Rachel Tam

Erlotinib and bevacizumab in patients with advanced non-small-cell lung cancer and activating EGFR mutations (BELIEF): an international, multicentre, single-arm, phase 2 trial.

BACKGROUND The tyrosine kinase inhibitor erlotinib improves the outcomes of patients with advanced non-small-cell lung carcinoma (NSCLC) harbouring epidermal growth factor receptor (EGFR) mutations. The coexistence of the T790M resistance mutation with another EGFR mutation in treatment-naive patients has been associated with a shorter progression-free survival to EGFR inhibition than in the absence of the T790M mutation. To test this hypothesis clinically, we developed a proof-of-concept study, in which patients with EGFR-mutant NSCLC were treated with the combination of erlotinib and bevacizumab, stratified by the presence of the pretreatment T790M mutation. METHODS BELIEF was an international, multicentre, single-arm, phase 2 trial done at 29 centres in eight European countries. Eligible patients were aged 18 years or older and had treatment-naive, pathologically confirmed stage IIIB or stage IV lung adenocarcinoma with a confirmed, activating EGFR mutation (exon 19 deletion or L858R mutation). Patients received oral erlotinib 150 mg per day and intravenous bevacizumab 15 mg/kg every 21 days and were tested centrally for the pretreatment T790M resistance mutation with a peptide nucleic acid probe-based real-time PCR. The primary endpoint was progression-free survival. The primary efficacy analysis was done in the intention-to-treat population and was stratified into two parallel substudies according to the centrally confirmed pretreatment T790M mutation status of enrolled patients (T790M positive or negative). The safety analysis was done in all patients that have received at least one dose of trial treatment. This trial was registered with ClinicalTrials.gov, number NCT01562028. FINDINGS Between June 11, 2012, and Oct 28, 2014, 109 patients were enrolled and included in the efficacy analysis. 37 patients were T790M mutation positive and 72 negative. The overall median progression-free survival was 13·2 months (95% CI 10·3-15·5), with a 12 month progression-free survival of 55% (95% CI 45-64). The primary endpoint was met only in substudy one (T790M-positive patients). In the T790M-positive group, median progression-free survival was 16·0 months (12·7 to not estimable), with a 12 month progression-free survival of 68% (50-81), whereas in the T790M-negative group, median progression-free survival was 10·5 months (9·4-14·2), with a 12 month progression-free survival of 48% (36-59). Of 106 patients included in the safety analysis, five had grade 4 adverse events (one acute coronary syndrome, one biliary tract infection, one other neoplasms, and two colonic perforations) and one died due to sepsis. INTERPRETATION The BELIEF trial provides further evidence of benefit for the combined use of erlotinib and bevacizumab in patients with NSCLC harbouring activating EGFR mutations. FUNDING European Thoracic Oncology Platform, Roche.

Identification and Analysis of In Vivo VEGF Downstream Markers Link VEGF Pathway Activity with Efficacy of Anti- VEGF Therapies

Purpose: The aim of this study was to identify conserved pharmacodynamic and potential predictive biomarkers of response to anti-VEGF therapy using gene expression profiling in preclinical tumor models and in patients. Experimental Design: Surrogate markers of VEGF inhibition [VEGF-dependent genes or VEGF-dependent vasculature (VDV)] were identified by profiling gene expression changes induced in response to VEGF blockade in preclinical tumor models and in human biopsies from patients treated with anti-VEGF monoclonal antibodies. The potential value of VDV genes as candidate predictive biomarkers was tested by correlating high or low VDV gene expression levels in pretreatment clinical samples with the subsequent clinical efficacy of bevacizumab (anti-VEGF)-containing therapy. Results: We show that VDV genes, including direct and more distal VEGF downstream endothelial targets, enable detection of VEGF signaling inhibition in mouse tumor models and human tumor biopsies. Retrospective analyses of clinical trial data indicate that patients with higher VDV expression in pretreatment tumor samples exhibited improved clinical outcome when treated with bevacizumab-containing therapies. Conclusions: In this work, we identified surrogate markers (VDV genes) for in vivo VEGF signaling in tumors and showed clinical data supporting a correlation between pretreatment VEGF bioactivity and the subsequent efficacy of anti-VEGF therapy. We propose that VDV genes are candidate biomarkers with the potential to aid the selection of novel indications as well as patients likely to respond to anti-VEGF therapy. The data presented here define a diagnostic biomarker hypothesis based on translational research that warrants further evaluation in additional retrospective and prospective trials. Clin Cancer Res; 19(13); 3681–92. ©2013 AACR.

