared to lung small cell carcinoma (p<0.001).

Conclusion:
The value of PD-L1 expression based on mRNA sequencing in predicting survival in anti PD-1 naïve patients appears to be limited. However, the levels of PD-L1 expression in various disease stages and subgroups of lung cancer patients provide rational for neoadjuvant or window-of-opportunity immunotherapy trials, which would enable us to sort out the mechanisms and to identify patients best suited for immunotherapy.

Background:
Immunotherapy in lung cancer is currently experiencing huge interest. The greatest efficacy demonstrated the antibodies against immune checkpoints receptors (PD-L1, CTLA-4 or LAG-3). However, an effective anti-tumor response also required tumor antigen presentation to lymphocytes in peripheral lymph nodes. To enhance this effect, vaccination with synthetic tumor antigen was conducted in many clinical trials using antigen presenting cells (APC). Unfortunately, this type of active immunotherapy has not achieved spectacular success (START or MAGRIT studies). The explanation could lie in distorted presentation of tumor antigens in peripheral lymph nodes or in activation of lymphocytes with strong immunosuppressive properties. In our study, we checked which pathways of T helper differentiation are favored by autologous dendritic cells (DCs) after synthetic tumor antigens stimulation.

Methods:
Peripheral blood was acquired from twenty unresectable and treatment-naïve lung adenocarcinoma patients. Using CD14 magnetic beads, two cell populations were isolated. CD14-negative cells were used for CD3-positive cells isolation, which were frozen until further stimulation. CD14-positive cells were cultured in CellDCGrow medium supplemented with IL-4 and GM-CSF. After TNF-a stimulation, mature DCs were incubated with tumour-specific antigens: MUC1, MAGE-A3, EGFR and CMV (positive stimulator) or only with medium (negative control). Flow cytometry and specific monoclonal antibodies were used to analyse immunophenotype of DCs: CD1a, CD11c, CD83, CD80, CD86, B7-H1 (PD-L1), B7-DC (PD-L2). Mature autologous DCs were cultured with fraction of CD3-positive cells and after 48h of mixed cultures, the expression of intracellular factors specific for different T helper cells subpopulation (T-bet – Th1, STAT-6 – Th2, ROR-gT – Th17, FoxP3 – Treg) and expression of extracellular interleukin receptors (IL-12R, IL-4R, IL-23R, TGF-bR, IL-2R) were analysed.

Results:
Autologous DCs were well generated and showed phenotype specific for fully-mature DCs. In culture with DCs after MUC1 stimulation, we observed in lymphocytes the highest expression of ROR-gT and IL-23R (p<0.05) compared with lymphocytes from cultures with other synthetic tumour antigens. Expression of IL-4R on lymphocytes was also significantly (0<0.05) higher in the culture stimulated with MUC1. The highest expression of intracellular FoxP3 factor, TGF-bR (p<0.05) and IL-2R (p=0.009) on Th cells were observed in mixed culture with DCs after MAGE-A3 stimulation.

Conclusion:
Dendritic cells stimulated with synthetic tumour antigens could activate different T helper cell populations. It could depend on the nature of antigens and could influence the effectiveness of stimulation different lymphocyte subtypes in peripheral lymph nodes. Therefore, our study clarifies some failure reasons of large clinical trials using active antigen-specific immunotherapy.

Background:
Pulmonary Sarcomatoid Carcinoma (PSC) is a unique and highly aggressive subtype of non-small cell lung cancer (NSCLC), characterized by a component of sarcoma or sarcoma-like differentiation. It is generally resistant to platinum-based chemotherapy. However, frequent KRAS and the MET exon 14 skipping mutations have been reported in this subtype. Recently, immunotherapy with antibodies against PD-1/PD-L1 has led to clinical benefits in managing NSCLC. Immune biomarker expression of PD-L1 in differential components of PSC and its relationship with other molecular markers has not been defined and thus there is critical need to investigate its expression profile in this deadly disease.

Methods:
We investigated PD-L1 expression by immunohistochemistry (IHC) using tissue microarrays from a cohort of 41 patients with PSC (antibody: PD-L1/CD274 (SP142)). Positive cases were defined as PD-L1 ≥ 1%. The clinical and molecular characterization of these cases was previously reported (PMID: 26215952). Among the 41 cases, 36 had sufficient tumor tissue for PD-L1 assessment, 8 (20%) were identified with MET exon 14 skipping mutation and 6 (15%) were found to have KRAS mutations.

Results:
The PD-L1 expression was positive in 75% (27/36) of cases. Among the positive cases, 78% expressed PD-L1 ≥ 50%, whereas 22% had PD-L1 staining of 1-49%. Interestingly, only 33% (9/27) had PD-L1 expression in both epithelial and sarcomatoid components, whereas the remaining 66% detected PD-L1 in just one component. The PD-L1 expression was statistically different between the two components (P=0.007). Moreover, all 8 patients with MET exon 14 skipping mutation were PD-L1 positive (6 with PD-L1 ≥ 50%), whereas 5 of 6 patients with KRAS mutation expressed PD-L1 (2 with PD-L1 ≥ 50%). The average age at diagnosis was similar between the PD-L1 positive vs. negative groups (71 years). There was a trend of shorter overall survival in patients whose tumors were positive for PD-L1 (671 vs. 985 days).

Conclusion:
In our particular cohort of PSC patients, high PD-L1 (≥ 50%) was expressed in the large majority of cases which favors potential application of immunotherapy in this disease. In addition, it appears that the expression of PD-L1 is different in the epithelial and sarcomatoid components which could affect scoring in practice. Furthermore, the overall positivity of PD-L1 expression in the unique molecular subtype of PSC patients with MET exon 14 skipping mutation indicates potential interplay between the two biomarkers and suggests that exploration of combination MET and immunotherapy in this subtype is warranted.

Background:
Accumulating data suggests that the extent of lymphocytic infiltrations into the tumor provides prognostic value in non-small cell lung cancer (NSCLC). However, the factors that influence the status of tumor immune environment remain poorly defined and need investigation. The aim of this study was to assess whether the density of tumor infiltrating lymphocytes (TILs) was related to tumor molecular characteristics in completely resected stage IIIA(N2) lung adenocarcinoma.

Methods:
We retrospectively screened consecutive patients with pathologic stage IIIA(N2) pulmonary adenocarcinomas, who had undergone complete resection in our hospital between 2005 and 2012. Patients who received neoadjuvant chemotherapy and/or radiotherapy were excluded. DNA of EGFR and KRAS was extracted and purified from paraffin-embedded primary lung cancer tissue samples. EGFR (exons 18-21) and KRAS (exons 2, 3) mutation analyses were performed by the DNA sequencing. The density of TILs was evaluated by full-face hematoxylin- and eosin-stained sections from surgical specimens for each case by two specialized pathologists. The degree of lymphocyte infiltration into the tumor was scored as none, low, moderate, or high. Patients were stratified into TIL- (none to low infiltration) or TIL+ (moderate to high infiltration) group based on pathologic evaluation. We investigated the association of densities of TILs with tumor-cell mutation status.

Results:
Among the 192 eligible patients included, 96 (50%) patients were male and 123 (64%) were never/light ex-smokers, and the median age of all patients was 59 years (range, 22–84 years). There were 84 (43.8%) EGFR mutated and 21 (10.9%) KRAS mutated cases. Among the 84 patients with activating EGFR mutations, there were 30 patients harboring mutations in the exon 19 deletions, 35 patients in the exon 21 L858R mutations, and 1 patient with concurrent exon 19 deletion and L858R mutation. The proportion of patients who had higher density of TILs (TIL+) was lower in the EGFR mutation-positive subgroup (42.9% versus 53.7%, P=0.14) and this difference was significantly observed in the patients with L858R mutation and/or exon 19 deletion subgroup (P=0.026). The proportion of patients who had higher density of TILs (TIL+) was significantly lower in the 66 patients harboring L858R mutation and/or exon 19 deletion (37.9% versus 54.8%, P=0.026).

Conclusion:
There existed association between the lower density of TILs and activating EGFR mutation status in lung adenocarcinoma, underlying the interactions between cancer cells and their microenvironment. Further studies are warranted to validate these results and clarify the potential molecular mechanisms responsible for regulation of lymphocytic infiltration by activating EGFR pathway.

Background:
Recent reports convey that the abundance and patterns of DNA mutational features in human cancers could modulate their antigenicity and sensitivity to immune checkpoint blockade. We thus sought to comprehensively characterise the immunogenomic landscape of NSCLC.

Methods:
We used publicly-available molecular (DNA mutation and RNA expression profiles) and clinical data of 658 NSCLC patients from The Cancer Genome Atlas project. HLA-type was inferred using POLYSOLVER, and we identified neoantigens with patient-specific HLA-binding affinity of IC50<500nm using NetMHC. The relative tumour infiltration by 22 immune cell types was enumerated using CIBERSORT. Finally, we developed and applied MUTPROFILER, a computational approach for mutational signature analysis capable of decrypting sequence alterations across 96 trinucleotide contexts and indels of varying lengths.

Results:
This analysis recapitulated a pervasive and prominent association between neoantigen levels and the molecular smoking signature (that is characterised by C:G>A:T transversions; Spearman ρ=0.78, P=1.37×10[-69]), and identified several novel and powerful immunogenetic associations in NSCLC. For instance, the proportion of activated natural killer (NK) cells was greater in tumours which showed a higher abundance of 1-3bp insertion/deletions (indels; ρ=0.23, P=1.34×10[-9]). Furthermore, small (1bp) indels were associated with increased expression of markers of immune cytolytic activity, including TNFA (TNFα; ρ=0.30, P=4.77×10[-15]), GZMA (ρ=0.21, P=4.92×10[-8]) and PRF1 (ρ=0.15, P=1.84×10[-4]). Fourteen trinucleotide alterations, including GCA>GTA, TCG>TAG and TCG>TGG, were more strongly correlated with PDCD1LG2 (PD-L2) expression compared to small indels (q<5×10[-8], Fisher’s Z-transformation). Interestingly, the number of small frameshifting indels was associated with downregulation of the antigen-presenting machinery (APM) such as TAP1 (ρ=–0.22, P=7.93×10[-9]) and TAP2 (ρ=–0.23, P=1.69×10[-9]), suggesting a potential immunoediting mechanism by which NSCLC tumours co-opt APM pathways to prevent neoantigen-recognition. Finally, by analysing the mutational signatures of the AID/APOBEC family, which has important roles in adaptive and innate immunity, we identified a potential novel mutagenic contribution of APOBEC3H, whose expression levels was associated with a pattern of C>A (ρ=0.18, P=4.76×10[-6]) and C>G (ρ=0.14, P=3.67×10[-4]) within TC motifs (with the mutated base underlined).

Conclusion:
Our study portrays an atlas of immunogenetic features in NSCLC. The results sheds light on the dynamics of tumour-immune cell interactions which are likely to form the driving force behind the clinical activity of novel immunologic strategies, and may lead to new biomarkers and targets for cancer immunotherapy.

Background:
Glycodelin (gene name: progesterone-associated endometrial protein, PAEP) is a protein initially described as an immune system modulator during the establishment of pregnancy. Former studies determined an atypical expression and secretion of glycodelin in non-small cell lung cancer (NSCLC), the most common type of lung cancer. To date, there is not much known about the signaling pathway which regulates PAEP/glycodelin expression in cancer. However, initial experiments already revealed an inducing effect of epidermal growth factor (EGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), lysophosphatidic acid (LPA) and Phorbol 12-myristate 13 acetate (PMA) on PAEP/glycodelin expression in two NSCLC cell lines (H1975 and 2106T). In this study, we analyzed an extended number of possible regulatory candidates to acquire a more detailed view on the regulatory pathway of PAEP/glycodelin in NSCLC.

Methods:
A lung adenocarcinoma (H1975) and a lung squamous cell carcinoma cell line (2106T) were transfected with siRNA targeting nuclear factor κB1 (NFκB1) or treated with human chorionic gonadotropin (hCG), transforming growth factor-β (TGF-β) 1, -2, -3, protein kinase C (PKC) activator bryostatin 1 and PKC inhibitor GF109203x, respectively. Additionally, combined treatments with GF109203x and TGF-β 1,-2, EGF or HB-EGF were performed. PAEP expression in the manipulated cells was determined by quantitative polymerase chain reaction (qPCR), while glycodelin expression or secretion was detected by western blot analysis.

Results:
NFκB1 siRNA transfection resulted in decreased PAEP and glycodelin amounts (H1975 and 2106T), whereas hCG (H1975 and 2106T) and TGF-β 1, -2, -3 (2106T) treatment led to higher levels. In bryostatin treated cells (H1975 and 2106T), PAEP/glycodelin expression was upregulated. The contradictory effect could be demonstrated for cells treated with the PKC inhibitor GF109203x alone and in combination with TGF-β 1,-2, EGF or HB-EGF (H1975 and 2106T).

Conclusion:
This study revealed that there are different regulation mechanisms of PAEP/glycodelin induction in NSCLC. Especially, PKC seems to be involved as a key molecule. The investigated candidates which play a crucial role in driving this signaling pathway are all known to promote the development of cancer. Elucidating the regulatory pathway of the immune system modulating protein glycodelin might reveal a potential strategy to weaken the immune system defense of lung tumors.

Background:
Blockade of programmed death receptor-1 (PD-1) pathway has been shown to be effective against solid tumors including lung cancer. Although PD-ligand 1 (PD-L1) expression on tumor tissue is expected as a potential predictive biomarker, its detection remains challenging due to its dynamic and unstable status. Here, we evaluated the PD-L1 expression on circulating tumor cells (CTCs) in patients with lung cancer and investigated its concordance with that on tumor tissues.

Methods:
CTCs were captured and immune-stained using microcavity array system. CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. PD-L1 expression on CTCs was evaluated by addition of the process of PD-L1 immunocytochemistry. For CTCs detection, 3 ml of peripheral whole blood was collected from the patients who consented in written form and PD-L1 immunohistochemistry was performed using corresponding tumor tissues.

Results:
Sixty-seven lung cancer patients were enrolled in the study between July 2015 and April 2016 at Wakayama Medical University. Patient characteristics were as follows: median age 71 (range, 39 to 86); male 72%; stage II-III/IV, 15/85%; non-small cell lung cancer (NSCLC)/small cell lung cancer (SCLC)/Other, 73/21/6%. CTCs were detected in 66 out of 67 patients (median 19; range, 0 to 115) and more than 5 CTCs were detected in 78% of patients. PD-L1 expressing CTCs were detected in 73% of patients and the proportion score (PS) of PD-L1 expressing CTCs ranged from 3% to 100%, suggesting intra-patient heterogeneity of PD-L1 expression on CTCs. Significantly more PD-L1 expressing CTCs were detected in patients without EGFR mutations than those with EGFR mutations (P = 0.0385). Tumor tissues were available from 27 patients and were immune-stained for PD-L1. No positive correlation was observed on PD-L1 expression between tumor tissues and CTCs based on PS (R[2 ]= 0.0103). Three adenocarcinoma cases with PD-L1-positive tumor tissue did not harbor any PD-L1 expressing CTCs and conversely, three adenocarcinoma cases with PD-L1-negative tumor tissue harbored PD-L1 expressing CTCs, showing the total discrepancy between tumor tissues and CTCs. It is also noteworthy that SCLC patients had perfect agreement on PD-L1 expression between tumor tissues and CTCs.

Conclusion:
PD-L1 expression was detectable on CTCs in lung cancer patients and intra-patient heterogeneity of its expression was observed. There was no agreement between tumor tissues and CTCs on PD-L1 expression though it may differ among tumor histologies. Further investigation is warranted to better understand the clinical significance of PD-L1 expressing CTCs.

Background:
The understanding of the co-expression of immune checkpoints in non-small cell lung carcinoma (NSCLC) is important to potentially design combinatorial immunotherapy approaches in this disease. We examined the expression of a panel of immune checkpoints markers by immunohistochemistry (IHC) and quantitative image analysis in a large cohort of surgically resected NSCLCs, and correlated those findings with patients’ clinicopathological features and tumors’ inflammatory cells infiltrate and molecular characteristics.

Methods:
We studied 225 formalin fixed and paraffin embedded (FFPE) tumor tissues from stage I-III NSCLCs, including 123 adenocarcinomas (ADC) and 83 squamous cell carcinomas (SCC), placed in tissue microarrays (TMAs). Nine immune checkpoints markers, 4 (PD-L1, B7-H3, B7-H4, IDO1) expressed predominantly in malignant cells (MCs), and 5 (ICOS, VISTA, TIM3, LAG3 and OX40) expressed mostly in stromal tumor associated inflammatory cells (TAICs). All IHC markers were examined using quantitative image analysis system (Aperio).

Results:
Using > median value of the immune checkpoint expressions as positive expression we observed that MCs H-score expressing PD-L1, B7-H3, B7-H4 and IDO1was higher in SCC than ADC, with 3 out of 4 markers showing statistically significant (P<0.05) differences. In contrast, density of TAICs expressing ICOS, VISTA, OX40, LAG3 and TIM3 was higher in ADC than SCC, with 3 out of 5 markers demonstrating significant (P<0.05) differences. Furthermore, we identified frequent co-expression of markers: a) 11% ADC (13/123) and 10% SCC (8/83) co-expressed 8 to 9 markers; b) 45% ADC (55/123) and 32% SCC (27/83) co-expressed 6 to 7 markers, c) 28% ADC (35/123) and 40% SCC (33/83) co-expressed 4 to 5 markers, and d) 16% ADC (20/123) and 18% SCC (15/83) co-expressed 2 to 3 markers. In ADC, higher number of TAICs expressing OX40 and lower levels of MCs expressing B7-H4 were detected in tumors with EGFR (median, 7.49 vs. 1.16, P=0.021) and KRAS (median, 6.88 vs. 0.67, P=0.033) mutation compared with wild-type tumors, respectively. Univariate analysis demonstrated that high B7-H4 and low OX40 expression in MCs and in TAICs respectively correlated with worse overall survival (OS; P=0.016 and P=0.037, respectively) in ADC patients.

