James Ranger-Moore

PD-L1 Immunohistochemistry Assays for Lung Cancer: Results from Phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project

Introduction: The Blueprint Programmed Death Ligand 1 (PD‐L1) Immunohistochemistry (IHC) Assay Comparison Project is an industrial‐academic collaborative partnership to provide information on the analytical and clinical comparability of four PD‐L1 IHC assays used in clinical trials. Methods: A total of 39 NSCLC tumors were stained with four PD‐L1 IHC assays (22C3, 28‐8, SP142, and SP263), as used in the clinical trials. Three experts in interpreting their respective assays independently evaluated the percentages of tumor and immune cells staining positive at any intensity. Clinical diagnostic performance was assessed through comparisons of patient classification above and below a selected expression cutoff and by agreement using various combinations of assays and cutoffs. Results: Analytical comparison demonstrated that the percentage of PD‐L1–stained tumor cells was comparable when the 22C3, 28‐8, and SP263 assays were used, whereas the SP142 assay exhibited fewer stained tumor cells overall. The variability of immune cell staining across the four assays appears to be higher than for tumor cell staining. Of the 38 cases, 19 (50.0%) were classified above and five (13%) were classified below the selected cutoffs of all assays. For 14 of the 38 cases (37%), a different PD‐L1 classification would be made depending on which assay/scoring system was used. Conclusions: The Blueprint PD‐L1 IHC Assay Comparison Project revealed that three of the four assays were closely aligned on tumor cell staining whereas the fourth showed consistently fewer tumor cells stained. All of the assays demonstrated immune cell staining, but with greater variability than with tumor cell staining. By comparing assays and cutoffs, the study indicated that despite similar analytical performance of PD‐L1 expression for three assays, interchanging assays and cutoffs would lead to “misclassification” of PD‐L1 status for some patients. More data are required to inform on the use of alternative staining assays upon which to read different specific therapy‐related PD‐L1 cutoffs.

Correlation between MET Gene Copy Number by Silver In Situ Hybridization and Protein Expression by Immunohistochemistry in Non-small Cell Lung Cancer

Purpose: The MET receptor is involved in the pathogenesis and progression of non-small cell lung cancer (NSCLC). Clinical trials with MET inhibitors in NSCLC are planned with patient selection based on immunohistochemistry (IHC) and/or gene copy number assessment. Therefore, a detailed understanding of relationship between these markers and prognosis is essential. Methods: This study included tumors from 189 patients with NSCLC who underwent pulmonary resection (median follow-up, 5.3 years). MET expression was evaluated by IHC on tissue microarrays and scored according to hybrid (H) score (range: 0–400) and by scoring system used in the MetMAb trial (≥50% of cells with moderate or strong staining). MET gene copy number was assessed by silver in situ hybridization (n =140 patients). Results: Median MET IHC H score was 60 (range: 0–400; n =174). There were no associations between clinical and pathological characteristics, disease-free survival, and overall survival according to median value (p =0.36 and p =0.38, respectively), or other cut-points. According to MetMAb scoring criteria, IHC positivity rate was 25%, again with no associations to clinicopathological features or survival. In 140 tumors evaluable for MET copy number, 3 (2.1%) showed gene amplification and 14 (10%) had tumors with average of 5 or more copies per nucleus. There were no associations of MET copy number with clinical characteristics, disease-free survival, or overall survival with any analyzed cut-points. Correlation between MET copy number and protein expression was significant (Pearsons r =0.42, p < 0.0001). Conclusions: There is a significant correlation between MET protein expression and MET gene copy number in operable NSCLC, but neither is associated with prognosis.

Increased Insulin-Like Growth Factor 1 Receptor Protein Expression and Gene Copy Number in Small Cell Lung Cancer

