Murry W. Wynes

Increased ALK Gene Copy Number and Amplification are Frequent in Non-small Cell Lung Cancer

Introduction: Translocation of the anaplastic lymphoma kinase (ALK) gene is involved in the tumorigenesis of a subset of non-small cell lung carcinomas (NSCLCs) and identifies patients sensitive to ALK inhibitors. ALK copy number changes and amplification, which plays an oncogenic role in tumors such as neuroblastoma, are poorly characterized in NSCLC. We aimed to study the prevalence of ALK copy number changes and their correlation to ALK protein expression, epidermal growth factor receptor (EGFR) status, and clinicopathological data in patients with NSCLC. Methods: ALK status was evaluated by fluorescence in situ hybridization (FISH). Specimens with ALK translocation were studied for echinoderm microtubule-associated protein-like 4 (EML4), KIF5B, and TFG status. ALK expression was assessed by immunohistochemistry. EGFR gene and protein status were evaluated in adenocarcinomas. Survival analysis was performed. Results: One hundred seven NSCLC cases were evaluated. There were two cases of EML4-ALK translocation and one with an atypical translocation of ALK. Both cases of EML4-ALK translocation had ALK protein expression, whereas in the rest, ALK was undetected. Eleven cases (10%) exhibited ALK amplification and 68 (63%) copy number gains. There was an association between ALK amplification and EGFR FISH positivity (p < 0.0001) but not with prognosis. In conclusion, EML4-ALK translocation is a rare event in NSCLC. Conclusion: The study reveals a significant frequency of ALK amplification and its association with EGFR FISH positivity in lung adenocarcinomas. Based on these findings, a potential role of ALK amplification in the response to ALK inhibitors alone or combined with EGFR inhibitors in NSCLC merits further studies.

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.

Next-Generation Sequencing Identifies and Immunohistochemistry Confirms a Novel Crizotinib- Sensitive ALK Rearrangement in a Patient with Metastatic Non-Small-Cell Lung Cancer

The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) gene fusion occurs in 2–7% of NSCLC cases[1,2] and is typically identified by Vysis ALK Break-Apart fluorescent in situ hybridization (FISH) assay. Tumors expressing this fusion respond to treatment crizotinib[1].

FGFR1 mRNA and Protein Expression, not Gene Copy Number, Predict FGFR TKI Sensitivity across All Lung Cancer Histologies

Purpose: FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer. Experimental Design: Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray composed of resected lung tumors was submitted to FGFR1 GCN, and mRNA analyses and the results were validated with The Cancer Genome Atlas (TCGA) lung cancer data. Results: Among 58 cell lines, 14 exhibited ponatinib sensitivity (IC50 values ≤ 50 nmol/L) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations. Conclusions: FGFR1 dependency is frequent across various lung cancer histologies, and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types. Clin Cancer Res; 20(12); 3299–309. ©2014 AACR.

An International Interpretation Study Using the ALK IHC Antibody D5F3 and a Sensitive Detection Kit Demonstrates High Concordance between ALK IHC and ALK FISH and between Evaluators

Introduction: The goal of personalized medicine is to treat patients with a therapy predicted to be efficacious based on the molecular characteristics of the tumor, thereby sparing the patient futile or toxic therapy. Anaplastic lymphoma kinase (ALK) inhibitors are effective against ALK-positive non–small-cell lung cancer (NSCLC) tumors, but to date the only approved companion diagnostic is a break-apart fluorescence in situ hybridization (FISH) assay. Immunohistochemistry (IHC) is a clinically applicable cost-effective test that is sensitive and specific for ALK protein expression. The purpose of this study was to assemble an international team of expert pathologists to evaluate a new automated standardized ALK IHC assay. Methods: Archival NSCLC tumor specimens (n =103) previously tested for ALK rearrangement by FISH were provided by the international collaborators. These specimens were stained by IHC with the anti-ALK (D5F3) primary antibody combined with OptiView DAB IHC detection and OptiView amplification (Ventana Medical Systems, Inc., Tucson, AZ). Specimens were scored binarily as positive if strong granular cytoplasmic brown staining was present in tumor cells. IHC results were compared with the FISH results and interevaluator comparisons made. Results: Overall for the 100 evaluable cases the ALK IHC assay was highly sensitive (90%), specific (95%), and accurate relative (93%) to the ALK FISH results. Similar results were observed using a majority score. IHC negativity was scored by seven of seven and six of seven evaluators on three and two FISH-positive cases, respectively. IHC positivity was scored on two FISH-negative cases by seven of seven readers. There was agreement among seven of seven and six of seven readers on 88% and 96% of the cases before review, respectively, and after review there was agreement among seven of seven and six of seven on 95% and 97% of the cases, respectively. Conclusions: On the basis of expert evaluation the ALK IHC test is sensitive, specific, and accurate, and a majority score of multiple readers does not improve these results over an individual reader’s score. Excellent inter-reader agreement was observed. These data support the algorithmic use of ALK IHC in the evaluation of NSCLC.