Conclusion:
We detected different patterns of immune checkpoints expression in NSCLC with higher level of markers found in malignant cells of SCC and in stromal inflammatory cells of ADC. Immune checkpoints expression correlated with the outcome of NSCLC patients. Importantly, co-expression of several immune checkpoints is a frequent event in NSCLC (Supported by CPRIT MIRA and UT Lung SPORE grants).

Background:
The number and function of tumor infiltrating lymphocytes (TILs) represent an important prognostic factor in cancer. Among the multiple immune escape mechanisms triggered by cancer, the PD-1/PD-L1 checkpoint seems to play a central role. Accordingly, PD-1/PD-L1 inhibitors have shown significant clinical results in multiresistant NSCLC. However, the role of this immune checkpoint on tumor biology and clinical outcome remains to be determined. To this end, the number and distribution of subpopulations of TILs together with the quantification of PD-L1 expression were immunohistochemically assessed in NSCLC and their impact on patients survival evaluated.

Methods:
Histologic sections from 106 NSCLC (46 ADC,60 SCC) were morphometrically analysed after immunohistochemical assessment of the incidence of CD3+, CD8+ and PD-1+ TILs and their proximal or distal location with respect to neoplastic cells. A comparative evaluation between immuneperoxidase and immunofluorescence (IF) on control tissues and on serial sections from the same cases was undertaken using three different anti-PD-L1 antibodies (clones:28-8,SP142 and M4420). Following suitability criteria, PD-L1 was measured by confocal quantitative IF. Neoplastic and stromal expression of PD-L1 was ascertained by the simultaneous IF detection of Cytokeratin (CK). Morphometric data and clinical records were subjected to Kaplan Meier estimation.

Results:
The gradient of lymphocyte subsets according to their hierarchical phenotype was maintained in both NSCLC, however, the number of CD3[+] TILs was 1.8-fold higher in ADC vs SCC in the presence of similar density of CD8[+] and PD-1[+ ]cells. EGFR and K-RAS mutations conditioned the ADC immune microenvironment by altering CD8[+] and PD-1[+] distribution. High intra- and inter-patients variability in PD-L1 levels was expectedly observed although the average value in SCC samples was higher compared to ADC . K-RAS and to a less extent EGFR mutations were associated with a lower PD-L1 expression. Significant PD-L1 labelling of stromal cells was present in 10% of cases. Interestingly, a lower expression of CK in cells with high PD-L1 signal and the occasional presence of neoplastic plugs overexpressing PD-L1 and lacking CK were documented. Although the number of TILs and PD-L1 levels tended to positively correlate with OS in the entire population of NSCLC, in the individulal cohort of ADC and SCC patients only low number of intratumor PD-1[+] lymphocytes was statistically associated with a significant increased (>10months) OS.

Conclusion:
High levels of PD-L1 and reduction of its cellular target are associated with improved clinical outcome in NSCLC suggesting that adoption by TILs of local escape from PD-L1 pressure delays tumor progression.

Background:
Predictive biomarkers research in anti-PD-1/PD-L1 immunotherapy treatment is still at an early stage of development. Recently, amplification of PDL1 gene (9p24.1) has been described in NSCLC in correlation with PD-L1 protein expression. In addition, other tumor-constitutive alterations such as JAK2 amplifications (322kb upstream of PDL1) or PTEN deletions are also known to modulate PD-L1 expression. We aimed to determine PDL1, JAK2 and PTEN copy number alterations (CNAs) and subsequent PD-L1 protein expression in NSCLC.

Methods:
A total of 171 NSCLC patients (121 ADC and 50 SCC) were included. Clinical, histopathological and molecular data were collected. In resected early-stage diseases, two distinct histologic areas from FFPE tumoral tissue were included for each patient in 8 tissue microarrays (TMAs). PD-L1 expression analysis was assessed by IHC using PD-L1 #SP142 clone (Ventana) and positive cut off was defined at >1%. Moreover, H-score semi-quantitative approach was used to generate a score from 0 to 300. PDL1, JAK2 and PTEN CNAs were studied by FISH using commercial and non-commercial probes hybridized with respective centromere enumeration probes. Amplifications were defined as mean gene by mean centromere ratio ≥2.0, deletions as mean gene by mean centromere ratio ≤0.8, gains as mean gene ≥2.5, and high gains as mean gene ≥4.0.

Results:
PD-L1 expression was positive in 40 out of 171 cases (23.3%), with an average H-score of 177 and significantly associated with ADC solid histological pattern (p=0.012), KRAS mutations (p=0.001), the presence of TILs (p=0.001), and active smoking status (p=0.031). PDL1 gene CNAs were seen in 68/159 assessable cases (42.8%). We found 14 tumors with PDL1 amplification (8.8%), 21 PDL1 high gains (13%) and 33 PDL1 gains (20.8%). Twelve out of 14 FISH amplified cases had PD-L1 positive expression. Thus, FISH predicted positive PD-L1 IHC result with a low sensitivity (31.6%) but a high specificity (98.6%). Among PD-L1 expressing tumors (n=40), seven cases had JAK2 amplifications (6 of them with PDL1 gene coamplification) and eight showed PTEN deletions (3 of them were PDL1 amplified). Differences in H-score intensity between these groups were observed: JAK2-PDL1 coamplified cases had near 2-fold increase in PD-L1 expression than PDL1 alone (average H-score: 282 vs. 148).

Conclusion:
PDL1 gene amplification is synergistically regulating PD-L1 protein expression. In addition, JAK2 amplification upregulates PD-L1 expression, following the concept of cooperative oncogenic effects of genes within the PDJ amplicon. PDL1, JAK2 and PTEN CNAs analysis may be relevant for anti-PD-1/PD-L1 patient selection.

Background:
Although it has been proposed that the number of somatic mutations, together with high PD-L1 and CD8 expression, defines a type of tumour microenvironment predictive of response to immune checkpoint inhibitors, this data has been challenged because the methodologies are difficult to reproduce (e.g. tissue microarrays, whole exome sequencing or complicated scoring approaches). This situation prompted us to investigate possible alternatives that are easier to reconcile with daily practice.

Methods:
A total of 40 consecutive patients with early stage lung squamous cell carcinoma who underwent surgery at HM Sanchinarro University Hospital were considered. Automated immunohistochemistry (IHC) for PD-L1 expression was performed on whole tissue sections (Benchmark ULTRA, OptiView, Ventana Medical Systems, USA) with three different antibodies: SP142 (Ventana), SP263 (Ventana), and E1L3N (Cell Signaling). PD-L1 IHC was considered positive according to the criteria used in the corresponding clinical trials. P53 aberrant IHC expression was used as a surrogate marker for TP53 mutations. Whole tissue sections were also automatically scored for CD8+ tumour-infiltrating lymphocytes (TILs) density (off-label algorithm on the iScan Coreo). Afterwards, we performed targeted next-generation sequencing (NGS) in 52 genes (Oncomine Focus Assay[TM], Life Technologies, USA). The prognostic impact of all these variables was also evaluated.

Results:
Correlation between E1L3N and SP142 or SP263 was similar when scoring tumour cells (TCs) (0.94). There was a significant association between the intraepithelial and peritumour stromal CD8+ TILs density and overall survival when using the image analysis software (p=0.032). The presence of >247 CD8+ cells/mm[2] had a 94% specificity and a 67% sensitivity for identifying patients with at least 5% SP142 PD-L1+ TCs. The highest percentage of PD-L1+ TCs were found in samples with CDK6 (2.6%) or MYC (2.6%) amplifications. Cases with FGFR1 amplification (7.9%) were negative for all PD-L1 antibodies. P53 aberrant expression and PD-L1 expression in TCs also seem to be related.

Conclusion:
The methodology presented herein could help align the use of targeted NGS and the immune microenvironment assessment to increase the clinical value of immune checkpoint inhibitors. Acknowledgements This study was partially funded by Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitarias (FIS), Fondos FEDER-Plan Estatal de I+D+I 2013-2016 (PI14-01176).

Background:
How clinical-genomic features of the lung tumour microenvironment (TME) influence immune-checkpoint-blockade therapy is not well understood. Immunohistochemistry (IHC) is necessary to decipher cell-cell relationships that cannot be observed by bulk tumour profiling. In this pilot study, we assess whether immune cell phenotypes and spatial relationships differ between lung adenocarcinoma (LUAD) from smokers/non-smokers, KRAS/EGFR mutation, or with stage and tumour size using a novel multicolour IHC quantitative pathology method that enables in situ single cell profiling within the TME.

Methods:
Two consecutive sections from 21 cases of LUAD were stained with multicolour IHC panels to assess immune cell composition (CD8, CD3, CD79a) and T-cell exhaustion (CD8, PD1, PDL1). Hyperspectral images were captured as directed by a pathologist and analyzed using software developed in-house. The software segments individual cell boundaries based on haematoxylin stain. IHC stain positivity thresholds were applied based on intensity. Tumour and immune cells were classified into groups based on IHC staining. Interactions between specific groups were quantified by assessing the frequency and variance of the spatial relationship of each group vs. all other groups. Voronoi tessellation, based on cell centres, was used to define “next to”. Group counts and relationships were then compared with clinical features using a Student’s t-test or Kruskal-Wallis test.

Results:
A greater number of cells expressed PDL1 in KRAS+ LUAD. While the total number of CD8+PD1+ T-cells did not differ between KRAS+ and EGFR+ LUAD, there was an observed increased proximity between PDL1+ cells and CD8+PD1+ T-cells in KRAS+ LUAD. In EGFR+ LUAD, CD8+ T-cells that did not express PD1 were primarily localized in PDL1 negative regions. Both EGFR+ LUAD and never smokers harbored a higher proportion of CD8- T-cells and CD3-CD8+ immune cells. Both immune cell types were frequently localized in clusters with CD8+ T-cells. KRAS+ LUAD and smokers had increased B-cell counts. No significant associations of PD1 and PDL1 expression were found with stage; however, there was a statistically significant increase in proximity between varied immune cell types as stage and tumour size increased.

Conclusion:
Our method enabled identification of specific cell-cell spatial relationships within LUAD that are associated with smoking history and KRAS/EGFR mutation. Despite limited sample size, we observed an increased proximity between PDL1+ cells and CD8+PD1+ T-cells in KRAS+ LUAD. TME single cell profiling and cell sociology is a promising method to improve stratification of patients for immune-checkpoint-blockade therapies and opens new avenues to explore the complex cell-cell interactions within the TME.

Background:
Anti-PD1 monoclonal antibodies have demonstrated survival advantage over conventional chemotherapy in progressive metastatic non-small cell lung cancer (NSCLC). The prognostic role of tumoral expression of PD-L1 in NSCLC remains conflicting. We performed this study to evaluate the impact of PD-L1 expression as a prognostic marker in non-metastatic NSCLC.

Methods:
NSCLC patients (pts) who underwent curative resection between 1998 and 2006 in St. James’s Hospital, Dublin were included. PD-L1 status was assessed using Ventana SP124 antibody on archival FFPE surgical tumor specimens, arrayed on tissue microarrays (TMAs) with triplicate 0.6 mm cores. PD-L1 was scored as positive if membranous staining was present in >1% of tumor cells aggregated across the replicate cores to address heterogeneity. Clinical characteristics of the pts were obtained from the hospital electronic database including age, gender, histological subtype, smoking status, grade, tumor size, nodal status, stage and survival data.

Results:
One-hundred and forty-seven patients from our institutional database were included, of which 92 (63.0%) were males, with a median age of 65 years (range: 42-82). 53.1% (n=78) with squamous histological subtype, 43.5% (n=60) were ex-smoker and 50.3% (n=74) had Stage I disease. PD-L1 positivity vs negativity among non-smoker, ex-smoker and current smoker were 13.0% vs 20.9%, 47.8% vs 43.3% and 39.1% vs 35.8% respectively, (p=0.708). PD-L1 expression by IHC was significantly higher in squamous NSCLC compared to non-squamous NSCLC (34.7% vs 14.6%, p=0.030). We also noted increased PD-L1 positivity with rising tumor T stage (T1 vs T2 vs T3 vs T4: 7.1% vs 30.6% vs 0% vs 60%, p=0.023) and grade of differentiation (G1 vs G2 vs G3: 11.1% vs 19.6% vs 44%, p=0.039). There was no correlation between nodal status and PD-L1 expression (N0 vs N1 vs N2: 25.5% vs 25% vs 26.3%, p=0.995). PD-L1 expression appears to be independent of overall disease stage (I vs II vs III: 27.3% vs 22.7% vs 25.0%, p=0.921). The median overall survival for PD-L1 positive vs negative pts was 22.1 vs 20.8 months with HR of 0.64 (95% CI: 0.34-1.12, p=0.123). Overall survival rates of pts with PD-L1 positive vs negative tumors at 2 years were 47.8% vs 44.8% and at 5 years were 43.5% vs 26.9%.

Conclusion:
In our cohort, PD-L1 expression was not associated with poorer survival among resected NSCLC pts. Tumour size and grade of differentiation appear to correlate with PD-L1 expression which warrants further validation in future studies.

Background:
CEA, CYFRA21-1 and NSE are tumor markers acknowledged as useful predictors of response to chemotherapy for advanced adenocarcinoma, squamous and small-cell lung cancer, respectively. However, their role in cancer immunotherapy needs to be investigated.

Methods:
We analyzed 56 patients with advanced NSCLC treated with nivolumab (3 mg/kg) every 14 days within a single-institutional translational research study. Blood samples were collected at baseline and at each cycle up to 5 cycles, and then every two cycles. All patients underwent a CT-scan every 4 cycles and responses were classified according to RECIST and Immune-Related Response Criteria (irRC). The serum level of CEA was measured with a Chemiluminescent Microparticle Immunoassay while CYFRA21-1 and NSE with an Immuno Radiometric Assay. The markers levels at baseline and after 4 cycles were used to analyze the relationship between their median variation and the objective response rate (ORR). The performance of tumor markers in predicting ORR was analyzed by ROC analysis and a reduction of 20% was used as cut-off level.

Results:
Forty-eight patients were evaluated: median age: 71 years (44-85); male/female: 73%/27%; current or former smokers: 87.5%; non-squamous/squamous histology: 79%/21%. Baseline median levels were 4.8 ng/ml for CEA, 3.47 ng/ml for CYFRA21-1 and 7.51 ng/ml for NSE. At baseline, values over the upper normal limit of CEA, CYFRA21-1 and NSE were detected in 23 (48%), 26 (54%), and 7 (14%) patients respectively. Significant differences were observed between responders and non-responders and CEA variation (-9% vs.+41%, p=0.003 for RECIST; -10% vs.+31%, p=0.015 for irRC), CYFRA21-1variation (-39% vs.+92%, p<0.001 for RECIST; -35% vs.+72%, p=0.003 for irRC) and NSE variation (-30% vs.+23%, p=0.005 for RECIST; -23% vs.+36%, p=0.004 for irRC). Significant correlations were observed between CEA and CYFRA21-1 decrease with RECIST or irRC: with RECIST, a decrease of 20% of CEA was achieved in 43% of responders and in 8% of non-responders (p=0.013), while a decrease of 20% of CYFRA21-1 occurred in 67% of responders and in 8% of non-responders (p<0.007). With irRC, a decrease of 20% of CEA was achieved in 42% of responders and in 9% of non-responders (p=0.018), while a decrease of 20% of CYFRA21-1 occurred in 58% of responders and in 14% of non-responders (p=0.002). Multivariate analysis confirmed the positive association between CYFRA 21-1 (≤20%) and ORR (RECIST: p=0.004; irRC: p=0.016).

Conclusion:
The reduction in serum level of CEA and CYFRA21-1 might be a reliable biomarker to predict immunotherapy efficacy in NSCLC patients.

Background:
The communication between epithelial cells and their underlying stroma is an important but poorly understood aspect of organismal biology. If aberrantly regulated, these interactions can prove to be tumorigenic. Although it has been known for years that cancer-associated fibroblasts (CAFs) promote and sustain the growth of tumors, the underlying mechanisms remain incompletely understood. Previous work in our lab has identified a novel mechanism of communication in which CAFs secrete cardiotrophin-like cytokine factor 1 (CLCF1), a cytokine that binds ciliary neurotrophic factor receptor (CNTFR) on tumor cells and promotes neoplastic growth. CNTFR is a component of the tripartite receptor complex formed by CNTFR-gp130-LIFR and is capable of activating several oncogenic signaling cascades, including JAK-STAT.

Methods:
Patient tumor and normal samples were collected and plated as CAFs and normal lung fibroblasts (NLFs), respectively. The effect of CNTFR knockdown was assessed by shRNA in A549 xenografts, and the CNTFR decoy receptor was evaluated using patient-derived xenograft (PDX) models.

Results:
Independent TCGA analyses of CNTFR and CLCF1 expression levels in non-small cell lung cancer (NSCLC) patients revealed that increased levels of both genes correlate with poor patient outcomes. In isolated pairs of human CAFs and NLFs from the same patient, gene expression analyses consistently demonstrated a higher level of CLCF1 in CAFs. In vitro studies using three NSCLC cell lines–A549, H23, and H358–showed that CNTFR knockdown decreases proliferation whereas exogenous recombinant CLCF1 increases growth. We repeatedly observed decreased protein levels of phosphorylated STAT3 and phosphorylated ERK upon CNTFR knockdown, thus implicating two canonical oncogenic pathways: Jak-STAT and Ras-Raf-MEK-ERK. Using xenograft models, we found that CLCF1 overexpression by CAFs increases tumor growth while knockdown of CNTFR in lung tumor cells decreases overall growth. With the use of advanced protein engineering technology, we generated a high-affinity CNTFR decoy that inhibits CLCF1-CNTFR signaling and are currently testing this novel reagent to elucidate the mechanism by which CNTFR activation alters intercellular signaling to increase tumor cell growth. Through in vivo studies with cell lines and PDXs, we are also exploring the efficacy of this CNTFR decoy as a form of lung cancer therapy.

Conclusion:
In sum, these data indicate that CLCF1-CNTFR signaling is important for NSCLC tumor growth and is a viable therapeutic target.

