Virtual Library

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Abstract:
The treatment of lung adenocarcinomas has improved with the identification of driver gene mutations and the development of drugs tackling the altered driver gene proteins [1]. The interrogation of EGFR mutations as well as ALK and ROS1 translocations is nowadays part of the routine diagnostic workup of lung adenocarcinomas. Further therapies aiming at mutations in driver genes like RET, HER2, BRAF and MET are emerging. The main techniques currently employed in molecular pathology laboratories for mutation detection are mutation specific PCR, conventional capillary (Sanger) sequencing and next generation sequencing (NGS) [2]. The basic principles of these methods, advantages and disadvantages with a special emphasis on NGS and its potency for cancer diagnostics will be discussed. Recently the detection of driver gene mutations in DNA that is derived from cancer cells and released into the blood has become feasible (so-called „liquid biopsy“) [3]. It has already entered routine diagnostics with the detection of the T790M mutation in circulating tumor DNA of primary EGFR mutated lung cancers, that developed resistance to first or second generation tyrosine kinase inhibitors [4]. The verification of a T790M mutation either by liquid biopsy or the analysis of tumor tissue is a prerequisite for therapy of lung adenocarcinomas with third generation tyrosine kinase inhibitors like osimertinib. The principles of liquid biopsy, employed techniques and potential applications will be introduced. The recent advent of cancer immunotherapy that interferes with immune checkpoint molecules like programmed death 1 (PD-1) offers a new treatment for subgroups of lung cancer patients. A biomarker that would predict responsiveness to this expensive therapy is greatly needed. The expression of the PD-1 ligand (PD-L1) by tumor or immune/stromal cells is a potential biomarker, however its utility is heavily debated [5]. The confusion on PD-L1 as a biomarker is partly caused by technical difficulties in the determination of expression, such as the antibody used for immunohistochemistry and the determination of a treshold of expression that correlates with response [6]. A further biomarker that is determined by immunohistochemistry is the expression of EGFR in lung squamous cell carcinoma. A therapy with the recently approved anti-EGFR antibody necitumumab requires the demonstration of EGFR expression by the cancer cells [7]. The methods and pitfalls in PD-1 and EGFR immunohistochemistry will be presented. The requirement for biomarkers increases. This poses a challenge for diagnostics in respect to availabiltity of techniques, infrastructure and budget. Particularly in lung cancer often very little tissue is available, but different assays should be run. To fulfill the increasing demand for a plethora of biomarker analysis multiplexing techniques that simultaneously interrogate a large number of gene mutations, gene fusions, gene amplification and deletions, as well as RNA and protein expression will be needed. An outlook on available and emerging multiplexing techniques will be provided. References 1. The Cancer Genome Atlas Research N (2014) Comprehensive molecular profiling of lung adenocarcinoma. Nature 511 (7511):543-550. doi:10.1038/nature13385 2. Buermans HP, den Dunnen JT (2014) Next generation sequencing technology: Advances and applications. Biochimica et biophysica acta 1842 (10):1932-1941. doi:10.1016/j.bbadis.2014.06.015 3. Diaz LA, Jr., Bardelli A (2014) Liquid biopsies: genotyping circulating tumor DNA. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32 (6):579-586. doi:10.1200/JCO.2012.45.2011 4. Thress KS, Paweletz CP, Felip E, Cho BC, Stetson D, Dougherty B, Lai Z, Markovets A, Vivancos A, Kuang Y, Ercan D, Matthews SE, Cantarini M, Barrett JC, Janne PA, Oxnard GR (2015) Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat Med 21 (6):560-562. doi:10.1038/nm.3854 http://www.nature.com/nm/journal/v21/n6/abs/nm.3854.html - supplementary-information 5. Sunshine J, Taube JM (2015) PD-1/PD-L1 inhibitors. Current opinion in pharmacology 23:32-38. doi:10.1016/j.coph.2015.05.011 6. Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, Patnaik A, Aggarwal C, Gubens M, Horn L, Carcereny E, Ahn M-J, Felip E, Lee J-S, Hellmann MD, Hamid O, Goldman JW, Soria J-C, Dolled-Filhart M, Rutledge RZ, Zhang J, Lunceford JK, Rangwala R, Lubiniecki GM, Roach C, Emancipator K, Gandhi L (2015) Pembrolizumab for the Treatment of Non–Small-Cell Lung Cancer. New England Journal of Medicine 372 (21):2018-2028. doi:doi:10.1056/NEJMoa1501824 7. Thatcher N, Hirsch FR, Luft AV, Szczesna A, Ciuleanu TE, Dediu M, Ramlau R, Galiulin RK, Balint B, Losonczy G, Kazarnowicz A, Park K, Schumann C, Reck M, Depenbrock H, Nanda S, Kruljac-Letunic A, Kurek R, Paz-Ares L, Socinski MA (2015) Necitumumab plus gemcitabine and cisplatin versus gemcitabine and cisplatin alone as first-line therapy in patients with stage IV squamous non-small-cell lung cancer (SQUIRE): an open-label, randomised, controlled phase 3 trial. The Lancet Oncology 16 (7):763-774. doi:10.1016/s1470-2045(15)00021-2