A Phase I/II Study of Erlotinib in Combination with the Anti-Insulin-Like Growth Factor-1 Receptor Monoclonal Antibody IMC-A12 (Cixutumumab) in Patients with Advanced Non-small Cell Lung Cancer

Introduction: This phase I/II study evaluated the safety and antitumor effect of the combination of erlotinib with cixutumumab, a recombinant fully humanized anti-insulin-like growth factor-1 receptor IgG1 monoclonal antibody, in advanced non-small cell lung cancer (NSCLC). Methods: Patients with advanced NSCLC were treated in an initial safety-lead and drop-down cohorts using erlotinib 150 mg/d with cixutumumab 6 or 5 mg/kg on days 1, 8, 15, and 22 in 28-day cycles (cohorts 1 and 2). Emerging pharmacokinetic data led to an additional cohort (3 + 3 design) with cixutumumab at 15 mg/kg on day 1 in 21-day cycles (cohort 3). Results: Eighteen patients entered the study (6 at 6 mg/kg, 8 at 5 mg/kg, and 4 at 15 mg/kg), with median age of 65 years. Four of six patients at 6 mg/kg experienced dose-limiting toxicities (DLTs), whereas at 5 mg/kg, one of eight patients experienced DLT but three of eight patients still required a dose delay during cycle 1. At 15 mg/kg every 21 days, two of four patients experienced DLTs. In all cohorts, DLTs were either G3 rash or fatigue. Five patients had stable disease as best response and 14 patients had progressive disease. The median progression-free survival was 39 days (range 21–432+ days). Biomarkers analyses showed a trend toward better progression-free survival seen with higher free baseline insulin-like growth factor-1 levels as seen with other insulin-like growth factor-1R inhibitors. Conclusions: The combinations of cixutumumab at 6 mg/kg every 7 days and 15 mg/kg every 21 days and full-dose erlotinib are not tolerable in unselected patients with NSCLC, as measured by DLT. Cixutumumab at 5 mg/kg every 7 days was tolerable per DLT, but dose delays were common. Efficacy in unselected patients with NSCLC seems to be low.

Folylpoly-Glutamate Synthetase Expression Is Associated with Tumor Response and Outcome from Pemetrexed-Based Chemotherapy in Malignant Pleural Mesothelioma

Background: Pemetrexed-based chemotherapy represents the standard of care in first-line treatment of advanced malignant pleural mesothelioma (MPM). However, there are no established predictors of clinical benefit. Pemetrexed inhibits multiple enzymes involved in pyrimidine and purine synthesis, but the main target is thymidylate synthase (TS). After cellular uptake pemetrexed is converted into more effective polyglutamated forms by folylpoly-&ggr;-glutamate synthetase (FPGS). We hypothesized that FPGS and TS protein expressions are associated with clinical outcome after pemetrexed-based chemotherapy. Methods: Pretreatment tumor samples from 84 patients with histologically confirmed MPM, who received pemetrexed combined with platinum (79 of 84) or single-agent pemetrexed (5 of 84) as first-line treatment, were retrospectively analyzed. FPGS and TS protein expressions were semiquantitatively assessed by using the Hybrid (H)-scoring system (range, 0–300). H-scores were correlated with radiological response according to modified Response Evaluation Criteria in Solid Tumors, progression-free survival (PFS) and overall survival (OS). Results: Median H-score of the entire cohort was 230 for FPGS (range, 100–300), and 210 for TS (range, 100–300). High FPGS protein expression was significantly associated with longer PFS (pCOX = 0.0337), better objective tumor response (partial response versus stable disease + progressive disease; pKW = 0.003), and improved disease-control rate (partial response + stable disease versus progressive disease; pKW = 0.0208), but not with OS. In addition, high TS protein expression was associated with progressive disease under pemetrexed-based therapy (p = 0.0383), and shorter OS (pCOX = 0.0071), but no association with PFS was observed. Conclusion: FPGS and TS expressions were associated with clinical response and outcome to pemetrexed-based first-line chemotherapy in MPM. Prospective evaluation of FPGS and TS expressions and their prognostic/predictive power in MPM patients is warranted.

Contributions of KRAS and RAL in Non–Small-Cell Lung Cancer Growth and Progression

Introduction: KRAS mutations are poor prognostic markers for patients with non–small-cell lung cancer (NSCLC). RALA and RALB GTPases lie downstream of RAS and are implicated in RAS-mediated tumorigenesis. However, their biological or prognostic role in the context of KRAS mutation in NSCLC is unclear. Methods: Using expression analysis of human tumors and a panel of cell lines coupled with functional in vivo and in vitro experiments, we evaluated the prognostic and functional importance of RAL in NSCLC and their relationship to KRAS expression and mutation. Results: Immunohistochemical (N = 189) and transcriptomic (N = 337) analyses of NSCLC patients revealed high RALA and RALB expression was associated with poor survival. In a panel of 14 human NSCLC cell lines, RALA and RALB had higher expression in KRAS mutant cell lines whereas RALA but not RALB activity was higher in KRAS mutant cell lines. Depletion of RAL paralogs identified cell lines that are dependent on RAL expression for proliferation and anchorage independent growth. Overall, growth of NSCLC cell lines that carry a glycine to cystine KRAS mutation were more sensitive to RAL depletion than those with wild-type KRAS. The use of gene expression and outcome data from 337 human tumors in RAL-KRAS interaction analysis revealed that KRAS and RAL paralog expression jointly impact patient prognosis. Conclusion: RAL GTPase expression carries important additional prognostic information to KRAS status in NSCLC patients. Simultaneously targeting RAL may provide a novel therapeutic approach in NSCLC patients harboring glycine to cystine KRAS mutations.