Purpose: Identification of new therapies in small cell lung cancer (SCLC) is urgently needed. Insulin-like growth factor 1 receptor (IGF1R) is a tyrosine kinase receptor implicated in the pathogenesis of several malignancies and is potentially an attractive target for anticancer treatment. Knowledge about IGF1R protein expression, gene copy number, and the prognostic relevance of these features in SCLC is limited. Methods: We analyzed IGF1R protein expression and gene copy number in primary tumors from 90 patients with SCLC (67 men and 23 women) who underwent pulmonary resection. IGF1R expression assessed by immunohistochemistry with H scores from 0 to 400 was evaluable in 84 patients and IGF1R gene copy number assessed by silver in situ hybridization technique in 81 patients. Results: Median H score for IGF1R protein expression was 88 (range, 0–400), and the proportion of positive immunostaining using cutoff H score of 10 was 74%. Increased IGF1R gene copy number (an average of four or more copies per cell) was found in 15 cases (18.5%), five of whom (6.2%) showed gene amplification. There was a significant correlation between protein expression and gene copy number (r = 0.49, p < 0.005). IGF1R expression and gene copy number did not associate with clinicopathological factors such as patient age, tumor size, lymph node involvement, stage, and survival. Conclusions: SCLC is characterized by frequent high-IGF1R protein expression, increased gene copy number, and occasional occurrence of true gene amplification. These features may have important implications for future anti-IGF1R therapeutic approaches.

Thymidylate Synthase Protein Expression by IHC and Gene Copy Number by SISH Correlate and Show Great Variability in Non–Small Cell Lung Cancer

Introduction: Increased expression of thymidylate synthase (TS) is thought to be associated with resistance to TS-targeting drugs, e.g., pemetrexed. Methods: TS protein expression (PE) and gene copy number (GCN) were assayed using immunohistochemistry and silver in situ hybridization, respectively, on primary tumors of 189 resected non–small cell lung patients. Associations with pathological and clinical features and prognosis were explored. Results: Median immunohistochemistry H-score was 220 (range, 10–380) on a 0 to 400 scale; 17% of the patients had a TS expression of 300 or more. TS PE expression did not significantly differ by histology and did not significantly associate with disease-free survival (DFS) or overall survival (OS). However, there was a tendency for increased DFS (p = 0.12) and OS (p = 0.12) in PE positive (>median) squamous-cell carcinoma (SCC) patients. Median GCN was 2.5 genes/nucleus (range, 1.4–9.6); 29% of patients had GCN of 3 or more, 7% of 4 or more and 0.8% amplification. GCN differed by histology (p = 0.015); 50% of SCCs having GCN more than 2.5 versus 32% of adenocarcinomas. There was no significant relationship between TS GCN and DFS or OS; however, a trend toward better DFS (p = 0.18) and OS (p = 0.10) with increased GCN in SCCs was observed. TS GCN was significantly correlated with PE (r = 0.30, p = 0.0009). Conclusions: TS PE and GCN vary widely in non–small cell lung and correlate significantly with each other. TS GCN is higher in SCCs, whereas TS PE does not associate with histological subtypes, clinical features, or survival. Variability of TS PE and GCN may indicate potential benefit from pemetrexed therapy in selected SCC patients.

Abstract B31: Fibroblast growth factor receptor-1 (FGFR1) gene amplification is frequent in lung squamous cell carcinoma and high gene copy number indicates a better prognosis in early stage NSCLC.

Introduction: Lung cancer is the leading cause of cancer death globally. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers. Histologically NSCLC is divided into five subtypes, with adenocarcinoma (AD) and squamous cell carcinoma (SCC) representing the predominant subclasses, 40% and 30%, respectively. Several molecular targets have been identified in AD but such targets in SCC are lacking and there are no FDA-approved targeted agents for SCC. FGFR1 is a druggable receptor tyrosine kinase purported to be an oncogenic driver in lung cancer and the gene has been shown to be amplified in SCC. Methods: Resection specimens from 32 SCC and 27 non-SCC tumors (22 AD) were evaluated for FGFR1 and chromosome (CH) 8 copy number status using dual-color silver in situ hybridization, black and red, respectively. FGFR1 gene copy number (GCN) and the ratio of FGFR1:CH8 was compared to clinical and pathological characteristics and related to survival. Results: Of the 59 cases 2 were non-evaluable due to weak staining (one SCC specimen) and lack of sufficient tumor (one non-SCC specimen). The mean and median GCN for FGFR1 were 2.81 (SD±1.17) and 2.36 (range 1.18–6.64) genes/nucleus, respectively, and 1.34 (±0.73) and 1.14 (0.52–5.19) for the FGFR1:CH8 ratio. The average FGFR1 GCN for SCC vs. non-SCC tumors was 3.16 vs 2.38, p=0.02, and the average FGFR1:CH8 ratio was 1.54 vs. 1.10, p=0.03 There were 5 tumors with true FGFR1 amplification, as defined by clusters (all of these SCCs), with an average GCN >4/nucleus. Interestingly, only 3 of these amplified tumors had a FGFR1:CH8 ratio >2. There were 10 tumors, 7 of which were SCC, that contained FGFR1 GCN >3, however only 2 of these had a FGFR1:CH8 ratio >2. There were no associations of FGF1R GCN with age, sex, smoking status, stage or grade. There was a positive association between increased FGFR1 GCN and both DFS and OS (log-rank p= 0.01 and p=0.03, respectively) when using the median (2.36) as the cut-point. However, no associations were seen when evaluating the median (1.14) of the FGFR1:CH8 ratio (log-rank p= 0.93 and p=0.98, respectively). Conclusions: FGFR1 GCN is highly increased in SCC with 16% of the SSC tumors showing amplification and 48% showing gain (>3). Increased FGFR1 GCN associated with increased DFS and OS but a larger study is warranted to validate this finding. FGFR1 GCN alone appears better at showing gene gain compared to the FGFR1:CH8 ratio. FGFR1 SISH may be a feasible assay to validate whether FGFR1 is a bona fide biomarker for prediction of response and/or outcome to FGFR1 inhibitor therapy in SCC of the lung. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B31.