Background:
It is known that in many cancers recorded violations in the erythroid hematopoiesis. In order to determine these deviations predicative of significance in the assessment and prediction of malignancy conducted studies on patients with lung cancer.

Methods:
The study group included 28 men aged 45 to 72 years and three women aged 45 - 50 years; the tumor stages: T1N0M0 - 1; T2N0M0 - 10; T2N1Mh -6; T2N2M0 -4; T3N0M0 - 6; T3N0M1 - 2; T3N2M0 - 2. All patients underwent specific anticancer therapy. As a comparison group used clinically healthy people of the same sex and age. Characteristics of red blood was obtained for clinical analysis using an automated hematology analyzer. On the kinetics of acid hemolysis of red blood cells (Gitelzon test) evaluated their resistance and the volume fraction of young erythrocytes. By enzyme immunoassay the concentration of hypoxia-induced factor 1 α in serum using commercial kits Human hypoxia-inducible factor 1α («Cusabio», France).

Results:
As an example, the data obtained during the examination preoperatively patient 50 years old, diagnosed with lung cancer at stage T1N0M0. It was registered a significant increase in serum concentrations of HIF 1 α, exceeding the number of red blood cells and hemoglobin concentration of peripheral blood when compared with reference values ​​of parameters. Against this background, it increases resistance to acid lyse erythrocytes, and the volume fraction of young erythrocytes increased to 46% to 16% of clinically healthy individuals. Stimulation of erythroid hematopoiesis additionally recorded and release into the blood reticulocytes with a low degree of maturity. Additionally, peripheral blood decreased platelet count, and the postoperative period was complicated by bleeding without an identified source. Reaction red germ hematopoiesis and coagulation in this patient may be due to the influence of factors released into the blood tumor.

Conclusion:
These results confirm the fact that tumors are able to exert influence on the state of a number of functional systems, and thus are able to exacerbate the underlying disease. A pooled analysis of hematological and biochemical parameters of blood can yield information on the formation of an aggressive tumor phenotype.

Background:
Patients with borderline performance status (PS), multiple co-morbidities or who are frail have increased chemotherapy toxicity. With an ageing population, more NSCLC patients are presenting with these characteristics. Improved assessment is required to distinguish those patients likely to benefit from therapy from those where treatment precipitates significant functional decline. The Newcastle 85+ study identified circulating biomarkers associated with frailty in a cross-section of adults aged 85 years (n=845). Their utility in NSCLC is not known.

Methods:
Samples from 161 patients (median age 65; range 33 to 81) with advanced NSCLC (stage 3B/4) and PS adequate to consider systemic therapy (34% PS0; 53% PS1; 13% PS2) were analysed for biomarkers of frailty at Northern Institute for Cancer Research and Newcastle Institute for Ageing , Newcastle UK. Biomarkers included telomere length, adiponectin, high-sensitivity CRP (hsCRP), IGFBP1, TGF-β, Alpha-1 acid Glycoprotein (AAG) and FLT3 ligand. Full blood count, liver function tests, serum creatinine, CR-51 EDTA glomerular filtration rate and survival were extracted from patients’ clinical notes.

Results:
Age and line of therapy were not predictive of survival. As expected PS 2 was associated with significantly worse survival (70 days vs 315 days (PS 0-1), p<0.0001) and was associated with significantly higher biomarkers of inflammation (high hsCRP, AAG, TGF- β and neutrophils, low albumin and haemoglobin). In PS 0-1 patient’s high hsCRP, shorter telomere length and low adiponectin were associated with poor survival. An exploratory risk score combining PS and biomarkers was a stronger predictor of prognosis than PS alone (Figure 1); HR 3.2 (95%CI, 2.0 to 5.2), p<0.0001, and seemed to be particularly useful in patients >65years; HR 4.1 (95%CI, 2.0 to 8.4), p<0.0001.Figure 1



Conclusion:
Circulating biomarkers of frailty are associated with a poor prognosis in NSCLC patients and may give additional information over PS. Prospective validation and assessment of the utility in guiding therapy choices is now needed.

Background:
Epithelial cell transforming 2 (ECT2) is a guanine nucleotide exchange factor (GEF) for Rho family GTPases including RhoA, Rac1, and Cdc42. In normal cells, ECT2 is localized in the nucleus,where it regulates dynamic processes including the cell cycle and cytokinesis. On the other hand, several studies have suggested that ECT2 signaling promotes tumor proliferation, migration, and invasion in non-small cell lung cancer. Recently, Murata et al. demonstrated that ECT2 is amplified in early invasive adenocarcinoma but not in situ adenocarcinoma (Cancer Sci, 105:490, 2014). However, the oncogenic mechanism whereby ECT2 drives cell transformation in lung adenocarcinoma is still unknown

Methods:
Cellular fractionation assay was conducted using nine lung adenocarcinoma cell lines Calu-3, A549, RERF-LC-KJ, NCI-H1650, PC-9, NCI-H23, NCI-H1975, LC-2/ad, and HCC827. Immunoblotting, Immunofluorescence, and Immunohistochemistry assays were used to evaluate the expression and localization of ECT2. For ECT2 amplification, nine lung adenocarcinoma were genetically examined using Quantitative Real-Time PCR. Immunoprecipitation was used to examine the interaction between ECT2 and PKCι. And ECT2 siRNA was confirmed the effect of ECT2 on the downstream singling pathway.

Results:
In this study, we showed that ECT2 was localized predominantly in the nucleus of normal lung epithelial cells, whereas tumor cells in nine lung adenocarcinoma cell lines expressed ECT2 protein to differing degrees in their cytoplasm. Importantly, high expression of cytoplasmic ECT2 in surgically resected materials was significantly associated with poor outcome. Moreover, our data showed that overexpression of ECT2 mRNA was roughly correlated with ECT2 amplification in lung adenocarcinoma cell lines. We then investigated the mechanism underlying the cytoplasmic localization of ECT2 and its oncogenic activity in lung adenocarcinoma using the lung adenocarcinoma cell lines Calu-3, A549, RERF-LC-KJ, NCI-H1650, PC-9, NCI-H23, NCI-H1975, LC-2/ad, and HCC827. We found that the cytoplasmic ECT2 was phosphorylated and bound to protein kinase C iota (PKCι) in the cytoplasm. We also observed that the overexpression of cytoplasmic ECT2 greatly increased its degree of phosphorylation and enhanced its interaction with PKCι, resulting in significant promotion of tumor growth through activation of the Mek1,2/Erk1,2 cytoplasmic downstream signaling pathway.

Conclusion:
These results indicate that aberrant cytoplasmic localization of ECT2 is a specific feature of lung adenocarcinoma and important for its malignant progression. This finding offers new insight into the molecular mechanism responsible for aberrant cytoplasmic localization of ECT2, which is correlated with the progression of malignancy, and highlights cytoplasmic ECT2 expression as a new prognostic biomarker in lung adenocarcinoma.

Background:
Mucin 1 (MUC1) is a cell membrane glycoprotein overexpressed in many human cancers, including non­small cell lung cancer (NSCLC). Its role has been implicated in carcinogenesis of premalignant lung lesions in animal models and in clinical association with prognosis in NSCLC. Thus, MUC1 has been a target of interest for vaccine strategies as an mmunomodulatory approach to lung cancer treatment and prevention.

Methods:
Tumor samples from 38 patients with biopsy­-proven NSCLC were assessed for MUC1 expression as determined by immunohistochemistry, expressed on a 0 to 3 scale. Levels of MUC1 expression in areas of dysplasia, metaplasia, bronchoalveolar carcinoma (BAC) and carcinoma present within the same tissue sample were characterized independently and compared using the paired t­test. Clinical data including patient characteristics, staging, treatment and survival were also assessed for correlation with MUC1 expression.

Results:
16 patients with squamous and 19 patients with glandular lesions had tumor samples that were satisfactory for analyses. Among squamous lesions, there was a significant increase in MUC1 expression score in dysplastic compared with metaplastic areas (mean difference = 0.83, 95% CI, 0.21 to infinity; p = 0.021). We also observed an increase in squamous cell carcinoma compared with dysplastic areas (mean difference = 0.44, 95% CI, ­0.006 to infinity; p = 0.052). Among glandular lesions, there was a non­significant increase in MUC1 expression in adenocarcinoma compared with BAC (mean difference = 0.20, 95% CI, ­0.055 to infinity; p = 0.094). According to the Spearman correlation test (p = 0.020 for carcinoma score; p = 0.008 for dysplasia score), a significant positive correlation was observed between MUC1 expression and survival in patients with squamous lesions; however, no significant correlation was observed between MUC1 expression and survival in patients with adenocarcinoma.

Conclusion:
MUC1 overexpression appears to be increased with the progression of premalignant lung lesions to invasive carcinoma in patients with NSCLC. This supports the rationale for MUC1 as a therapeutic target in tumor vaccines that could be ultimately used to prevent or reverse precancerous lesions and treat lung cancer.

Background:
Lung adenosquamous carcinoma (ASC) is a rare malignant tumor with an adenocarcinoma and a squamous cell carcinoma component, and associated with a lower 5-year survival rate than lung squamous cell carcinoma and lung adenocarcinoma. Surgical specimen histology revealed inadequacy of conventional transbronchial needle aspiration sample in the diagnosis of lung ASC. Most of lung ASC patients are not suitable to receive surgery, and it is difficult to diagnosis ASC.This study is to explore the possibility of using serum carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC) as a supplementary diagnostic test for ASC.

Methods:
We retrospectively analyzed the preoperative serum CEA and SCC levels in 34 patients with lung ASC, 35 cases of lung adenocarcinoma patients, 35 cases of lung squamous cell carcinoma patients. 36 cases of lung benign disease patients and 35 cases of healthy people as control group were also retrospectively collected and analyzed from January 2012 to December 2014 at the Zhejiang Cancer Hospital, China. The differences of CEA and SCC among the groups were evaluated, and the area under the curve (AUC), sensitivity and specificity were calculated.

Results:
The levels of SCC and CEA in lung ASC group were significantly higher than those in healthy control group and benign disease group (P<0.05), and SCC level in lung ASC group was significantly higher than that in lung adenocarcinoma group (P<0.05). CEA and SCC had good diagnostic sensitivity and specificity compared with the healthy control group, and the difference was statistically significant (P<0.05).

Conclusion:
Our retrospective study suggested a role for serum CEA and SCC levels as reference markers in the diagnosis of lung ASC. If the patients which CEA and SCC levels were elevated and diagnosed as lung adenocarcinoma by limited biopsy materials should be offered further work-up to reach an accurate diagnosis and treatment.

Background:
Aminoacyl t-RNA synthetase-interacting multi-functional proteins (AIMPs) are scaffolding protein required for the assembly of the t-RNA synthetase complex, forming multisynthetase complex. Besides their inherent roles, AIMPs translocate to the other cellular compartments and involve in various cellular pathways. On the other hands, its alternative spliced form lacking exon2 (AIMP2-DX2) compromises the tumor suppressive activity of AIMP2. Recently, presence of autoantibodies against AIMP2-DX2 and AIMP2 were identified in the human blood but its clinical implication is unknown.

Methods:
The diagnostic usefulness of blood autoantibody against AIMP2-DX2 and AIMP2 was investigated in 80 lung cancer cases and 1:1 age, gender and smoking status matched control cases using ROC curve. To exploit their clinical implication, blood level of autoantibody against AIMP2-DX2, AIMP2 and AIMP2-DX2/AIMP2 ratio was analyzed with clinical parameters in 165 lung cancer patients.

Results:
There was no statistically significant difference in the blood autoantibody level against AIMP2-DX2 and AIMP2 between lung cancer and control cases. However AIMP2-DX2/AIMP2 ratio was higher in lung cancer patients (30.7±12.6 vs. 39.1±18.4, P=0.001, t-test). When their diagnostic usefulness was evaluated by ROC curve generation, the AUC of auto-antibody level of AIMP2-DX2, AIMP2, and AIMP2-DX2/AIMP2 ratio were 0.416, 0.579 and 0.357 respectively, suggesting their diagnostic value in lung cancer is limited. A total 165 lung cancer patients were classified into 2 groups, high and low group, on the basis of median value of AIMP2-DX2, AIMP2, and AIMP2-DX2/AIMP2 ratio, respectively, and then further analyzed. There was no statistical difference in the gender, smoking, pathologic diagnosis and stage between high and low group of AIMP2-DX2, AIMP2, and AIMP2-DX2/AIMP2 ratio. But AIMP2-DX2 high group was older than those with lower AIMP2-DX2 group (66.8±8.4 vs. 63.8±10.1 years, P-value=0.040, t-test). When the relationship with CEA and CYFRA-21 was evaluated, the AIMP2-DX2/AIMP2 high group showed higher CYFRA-21 level (7.9±12.1 vs. 4.3±4.3 ng/mL, P-value=0.020, χ[2]-test). There was no significant relationship between AIMP2-DX2 and AIMP2 concentration with progression free survival and overall survival. But the patients with high AIMP2-DX2/AIMP2 ratio showed significant short overall survival (18.6 (95% CI: 15.19~22.00) vs. 48.9 (95% CI: 14.89~82.91 months), P-value=0.021, Log-Rank Test).

Conclusion:
Autoantibodies against AIMP2-DX2 and AIMP2 exist at detectable level in human blood. Increased AIMP2-DX2/AIMP2 ratio is closely related to the poor clinical outcome of lung cancer patents, indicating those are warranted for further study for development as biomarkers in lung cancer.

Background:
Previous data on the prognostic value of vascular endothelial growth factor (VEGF), E-cadherin and CD44 in non-small cell lung cancer (NSCLC) remain limited and largely controversial. The primary aim of this study was to further investigate these proteins, along with other well-studied biomarkers of prognosis, as predictors of overall survival (OS) in NSCLC.

Methods:
Formalin-fixed and paraffin-embedded tissue specimens from 77 surgical and 41 autopsy cases, were retrieved and evaluated by immunohistochemistry (IHC) for the expression of VEGF, E-cadherin, CD44, p53, Ki-67 and thyroid transcription factor -1 (TTF-1). Immunohistochemical findings were correlated with gender, age, primary tumor location/side, tumor histology and grade and disease stage at diagnosis. The association of clinicopathologic variables and IHC results with overall survival (OS) was assessed –only in the surgical subgroup- by univariate and multivariate Cox regression analysis.

Results:
Mean age of all cases (N=118) was 64.8 years (SD=11.1 years), while the majority were men (104/118, 88.1%). Adenocarcinoma was the predominant histological type (38.1%), while most cases (62.6%) had stage II disease at diagnosis. E-cadherin and CD44 expression were significantly correlated with lower tumor grade and disease stage at diagnosis, both in the total sample and in the surgical and autopsy subgroups. Positive VEGF expression was also correlated with lower grade and stage, in the total sample and the autopsy subgroup, but not in the surgical subset of cases. Positive E-cadherin and CD44 expression were associated with improved OS, both in univariate analysis (p=0.006 and p=0.011, respectively), as well as in the multivariate model, after adjusting for sex, age, tumor location, histology, grade and stage (HR=0.08, 95% CI: 0.09-0.65, p=0.019 and HR=0.07, 95% CI: 0.09-0.63, p=0.017, respectively).

Conclusion:
Our study findings suggest that positive E-cadherin and CD44 immunostaining may represent independent predictors of an improved survival in NSCLC. Larger prospective studies are nevertheless warranted to confirm the independent prognostic value of these candidate biomarkers.

Background:
Embryonic antigens, such as carcinoembryonic antigen (CEA) and alfa-fetoprotein (AFP), are routinely employed as serum and immunohistochemical tumor markers in clinical medicine. Since they are not expressed in adult human tissues, it is reasonable to conclude that embryonic antigens are extremely specific tumor markers. However, no systematic studies have yet identified clinically useful embryonic antigens for tumor diagnosis. In the present study, we developed a strategy for systematic identification of lung adenocarcinoma markers using monoclonal antibodies generated from embryonic swine tissue. Swine mRNA shows more than 80% homology with human mRNA, and embryonic swine tissue is thought to be a useful material for detection of human embryonic markers.

Methods:
In order to produce mouse monoclonal antibodies, we immunized BALB/c mice by injection of fetal swine lung nuclear fraction into the hock, and used a human lung adenocarcinoma nuclear fraction for the second immunization. We recovered lymph nodes from the mice, and fused mouse B lymphocytes with the murine myeloma cell line SP2/0 using polyethylene glycol. The resulting hybridomas were then selectively cultured. For selection of interesting hybridoma clones, we performed immunohistochemical staining using the supernatant from each one, with tissue microarray loading swine fetal and adult lung, human lung cancer and normal lung tissue.

Results:
Immunohistochemical screening of 284-clones showed that the antibodies derived from four of them were strongly reactive with the cytoplasm and cytomembrane of fetal swine lung and human lung cancer. We then focused on one clone (B246) whose antibody reacted most clearly with human lung adenocarcinoma cells . Protein microarray analysis confirmed that B246 reacted specifically with “drebrin”, one of the actin binding proteins, originally identified in neuronal cells. There are two drebrin isoforms in human tissue: drebrin E (embryonic) is abundant in the developing neurons, and drebrin A (adult) is expressed in adult brain. We then examined the association of the drebrin expression pattern with the pathological features and prognosis of lung adenocarcinoma using 200 selected cases for which formalin-fixed and paraffin-embedded samples were available. Drebrin immunohistochemistry delineated the samples into those with strong (n=85) and weak (n=115) drebrin expression. Kaplan-Meier analysis demonstrated a significant difference in disease-free survival (DFS) between the groups with strong and weak drebrin expression (p=0.033) .

Conclusion:
Drebrin is expressed specifically in lung adenocarcinoma and is associated with outcome. The present findings indicate that drebrin is a new marker of lung adenocarcinoma and indicative of prognosis.

Background:
Tumor markers such as CEA and CYFRA 21-1 have been shown to be effective in patients with non-small cell lung cancer as an aid in diagnosis and monitoring response to treatment. CYFRA 21-1 is a fragment of cytokeratin 19, presented in the cytoplasm of tumor cells of epithelial origin. CEA or Carcino-Embryonic Antigen is a cell surface protein.

