Valsamo Anagnostou

Programmed death ligand-1 expression in non-small cell lung cancer

Recent strategies targeting the interaction of the programmed cell death ligand-1 (PD-L1, B7-H1, CD274) with its receptor, PD-1, resulted in promising activity in early phase clinical trials. In this study, we used various antibodies and in situ mRNA hybridization to measure PD-L1 in non-small cell lung cancer (NSCLC) using a quantitative fluorescence (QIF) approach to determine the frequency of expression and prognostic value in two independent populations. A control tissue microarray (TMA) was constructed using PD-L1-transfected cells, normal human placenta and known PD-L1-positive NSCLC cases. Only one of four antibodies against PD-L1 (5H1) validated for specificity on this TMA. In situ PD-L1 mRNA using the RNAscope method was similarly validated. Two cohorts of NSCLC cases in TMAs including 340 cases from hospitals in Greece and 204 cases from Yale University were assessed. Tumors showed PD-L1 protein expression in 36% (Greek) and 25% (Yale) of the cases. PD-L1 expression was significantly associated with tumor-infiltrating lymphocytes in both cohorts. Patients with PD-L1 (both protein and mRNA) expression above the detection threshold showed statistically significant better outcome in both series (log-rank P=0.036 and P=0.027). Multivariate analysis showed that PD-L1 expression was significantly associated with better outcome independent of histology. Measurement of PD-L1 requires specific conditions and some commercial antibodies show lack of specificity. Expression of PD-L1 protein or mRNA is associated with better outcome. Further studies are required to determine the value of this marker in prognosis and prediction of response to treatments targeting this pathway.

GOLPH3 modulates mTOR signalling and rapamycin sensitivity in cancer

Genome-wide copy number analyses of human cancers identified a frequent 5p13 amplification in several solid tumour types, including lung (56%), ovarian (38%), breast (32%), prostate (37%) and melanoma (32%). Here, using integrative analysis of a genomic profile of the region, we identify a Golgi protein, GOLPH3, as a candidate targeted for amplification. Gain- and loss-of-function studies in vitro and in vivo validated GOLPH3 as a potent oncogene. Physically, GOLPH3 localizes to the trans-Golgi network and interacts with components of the retromer complex, which in yeast has been linked to target of rapamycin (TOR) signalling. Mechanistically, GOLPH3 regulates cell size, enhances growth-factor-induced mTOR (also known as FRAP1) signalling in human cancer cells, and alters the response to an mTOR inhibitor in vivo. Thus, genomic and genetic, biological, functional and biochemical data in yeast and humans establishes GOLPH3 as a new oncogene that is commonly targeted for amplification in human cancer, and is capable of modulating the response to rapamycin, a cancer drug in clinical use.

Personalized genomic analyses for cancer mutation discovery and interpretation

Analysis of matched tumor and normal DNA from the same patient improves accuracy of identification of actionable mutations, allowing better targeting of potential treatments. Will the real mutation please stand up? When a patient is diagnosed with cancer, a sample of the tumor is often analyzed to look for mutations that might guide the approach to targeted treatment of the disease. Jones et al. analyzed samples from more than 800 patients with 15 different cancer types and showed that this standard approach is not necessarily accurate without also analyzing a matched sample of normal DNA from the same patient. The authors found that, compared to analysis of paired samples, the standard tumor-only sequencing approach frequently identified mutations that were present in the patient’s normal tissues and were therefore not suitable for targeted therapy or, conversely, missed useful new mutations in the tumor. Massively parallel sequencing approaches are beginning to be used clinically to characterize individual patient tumors and to select therapies based on the identified mutations. A major question in these analyses is the extent to which these methods identify clinically actionable alterations and whether the examination of the tumor tissue alone is sufficient or whether matched normal DNA should also be analyzed to accurately identify tumor-specific (somatic) alterations. To address these issues, we comprehensively evaluated 815 tumor-normal paired samples from patients of 15 tumor types. We identified genomic alterations using next-generation sequencing of whole exomes or 111 targeted genes that were validated with sensitivities >95% and >99%, respectively, and specificities >99.99%. These analyses revealed an average of 140 and 4.3 somatic mutations per exome and targeted analysis, respectively. More than 75% of cases had somatic alterations in genes associated with known therapies or current clinical trials. Analyses of matched normal DNA identified germline alterations in cancer-predisposing genes in 3% of patients with apparently sporadic cancers. In contrast, a tumor-only sequencing approach could not definitively identify germline changes in cancer-predisposing genes and led to additional false-positive findings comprising 31% and 65% of alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic and germline alterations and have important implications for the diagnostic and therapeutic management of cancer patients.