Abstract:
Early adaptive resistance in EGFR mutant NSCLC Rafael Rosell Small molecule inhibitors are the current treatment for non-small-cell lung cancer (NSCLC), especially for tumours harbouring an active mutation of epidermal growth factor receptor (EGFR). Approximately 90% of EGFR mutations are exon 19 deletions or exon 21 single-point L858R substitutions and are associated with sensitivity to EGFR tyrosine kinase inhibitors (EGFR TKIs), like gefitinib or erlotinib. However, less than 5% of EGFR-mutant NSCLC patients achieve a complete response to EGFR TKI and the overall median progression-free survival is no longer than 9-11 months. Accumulated studies report several mechanisms of early adaptive resistance that can occur as early as two hours after starting EGFR TKI therapy. The activation of signal transducer and activator of transcription 3 (STAT3) signalling is among these mechanisms of resistance. Furthermore, EGFR blockage enriches lung cancer stem cells through Notch3-dependent signalling pathway. We have previously demonstrated that NF-κB contributes to gefitinib resistance in EGFR-mutant NSCLC. The efficacy of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is jeopardized by the activation of signalling pathways. We examined the relevance of co-targeting EGFR, signal transducer and activator of transcription 3 (STAT3) and Src-YES-associated protein 1 (YAP1) signalling. We conducted clinical and preclinical studies of key components of signalling pathways limiting EGFR TKI efficacy in EGFR-mutant NSCLC. High levels of STAT3 or YAP1 mRNA expression were associated with worse outcome to EGFR TKI in two independent cohorts of EGFR-mutant NSCLC patients. In the initial cohort of 64 patients, median progression-free survival was shorter among the patients with high STAT3 than among those with low STAT3 (hazard ratio [HR] for disease progression, 3·02; 95% confidence interval [CI], 1·54-5·93; P=0·0013). Median progression-free survival was shorter among the patients with high YAP1 than among those with low YAP1 (HR for disease progression, 2·57; 95%CI, 1·30-5·09; P=0·0067). The results were similar in the validation cohort of 55 patients. We demonstrated that gefitinib augments STAT3 signalling in EGFR-mutant NSCLC cells. Gefitinib with TPCA-1 (STAT3 inhibitor) blocked STAT3, but not the YAP1 phosphorylation on tyrosine residue 357 by Src family kinases (SFKs) that occurs downstream of IL-6. The triple combination of gefitinib, TPCA-1 and AZD0530 (SFK inhibitor) ablated both STAT3 and YAP1 phosphorylation and markedly and safely suppressed tumour growth. Added value of our research: We found that EGFR TKI therapy activates STAT3 and that EGFR blockage enriches lung cancer stem cells with up-regulation of the YAP1 and Notch downstream effectors connective tissue growth factor (CTGF) and hairy-enhancer of split-1 (HES1), respectively. We sought to demonstrate that EGFR TKI treatment cannot abrogate STAT3 and Src-YAP1-Notch activation in EGFR-mutant NSCLC cell lines, leading us to examine whether the combination of gefitinib with compounds targeting STAT3 and Src, supresses the mechanisms of resistance. Nine days after gefitinib treatment, STAT3 mRNA level was significantly increased, as well as the fraction of ALDH positive cells. TPCA-1, a compound that targets STAT3, increases sensitivity to gefitinib in PC-9 and H1975 cells; however, neither gefitinib nor TPCA-1 inhibits Src or YAP1. The addition of the Src inhibitor, saracatinib, to the doublet of gefitinib and TPCA-1, was highly synergistic and abrogated STAT3, and Src-YAP1-Notch signalling. Implications: Treatment with single EGFR TKI can no longer be considered adequate for patients with EGFR mutant NSCLC. Our findings ultimately suggest that a clinical trial evaluating the co-targeted inhibition of STAT3 and Src is warranted. As a result, STAT3 and YAP1 mRNA levels could become important predictive biomarkers. References: We searched PubMed for English language reports published up to December, 2015 using the terms “non-small-cell lung cancer”, “STAT3”, “interleukin-6”, “NF-κB”, “aldehyde-dehydrogenase (ALDH)”, “integrin-linked kinase (ILK)”, “glycoprotein 130 (gp130)”, “Src-homology 2 domain-containing phosphatase 2 (SHP2)”, “the complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing protein-1 (CDCP1)”, “AXL”, “ephrin type-A receptor-2 (EphA2)”, “Src family kinases (SFK)”, “YES-associated protein 1 (YAP1)”, “Notch”, “cell migration, invasion and metastases” and “STAT3 inhibitors”.