Methods:
We recruited advanced non-small cell lung cancer patients who received first line treatment with standard chemotherapy between February and September 2015 in Maharaj Nakorn ChiangMai hospital. Excluded criteria were patients who had history of other active cancers and who had end stage renal disease ( confounding factors to serum CYFRA measurement). Clinical data was obtained and a blood sample was collected at baseline, after start chemotherapy at cycle 2nd and at the end of treatment. Serum tumor marker levels were determined with a test kit (Roche Diagnostics Corp) using a Cobas e 411 analyzer. Primary objective outcome is correlation in dynamic change of serum CYFRA and CEA level compare to clinical response from radiologic assessment. Secondary objective outcome Secondary objective outcome is optimal cut point to predict the treatment outcome from sensitivity and specificity analysis. Statistical Analysis Multivariable logistic regression analysis was used to identify the effect of changed value of CYFRA-21. C-statistic was used to identify the optimal cut of point of changed value of CYFRA-21 to predict the outcome of treatment demonstrated by area under receiver operating characteristic curve (AuROC). The p-value < 0.05 was considered statistically significant. All statistical analysis was performed using STATA program (version 12.0).

Results:
Forty patients (24 males and 16 females) were enrolled .The median age was 59.8 years. Histology subtypes were adenocarcinoma (70%), squamous cell (22.5% ), large cell carcinoma (5%) and NOS(not otherwise specified) (2.5%).The treatment responses were partial response (50%), stable of disease (27.5%) and progression of disease (22.5%). The result demonstrated significant correlation between dynamic change of serum CYFRA level and clinical benefit from radiologic assessment. Mean change value of CYFRA-21 before and after treatment between clinical beneficial group (PR + SD) and PD group were -7.7±9.2 and 12.5±23.2 respectively (p<0.001). In contrast dynamic change of serum CEA did not show significant correlation.For secondary end point, at cut point of 2 ng/ml reduction of CYFRA level after treatment had the most accepted from AuROC curve.

Conclusion:
CYFRA 21-1 have capability to predict benefit of treatment from chemotherapy in non-small cell lung cancer.

Background:
Approved anti-programmed cell death-1 (PD-1) therapies have produced durable responses in advanced non-small cell lung cancer (NSCLC), but objective response rates in unselected populations remain modest at approximately 20%. As a result, therapies targeting other immune checkpoints are currently being investigated as monotherapy or in combination with anti-PD-1 therapy. One such immune checkpoint is T-cell immunoglobulin and mucin-domain containing 3 (TIM-3), which is involved in T-cell exhaustion and has also been found on NSCLC tumor cells, more frequently in adenocarcinoma. The present study sought to further characterize the expression of TIM-3 in resected NSCLC specimens via microarray analysis.

Methods:
Gene expression microarray analysis was performed using the Agilent Whole Human Genome 4x44K 2-color platform for 319 NSCLC and 15 normal lung resection specimens. The reference sample was an equal mixture of 258 of the NSCLC samples included in the study. Microarray data was imported into Rosetta Resolver for analysis. Samples with expression significantly greater than the reference level were classified as high, samples with expression unchanged from the reference were classified as moderate, and samples with significantly lower levels were classified as low (P<0.01). Relationships between TIM-3 expression and smoking status, histology, T stage, and gender were evaluated with the chi-square test. The three survival curves based on TIM-3 expression were compared and a single p-value based on chi-square test was determined using Statistica 13.0.

Results:
Within the 319 NSCLC tissue samples, 90 samples (28%) had high TIM-3 expression, 150 samples (47%) had moderate expression, and 79 samples (25%) had low expression. Interestingly, 47% (7/15) of normal lung samples evidenced high TIM-3 expression, while none had low TIM-3 expression. Tumors with adenocarcinoma histology had a greater percentage of samples with high TIM-3 expression, 34%, compared to those with squamous cell histology, 17% (p=0.03). Gender and T stage were not significantly related to TIM-3 expression level, while a trend towards high TIM-3 levels was observed in smokers compared to non-smokers (p=0.10). In this surgical cohort, TIM-3 expression did not appear to be prognostic for survival.

Conclusion:
Our findings suggest that high TIM-3 expression occurs frequently in resected NSCLC, supporting the ongoing evaluation of anti-TIM-3 therapy in NSCLC. Additionally, TIM-3 expression was more frequently high in adenocarcinoma, normal lung, and a trend towards high expression was noted in smokers. Future efforts will focus on identifying cell type specific TIM-3 expression via immunohistochemistry analysis and selecting patients for anti-TIM-3 clinical trials.

Background:
Lung cancer and chronic obstructive pulmonary disease (COPD) share a common etiology. Despite the known associations of alpha-1 antitrypsin (AAT) deficiency with COPD and COPD with lung cancer, few studies examined the association of AAT and lung cancer. We have investigated AAT serum levels in PiMM non-small cell lung cancer (NSCLC) patients with respect to PiMM controls with COPD and benign lung nodules since AAT is an acute-phase protein. The AAT tumor tissue expression was analyzed in NSCLC group to evaluate the potential contribution of cancer cells in AAT production.

Methods:
Serum and matched FFPE tissue samples were collected from 194 NSCLC patients (stages I-IV) with PiMM phenotype of AAT. The serum concentrations of AAT and CRP were measured by nephelometry. The AAT protein expression in NSCLC tumor cells was assessed by immunohistochemistry. Reference groups consisted of 183 PiMM COPD and 50 PiMM patients with benign lung nodules (hamartoma, tuberculoma, granuloma and other).

Results:
In NSCLC patients mean AAT serum concentration (195.5 mg/dl) was significantly higher than in COPD group (171 mg/dl) and patients with benign lung nodules (154 mg/dl; p<0.0001). AAT concentration was significantly higher in SQC type (202 mg/dl) than ADC (175 mg/dl; p<0.029) patients, and in advanced (IIIb-IV, 247 mg/dl) versus early stage disease (I-IIIa, 190 mg/dl, p<0.0001). AAT levels significantly correlated with CRP (R=0.6; p<0.0001), however CRP level did not differentiate NSCLC from COPD. Importantly, the strong AAT expression observed in tumor tissue was positively associated with the higher AAT blood levels, while weak or no AAT expression directly correlated with the lower AAT blood levels (p<0.0079).

Conclusion:
We have demonstrated for the first time that the local production of AAT by tumor cells significantly contributes to the high levels of AAT in blood of NSCLC patients. The significant association of serum AAT levels with stage and histology of NSCLC may implicate clinical use of AAT as a biomarker or therapeutic target.

Background:
Nuclear grading can prognostically estimate inter-observer reproducibility in pulmonary adenocarcinoma (Nakazato Y. et al, Cancer, 2010 and JTO, 2013). However, no correlation has been shown between pathologic prognostic marker, number of cancer cells, and survival in pulmonary adenocarcinoma cases. Immunohistochemistry for phos­phorylated histone 3 (pHH3), which is present during early prophase, is a reliable mitosis-specific marker. We evaluated the correlation between pHH3-stained mitotic figures (PHMFs) and clinical outcome, comparing the results with those of PHMFs, Ki-67 labeling index, and number of cancer cells.

Methods:
Primary tumors were obtained from 104 patients with pulmonary adenocarcinomas (≤2 cm maximum dimension) who were treated surgically between January 2006 and December 2010 at Dokkyo Medical University Hospital. Specimens were stained with hematoxylin and eosin and pHH3 and anti-Ki-67 antibodies. Cells were enumerated with a NanoZoomer[®] Digital Pathology. Results were evaluated using receiver operating characteristic (ROC) curve analysis, the Kaplan–Meier method and Cox proportional hazards regression.

Results:
Cases judged negative by nuclear grading had significantly improved prognoses compared with positive cases (mean overall survival, 8.923 vs. 7.884 years; p=0.03). ROC curve analysis showed a cut-off of 400/10 hpf (area under the curve = 0.743; 95% CI = 0.594-0.891). Cancercell index, defined as the number of cancer cells within 10 hpf, of ≥400 tended to be positive, and of <400 tended to be negative. PHMF/cancercell index of ≥0.01 tended to be positive, and of <0.01 tended to be negative. PHMF/cancercell index (HR, 6.022), cancercell index (HR, 6.399), and lymphatic invasion (HR, 5.308) were correlated with prognosis (p<0.02). The number of cancer cells was correlated with Noguchi’s classification and WHO pathologic type (figure).Figure 1



Conclusion:
PHMF/cancer cell index is useful for prognostic evaluation of pulmonary adenocarcinoma. PHMF/cancercell index, cancercell index, and lymphatic invasion are strongly correlated with prognosis. The number of cancer cells correlates with Noguchi’s classification and WHO pathologic types.

Background:
Lung cancer is the leading cause of cancer-related mortality worldwide with a 5 year survival rate of 15%. Non-small cell lung cancer (NSCLC) is the most commonly diagnosed form of lung cancer. Cisplatin-based regimens are currently the most effective chemotherapy for NSCLC, however, chemoresistance poses a major therapeutic problem. New and reliable strategies are required to avoid drug resistance in NSCLC. Cell division cycle associated 3 (CDCA3) is a key regulator of the cell cycle. CDCA3 modulates this process by enabling cell entry into mitosis through degradation of the mitosis-inhibitory factor WEE1. CDCA3 itself is also degraded in G1 yet re-expressed in G2/M phase, to allow successful progression through the cell cycle. Herein, we describe CDCA3 as a novel prognostic factor in NSCLC and target to delay or prevent cisplatin resistance in NSCLC.

Methods:
CDCA3 expression was investigated in squamous and non-squamous NSCLC using several approaches including bioinformatic analysis of publicly available datasets, immunohistochemistry of a tissue microarray and western blot analysis of matched tumour and normal tissue and NSCLC cell lines. CDCA3 function in NSCLC was determined using several in vitro assays by siRNA depleting CDCA3 in a panel of three immortalized bronchial epithelial cell lines (HBEC) and seven NSCLC cell lines.

Results:
CDCA3 transcripts and protein levels are elevated in NSCLC patient tissue and highly expressed in tumour cells relative to proximal normal cells. High mRNA levels are associated with poor survival in resected NSCLC. Depletion of CDCA3 in vitro markedly impairs proliferation in seven NSCLC cell lines by inducing a mitotic cell cycle arrest, ultimately resulting in p21-dependent cellular senescence. Importantly, silencing of CDCA3 also greatly sensitises NSCLC cell lines to cisplatin. In line with these in vitro data, NSCLC patients that have elevated levels of CDCA3 and are treated with cisplatin have a poorer outcome than patients with reduced levels of the protein. To improve patient response to cisplatin, we are exploring novel strategies to suppress CDCA3 expression in tumour cells.

Conclusion:
Our data highlight CDCA3 as a novel factor in mediating NSCLC. We propose that evaluating novel strategies to target CDCA3 may prove a useful strategy is enhancing the anti-tumour activity of platinum-based chemotherapy and may ultimately benefit patient outcomes by preventing cisplatin resistance.

Background:
Mitofusin-2(MFN2) was initially identified as a hyperplasia suppressor in hyper-proliferative vascular smooth muscle cells of hypertensive rat arteries, which has also been implicated in various cancers. There exists a controversy in whether it is an oncogene or exerting anti-proliferative effect on tumor cells. Our previous cell cycle analysis and MTT assay showed that cell proliferation was inhibited in MFN2 deficient A549 human lung adenocarcinoma cells, without investigating the changes in regulatory network or addressing the underlying mechanisms.

Methods:
We performed expression profiling in MFN2 knock-down A549 cells. Furthermore, we compared the expression profiling of a cohort consisting of 61 pairs of tumor-normal match samples from The Cancer Genome Atlas(TCGA).

Results:
The expression profiling in MFN2 knock-down cells suggested that cancer related pathways were among the most susceptible pathways to MFN2 deficiency. Next, we teased out the specific pathways to address the impact that MFN2 ablation had on A549 cells, as well as identified a few genes whose expression level associated with clinicopathologic parameters. In addition, transcriptional factor target enrichment analysis identified E2F as a potential transcription factor that was deregulated in response to MFN2 deficiency. Figure 1 Figure 2





Conclusion:
The anti-proliferative effect of MFN2 deficiency and its risk in lung adenocarcinoma were found by transcriptional profiling.

Background:
The 2011 IASLC/ATS/ERS pathological calssification of pulmonary adenocarcinoma(ADC) gives new direction to clinical individualised treatment strategies and prognostic evaluation.We analyzed the prognostic effect of invasive ADC sub-types according to the new classification system.

Methods:
150 invasive lung ADCs resected in West China Hospital from 2008 to 2013 was analyzed in 5% increments, and classified and graded according to their predominant patterns, as proposed by the IASLC/ATS/ERS. Clinical data,including smoking status, chemotherapy/ radiotherapy after surgery and patient outcomes were collected.Overall survival was evaluated.

Results:
Tumor necrosis (p=0.033), poor differentiation (p=0.027), lymph node metastasis (p<0.001), surgical procedures (p=0.010), tumor diameter (p<0.001),and TNM stage (p<0.05） were significantly associated with overall survival (OS). Solid predominant ADCs (SPA) had a shorter OS than non-SPAs (43.5 vs 65.3 months, p=0.014). High-risk group (including SPA and micro-papillary predominant ADCs, MPA for short) had a poorer prognosis than low-risk group (including lepidic predominant , acinar predominant and papillary predominant ADCs, LPA,APA and PPA for short respectively) (44.4 vs 65.1 months, p=0.025). ADCs with papillary growth patterns (PP) had a better OS than those without PP (67.1 vs 42.5 months, p=0.001).In patients treated with chemo-or radiotherapy after surgery, OS of SPA and ADCs with PP were comparable to that of non-SPA and ADCs without PP ,respectively (p value>0.05). Smoking also increased the risk of poor OS in certain subtypes significantly. Multivariate analysis showed SPA, high-risk group and ADCs with PP were independent prognosis factors for OS.

Conclusion:
Growth pattern and grading system are effective prognosticators of OS in invasive lung ADCs, which also influenced by other factors like post-operative chemo-/radio-therapy and smoking status.These results will give an instruction to the future individualized treatment of lung ADCs.

Background:
Cancer-associated inflammation develops resistance to the epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancers (NSCLCs) harboring oncogenic EGFR mutations. The molecular mechanisms how the cytokines produced and activated in the tumor microenvironment such as transforming growth factor-β (TGF-β) and IL-6 regulate EGFR-TKI resistance remain largely unknown.

Methods:
To determine the mechanisms how Smad-mediated TGF-β signaling and STAT3-mediated IL-6 signaling regulate sensitivity and resistance to gefitinib, we treated HCC827 adenocarcinoma cell line harboring an oncogenic deletion within the EGFR (delE746-A750) with gefitinib, an activin receptor-like kinase5 (ALK5) inhibitor, EW-7197 and/or IL-6.

Results:
IL-6 and a TGF-β antagonist, EW-7197 synergized to suppress gefitinib-induced apoptosis of HCC827. Treatment with gefitinib induced interaction between unphosphorylated Smad2 and STAT3 in cytoplasm. IL-6 and/or EW-7197 significantly upregulated phosphorylation of Smad2 linker region. Linker-phosphorylated Smad2 at serine 245 and 255 residues interacted with phosphorylated STAT3 at tyrosine 705 and serine 727 residues to suppress gefitinib-induced apoptosis of HCC827. In contrast with Smad2, IL-6 and EW-7197 synergized to downregulate the expression of Smad3.

Conclusion:
Our data suggest that inhibition of phosphorylation of Smad2 linker region and STAT3 could prevent EGFR-TKI resistance in NSCLCs.

Background:
Lung cancer is the worldwide leading cause of death from cancer. Epigenetic silencing of tumor suppressor genes (TSG) contributes to the development and progression of lung cancer. In this prospective study we assess the methylation profile of locally advanced and oligometastatic versus widespread metastatic Non-Small Cell Lung Cancer (NSCLC). We will determine the ability of a panel of tumor suppressor genes (TSG) to discriminate these clinical phenotypes

Methods:
Patients (≥18 years) were eligible for inclusion if they had histologically confirmed unresectable non-pretreated stage III/IV NSCLC. Paraffin-embedded blocks from patients from this study cohort were macro-dissected based on hematoxylin-eosin evaluations to ensure a minimum of 75% of tumor cells. DNA was extracted. Promoter methylation status of TSG (SFRP5, TIMP3, HLTF, RUNX3, ID4) will be evaluated by EpiTect Methyl II Signature PCR (Qiagen). Data analysis was done using integrated Excel-based templates, which provide gene methylation status as percentage unmethylated (UM) and percentage methylated (M) fraction of input DNA. Methylation levels were described as: ³1-19%: low methylation level, 20-59%: moderate methylation level and ³60%: high methylation level. Presence of intrathoracic disease and limited extrathoracic disease, (M1b, proposed 8th TNM edition) vs multiple metastatic disease (M1c) was described for clinical and molecular analysis.

Results:
From March 2015 to June 2016, 40 out of 60 sixty patients had enough tissue to be included. Intrathoracic disease was present in 19 patients (47.5%), M1b disease in 8(20%) and 13 patients (32.5%) had multiple extrathoracic disease. The methylation profile for intrathoracic disease was: SFRP5 was found in 4/19 patients (21.1%), ID4 7/19 (36.6%), HLTF 12/19 (63.2%), RUNX3 19/19 (100%) and TIMP3 10/19 (52.6%); for M1b disease: SFRP5 was found in 4/8 patients (50%), ID4 3/8 (37.5%), HLTF 6/8 (75%), RUNX3 6/8 (75%) and TIMP3 7/8 (87.5%); for M1c disease: SFRP5 6/13 patients (46.2%), ID4 9/13 (69.2%), HLTF 11/13 (84.6%), RUNX3 13/13 (100%) and TIMP3 8/13 (61.5%). Overall survival was shortened for the group of patients with methylation of ID4³20% (14.5 vs 19 months, p=0.010 Log Rank Test). In the subgroup analysis this difference was sustained for patients with oligoprogressive disease (Intrathoracic plus IVb) vs IVc (9.5 vs 17.5 months, p=0.05 Log Rank Test vs 14 vs 19 months , p=0.51, respectively)

Conclusion:
Although no definitive conclusions can be done because of the sample size, it seems that patients with methylation of ID4 of 20% or more have worse prognosis. ID4 could also help to differentiate oligometastatic vs widespread NSCLC

Background:
Axl receptor tyrosine kinase (RTK) plays a critical role in cell growth, proliferation, and anti-apoptosis. In this study, we demonstrated the effect of luteolin, a non-toxic flavonoid widely found in various plants, on expression and activation of Axl RTK in NSCLC, H460, and its cisplatin-resistant cell, H460/CisR.