Gefitinib or Placebo in Combination with Tamoxifen in Patients with Hormone Receptor-Positive Metastatic Breast Cancer: a Randomized Phase II Study

Purpose: Increased growth factor signaling may contribute to tamoxifen resistance. This randomized phase II trial assessed tamoxifen plus placebo or the epidermal growth factor receptor inhibitor gefitinib in estrogen receptor (ER)–positive metastatic breast cancer. Experimental Design: Patients with newly metastatic disease or recurred after adjuvant tamoxifen (stratum 1), or recurred during/after adjuvant aromatase inhibitor (AI) or after failed first-line AI (stratum 2), were eligible. Primary variables were progression-free survival (PFS; stratum 1) and clinical benefit rate (CBR; stratum 2). A 5% or more improvement in response variables with gefitinib was considered to warrant further investigation. Outcome was correlated with biomarkers measured on the primary tumor. Results: In stratum 1 (n = 206), the PFS HR (gefitinib:placebo) was 0.84 (95% CI, 0.59–1.18; median PFS 10.9 versus 8.8 months). In the stratum 1 endocrine therapy–naïve subset (n = 158) the HR was 0.78 (95% CI, 0.52–1.15), and the prior endocrine-treated subgroup (n = 48) 1.47 (95% CI, 0.63–3.45). In stratum 1, CBRs were 50.5% with gefitinib and 45.5% with placebo. In stratum 2 (n = 84), CBRs were 29.2% with gefitinib and 31.4% with placebo. Biomarker analysis suggested that in stratum 1 there was greater benefit with gefitinib in patients who were ER-negative or had lower levels of ER protein. Conclusions: In stratum 1, the improved PFS with gefitinib plus tamoxifen met the protocol criteria to warrant further investigation of this strategy. In stratum 2, there was a numerical disadvantage for gefitinib; additional investigation after AI therapy is not warranted. Studies of predictive biomarkers are needed to subset appropriate patients. Clin Cancer Res; 17(5); 1147–59. ©2011 AACR.

The genomic landscape of response to EGFR blockade in colorectal cancer

Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.

Evolution of Neoantigen Landscape during Immune Checkpoint Blockade in Non–Small Cell Lung Cancer

Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non-small cell lung cancer after initial response to immune checkpoint blockade with anti-PD-1 or anti-PD-1/anti-CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens.Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of neoantigen reactivity may mitigate the development of acquired resistance. Cancer Discov; 7(3); 264-76. ©2017 AACR.See related commentary by Yang, p. 250This article is highlighted in the In This Issue feature, p. 235.

Clinical implications of genomic alterations in the tumour and circulation of pancreatic cancer patients

Pancreatic adenocarcinoma has the worst mortality of any solid cancer. In this study, to evaluate the clinical implications of genomic alterations in this tumour type, we perform whole-exome analyses of 24 tumours, targeted genomic analyses of 77 tumours, and use non-invasive approaches to examine tumour-specific mutations in the circulation of these patients. These analyses reveal somatic mutations in chromatin-regulating genes MLL, MLL2, MLL3 and ARID1A in 20% of patients that are associated with improved survival. We observe alterations in genes with potential therapeutic utility in over a third of cases. Liquid biopsy analyses demonstrate that 43% of patients with localized disease have detectable circulating tumour DNA (ctDNA) at diagnosis. Detection of ctDNA after resection predicts clinical relapse and poor outcome, with recurrence by ctDNA detected 6.5 months earlier than with CT imaging. These observations provide genetic predictors of outcome in pancreatic cancer and have implications for new avenues of therapeutic intervention.

Cancer Immunotherapy: A Future Paradigm Shift in the Treatment of Non–Small Cell Lung Cancer