Abstract B45: Analysis of c‐Met gene copy number by dual‐color silver in situ hybridization (SISH) in non‐small cell lung cancer (NSCLC)

Background: c‐Met is a receptor capable of activating downstream proliferative, survival and invasive pathways. Numerous human malignancies, including NSCLC, show over‐expressed and amplified c‐Met. Elevated expression of c‐Met and its ligand, HGF, have been associated with inferior prognosis. The aim of this study was to evaluate the prognostic relationship of c‐Met gene copy number in surgically‐treated NSCLC patients (pts) using dual‐color silver in situ hybridization. SISH is advantageous because bright field microscopy allows visualization of intact malignant and non‐malignant cell morphology. Methods: Experimental SISH probes, Ventana Medical Systems, Inc. (Tucson, AZ), were used to evaluate c‐MET and CH 7 copy numbers on a tissue microarray containing triplicate samples from 189 pts (median follow‐up 5.3 yrs and 5‐yr survival probability 38%). Evaluable results, at least one core with valid c‐Met and CH 7 counts, were obtained for 140 pts. There were 109 males; 80 squamous cell carcinomas; and 37 adenocarcinomas. The pathological stages were I: 50, II: 33 and III: 46, IV: 8 and unknown: 3. A certified pathologist counted signals (1 for individual signal, 6 for small cluster and 12 for big cluster) in 50 nuclei for each core and the mean number of c‐Met and CH 7 copies/nucleus/core were determined, along with the ratio. The triplicate cores were averaged and compared to the core with the highest score. Results: There was very high correlation between the highest core and the average of the triplicate cores: c‐Met (r=0.98), CH 7 (r=0.96), and ratio (r=0.98). The highest core value was used for further analysis. The median copy numbers were: c‐Met 3.1 (1.7–11.8), CH7 2.4 (1.5–2.4), and ratio 1.3 (0.6–6.3). There were 14 pts with greater than or equal to five c‐Met genes/nucleus and 3 pts with clusters. Using the median as a cut‐off there was no statistical difference related to age, gender, pathological stage, histology, grade or smoking. Kaplan‐Meier analysis revealed no significant differences in PFS or OS using the median scores, however there was a trend for increased PFS and OS in patients with high copy numbers. Cox regression analysis examining copy number as a continuous variable, and accounting for other variables, showed a similar trend of protection but statistical significance was not achieved: c‐Met (PFS‐ HR 0.88 (0.72–1.07, p=0.19, OS‐ HR 0.90 (0.73–1.10), p=0.29); CH7 (PFS‐ HR 0.88 (0.57–1.34, p=0.54, OS‐ HR 1.02 (0.66–1.57), p=0.95): ratio (PFS‐ HR 0.84 (0.54–1.30, p=0.43, OS‐ HR 0.79 (0.49–1.28), p=0.33). Analysis of the data using a cut‐off of 5 for c‐Met gave similar results. Conclusions: This study shows the clinical feasibility of SISH technology for c‐Met gene copy number assessment. There was a trend for increased PFS and OS in pts with increased c‐Met, CH 7 or the ratio between the two. Further studies are needed to determine the predictive value of c‐Met gene copy number in pts receiving molecular targeted therapeutics. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B45.