Methods:
1. Cell viability measurement & Clonogenic assay. 2. Western blot analysis. 3. Promoter activity test. 4. Ectopic expression of Axl. 5.siRNA trasnfection for Axl knockdown.

Results:
Luteolin treatment of H460 and H460/CisR cells was found to cause a dose‑dependent decline of Axl protein as well as mRNA levels. Axl promoter activity was also reduced by luteolin, suggesting the transcriptional down-regulation of Axl expression by luteolin. Axl phosphorylation upon its ligand, Gas6, was inhibited by luteolin, indicating that luteolin also abrogates Gas6-induced Axl phosphorylation. Next, it was found that treatment of both H460 and H460/CisR cells with luteolin decreased the cell viability and clonogenic ability in dose‑dependent manner. We further observed the synergistic anti-proliferative effect of luteolin in cells transfected with Axl specific siRNA, while the reduction of its cytotoxic effect in Axl RTK overexpressing cells, confirming that luteolin exerts its anticancer potential via interference of Axl expression. In addition, luteolin was found to result in the increase of p21, a cyclin-dependent kinase inhibitor, in H460 and H460/CisR cells.

Conclusion:
In summary, our data demonstrate that luteolin inhibits Axl expression and the activation which are associated with its anti-proliferative activity in both parental and cisplatin-resistant NSCLC cells. Thus, Axl seems to be a potent therapeutic target of luteolin to inhibit cell proliferation and to overcome chemoresistance of NSCLC cells.

Background:
The aim of study to investigate prognostic significance of carbonic anhydrase IX gene (CA IX) mRNA expression in stage III NSCLC patients received neoadjuvant treatment

Methods:
We have studied Carbonic Anhydrase IX (CA IX) mRNA expression at biopsy or surgical pathology tissues of 77 patient with Stage III A/B NSCLC received neoadjuvan treatment. CA IX mRNA expression were evaluated with 50 control .Total RNA were isolated from FFPE tissue from patients while the controls were isolated from the peripheral blood.

Results:
Median age is 56.Patients histology is 47 pts(61%)squamous cell carcinoma (SCC),30 pts(39%) adenocarcinoma(AC).Neoadjuvan Chemotherapy (NeoAdj CT)was given 45 pts(58,4%);Neoadjuvant chemoradiotherapy(neoAdjCT/RT) was given 32pts(41,6%).Median Neoadjuvan chemotherapy cycles is 3(2-6).Radiotherapy median dose was 60 Gy(45-66).Surgery outcomes is Lobectomy 38pts(49,4%);sleeve lobectomy 11 pts(14,3%),Bilobectomy 6pts(7,8%) Left pnemonectomy (18.2%),right pnemonectomy 8pts(10,4%). Recurrence rate was 39 pts(50,6%).Two year disease free (DFS) and overall survival (OS)was 59,6% and 71,4%. There was OS and DFS difference in favor of Neoadj CT 4-6 cycles versus 2-4 cycles(p=0.009 and p=0.034) There is no statistical difference(p=0,344) for CA IX mRNA expression between SCC and AC. There is no statictical difference for CA IX mRNA expression in Neoadj CT and NeoadjCT/RT groups(p=0.199). There is no statistical difference for OS between ≤Median CA IX versus ≥median groups( 20 events/39 versus 20/38 events p=0,799) There is no statistical difference for DFS between ≤median CA IX versus median groups ≥ (19 events/39 versus 20events /38)

Conclusion:
There is no any prognostic significance of Carbonic Anhydrase IX expression on DFS and OS in Stage III A/B NSCLC patients received neoadjuvat treatments.

Background:
The prenylation inhibitor zoledronic acid is a standard-of-care therapeutic option in bone metastasis. Recent studies suggest that prenylation inhibition using novel lipophilic bisphosphonates might be active against various malignancies outside the bone metastatic setting. Since prenylation is an important posttranslational modification in RAS protein function we explored the sensitivity of a panel of lung adenocarcinoma cells representing various oncogenic driver mutations.

Methods:
8 human lung adenocarcinoma cell lines were investigated in vitro to assess the short-term viability and long-term clonogenic potential following zoledronic acid and BPH-1222 treatments. The eight lung cancer cell lines represented wild type (HCC78, CALU3), EGFR- (H1650, H1975) KRAS- (A549, H358), BRAF- (CRL5885) and BRAF + NRAS double mutant (CRL5922) molecular subtypes. Effect on short and long term proliferation were measured with a SRB based photometric assay.

Results:
Neither short-term nor long-term treatment showed significant differences between the proliferation inhibitory effect of the hydrophilic zoledronic acid and novel lipophilic bisphosphonate BPH-1222. Interestingly, we found that the two KRAS mutant lung adenocarcinoma cell lines were more sensitive in the long-term bisphosphonate treatment assays than non-KRAS mutant cell lines. BRAF or EGFR mutations did not show a differential response against these inhibitors.

Conclusion:
In vitro proliferation inhibitory efficacies of hydrophilic and lipophilic bisphosphonates were not different in lung adenocarcinoma cells. Nevertheless, due to the different bone accumulation properties of zoledronic acid and lipophilic bisphosphonates further in vivo preclinical studies are warranted to evaluate the inhibitory effect in a more physiological setting.

Background:
AEG‑1 is important in the aggressiveness of NSCLC and also contributes to induce chemoresistance in treatment of NSCLC. In this study, we will assess the predictive and prognostic values of AEG-1 expression on tumor response and survival according to mRNA concentration by liquid biopsy in NSCLC patients treated with chemotherapy.

Methods:
Patients were diagnosed with a advanced NSCLC (stage IIIB and IV). Patients were enrolled to be treated by chemotherapy as first-line treatment or for metastatic or recurrent disease with Eastern Cooperative Oncology Group (ECOG) performance status of 0–1. All patients underwent blood sampling before any cancer treatment and at first response evaluation. Response to chemotherapy was assessed using RECIST criteria. mRNA was extracted from plasma samples using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA, USA) and quantification of mRNA was performed by real-time PCR (ABI 7900) with SYBR GREEN reagent expression assay for AEG-1.

Results:
A total of 12 patients (9 male and 3 female) with advanced NSCLC received platinum based doublets chemotherapy. Chemotherapy regimens included 8 cisplatin and 4 carboplatin with 5 pemetrexed, 4 paclitaxel, 2 gemcitabine and 1 vinorelbine. 7 of 12 (58.3%) were adenocarcinoma. The initial response rate of chemotherapy included 6 partial responses, 1 stable disease and 5 progressive disease. The expression level of AEG-1 in patients with disease progression after chemotherapy increased significantly compared with the expression level at pre-chemotherapy (AEG-1, relative quantification of mRNA, post-progression, range: 2.14 - 8.61, p = 0.035). In the group of patients with responsive chemotherapy, the mRNA expression level of AEG-1 was not increased compared to the baseline expression (Figure 1). Figure 1



Conclusion:
This result suggests that mRNA concentration of AEG-1 from liquid biopsy could be a predictive biomarker of tumor response. Increased expression of AEG-1 contributed to the chemoresistance and caused lung cancer progression.

Background:
Platinum-based antineoplastic therapies (platins) are a first line treatment for non-small cell lung cancer (NSCLC) that generate DNA adducts leading to the formation of both single and double stranded DNA breaks. Effective DNA damage response pathways contribute to cell survival and resistance to these agents. Ataxia telangiectasia mutated (ATM) is an important mediator of the DNA damage response involved in the activation of key components of DNA repair, cell cycle arrest, and apoptosis. Our lab has demonstrated that cell lines lacking ATM show increased sensitivity to platins. We hypothesize that platin exposure will activate ATM and that cells deficient in ATM will be innately sensitivity to platins due to an impaired DNA damage response. Here we assess the molecular action of ATM in response to platins to determine if ATM-deficiency is predictive of platin sensitivity.

Methods:
ATM status was determined in five NSCLC cell lines using western blotting and rt-qPCR. Cell lines were treated with varying concentrations cisplatin, carboplatin and oxaliplatin for 18 hours and assessed for ATM phosphorylation by western blot. Additionally, downstream targets of ATM (KAP-1, p53, and g-H2AX) were investigated to determine ATM pathway activation. Finally, transient and stable ATM knockdowns were generated using siATM and shATM. These cells were then tested for platin sensitivity by trypan blue viability or clonogenic assays.

Results:
NSCLC cell lines NCI-H226, NCI-H460, and NCI-H522 were found to be ATM-proficient whereas cell lines NCI-H23 and NCI-H1373 were found to be ATM-deficient. ATM-proficient cell lines demonstrated an increased level of phosphorylated-ATM in response to platins. In addition, KAP-1, a downstream target of ATM showed increased phosphorylation in response to these treatments when compared to non-treated controls. In contrast, ATM-deficient cell lines showed no increased levels of phosphorylated ATM or KAP-1 in response to platins. Preliminary analysis of siATM transient knockdowns in NCI-H226 shows an increased sensitivity to cisplatin.

Conclusion:
It is clear that platin exposure activated an ATM mediated signalling response and that cells lacking ATM showed deficiencies in the phosphorylation of key downstream targets of this pathway. Cells deficient in ATM may therefore be more susceptible to platin therapy due to an impaired DNA damage response. This data suggests that individuals with low or non-functioning ATM may be candidates for precision low does therapies that exploit this deficiency.

Background:
Ligands of Toll-like receptors (TLR) are often used as adjuvants in order to enhance the immunogenicity of vaccines in therapy of lung cancer. Such ligands are cell wall biopolymers of gram-positive microorganisms Staphylococcus aureus – techoic acids (TAs). They play a significant role as immunomodulators. Nowadays, agents which, in addition to specific effect on cellular and molecular targets of tumor growth and ischemia, posses the ability to inhibit angiogenesis, are attractive for therapeutic angiogenesis-dependent correction of pathological states, which has vascular-dependent outcome.

Methods:
In order to determine possible mechanisms of TAs+PO244 impact on tumor through immune cells we studied primary Lewis lung carcinoma (LLC) culture after the impact of macrophages from LLC-bearing mice at the last stage of carcinogenesis. Both TAs and PO244 were administrated on 8th day after tumor cell inoculation. After the therapy macrophages were contactless co-cultivated with primary LLC culture during 48 h. Mononuclear phagocyte fraction from peritoneal exudate of mice was obtained by standard Pietrangeli's procedure. Apoptotic index and distribution of LLC cells in phases of cell cycle were assessed by flow cytometry. An adhesive potential was assessed with crystal violet.

Results:
Aforementioned combination revealed in 2-times increasing of LLC cells apoptotic level in comparison with primary LLC cells (without co-culture) and LLC cells under condition of co-cultivation with macrophages from mice without therapy. TAs+PO244 therapy decreased population of LLC cells in proliferative pool (G2/M+S phase) to 40%, whereas control rates were 65% and 60% in LLC cells without co-culture and LLC cells with macrophage co-culture from mice without therapy, respectively. As the adhesive potential inversely correlates with cell ability to migrating, the in vitro data indicated that migration and tumor infiltration can be activate when tumor growing in vivo. We have shown it in combined therapeutic scheme application of TA and PO244 on LLC. Monotherapy by TA stimulates tumor infiltration by lymphocytes insignificantly, whereas in combined therapy with PO244 this parameter is increased 2.4 times (p<0.05).

Conclusion:
Cytotoxic/cytostatic influence, which was expressed in increasing of apoptotic level and decreasing of cell population of proliferative pool was defined after co-cultivation of macrophages from LLC-bearing mice treated by TAs+PO244 with primary LLC culture. This effect can be one of the possible mechanisms of TAs+PO244 impact on the lung cancer.

Background:
Protein arginine methyltransferase 5 (PRMT5), a member of the protein arginine methyltransferase family, has important regulatory function in many cellular processes through epigenetic control of target gene expression. Because of its overexpression in a number of human cancers and its essential role in cell proliferation, transformation and cell cycle progression, PRMT5 has been recently proposed to function as an oncoprotein in cancer cells. In this study, we explore prognostic and predictive value of PRMT5 expression in lung cancer. Impact of PRMT5 inhibition in the setting of radiation therapy and platin-based chemotherapy was investigated.

Methods:
PRMT5 expression levels in lung tumors as well as their paired normal tissue obtained from TCGA public databases were compared. The impact of PRMT5 expression on lung cancer patient survival was investigated by using “Director’s challenge Consortium for the Molecular Classification of Lung Adenocarcoma” and JBR10 datasets. SiRNA designed to target PRMT5 was used to transiently knockdown (KD) PRMT5 expression in several lung cancer cell lines. Clonogenic survival assays of lung cancer cell lines with increasing doses of cisplatin or radiation were performed in cells with normal endogenous PRMT5 expression or in cells after siRNA knockdown. Impact of PRMT5 knockdown in cell cycle, apoptosis, DNA damage response was investigated through cell cycle analysis, Annexin/PI flow cytometry, ɣH2A foci measurements in lung cancer cells with normal or reduced PRMT5 expression.

Results:
PRMT5 expression is significant higher in lung tumors compared to parired normal tissue in TCGA datasets (LUAD and LUSC) with p value ≤0.0001. Patients with high PRMT5 expression portend lower overall survival at 3 years (p=0.02) from director’s challenge lung cancer study. Patients with low PRMT5 expression had significantly better DFS at 5 years (p=0.3) if they received cisplatin while patients with high PRMT5 expression did not benefit from cisplatin treatment (p=0.7). In several lung cancer cell lines, we observed >90% PRMT5 KD in transiently transfected cells at 48 h and 72 h post transfection as verified by western blot analysis. This inhibition of PRMT5 activity achieved by transient KD lead to a significant decrease in colony survival after radiation and cisplatin. There is an increase of cell population in G1 arrest in PRMT5 transient KD cells.

Conclusion:
High PRMT5 expression is associated with worse survival in lung cancer patients. Inhibition of PRMT5 in lung cancer cells results in sensitization to cisplatin and radiotherapy,

Background:
A subset of NSCLCs (approx. 5%), present alterations in ALK gene. This produces abnormal ALK proteins that induce cells to grow and spread. Different generation of ALK inhibitors are available for targeted therapy and their indication depends on the detection of ALK alterations in the tissue. Thus, it is mandatory to develop new techniques that allow us to demonstrate ALK alterations in peripheral blood. The purpose of this study is to analyze the feasibility to determine ALK alterations in exosomes (Exo-ALK) in NSCLC patients and determine the sensitivity and specificity of the technique.

Methods:
This study is performed in blind in a cohort 19 NSCLC with and without known alterations of ALK in tumoral tissue. ALK-positive tissue samples were identified by FISH or IHQ and patients were included independently of stage and time of disease. Exosomal RNA is isolated by exoRNeasy Serum/Plasma (Qiagen) and retrotranscripted by ProtoScript II First Strand cDNA Synthesis kit. The ALK gene present in the exosomes was determined by NGS and bioinformatic analysis by OncoDNA. Samples and data from patients included in the study were provided by the Biobank of the University of Navarra and were processed following standard operating procedures approved by the Ethical and Scientific Committees, were provided also by UZA Biobank and by the University of Naples Federico II.

Results:
The analyzed samples have been 16 ALK-EML4 tissue positive patients and 3 ALK-EML4 tissue negative, defined in this case by FISH. After analysis, we have been able to detect 9 positive ALK-EML4 patients, 8 negative samples and 2 samples where the RNA was degraded. Looking at the clinical data, the 9 positive samples detected in the exosomal RNA were positive also for ALK-EML4 translocation in the tissue, and comparing the 8 negative samples, 3 were tissue negative and 5 tissue positive. These data show a specificity of 64% and a specificity of 100%. No correlation has been found comparing naïve patients with treated patients.

Conclusion:
Exosomes are raising as one of the most promising tools to understand the tumor due to their stability in the blood and their similarity to the cells of origin. Our preliminary results show a high specificity and sensitivity for a proof of concept analysis. Further studies with a bigger number of patients and a crossvalidation analysis are required, but as we represent in this abstract, exosomes can represent an important tool for the clinical management of this specific NSCLC population.

Background:
The molecular composition NSCLC is heterogeneous and clinically manifested as differential therapeutic responsiveness. It is increasingly appreciated that changes in high order chromatin (HOC) structure may play an important role in controlling gene expression and may be one of the fundamental events in carcinogenesis. However, while several HOC regulators are altered in lung cancer (e.g. Arid1a) from the cancer genome atlas work (TCGA), these occur in a minority of tumors suggesting involvement of other modulators. Recently, SA-1 (Stag-1), a member of the cohesin family, has been shown to be a HOC regulator in cancer via controlling chromatin looping and hence gene expression. Since no previous reports on cohesin in lung cancer, we therefore, focused on the role of SA-1 in NSCLC.

Methods:
We performed immunohistochemical analysis (IHC) of 190 cancers and compared to benign tissue through standard techniques. We also extracted SA-1 data from the TCGA databases (Nature 2012 & 2014).

Results:
SA-1 was markedly (~2-3 fold) overexpressed in all types of NSCLC (p<0.01) versus benign tissue (Figure 1). This increase was striking at stage 1 NSCLCs with minimal further increase noted at higher stages. TCGA data demonstrated amplification/mutation in ~17% of squamous but only 3% of adenocarcinomas. This suggested that epigenetic regulations was paramount. Kaplan-Meier analysis showed major impact of SA-1 alterations on survival. For instance, in squamous cancers, median disease-free survival with versus without SA-1 amplification was 8 vs 38 months, respectively. Figure 1



Conclusion:
We show for the first time that SA-1 is overexpressed early in NSCLC consonant with status as a proto-oncogene. This upregulation occurs predominantly epigenetic with some contributions with genetic factors. Moreover, SA-1 may have important prognostic markers underscoring its importance of HOC regulation in NSCLC. Further studies are ongoing to elucidate the precise role of SA-1 in the pathogenesis and natural history of NSCLC.