Background:
High-throughput gene expression profiling led to proposal of multiple expression-based prognostic signatures for squamous cell lung carcinoma (SCC), but none has been validated. A multi-institutional squamous lung cancer consortium of investigators is developing prognostic signatures through the US NCI Lung SPECS (Strategic Partnership for Evaluation of Cancer Signatures) program. Six institutions contributed tumor specimens and published/unpublished expression-based prognostic signatures for validation using standardized sample cohorts (a primary validation cohort comprising institutional cases, and additional validation cohorts from two prospective cooperative group studies) and quality controlled assessment in independent laboratory and statistical cores. Here, we report the results of the primary validation.

Methods:
Cases of primary SCC (by central pathology review) meeting clinical (Stage I-II; surgical treatment only; 3-year followup) and specimen quality criteria (Tumor cellularity >= 50%; necrosis <= 20%) were submitted. Clinical, pathological and outcome data were uploaded to a central database. Frozen tumor samples underwent centralized mRNA extraction (Qiagen Symphony), quality control (RIN >= 6.0) and microarray profiling (Affymetrix U133) in core labs. An independent statistical core assessed validation of 7 pre-existing mRNA signatures and generated new models using MCP clustering.

Results:
Among 250 cases meeting entry criteria, median age was 70 (43-92), 161 (65%) were male, and most were former (70%) or current (28%) smokers. Surgery was pneumonectomy: 5%; bilobectomy: 2%; lobectomy: 74%; sublobar: 18%. Pathologic staging was T1: 49%; T2: 50%; T3: 1%; N0: 88%; N1: 12%, and grade was G1: 4%; G2: 50%; G3: 44%. At followup, 148 (59%) were deceased. Three mRNA signatures demonstrated significant univariable association with OS and added independent prognostic value (see Figure) to a multivariable model accounting for age, sex and stage (c-index = 0.641).

Conclusion:
The validated signatures, along with two novel signatures generated from the current dataset, are currently undergoing further validation studies using two prospective co-operative group cohorts. Figure 1

Background:
A number of studies have characterized TAICs in lung cancer and associated their levels of infiltration with patients’ outcome. There is limited information about the correlation of TAICs infiltration with clinical and pathological features of lung cancer. We investigated the association between patterns of tumor infiltrating lymphocytes and macrophages with detailed clinical and pathological features in lung adenocarcinoma.

Methods:
We studied archival tumor tissue from 93 surgically resected lung adenocarcinomas, stages I to III. Density of TAICs expressing CD3, CD4, CD8, and CD68 was evaluated using immunohistochemistry (IHC) and image analysis. TAICs density was correlated with tumor’s histological characteristics and patients’ characteristics.

Results:
We found significant differences in the TAICs infiltrate density of lung adenocarcinomas based on patients’ characteristics. Overall: a) females showed higher levels of CD8+ (P=0.01) and CD3+ (P=0.03) cell density than males; b) smaller tumors (<3cms) showed more CD4+ (P=0.01) and CD3+ (P=0.03) cells than larger tumors; and, c) tumors with solid histology pattern showed higher levels of CD8+ (P=0.03) cells than non-solid pattern. No overall significant differences on TAICs infiltrates were detected by age, tobacco exposure by pack-years and TTF-1 IHC expression score. However when TAICs density of tumors was examined by sex we found the following: a) in larger tumor (>3cms), females demonstrated higher levels of CD8+ cells (P=0.0007) than males; b) tumors from females older than the median age (63 years) showed more CD4+ (P=0.04), CD8+ (P=0.009) and CD3+ (P=0.042) cells than males; c) tumors from females with <40 pack-years of tobacco history showed significantly higher levels of CD3+ (P=0.004) and CD68+ (P=0.004) cells than males; d) tumors from females with high levels of TTF1 expression (score >150) showed higher levels of CD8+ cells(P=0.03) than males; e) females with tumors having non-solid histology pattern showed higher CD8+ (P=0.02) and CD3+ (P=0.02) cells than males. Finally, tumors expressing low levels of TTF-1 and lower CD4+ cells correlated significantly with worse overall recurrence free survival and overall survival in both males (P<0.0001) and females (P=0.0072).