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.

Detection of Circulating Lung Cancer Cells with Strong Thymidylate Synthase Reactivity in the Peripheral Blood of a Patient with Pulmonary Adenocarcinoma Treated with Pemetrexed

Circulating tumor cells (CTCs) can be collected from peripheral blood and may provide pharmacodynamic information to aid therapeutic decision making.1 Furthermore, CTCs as a “virtual and real-time biopsy” have clear potential to facilitate exploration of the same prognostic or predictive biomarkers as those found in the primary tumor.2,3 The multitarget antifolate agent pemetrexed (PMX) currently administered to patients with nonsquamous non-small cell lung cancer (NSCLC). PMX primarily targets thymidylate synthase (TS), and TS is currently being investigated as a predictive biomarker for PMX-based chemotherapy.4,5 In November 2010, a 50-year-old woman was diagnosed with metastatic adenocarcinoma of the right lower lobe (cT4 cN3 cM1b, epidermal growth receptor factor wild type). First-line treatment with cisplatin and PMX was scheduled for December 3, 2010. Before administration, we collected 20 ml of peripheral venous blood to isolate CTCs. Mononuclear cells including CTCs were enriched using modified buoyant density gradient centrifugation method in Percoll PLUS solution (GE Healthcare GmbH, Munich, Germany). Hematopoietic cells were negatively selected from the sample using immunomagnetic beads with anti-CD15 and anti-CD456 monoclonal antibodies. Epithelial tumor cells were enriched using magnetic beads (Dynabead Epithelial Enrich, Invitrogen GmbH, Darmstadt, Germany) coated with the monoclonal antibody BerEP41 against the human epithelial antigen EpCAM.6 The resulting CTC-enriched cell population was dissolved in ThinPrep CytoLyt solution and was centrifuged at 150g for 10 minutes. The sediment was dissociated in fixating solution consisting of 50% ethanol and 50% phosphate-buffered saline-buffered formalin. Cells were fixed for a minimum of 20 minutes before being mounted on a ThinPrep slide. TS protein expression was evaluated using the BenchMark XT autostainer (Ventana Medical Systems, Inc., Tucson, AZ). After pretreatment, CTC containing slides and 4 μm sections from the formalin-fixed paraffin-embedded primary tumor were incubated for 1 hour at 25°C with a mouse monoclonal anti-TS antibody (clone 4H4B1, Invitrogen, Carlsbad, CA) and counterstained with hematoxylin for 4 minutes. Negative control was obtained by using a nonimmunized ready-to-use mouse antibody as the primary antibody. Tissue section of colorectal carcinoma served as positive control. Microscopic examination revealed several CTCs with varying TS expression ranging from weak to strong (Figures 1A–C). For estimation of the staining intensities, we compared the stained CTCs with TS-stained primary tumors (Fig. 2). In addition, a strongly TS-positive CTC adjacent immune magnetic beads was detected, whose shape is similar to a dividing tumor cell in the late prophase stage of mitosis (Figure 1A). This dividing CTC presents chromosome maturation with condensed chromosomes, which has rarely been observed in CTCs. To the best of our knowledge, this is the first report of circulating NSCLC cells in the peripheral blood, most of which stained strongly for TS. FIGURE 1 Circulating tumor cells (CTCs) isolated from the peripheral blood of a patient suffering from an adenocarcinoma of the lung stained with an antibody against thymidylate synthase (TS). A, Strongly TS expressing CTC which resembles a proliferating CTC in ... FIGURE 2 Adenocarcinoma cells of the lung from the same patient stained with hematoxylin/eosin (HE) or an antibody against thymidylate synthase (TS) on a 4-μm section of the formalin-fixed paraffin-embedded primary tumor. A, HE staining. B, TS staining. ... As high TS expression is associated with inferior clinical outcome to PMX-based therapy, we followed the clinical course of this patient. Until February, she received four cycles of combined chemotherapy with cisplatin and PMX resulting in a partial response per RECIST. Disease progression was diagnosed in multiple sites on May 9, 2011, which was 157 days after initiation of treatment and 70 days after the end of first-line treatment. The progression-free survival is compatible with the median progression-free survival reported in a large phase III trial of cisplatin and PMX.7 Despite receiving four additional lines of treatment (erlotinib, docetaxel, gemcitabine/vinorelbine, and mitomycin), the patient never achieved a second objective treatment response and showed disease progression notably under each line within 6 weeks. She was still alive on August 8, 2011. In conclusion, our report established that immunocytochemical detection of biomarkers in CTCs from the peripheral blood of NSCLC patients is feasible, and CTCs acting as a surrogate for tumor biopsies hold promise for “real-time” monitoring of personalized systemic treatments for lung cancer.