Background:
Large cell lung cancer (LCLC) is a newly recognized clinicopathologic entity. The clinical criteria and optimal treatment for patients with LCLC are not yet established. The aim of this study is to understand the clinicopathologic criteria of LCLC.

Methods:
Among enrolled NSCLC cases attending National Cancer Institute –Cairo (NCI) between 2012-2014, we retrospectively reviewed those had LCLC. Data regarding demographics, ECCOG-performance status(PS), tumor histology, grade and stage, chemotherapy type, number of cycles, response to chemotherapy, overall and progression free survival(OS, PFS) were retrieved. Pearson’s(X[2])test and Kaplan-Meier survival curves were used in statistical analysis.

Results:
Among 99 NSCLC cases, we identified squamous cell carcinoma (35.4%), adenocarcinoma(29.3%), ,undifferentiated(20.2%),large cell carcinoma (12.1%) and adenosquamous carcinoma(3%). Among 12 LCLC cases; median age was 52years (range;41-62years), Male : Female was 3:1. Three-quarters of our cohort were PS=1. Progressive disese occurred in 58.3%. All were grade 3. Near 60% were stage IV while stage IIIB represents the remaining. Median OS was not reached while mean OS was 16.2 months. Median PFS was 6 months. Nearly 90% of disease progression were found within 1 year after start of chemotherapy. There was no difference in median OS or PFS in LCLC vs other NSCLC(OS= not reached vs 13 months in other NSCLC(p=0.372) while PFS=6months in both groups(p= 0.915)). Further analysis by stage was conducted and revealed same results(in STAGE III; median OS not reached,mean OS was 18.2 vs 19.1for other NSCLC(p=0.400),median PFS was 8 vs 6monthsin other NSCLC(p=0.948). In STAGE IV; median OS was 12 vs 9 months (p=0.511), median PFS was 5months in both groups (p=0.956) See figure.

Conclusion:
Most cases of LCLC represents high grade tumors and indeed aggressive treatment is warranted. Although previously reported data revealed poor prognosis of LCLC (stage-I) in comparison to other NSCLC, our cohort represents similar prognosis in both groups in advanced stagesFigure 1

Background:
Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factor, which promotes endothelial cell growth and tumor neovascularization. VEGF expression is a marker of invasiveness and tumor progression in various cancers including in NSCLC. The aim of this study was to analyze association between VEGF gene functional polymorphisms and clinical and pathological characteristics of NSCLC.

Methods:
A total of 276 people with histological diagnosis of squamous cell carcinoma (SCC) and adenocarcinoma (AC) were included in this study. All patients gave their informed consent. VEGF gene polymorphisms were determined by PCR-RFLP analysis. Statistical analysis of the material was carried out using SNPStats online program.

Results:
Analysis of rs699947 polymorphism association with clinical and pathological characteristics of the tumor showed that patients with -2578CC genotype are more likely to have a greater extent of the primary tumor (T2-T4) than a small non-invasive cancer (T1): p = 0.005; OR = 2.54, 95% CI: 1.35-4.77. Association between this polymorphism and regional lymph node metastasis and the stage of the disease was found. A-allele is protective against a more aggressive course of the disease: in patients with -2578AA genotype regional lymph node metastases (N1-3) occur less frequently as compared to -2578CC genotype carriers (p = 0.0098; OR = 0.34, 95% CI: 0.17-0.69). -2578A-allele carriers are less likely to have stages of the disease III and IV (p = 0.012 and 0.015 respectively). A tendency of rs3025039 polymorphism influence on the histological type of the tumor was identified. + 936TT genotype is more common in patients with SCC as compared to AC (p = 0.055; OR = 4.78, 95% CI: 1.01-22.71). For rs2010963 polymorphism the association with clinical and pathological characteristics of NSCLC was not identified. Haplotype analysis of three studied polymorphisms of VEGF gene showed a significant association between -634C/-2578C/+936C haplotype and a small non-invasive cancer (p = 0.0068). -634G/-2578C/+936C haplotype carriers showed high aggressiveness of the disease (stage - p = 0.0061; regional lymph node metastasis - p= 0.0014).

Conclusion:
-2578CC genotype of rs699947 polymorphism VEGF gene is associated with a large size of the primary tumor focus, the occurrence of regional lymph node metastasis and a greater stage of the disease. High aggressiveness of the disease was revealed in -634G/-2578C/+936C haplotype carriers. A significant association between -634C/-2578C/+936C haplotype and small non-invasive cancer was showed.

Background:
Our goal was to evaluate the prognostic significance of circulating tumour markers in locally advanced squamous cell carcinoma of lung (LA-SCCL).

Methods:
Eligible patients included those with histologically proven LA-SCCL, available baseline tumour marker panel analysis (carcino-embryonic antigen [CEA], carcinoma antigen 125 [CA125], squamous cell carcinoma antigen [SCC], cytokeratin-19 [Cyfra 21-1] and neuron-specific enolase [NSE]) and receiving definitive radiotherapy. Age, gender, radiation dose, baseline KPS, smoking history, weightless, TNM stage, PET staging, RT technique and treatment modality (radiotherapy alone vs. sequential chemoradiotherapy vs. concurrent chemoradiotherapy) were also retrospectively collected. To dichotomise the continuous values of tumour markers into categorical variables, ROC analysis was adopted to identify the optimal cutoff values using the progression within 2 years after diagnosis as the endpoint. Cox regression based multivariate analyses were used to select independent factors correlated with various survival endpoints. Overall survival (OS), local regional progression free survival (LRPFS) and distant metastasis free survival (DMFS) were defined as the time from diagnosis until the first occurrence of specific event: death, local-regional recurrence or distant metastasis, respectively. Progression free survival (PFS) was defined as the duration between the cancer diagnosis and the date of any progression or cancer related death.

Results:
A total of 216 patients with LA-SCCL were analyzed. The optimal discriminative values for CEA, CA125, SCC, Cyfra 21-1 and NSE in predicting 2-y progression were 5.3 ng/ml, 17.0 U/ml, 2.5 ng/ml, 5.2 ng/ml and 17.8 ng/ml, respectively. Univariate analyses showed that increased Cyfra 21-1 was associated with inferior OS, LRPFS, DMFS and PFS. Increased NSE was predictive of poor OS, DMFS and PFS. CEA also presented significant correlation with OS. Under multivariate analysis involving all clinical and tumour markers, IIIA stage, better performance status, CEA ≤ 5.3 ng/ml and Cyfra 21-1 ≤ 5.2 ng/ml were independently associated with improved OS. IMRT technique, RT dose ≥ 60Gy and Cyfra 21-1 ≤ 5.2 ng/ml were correlated with better LRPFS. None-smoker, IIIA stage, NES ≤ 17.8 ng/ml were favourable predictors for DMFS. IIIA stage, KPS ≥ 80 and Cyfra 21-1 ≤ 5.2 ng/ml were advantageous factors related with favourable PFS.

Conclusion:
Baseline tumour marker panel including Cyfra 21-1, NSE and CEA can be prognostic of OS, local and distant tumor control for LA-SCCL, and should be recommended for baseline evaluation of tumour burden.

Background:
Enumeration and karyotyping of circulating tumor cells (CTCs) in therapeutic cancer patients is of particular clinical significance. The aim of this study is to evaluate therapeutic effect of neoadjuvant chemotherapy (NAC) by means of real-time monitoring of CTCs in locally advanced non-small cell lung cancer (NSCLC).

Methods:
Real-time monitoring of CTCs in the course of 2 cycles of platinum-based NAC was conduct in 34 locally advanced NSCLC patients. The integrated subtraction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) method was applied to detect and characterize CTCs in peripheral venous blood. Chest CT was used to evaluate therapeutic response with RECIST 1.1 as the evaluation criterion.

Results:
Of the 34 patients enrolled, 13 acquired partial response (PR) and 21 were stable diseases (SD) after NAC. The numbers of CTCs were found decrease in 70% of PR patients and only 25% of SD patients. The changes of CTC count were significantly different between PR and SD group (p=0.009). The positive rate of CTCs with triploidy of chromosome 8 increased after 2 cycles platinum-based NAC, and the elevation was even more remarkable in SD group.

Conclusion:
The changes of CTC count after NAC were in accordance with CT responses. Triploidy of chromosome 8 CTC was correlated with primary resistance to platinum-based chemotherapy. Real-time monitoring of CTC count and karyotype may be of clinical value in rapid evaluation of therapeutic effect and monitoring occurrence of chemo-resistance.

Background:
44% of acrometastasis are originated from primary lung tumors and metastasis to digits is seen in 0.2% of patients with lung cancer. After clinical staging, amputation or radiotherapy are most often therapeutic options for pain palliation. We want to present an oligo-acrometastasis of fourth proximal phalanx of left hand from EGFR-mutant non-small cell lung cancer.

Methods:
A 58-year-old man was admitted with pain and swelling in fourth finger of his left hand (Figure 1A). Magnetic resonance imaging (MRI) of left upper extremity showed a destruction by a soft tissue measuring about 30x17 mm on the distal part of fourth proximal phalanx of left hand (Figure 1B-C). Three phase bone scan with technetium-99m methylene diphosphonate (MDP) revealed increased radiotracer uptake in the fourth finger. Diffuse increased uptake is seen at the left wrist secondary to the old fracture and trauma in both blood pool and metabolic phases and hypertrophic osteoartropathy in both tibia (Figure1E-F). Computed thorax tomography (CTT) revealed a 25x21 mm lobulated contour lesion in the posterior segment of right lower lobe (Figure 1H). CT-guided biopsy was performed and pathological examination showed non-small cell lung carcinoma-not otherwise specified (NSCLC-NOS). A 24x27x24 mm mass with SUV-max value 9.85 in the right lower lobe, right tracheobronchial and right hilar lymphadenopathies 13 mm in diameter was detected (SUV-max: 7.51) on PET-CT. Patient was staged as T1bN2M1b with oligoacrometastasis. Figure 1



Results:
His finger was amputated from metacarpophalangeal level and surgery margin was negative for tumour. Pathological diagnosis was metastatic NSCLC-NOS harbouring EGFR-21L858R mutation. After curative treatment of acrometastasis, concurrent chemo-radiotherapy was planned for primary lung cancer as a therapeutic approach. He is still under treatment.

Conclusion:
Oligometastatic disease by acral involvement in NSCLC is extremely rare. Curative treatment approach should be consider for both primary tumour and metastasis side.

Background:
GNAQ is a stimulatory αq subunit of heterotrimeric G-proteins that is highly mutated in human melanoma and currently has no targeted treatment. GNAQ protein is similar to the RAS protein, in which activating mutations occurring within the catalytic (GTPase) domain confer constitutive signaling activity of the RAS pathway. However, GNAQ mutations have not been documented in Non-Small Cell Lung Cancer (NSCLC). Therefore, the aim of this study was to examine genomic alterations in GNAQ and correlate its mutation status with clinical characteristics of NSCLC patients.

Methods:
A cohort of 53 patients treated at the Thoracic Oncology Unit of the Instituto Nacional de Cancerologia (INCan) of Mexico were screened in a mutation analysis of the GNAQ oncogene by targeted next generation sequencing (NGS). All information from the patients was recorded in a database containing clinicopathological characteristics.

Results:
Patients characteristics included a median age of 66 years (36-82 years), with 77% of females, 39% of smokers, 51% with wood smoke exposure and the predominant histology was adenocarcinoma (86%) with intermediate grade (acinar-papillary) in 65% of cases. In this study, recurrent somatic mutations in GNAQ were found in 37/53 patients (70%) with a mutant allele (QNAQmut) frequency over 1%. GNAQ mutations were more frequently found in adenocarcinoma and stage IV (p=0.054 and p=0.098 respectively). The GNAQmut allele was associated with metastasis to the Central Nervous System (CNS) and bones (p < 0.001). This mutation was associated with a decrease in overall survival (69 vs. 12 months, p = 0.047). Additionally, two of these GNAQ-mutated patients having co-ocurring oncogenic mutations in GNAS and GNA11 exhibited faster disease progression and a poorer overall survival of only two months. There was no association between GNAQ and frequently mutated genes like EGFR, KRAS or MET.

Conclusion:
This is the first report of the presence of recurrent somatic mutations in GNAQ, GNA11 and GNAS oncogenes in NSCLC based on targeted NGS. We found a correlation between these genomic alterations and the patients response measured as disease progression and overall survival.

Background:
Since 1990s, the standard treatment for locally advanced non-small cell lung cancer (NSCLC) has been changed because the treatment by adding chemotherapy to thoracic radiation (TRT) was proved to gain a survival benefit over TRT alone. We conducted this study to report the outcome of combination treatment along with determine the factors that effecting survival.

Methods:
Medical records of 1,325 NSCLC patients who treated with radiotherapy in our division during 2008 to 2013 were reviewed. The patient characteristics, the management characteristics and outcome data were recorded. Univariate and multivariate analysis were performed to identify the prognostic factor for overall survival.

Results:
A total of 103 patients were included in the analysis. With a median follow up time 13.27 months, these patients had a median overall survival (OS) time of 21.4 months (95%-CI 17.6-25.2 months) and median progression-free survival (PFS) time of 11.67 months (95%-CI 9.69-13.65 months). The 2-year OS and PFS rate were 34.0 and 21.4%, respectively. For the patients treated by concurrent and sequential chemoradiation, the 2-year OS rate were 31.0% and 37.8% (p=0.349) and the 2-year PFS rate were 24% and 20.6% (p=0.690), respectively. The multivariate analysis revealed that age (hazard ratio (HR) 1.68; 95% CI: 1.06 – 1.69) and stage (HR 2.13; 95% CI: 1.43 – 3.39) were significant prognostic factors for overall survival.

Conclusion:
The treatment of locally advanced NSCLC in our hospital is feasible and the outcomes are comparable to others. The results of concurrent chemoradiation may improve further by careful patient selection.

Background:
About a quarter of patients with NSCLC have stage III disease. Standard treatment is cisplatin-based concomitant chemoradiotherapy, established in trials with participants younger and fitter than many patients seen in clinics. We have reviewed the treatment delivered to all patients registered on the South East Scotland Cancer Network (SCAN) database in 2011 to determine how many patients received standard of care therapy, and what might have influenced the decision not to administer this treatment.

Methods:
Individuals with stage III NSCLC presenting between January and December 2011 were identified from the SCAN database. Data were extracted on patient age, stage, histology, performance status, co-morbidities and treatment delivered.

Results:
154 patients were identified who presented with stage III NSCLC between January and December 2011. 11 patients declined treatment, one after initial surgical exploration, and 12 died before treatment could start. Only 48 of 130 (37%) patients received curative intent treatment, 13 (10%) with concomitant and 11(8%) with sequential chemoradiotherapy, 17 (13%) with radical radiotherapy and 7 (5%) with surgery. 44 (34%) received best supportive care, 33 (25%) palliative radiotherapy and 13 (10%) palliative chemotherapy. The strongest predictor of curative therapy was performance status (PS), with 41/70 (59%) PS 0-1 and 7/60 (12%) PS 2-4 (Χ[2] =30.5, p < 0.0001) respectively receiving this. Patients with 2 or more co-morbidities including emphysema (COPD), ischaemic heart disease (IHD), cerebrovascular disease (CVD) or second malignancy were also less likely to receive curative intent treatment (Χ[2] =6.4, p = 0.01) or chemotherapy (Χ[2] =4.4, p = 0.04). Absence of histological proof of disease and age did not affect treatment intent, although no patients over age 80 years received chemotherapy. Review of the 18 patients who were documented as PS 0-1 with one or fewer co-morbidity who did not receive curative intent treatment revealed 29 comorbidities between the 18 patients including 5 with thromboembolic disease, 4 with pulmonary fibrosis, 4 with COPD, 4 with IHD, 3 with atrial fibrillation 2 with second malignancy, 2 with CVD, 2 with hypertension and 1 each with diabetes, vasculitis and chronic kidney disease.

Conclusion:
Standard of care curative intent concomitant chemoradiotherapy is delivered to only a minority of patients with stage III NSCLC. Progress in improving patient outcomes in this disease requires not only the refinement of standard therapies, but research directed at patients with PS2 disease and those with multiple co-morbidities.

Background:
In the treatment of patients with locally-advanced non-small cell lung cancer (LA-NSCLC), we usually apply chemoradiotheraphy (CRT) consisted of docetaxel and cisplatin with concurrent 40-60 Gy radiation therapy. The radiation dose of 60 Gy is generally planned in the case of definitive CRT. On the other hand, the radiation dose of 46 Gy is planned in the case of induction CRT, considering the safety of surgery. In the induction CRT, if the treatment response is poor and complete resection is supposed to be difficult, additional radiation is performed. In this study, we examined the safety and clinical outcome of lung resection after induction CRT using high-dose radiation in patients with LA-NSCLC.

Methods:
One hundred and eighteen patients with LA-NSCLC who underwent induction CRT followed by surgery between March 1999 and December 2014 in our hospital were reviewed. We categorized those patients into low-dose radiation group who received less than 60 Gy of radiation (n=105) and high-dose radiation group who received more than 60 Gy of radiation (n=13). We compared postoperative outcomes between these two groups applying match-paired analysis with using propensity score.

Results:
One hundred and eighteen cases consisted of 91 males and 27 females, and the average age was 60 years. Eleven patients had stage IIB disease, 73 patients had stage IIIA disease, and 34 patients had stage IIIB disease before CRT. The background between low-dose group and high-dose group was similar. There were no significant differences in the mortality (0.8% vs 0% in low-dose group and high-dose groups), the incidence of postoperative complication (57% vs 77%), and post-operative hospital days (median 22 vs 28 days) between each group. In addition, there were no significant differences in the 5-year OS rates (73% vs 77% in low-dose group and high-dose groups, p =0.66), and the 5-year DFS rates (56% vs 77%, p =0.11) between each group, even when we applied matched-paier analyses.