Conclusion:
In lung adenocarcinoma, TAICs infiltration correlates with clinical characteristics of patients and pathological features of tumors, particularly, sex, age, size and TTF-1 expression. Compared with men, lung adenocarcinomas from females showed higher levels of TAICs, particularly at older age, larger tumor size, less exposure to tobacco, and more differentiated histological patterns. (UT Lung SPORE and MD Anderson Moon Shot Program).

Background:
The national lung cancer screening test (NLST) confirmed: low dose CT screening could reduce lung cancer mortality. However, the high false-positive rates of LDCT screening, especially the difficulty of diagnosis of micro-nodules with size less than 10 mm highlight the need of complementary biomarkers to discriminate micro-nodular lung cancer from benign pulmonary diseases.

Methods:
The blood gene expression profiles of 46 lung cancer patients, 38 pulmonary lesions and 51 healthy were investigated to identify the lung cancer-specific genetic signatures. The lung cancer patients containing micro-nodules less than 10 mm were surgically and pathologically diagnosed as lung adenocarcinoma in situ

Results:
A self-training logistic regression method was used to identify the lung cancer-specific gene signatures as we previously reported. Six genes, including DDX51, PSME2, ACTL6A, GMEB1, FAM200B, GEMIN6, were identified for discriminating lung adenocarcinoma in situ from health and benign pulmonary diseases. The performance of the six-gene panel for diagnosis of lung adenocarcinoma in situ identified was exhibited in Table 1. Through self-training SVM classifier, the logarithmic odds of each sample was calculated and exhibited, in which the cutoff value was set as zero in logarithmic odds for differentiating lung cancer from benign and control group. The predictive model based on 6-gene panel correctly classified 43 of 46 lung cancer, 39 of 42 benign pulmonary diseases with 93% accuracy, 94% sensitivity, and 93% specificity and 0.97 of ROC AUC.

Conclusion:
The predictive model based on 6-gene panel (DDX51, PSME2, ACTL6A, GMEB1, FAM200B, GEMIN6) can be used for discriminating between the malignant or benign nodules with size less than 10 mm

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Background:
Most human cancers express proteoglycans modified with distinct oncofetal chondroitin sulfate (CS) chains that are normally restricted to placental tissue. Oncofetal CS chains can be conveniently detected and targeted by recombinant VAR2CSA (rVAR2) proteins derived from the malaria parasite Plasmodium falciparum. In the present study, we have analyzed the expression landscape of oncofetal CS modifications in early-stage non-small cell lung cancer (NSCLC).

Methods:
Tissue microarrays from four separate patient cohorts representing a total of 493 clinically annotated stage I-II NSCLC cases were stained for oncofetal CS using rVAR2. Data were analyzed for correlation between low and high oncofetal CS presentation by immunohistochemical (IHC) staining of tumor and stroma compartments in respect to EGFR and KRAS mutations, as well as to clinical characteristics including relapse-free survival (RFS) and overall survival (OS).

Results:
There were 351 patients with low (IHC score 0-1) and 142 with high (IHC score 2-3) expressing tumors. We identified 331 adenocarcinomas, 145 squamous cell carcinomas, and 12 cases with other NSCLC subtypes. There were 314 stage I and 179 stage II cases by AJCC 7[th] edition. High oncofetal CS expression was significantly associated with shorter RFS (vs. high expressiors; 58 vs. 39 months, respectively, p=0.034) and OS (vs. high expressors; 69 vs. 51 months, respectively, p=0.044). High oncofetal CS expression was significantly associated with shorter RFS vs. low expression in men (p=0.024), smokers (p=0.011), and in patients with squamous cell tumors (p=0.012). High oncofetal CS was also significantly associated with shorter OS in men (p=0.005) and smokers (p=0.028). There were no significant RFS or OS differences in oncofetal CS expressions when stratifying the patients according to their EGFR or KRAS statuses. In multivariate survival analyses, histology, stage, and high oncofetal CS expression was significantly associated with shorter RFS vs. high expression (HR, 1.8; 95% CI, 1.32–2.48; p < 0.001).