Emerging evidence on the role of the antitumor activity of the immune system has generated great interest in immunotherapy even for tumors that were historically considered as nonimmunogenic. Immunotherapy is emerging as a major modality in non–small cell lung cancer (NSCLC) treatment focusing on vaccine approaches to elicit specific immune responses and development of inhibitors of the molecular mediators of cancer-induced immunosuppression (immune checkpoints) to boost antitumor immune responses. Amplification of the host response against evolving tumors through vaccination is being investigated in ongoing clinical trials with tumor cell vaccines; however, the clinical efficacy of these agents has been limited. Blocking inhibitory pathways such as the CTL antigen 4 (CTLA-4) and programmed cell death 1 (PD-1) checkpoint pathways with mAbs has generated antitumor immune responses that are transforming cancer therapeutics. PD-1 and programmed cell death ligand 1 (PD-L1) antibodies have shown durable responses in NSCLC, with a favorable safety profile and manageable side effects. The activity of immune checkpoint inhibitors is currently been assessed in treatment-naïve patients with PD-L1–positive advanced NSCLC. Combinatorial approaches with other immune checkpoint inhibitors, chemotherapy, or targeted agents are being explored in ongoing clinical trials, and may improve outcome in NSCLC. Clin Cancer Res; 21(5); 976–84. ©2015 AACR.

Thyroid Transcription Factor 1 Is an Independent Prognostic Factor for Patients With Stage I Lung Adenocarcinoma

PURPOSE Thyroid transcription factor 1 (TTF1) is a transcription factor that regulates the expression of multiple genes involved in lung development. It is preferentially expressed in adenocarcinomas of the lung and has been investigated as a potential prognostic parameter in patients with lung cancer, with conflicting results. We quantitatively assessed TTF1 protein expression in two large and independent data sets to investigate the impact of TTF1 nuclear expression on patient survival. PATIENTS AND METHODS Automated quantitative analysis, a fluorescent-based method for analysis of in situ protein expression, was used to assess a series of cell lines to find the threshold of detection of TTF1 expression. Then two independent cohorts (176 and 237 cases, respectively) were measured by the same technique, and TTF1 expression was correlated with survival. RESULTS Tumors expressed TTF1 in 45% and 58% of the cases in each cohort. TTF1 was consistently expressed in adenocarcinomas (n = 61 and 73; Spearman rho = 0.313 and 0.4 for the first and second set, respectively; P < .0001) independent of their differentiation and stage. Survival analysis showed that patients with stage I adenocarcinoma with TTF1 expression had a longer median overall survival than those without expression (n = 43, 44.3 v 26.2 months, P = .05 for the first cohort; n = 87; 49.7 v 38.5 months, P = .03 for the second cohort) Multivariate analysis revealed an independent lower risk of death for patients with stage I adenocarcinoma with TTF1-expressing tumors (hazard ratio = 0.479, 95% CI, 0.235 to 0.977; P = .043). CONCLUSION TTF1 expression defines a subgroup of patients with a favorable outcome and may be useful for prognostic stratification of patients with stage I lung adenocarcinoma.

Analytic Variability in Immunohistochemistry Biomarker Studies

Background: Despite the widespread use of immunohistochemistry (IHC), there are no standardization guidelines that control for antibody probe variability. Here we describe the effect of variable antibody reagents in the assessment of cancer-related biomarkers by IHC. Methods: Estrogen receptor (ER), epidermal growth factor receptor (EGFR) 1, and human epidermal growth factor receptor 3 (HER3) were evaluated by quantitative immunofluorescence. Correlations between ER clones 1D5, SP1, F10, and ER60c, and EGFR monoclonal 31G7, 2-18C9, H11, and 15F8, and polyclonal 2232 antibodies were assessed in 642 breast cancer patients. HER3 was measured by RTJ1, RTJ2, SGP1, M7297, RB-9211, and C-17 antibodies in 42 lung cancer patients. Survival analysis was done with the use of multiple cutoff points to reveal any prognostic classification. Results: All ER antibodies were tightly correlated (Pearsons r2 = 0.94-0.96; P < 0.0001) and western blotting confirmed their specificity in MCF-7 and BT474 cells. All EGFR antibodies but 2232 yielded specific results in western blotting; however, only 31G7 and 2-18C9 were strongly associated (Pearsons r2 = 0.61; P < 0.0001). HER3 staining was nonspecific and nonreproducible. High EGFR–expressing patients had a worse prognosis when EGFR was measured with H11 or 31G7 (log rank P = 0.015 and P = 0.06). There was no statistically significant correlation between survival and EGFR detected by 2-18C9, 15F8, or polyclonal 2232 antibodies. Conclusions: Antibody validation is a critical analytic factor that regulates IHC readings in biomarker studies. Evaluation of IHC proficiency and quality control are key components toward IHC standardization. Impact: This work highlights the importance of IHC standardization and could result in the improvement of clinically relevant IHC protocols. Cancer Epidemiol Biomarkers Prev; 19(4); 982–91. ©2010 AACR.