Conclusion:
This study showed that lung resection after induction CRT using high-dose radiation for LA-NSCLC patients had been performed safely with equivalent prognosis compared with that using low-dose radiation.

Background:
The influence of recurrence pattern on outcome in stage III NSCLC following definitive chemo-radiotherapy (CRT) has been scarcely addressed in the literature. Our aim was to analyze the relevance of oligoprogression (OP) in this clinical setting.

Methods:
Patients (pts) with stage III NSCLC who underwent concurrent CRT from 2010 to 2014 at the Catalan Institute of Oncology were retrospectively reviewed (n=170). Recurrence pattern at first progression was recorded. OP was defined as a single metastatic organ with up to 3 lesions. Overall Survival (OS) and Progression-Free Survival (PFS) were plotted using Kaplan Meier method, and multivariate Cox proportional hazards model was developed.

Results:
Median age 64 (37-87); male 87%; ECOG-PS≤1 92%; histology: adenocarcinoma 34%, squamous 43%, NOS+large cell 23%; cN0-1 21%, cN2 60%, cN3 19%. Platinum doublet: cisplatin 62%, carboplatin 38%. RT between 60-70 Gy (2Gy/fr): 94%. At a median follow-up of 38 months (m), 108 of 170 pts relapsed (63%) and 66% died. mPFS was 13m (95% CI 10-16), mOS was 28m (95% CI 22-34). Twenty-five of pts who relapsed (23%) developed OP. Sites involved: visceral 17, brain 4, lymph nodes 3, bone 1. Treatments delivered: local therapy with curative intent 9; palliative intention 12; no treatment 4 (table 1). Among pts who relapsed, mOS was longer in those with OP (32m) compared to pts without OP (18m, p=0.007). Pts with OP who received treatment, mOS according to curative or palliative intention was 53m versus 32m (p=0.1), respectively. In the multivariate Cox analysis of post-progression OS, OP remained a favourable prognostic factor (HR=0.36, 95% CI 0.17-0.74) independently of age, PS, stage, histology, smoking history, and platinum doublet.

Conclusion:
OP was associated with substantial better prognosis in this cohort of pts treated with concurrent CRT. Local ablative therapies in the context of OP yielded promising results in terms of survival and warrants further investigation.

Background:
The aim of the study is to determine the actual standard management of patients with stage III NSCLC in Central European centres/countries. The project is a multicentre, prospective, non-interventional registry.

Methods:
After ethical committee approval and signed informed consent, the data about diagnostic and therapeutic procedures of consecutive patients diagnosed with stage III NSCLC (UICC7) were collected in web-based registry organised by the IBA MUNI, Brno, Czech Republic.

Results:
With cut-off 30 June 2016, 509 patients from 7 countries/16 centres were enrolled, median number of patients per centre being 23 (range 6-99). There were 163 (32%) women and 37 (7%) never smokers. Performance status distribution was as follows: ECOG 0, 1, 2 and 3 in 29%, 56%, 12% and 3%, respectively. Squamous cancer was found in 52%, adenocarcinoma in 39%, not otherwise specified in 5% and others in 4% of cases. Genetic mutations were examined in 119 (23%) patients, predominantly EGFR in 111 subjects with 10 (8%) positive findings, while the ALK mutation in 64 patients with no positive finding. Regular staging procedures were X-Ray scan (97%), chest CT (96%) and bronchoscopy (89%). Staging was completed by abdominal CT in 66% of patients, abdominal US in 29%, PET/CT in 22%, bone scan in 17% and brain CT or MRI in 13%, respectively. Stage IIIA was found in 59% and stage IIIB in 41% of patients. N2/N3 nodes were diagnosed in 60%/22% of patients. Pathological mediastinal lymph-node positivity was confirmed in 109 (21%) patients (6% EBUS, 0.2% VATS, 1% mediastinoscopy, 1% transbronchial biopsy and 13% surgery). Median time from diagnosis to first treatment was 23 days (range 0–321). Treatment procedures were: surgery 138 (27%), chest radiotherapy 246 (48%) and chemotherapy 409 (80%) of subjects, respectively. Chemotherapy as only modality was given in 136 (27%) of patients. Surgery was combined with radiation in 6 cases, with chemotherapy in 79 (16%) cases and with both chemotherapy and radiotherapy in 37 (7%) patients. Chemotherapy plus radiotherapy was given in 159 (31%) patients including concurrent chemoradiotherapy in 67 (13%) cases. At the time of cut-off, 64% patients were alive, median survival time was not reached, and the 1-year estimated survival rate was 71%.

Conclusion:
The most prevalent histology was squamous cancer. Histopathological examination of mediastinal lymph-nodes was done in 21% of patients, mostly during surgery. Majority of patients (55%) were treated with combination therapy. Palliative chemotherapy only was given in 27% of patients. Survival data are not mature.

Background:
Chemo-radiotherapy is the standard of care for the treatment of inoperable locally advanced non-small cell lung cancer (LA-NSCLC). Cisplatin (P) plus vinorelbine is one of the chemotherapy (CT) regimens widely used concurrently with radiotherapy (RT). Since oral vinorelbine (oV) has achieved comparable results to the IV formulation, the optimal dose for the oV administration with P and concurrent RT is still being investigated. The aim of this study is to evaluate the efficacy and safety of full-dose oV combined with P and radical RT for LA-NSCLC patients (pts).

Methods:
Untreated pts between 18-70 years (y), with histologically proven inoperable LA-NSCLC (supraclavicular lymph node involvement excluded), V20<30%, adequate bone marrow, respiratory, hepatic and renal function, and ECOG PS0-1; received 4 cycles (cy) of oV 60 mg/m[2] D1 & 8 plus P 80 mg/m[2] D1, every 3 weeks, plus 2Gy/day of RT started on D1 of 2[nd] cy (total dose 66Gy). Primary endpoint was overall response rate (ORR) by RECIST 1.1. Secondary endpoints were: progression free survival (PFS), overall survival (OS) & safety profile. To guarantee a type-1 () error (one side) no greater than 0.05 and a type II (β) error 0.1 for the primary endpoint, a sample size of 45 eligible pts was planned. EudraCT 2009-010436-17.

Results:
Forty-eight pts were included between 02/2010-12/2011. Median age 61y [34-72], male 90%, PS1 58%; smokers 52%; squamous 63%; stage IIIB 54%. Main G3-4 toxicities (% cy) were: neutropenia 33%, febrile neutropenia 14.6%, anemia 12.5%, thrombocytopenia 16.6%, & esophagitis 12.5%. Two treatment-related deaths during the 1[st] cy. RT was administered to 87.5% of pts; 7.1% received less than 60Gy and 23.8% had delays due to adverse events. The ORR was 77.3% (2 complete responses). With a median follow-up of 28.2 months (m) [0.5-70.6], 33 pts (68.8%) have progressed and 32 (66.7%) have died. Median PFS and OS were 11.8m (CI~95%~ 7.2-16.5) and 29.8m (CI~95%~ 21.4-38.1), respectively. PFS at 1y was 48.8% pts (CI[95%] 33.9-63.7%). OS at 1 and 2y were 72.7% (CI95% 60-85.4%) and 57.3% (C95% 43-71.6%), respectively.

Conclusion:
This long-term analysis confirms the good efficacy results of the administration of full doses of oral vinorelbine combined with cisplatin and concurrent RT in patients with LA-NSCLC.

Background:
Standard care in inoperable stage III non-small-cell lung cancer (NSCLC) is concurrent chemoradiation. In the PROCLAIM trial, comparing concurrent pemetrexed-cisplatin (PemCis) and thoracic radiotherapy (TRT) followed by consolidation pemetrexed versus etoposide - cisplatin (EtoCis) and TRT followed by a consolidation platinum doublet in patients with locally advanced NSCLC, PemCis experienced significantly lower incidence of drug-related grade 3-4 adverse events (AE) and had similar resource use. Here, we estimate healthcare resource use costs associated within the PROCLAIM trial.

Methods:
Unit costs were applied to patient-level resource use (study drug, hospitalizations, radiotherapy, concomitant medications, laboratory tests, other procedures) to estimate total costs to a third-party US payer. Unit costs (in 2015 US dollars) were derived from publicly-available sources. Costs were compared using the nonparametric Wilcoxon rank sum test; sensitivity analyses were conducted. A subgroup analysis excluded patients with unusually long hospitalizations.

Results:
PemCis had significantly higher total costs than EtoCis (Table). While other medical costs were lower for PemCis in the concurrent phase, other medical costs were comparable for PemCis and EtoCis during the overall treatment mainly because PemCis patients remained in the trial longer (0.37 years) than EtoCis patients (0.29 years). Results were similar in the subgroup analysis.

Parameter	Overall Arm A Mean (SD)	Overall Arm B Mean (SD)	Concurrent Phase Arm A Mean (SD)	Concurrent Phase Arm B Mean (SD)
Years follow-up	0.37 (0.12)	0.29 (0.09)	0.20 (0.04)	0.19 (0.05)
Costs:
Total	$51,313.90 ($33,166.11)	$22,425.24 ($26,087.53)	$28,856.03 ($25,745.12)	$17,526.22 ($23,307.13)
Study treatment	$31,203.67 ($11,217.62)	$2,957.81 ($900.48)	$15,719.30 ($3,447.07)	$1,872.54 ($289.21)
Other medical[a]	$20,110.22 ($32,883.10)	$19,467.43 ($26,141.99)	$13,136.73 ($25,725.51)	$15,653.68 ($23,325.07)
Adverse-event-related[b]	$16,681.48 ($30,964.72)	$16,061.84 ($24,356.95)	$10,665.83 ($24,139.67)	$13,139.85 ($21,860.09)
Hospitalization	$15,141.15 ($29,937.04)	$13,562.54 ($23,156.58)	$9,839.42 ($23,488.47)	$11,778.76 ($21,007.19)
Concomitant medication	$3,158.12 ($3,615.92)	$4,238.32 ($5,242.10)	$2,032.67 ($2,064.07)	$2,498.43 ($2,997.28)
Monthly other-medical costs	$4,529.33	$5,594.09	$5,473.64	$6,865.65

SD = standard deviation. [a]Other medical includes hospitalizations, radiotherapy, supportive care, concomitant medications, laboratory/evaluation/radiology visits, and blood products. [b]Adverse-event-related includes concomitant medications, hospitalizations, and blood products associated with an adverse event specifically; subset of other medical costs.

Conclusion:
Higher total costs for PemCis compared to EtoCis were driven by study drug cost. However, other medical costs during the concurrent phase were lower for PemCis due to significantly lower hospitalization costs and lower concomitant medications use. When adjusting for overall treatment duration, other medical costs were favorable for PemCis. Pemetrexed patients may incur lower monthly other medical costs due to reduced hospitalization costs.

Background:
Administration of chemotherapy prior to surgical resection is one of the strategies for the treatment of locally advanced non-small cell lung cancer (NSCLC). Potential benefits of this approach include improved treatment tolerance, tumor downstaging, and the evaluation of tumor response. Utilizing the National Cancer Database (NCDB), we sought to compare short-term perioperative outcomes and treatment response of neoadjuvant chemotherapy followed by surgery with surgery alone.

Methods:
We queried the NCDB Participant User File (PUF) for patients with clinical stage IB-IIIA NSCLC who underwent definitive surgical resection for NSCLC between 2006-2013. We identified 83,274 patients with complete datasets who met the inclusion criteria. Patients were grouped by stage and perioperative outcomes were assessed, comparing those who underwent neoadjuvant therapy to surgery alone. Neoadjuvant therapy response was assessed by downstaging on final pathology in both unmatched and matched cohorts.

Results:
Neoadjuvant chemotherapy was administered to 11.9% (9,961/83,274) of potentially eligible patients. The incidence of neoadjuvant therapy increased with clinical stage; rates of 2.7% (995/37,453) for IB, 5.4% (724/13,435) for IIA, 15% (2,048/13,619) for IIB, and 33% (6,194/18,767) for IIIA. All cause 30-, and 90-day mortality was 3.1% and 6.3% vs. 3.1% and 6.0% for neoadjuvant vs. surgery alone across all stages, (p=0.159, p<0.001). The unplanned 30-day re-admission rates were 3.8% vs. 4.3% for neoadjuvant vs. surgery alone (p<0.001). Median length of hospital stay was similar between the groups, 7.6 vs. 7.2 days for neoadjuvant vs. surgery alone (p=0.015); stage specific analysis revealed similar results. Overall downstaging was seen in 29.5% in the neoadjuvant group compared to 17% in the surgery group (p<0.001). Primary tumor downstaging occurred in 31.5% vs 9.5% (p<0.001) and nodal downstaging in 23% vs 14.4% (p<0.001) for neoadjuvant and surgery groups respectively. Additionally, significantly improved R0 resection rate was achieved for stages IIIA and IIB in the neoadjuvant group 88.1% and 86.1% vs. 82.0% and 84.5% in the surgery alone group respectively (p<0.001 for IIIA and IIB).

Conclusion:
In this largest review of perioperative outcomes and downstaging effect of neoadjuvant chemotherapy prior to definitive surgical resection for NSCLC, we demonstrate that the treatment strategy of neoadjuvant chemotherapy followed by surgery is safe and effective. Tumor downstaging and increased R0 resection rate in locally advanced lung cancer stages support the utilization of this treatment paradigm.

Background:
Current evidence-based guideline-concordant care (GCC) is administration of platinum-based chemotherapy during thoracic radiotherapy (TRT) for locally advanced non-small cell lung cancer (NSCLC) patients with good performance status. This study evaluates factors associated with lack of GCC.

Methods:
Patients (pts) with unresected stage IIIA/IIIB NSCLC diagnosed from 2005 – 2013 and Charlson-Deyo Score 0 were identified from the National Cancer Data Base (NCDB). Primary outcomes measured were receipt of GCC, defined by administration of chemotherapy with TRT commencing within 2 weeks of each other and minimum TRT dose of 60 Gy, and overall survival (OS). Multivariable logistic regression (MLR) modeling was performed to identify variables associated with non-GCC. Cox proportional hazard modeling was utilized to examine OS.

Results:
Patient characteristics (n=37,809) included: mean age 67.8 years; 55% male; 13% African American; 3.4% Hispanic, 3.6% ‘other’ race/ethnicity; 66% government-insured; mean tumor size 5.0 cm; 38% adenocarcinoma; 32% squamous cell carcinoma (SCC); 30% large cell/other histology. In total, 28% of pts received GCC. On MLR analysis, Hispanic pts were more likely to receive non-GCC (OR=1.34, p <0.001) compared to non-Hispanic pts. Uninsured pts were more likely to receive non-GCC (OR=1.57, p<0.001) compared to privately-insured pts. Patients treated in the western, southern, or northeastern U.S. were more likely to receive non-GCC (OR= 1.43, 1.45, 1.21, all p values <0.001) compared to pts treated in the Midwest. Adenocarcinoma and large-cell/other histological types were more likely to receive non-GCC (OR= 1.71, 1.39, both p<0.001) compared to SCC. For every one-year increase in age or 50-mile increase in distance to treatment facility, patients had a 4% or 3% increased odds of not receiving GCC (OR=1.04, 1.03; p<0.001, p = 0.003, respectively). On hazard modeling, those receiving non-GCC had higher death rates compared to those receiving GCC (HR=1.42, p<0.001). Survival rates were lower for Hispanics receiving non-GCC versus GCC (HR=1.24, p=0.034). Other groups with lower OS for non-GCC versus GCC included: the uninsured (HR=1.61, p<0.001), treatment in the western, southern, or northeastern US (HRs= 1.56, 1.40, 1.33, respectively, p<0.001), adenocarcinomas and large cell/other histologies (both HR=1.40, p<0.001).

Conclusion:
Socioeconomic factors, including Hispanic ethnicity, lack of insurance, geographic location, and distance from treatment facility are associated with receipt of non-GCC. Patient and disease specific factors including increasing age and adenocarcinoma histology are also associated with non-GCC. Future interventions could target these groups to improve provision of GCC.

Background:
Chemo-radiotherapy (CT-RT) remains the standard therapy for locally advanced Non-Small Cell Lung Cancer (LA NSCLC) . Concurrent therapy is the choice for fit patients, without a proven benefit of either induction or consolidation therapies. However, sometimes, as in our Health system, RT is not readily available from the beginning so CT is started upfront and RT started when possible.

Methods:
Charts from every patient treated with CT-RT in our Hospital between January/2008 and December/2015 for LA-NSCLC have been reviewed. Patient and therapy characteristics have been assessed.

Results:
184 patients (p) were found: Median age 64 years (41-84), male 151p (82,1%), PS 0/1/2: 34,2/63,6/1,6%. Histology: adenocarcinoma 34,8%, squamous carcinoma 51,8%, NOS 2,7%, NSCLC with neuroendocrine features 10,9%. Stage IIIa 32,1%, IIIb 67,9%. CT included a platinum salt in 98.9% of cases: cisplatin in 57,6% and carboplatin in 41.3%. Most frequent companion drugs were vinorelbine (35.9% overall, 55.7% within patients treated with cisplatin) and paclitaxel (38.0%, 77.6% of those combined with carboplatin). Median number of CT courses was 4 (1-5), and median course when RT was started was third (1-4). Median survival was 22.5 months (18.3-26.7). It was longer in squamous carcinomas (23.1m), male patients (23.3m), stage IIIa (27.5m) and cisplatin-treated (23.5m) although these differences were non-significant. The only significant factor for survival was PS (0= 33.2 m, 1= 19.0m, p<0.001). No differences in patient characteristics existed with respect to stage, gender or histologic subtype between cis- or carboplatin-treated patients. More patients with PS=0 were treated with cisplatin (49/63= 77%) and carboplatin was preferred for PS=1 patients (59/117= 50.4%, p<0.001).