Conclusion:
This is the first study showing that high oncofetal CS expression is an independent prognostic factor of poor RFS in NSCLC and validates high oncofetal CS expression as a prognostic factor of poor OS. In contrast to non-smoker females, oncofetal CS appears to be a prognostic for OS in males and smokers. Our work promotes oncofetal CS as a candidate target for rVAR2-based therapeutic intervention in NSCLC patients with poor RFS/OS.

Background:
A clinically certified, 14-gene quantitative PCR expression assay has been found to assess mortality risk more accurately than clinicopathologic criteria in early-stage, non-squamous, non-small cell lung cancer (NSCLC). Clinically validated molecular stratification may provide a more informative approach to identify early stage NSCLC patients who are most likely to benefit from chemotherapy than current National Comprehensive Cancer Network (NCCN) high-risk clinicopathologic features.

Methods:
Prospective molecular risk-stratification by the 14-gene quantitative PCR expression assay was performed on 91 consecutive patients with stage I-IIA non-squamous NSCLC after complete surgical resection at a single institution. Information from molecular risk profiling was used in conjunction with pathologic stage and NCCN criteria to make adjuvant chemotherapy recommendations. Fisher’s exact test was used to compare recurrence rates, and Kaplan-Meier analysis and log-rank tests were used to evaluate differences in disease free survival.

Results:
Median age was 69 years, 57% were female and median follow up was 23±2 months. Among all patients, 33 (36%) met NCCN high-risk criteria for adjuvant chemotherapy and 27 (30%) were molecular high risk. Recommendations for adjuvant chemotherapy were discordant in 18 (55%) of NCCN high-risk patients and in 12 (44%) who were molecular high-risk. Twelve (44%) of molecular high-risk patients agreed to receive adjuvant chemotherapy. Whereas recurrence was observed in 33% of molecular high-risk patients who did not receive adjuvant chemotherapy, none of the molecular high-risk patients who underwent chemotherapy recurred (log-rank p=0.001).

Conclusion:
This prospective single-institution study demonstrates the clinical utility of molecular testing of early-stage NSCLC to supplement pathologic stage and NCCN guidelines in making adjuvant chemotherapy recommendations. Molecular risk scores better differentiated prospective recurrence rates than did NCCN risk criteria. This study provides preliminary evidence that molecular testing followed by adjuvant chemotherapy in molecularly high-risk patients may prevent a significant number of recurrences and improve outcomes.

Background:
Epidermal growth factor receptor (EGFR) gene mutation is a robust prognostic factor in patients with advanced lung adenocarcinomas. Recently, on the other hand, there are some reports proposing the difference of survival due to the type of EGFR mutation. In this study, we analyzed the difference of postoperative survivals between two most common mutations, that is, exon 19 deletions (DEL) and exon21 L858R (PM), using multi-institutional data of patients with surgically resected lung adenocarcinomas.

Methods:
We retrospectively collected 1,063 consecutive patients who underwent surgical resections for lung adenocarcinoma between 2005 and 2012 in five institutions, and who were examined their EGFR mutation status. The patients with minor EGFR mutations were excluded. We compared their clinicopathological characteristics among DEL, PM, and wild type (WT) group. We also analyzed postoperative recurrence-free survival (RFS) and overall survival (OS) according to the type of EGFR mutation.

Results:
The number of patients with DEL, PM, and WT was 218 (20.5%), 301 (28.3%), and 544 (51.2%) respectively, and their median follow-up period was 47.6 months. The patients of PM were older and earlier pathological staged than those with DEL, whereas no significant difference was observed among other clinicopathological factors. Five-year RFS and OS of DEL, PM, and WT were 67.3/85.9%, 76.4/88.6%, 59.2/71.5%, respectively, and both survivals of each mutant were significantly better than those of WT. Regarding the difference between DEL and PM, RFS curve of DEL was significantly worse than that of PM (p = 0.027), but OS curves of both mutant weren’t significantly different. (p = 0.16). In multivariate analysis, the type of EGFR mutation (DEL vs PM) was not an independent factor both in RFS and OS.

Conclusion:
Exon 21 L858R might be a more favorable recurrence-risk factor than exon 19 deletions in patients with surgically resected lung adenocarcinomas.

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