Conclusion:
Despite our limitations to start RT early in the treatment of LA-NSCLC our results in real-world clinical practice were comparable to those reported in clinical trials. This was at the cost of increasing the burden of CT up to 4 courses. Probably, proper selection of patients was crucial, with PS 0 patients benefiting most from this approach. No major differences existed according the CT regimen administered (either the use of cis- or carboplatin as backbone or the partner drug used). In our experience, squamous carcinomas remained the most frequent subtype in LA_NSCLC.

Background:
The detection of small-sized (≤ 1cm) non-small cell lung cancer (NSCLC) has increased with the development of high-resolution computed tomography. The reported 5-year survival rate of T1a (≤ 2cm) N0M0 patients is more than 80%, and that of p-T1a (≤ 2cm) N2M0 patients has also steadily improved.

Methods:
Between 1991 and 2011, a total of 917 patients with small-sized (≤ 2cm) NSCLC underwent curative pulmonary resection with systematic lymph node dissection at Tokyo Medical University Hospital and Tokyo Medical University Ibaraki Medical Center. We retrospectively evaluated their postoperative clinical outcomes and survival rates. Survival was analyzed using the Kaplan-Meier method and log-rank test.

Results:
There were 46 (5.0%) patients with mediastinal lymph node metastasis in pT1a (≤ 2cm). And there were 6 (0.6%) patients with pT1a (≤ 1cm) N2M0. The histological types were 3 cases of adenocarcinoma, 2 case of squamous cell carcinoma, and one large cell carcinoma. The respectively status of lymph node metastasis was single station in 2 cases and multiple station in 4 cases. Skip lymph node metastasis was observed in 2 cases. There were 26 cases (56.5%) that were upstaged from clinical diagnosis in pT1a (≤ 2 cm) N2M0 patients. There was one upstaging case from cT1a (≤ 1 cm) N0M0 to pT1a (≤ 2 cm) N2M0. The median overall survival period and 5-year survival of patients in pT1 (≤ 2 cm) N2M0 was 52.1 months and 45%. And patients with pT1a (≤ 1 cm) N2M0 has 29.8 months and 0% (3 year overall survival rate was 33.3%). The recurrence rate was 71.7% (5/6) and disease free survival was 13.2 months.

Conclusion:
This study showed that 5.0% of small-sized (≤ 2 cm) NSCLC had N2 disease and 0.6% of T1a (≤ 1 cm) NSCLC has pN2. Moreover, 56.5% of small-sized (≤ 2 cm) NSCLC was upstaged from clinical diagnosis to pathological diagnosis. The patients with pT1a (≤ 1 cm) N2M0 had worse survival data than the patients with pT1a (≤ 2 cm) N2M0. We recommend systematic lymph node dissection for local treatment as well as accurate diagnosis. As multiple mediastinal node metastases showed an unfavorable prognosis, surgery combined with systematic treatment is recommended.

Background:
The incidence of lung cancer increases with age and approximately 50% of non-small cell lung cancer (NSCLC) patients are over 70 years old. Combined modality therapy is standard of care for patients with unresectable locally advanced non-small cell lung cancer (NSCLC), however, despite the multitude of clinical trials performed, elderly patients have been under-represented in these studies. Objective: To investigate outcomes for elderly patients treated with chemoradiotherapy (CRT).

Methods:
Patients with locally advanced stage NSCLC admitted in a tertiary hospital, between 1th January 2014 and 31th may 2016, who received CRT were selected. Patients were divided in two groups by age (<70 vs. ≥70-years old). Clinical-demographic variables, overall survival (OS) and progression free survival (PFS) were compared between the two groups.

Results:
Fifty-one patients were included. The results are presented in the table:

	<70years n=23(45,1%)	≥70years n=28(54,9%)	p
Gender
Male[n;(%)]	19(82.6)	26(92.9)	0.390
Performance status (at diagnosis)[n;(%)]
0	6(26.1)	10(35.7)	0.172
1	16(69.6)	13(46.4)
2	1(4.3)	5(17.9)
Weight loss (at diagnosis)[n;(%)]
0%	14(60.9)	16(57.1)	0.895
5%	6(26.1)	7(25.0)
10%	3(13.0)	5(17.9)
Clinical stage[n;(%)]
IIIA	10(43.5)	17(60.7)	0.070
IIIB	13(56.5)	11(39.3)
Comorbidities[n;(%)]
Heart failure	-	4(14.8)	0.076
Hypertension	7(30.4)	20(74.1)	0.002
Dyslipidemia	5(21.7)	12(44.4)	0.091
Chemotherapy regimen[n;(%)]
Carboplatin	5(21.7)	20(71.4)	0.146
Cisplatin	18(78.3)	8(28.6)
CRT[n;(%)]
Sequential	6(26.1)	11(39.3)	0.320
Concurrent	17(73.9)	17(60.7)
Second line treatment[n;(%)]
No	16(69.6)	21(75.0)	0.320
Yes	7(30.4)	7(25.0)

Comparing with younger group the elderly group presented significant worse OS and longer PFS, although without statistical significance [respectively, median 7 vs. 12months (p=0.006) and median 11.5 vs. 8months (p=0.687)]. Elderly patients with higher PS presented worse survival (p=0.045). Patients submitted to a chemotherapy regimen with cisplatin presented better OS and PFS in both groups, although only statistical significant for the OS in patients under 70 years (p=0.023). There was no influence of other variables on OS and PFS.

Conclusion:
In our sample age was an important prognostic factor in patients submitted to CRT but other factors, as PS, also can influence prognosis. In both groups patients treated with cisplatin presented superior OS but less patients above 70 years received this treatment. Elderly patients could be considered for CRT treatment but each case should be analysed individually. More studies are needed to guide treatment in this population.

Background:
Background-Non small cell lung cancer in young adults appears to be increasing over recent years. It’s a devastating illness both for the patient and their family. It has got significant socioeconomic implications.

Methods:
Methods-Data were analysed for the period between 2010-2015 from the University Hospitals of Leicester data base. Young adults were defined as age less than 50 and further sub divided into 18-39 years and 40-50 years of age respectively. Data were extracted regarding the histological diagnosis of cancer, performance status, disease staging and the treatment received.

Results:
Results-From a total of 93 patient’s we found the majority had adenocarcinoma,with 56% in the 18-39 age group and 63.6% in 40-50 age group. A greater proportion of patients in each age group were found to have a performance status of 0.The number of male patients were noted to be slightly higher between 18-39 (55%), compared to the 40-50 age groups, where there was a female predominance (57%). The majority of patients in both age groups were found to have a good performance status and a larger proportion of patient’s eGFR status was negative. Young adults were more likely to have surgery and chemotherapy due to their better performance status.

Conclusion:
Conclusion: In our cohort of young adults with lung cancer, the majority of patients had a good performance status despite late stage disease.They were likely to be fit for treatment and have longer survival outcomes.

Background:
Optimal treatment in LA NSCLC patients is still debated. In fit patients concomitant radio-chemotherapy (RCT) seems to be the best treatment in terms of local control (LC), progression free survival (PFS) and overall survival (OS) while sequential RCT is a good alternative in unfit patients. Moderately hypofractionated radiotherapy improve OS in recent studies. Elderly patients often cannot be offered multimodality treatments. We report our experience with over 70 years old LA NSCLC patients deemed unfit for surgery.

Methods:

Patients' Characteristics
Age	Median	75
	Range	70-83
Gender	Male	50 (70%)
	Female	21 (30%)
Performance Status (ECOG)	0	29 (41%)
	1	36 (51%)
	2	6 (8%)
Histology	Adenocarcinoma	31 (44%)
	Squamous Cell Carcinoma	39 (55%)
	Large Cell Carcinoma	1 (1%)
Stage	IIa/IIb	12 (17%)
	IIIa	39 (55%)
	IIIb	20 (28%)
Chemotherapy	Concomitant	9 (13%)
	Sequential	62 (87%)
	Cycles: median	4
	Cycles: range	1-8
Radiotherapy	Median Dose	62,3 Gy
	Moderate hypofractionation	26 (37%)
	Conventional fractionation	45 (63%)

Characteristics of patients and treatments are summarized in table 1. All patients were treated with a platinum based doublet of chemotherapy (CT). RT target volumes included the primary lung tumor and involved mediastinal lymphnodes as defined on pre-treatment contrast enhanced CT scan. Elective nodal irradiation was not performed. Acute/late toxicities were reported in accordance to 4.0 CTCAE scale. Clinical response was evaluated according to RECIST criteria.

Results:
At a median follow up of 10 months clinical response was evaluable in 69/71 patients obtaining a partial response in 35 of them, stable disease in 17, progressive disease in 17 patients. Twenty six patients experienced a local relapse within RT primary tumor volume, while 13 on nodal volume (5 patients both tumor and nodal relapse). 22 patients developed metastatic disease. One and 3-year OS was 62.3%(SE±6.2%) and 24,5%(SE±7.8%) respectively, while 1- and 3 year PFS was 45.1%(SE±6.9%) and 9,7%(SE±5.7%) respectively. At univariate analysis, tumor dimension (p<0,002) was the only prognostic factor statistically significant for OS. G1-G2 acute toxicity was observed in 45 patients: 36/62 in sequential CRT (3/36 developed also chronic toxicities) and 9/9 in concomitant CRT; most events were G1 oesophagitis (27 patients) and G1 cough (17 patients). No G3-4 event was reported.

Conclusion:
CRT is feasible in elderly patients; multidisciplinary evaluation is needed in order to reserve CRT to very fit patients.

Background:
In stage III NSCLC concurrent chemoradiation (cCRT) compares favourably to sequential CRT (sCRT). No superior chemotherapy regimen has been identified regarding (extracranial)PD. Previously we reported that the specific chemotherapy did not influence symptomatic brain metastases incidence. Here we analyse whether the specific chemotherapy influences occurrence of extracranialPD as first PD site (ePD~first~) after CRT.

Methods:
This retrospective multicenter study included all consecutive stage III NSCLC patients that completed CRT between 01-2006 and 06-2014. Primary endpoint was ePD~first ~(with/without cranial PD). Differences between regimens were assessed using logistic regression modelling including known relapse risk factors (age, gender, stage, histology) and the specific chemotherapy: cCRT versus sCRT. Within cCRT: daily low dose cisplatin (LDC) versus cyclic dose polychemotherapy (CDC); LDC versus (non-)taxane CDC; LDC versus subgroups of ≥50 CDC patients).

Results:
838 patients (737 cCRT, 101 sCRT) from 5 institutions were eligible. Median follow-up [95% CI] was 45.1 [42.3-47.8] months. 530 (63.2%) had PD, of which 463 (87.4%) had ePD~first. ~Median time to ePD~first~ was 16.6 [14.5-18.7] months. Patients with ePD~first~ had more often squamous histology (p=0.04). ePD~first~% or median time to ePD~first~ was not different for sCRT versus cCRT (table 1). 461 (62.6%) patients had PD after cCRT, of whom 401 (87.0%) had ePD~first~. ePD~first~% or median time to ePD~first~ did not differ between LDC and CDC. The chemotherapy regimen (cCRT versus sCRT) did not influence ePD~first~ on multivariate analysis: OR 0.81 [0.52-1.24] (p=0.33). LDC versus CDC cCRT did not differ: OR 0.96 [0.72-1.29] (p=0.80). Comparable results were found for LDC versus CDC non-taxane (N=277) and CDC taxane regimens (N=69) and for cCRT regimens with ≥50 patients (LDC versus cisplatin/etoposide (N=188), cisplatin/vinorelbin (N=65), weekly cisplatin/docetaxel (N=60)).

Table1

	concurrent N=737	sequential N=101	p-value
PD N(%)	461 (62.6)	68 (68.3)	0.18
ePDfirst N(%) -patients with concomitant brain metastases	401 (87.0) -38 (9.5)	62 (89.9) -8 (12.9)	0.19
median time to ePD [95%CI] months	17.5 [15.0-20.0]	14.3 [11.9-16.7]	0.16
	LDC N=391	cyclic dose N=346	p-value
PD N(%)	245 (62.7)	216 (62.4)	0.33
ePDfirst N(%) -patients with concomitant brain metastases	211 (86.1) -17 (8.1)	190 (88.0) -21 (11.1)	0.80
median time to ePD [95%CI] months	17.4 [14.3-20.5]	18.3 [14.2-22.5]	0.59

Conclusion:
Sixty-three percent of stage III NSCLC patients developed PD, of whom 87% had ePD~first~. Incidence of ePD~first~ is independent of the specific chemotherapy regimen.

Background:
Non-small cell lung cancer (NSCLC) patients with clinical (c-) N3 M0 are conventionally regarded as inoperable. However, the role of surgery for such patients clinically down staged after chemo-radiotherapy has not been ascertained. We retrospectively compared the outcome after chemo-radiotherapy plus surgery for down staged patients versus only conventional chemo-radiotherapy.

Methods:
Patients treated at our institute from 2000 to 2016 for primary NSCLC with c-N3M0 were identified. Amongst them, six patients received lung resection surgery after chemo-radiotherapy was given and clinical evidence of downstaging found. Fifty patients received only conventional chemo-radiotherapy during the same period. Survival was estimated using the Kaplan-Meier method.

Results:
All of the 6 patients receiving chemo-radiotherapy plus surgery, are recurrence-free survival. The survival time ranged from 5 to 91 months. The 5-year overall survival for the patients receiving surgery was 100% compared with 24% for the 50 patients who did not receive surgery (p= 0.04).

Conclusion:
Our results suggest that the combination of chemo-radiotherapy plus surgery may improve survival for preoperatively down staged c-N3M0 NSCLC patients. These results should be validated by large-scale, prospective, randomized trials.

Background:
The role of neoadjuvant chemoradiation and surgery in patients with stage IIIB non-small cell lung cancer (NSCLC) is unclear. Previous studies have suggested that select patients may benefit from this trimodality approach. We retrospectively reviewed patients with stage IIIB NSCLC treated by trimodality intent with induction chemotherapy and concurrent full-dose radiation therapy followed by either surgery at our institution. Here we report survival and toxicity data in our cohort.

Methods:
Eight patients treated from 1999 to 2011 with neoadjuvant chemoradiation for stage IIIB NSCLC were included in the retrospective review. Five (63%) had pathologically proven N3 disease; 1 (13%) had radiographic evidence of N3 disease (2cm adenopathy with SUV>6); 2 (25%) had T4 disease due to involvement of multiple ipsilateral lobes. All 6 patients with N3 disease had minimal radiographically enlarged N3 nodes (fewer than 3) before treatment. Induction chemotherapy consisted of carboplatin or cisplatin doublet. Concurrent RT prescription consisted of 45Gy in 25 fractions to the mediastinum and primary tumor; most patients received a boost to at least 60Gy to gross disease. After re-evaluation, patients received surgery within three months of completion of induction therapy. Inoperable patients received consolidative chemotherapy.

Results:
Six patients (86%) received at least 59.4Gy to the primary tumor. Six patients underwent resection; 2 had pneumonectomy and 4 had lobectomy. A complete (R0) resection was achieved in all patients. Mediastinal nodal clearance (N2/3 negative) was seen in five (83%) patients. A complete pathological response was seen in three (50%) patients. With a median follow up of 45 months for all patients, the median overall survival (OS) was 52.8 months. The median progression-free survival (PFS) was 48.4 months. Median OS was 52.8 months for patients who achieved MNC, 5.7 months in one patient with residual mediastinal nodal disease (P=0.025), and 0.9 months in those who did not receive surgery. There was 1 grade 3 postoperative pulmonary complications and no treatment-related mortality within the follow up interval.

Conclusion:
Data from on our small cohort provide important preliminary evidence that neoadjuvant chemotherapy with concurrent full-dose radiation therapy followed by surgery may be a feasible treatment option for select patients with stage IIIB NSCLC. Toxicity is acceptable, and survival outcome compares favorably with that of patients with IIIA NSCLC treated with trimodality therapy.

Background:
The presence of malignant pleural effusion (MPE) frequently indicates locally advanced non-small cell lung cancer (NSCLC). The conventional management of MPE involves pleurodesis using a sclerosant substance. The Perng et al. reported injection into chest cavity using paclitaxel for 18 MPE cases. In this report overall response rate was 92.9%. No disease progression was noted among evaluable patients. Therefore we focused on Paclitaxel which had antitumor effect and a pleural effusion control, and planned clinical trials phase I and II for the treatment of MPE.

Methods:
The primary objective of this study was to evaluate the efficacy of paclitaxel pleurodesis, the side effects and systemic antitumor effect. Patients were enrolled cytologically proven malignant pleural effusion of NSCLC. Radiotherapy and systemic chemotherapy were also not allowed within 4 weeks. After adequate drainage and assurance of lung re-expansion, paclitaxel diluted in normal saline was infused through a double lumen catheter. The starting dose was 100 mg/m2 with dose escalation schedule of 125, 150, and 175mg/m2. The catheter was clamped for 24 hrs. Chest drainage was continued until daily drainage was under 200 ml, and the tube was removed. To measure paclitaxel concentration, serum and pleural fluid samples were collected 0.5, 1, 3, 6, 24, 72hrs after the end of drug instillation. The treatment response of malignant effusion was evaluated according to the following criteria: complete response (CR), no fluid reaccumulation for at least 4 weeks as determined by CT scan; partial response (PR), recurrence of effusion to less than 50% of the original effusion volume within 4 weeks after treatment; failure, recurrence of effusion greater than 50% of the original volume, patients were symptomatic and need for thoracentesis to relieve symptoms within 4 weeks. The criteria for main tumour lesion response to intrapleural paclitaxel treatment were in accordance with Response Evaluation Criteria in Solid Tumours(Revised RECIST guideline version 1.1).

Results:
From April 2009 to November 2012, 12 patients were enrolled. There were minimal local and systemic toxicities. The serum level of PXL of the cases with the effect of treatment was significantly higher than these of the cases without the effect. Over all response rate was 67%. No disease progression was noted among evaluable patients.

Conclusion:
We decided to plan a late phase II study of the therapy with 175 mg/m2, because it was not dosage-dependent even if it was a low dose and serum level-dependent.