Mutation Scanning Using MUT-MAP, a High-Throughput, Microfluidic Chip-Based, Multi-Analyte Panel

Targeted anticancer therapies rely on the identification of patient subgroups most likely to respond to treatment. Predictive biomarkers play a key role in patient selection, while diagnostic and prognostic biomarkers expand our understanding of tumor biology, suggest treatment combinations, and facilitate discovery of novel drug targets. We have developed a high-throughput microfluidics method for mutation detection (MUT-MAP, mutation multi-analyte panel) based on TaqMan or allele-specific PCR (AS-PCR) assays. We analyzed a set of 71 mutations across six genes of therapeutic interest. The six-gene mutation panel was designed to detect the most common mutations in the EGFR, KRAS, PIK3CA, NRAS, BRAF, and AKT1 oncogenes. The DNA was preamplified using custom-designed primer sets before the TaqMan/AS-PCR assays were carried out using the Biomark microfluidics system (Fluidigm; South San Francisco, CA). A cross-reactivity analysis enabled the generation of a robust automated mutation-calling algorithm which was then validated in a series of 51 cell lines and 33 FFPE clinical samples. All detected mutations were confirmed by other means. Sample input titrations confirmed the assay sensitivity with as little as 2 ng gDNA, and demonstrated excellent inter- and intra-chip reproducibility. Parallel analysis of 92 clinical trial samples was carried out using 2–100 ng genomic DNA (gDNA), allowing the simultaneous detection of multiple mutations. DNA prepared from both fresh frozen and formalin-fixed, paraffin-embedded (FFPE) samples were used, and the analysis was routinely completed in 2–3 days: traditional assays require 0.5–1 µg high-quality DNA, and take significantly longer to analyze. This assay can detect a wide range of mutations in therapeutically relevant genes from very small amounts of sample DNA. As such, the mutation assay developed is a valuable tool for high-throughput biomarker discovery and validation in personalized medicine and cancer drug development.

Next generation MUT-MAP, a high-sensitivity high-throughput microfluidics chip-based mutation analysis panel.

Molecular profiling of tumor tissue to detect alterations, such as oncogenic mutations, plays a vital role in determining treatment options in oncology. Hence, there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations. Although commercial assays are available to detect genetic alterations in single genes, only a limited amount of tissue is often available from patients, requiring multiplexing to allow for simultaneous detection of mutations in many genes using low DNA input. Even though next-generation sequencing (NGS) platforms provide powerful tools for this purpose, they face challenges such as high cost, large DNA input requirement, complex data analysis, and long turnaround times, limiting their use in clinical settings. We report the development of the next generation mutation multi-analyte panel (MUT-MAP), a high-throughput microfluidic, panel for detecting 120 somatic mutations across eleven genes of therapeutic interest (AKT1, BRAF, EGFR, FGFR3, FLT3, HRAS, KIT, KRAS, MET, NRAS, and PIK3CA) using allele-specific PCR (AS-PCR) and Taqman technology. This mutation panel requires as little as 2 ng of high quality DNA from fresh frozen or 100 ng of DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Mutation calls, including an automated data analysis process, have been implemented to run 88 samples per day. Validation of this platform using plasmids showed robust signal and low cross-reactivity in all of the newly added assays and mutation calls in cell line samples were found to be consistent with the Catalogue of Somatic Mutations in Cancer (COSMIC) database allowing for direct comparison of our platform to Sanger sequencing. High correlation with NGS when compared to the SuraSeq500 panel run on the Ion Torrent platform in a FFPE dilution experiment showed assay sensitivity down to 0.45%. This multiplexed mutation panel is a valuable tool for high-throughput biomarker discovery in personalized medicine and cancer drug development.

The molecular landscape of high-risk early breast cancer: comprehensive biomarker analysis of a phase III adjuvant population

Breast cancer is a heterogeneous disease and patients are managed clinically based on ER, PR, HER2 expression, and key risk factors. We sought to characterize the molecular landscape of high-risk breast cancer patients enrolled onto an adjuvant chemotherapy study to understand how disease subsets and tumor immune status impact survival. DNA and RNA were extracted from 861 breast cancer samples from patients enrolled onto the United States Oncology trial 01062. Samples were characterized using multiplex gene expression, copy number, and qPCR mutation assays. HR+ patients with a PIK3CA mutant tumor had a favorable disease-free survival (DFS; HR 0.66, P=0.05), however, the prognostic effect was specific to luminal A patients (Luminal A: HR 0.67, P=0.1; Luminal B: HR 1.01, P=0.98). Molecular subtyping of triple-negative breast cancers (TNBCs) suggested that the mesenchymal subtype had the worst DFS, whereas the immunomodulatory subtype had the best DFS. Profiling of immunologic genes revealed that TNBC tumors (n=280) displaying an activated T-cell signature had a longer DFS following adjuvant chemotherapy (HR 0.59, P=0.04), while a distinct set of immune genes was associated with DFS in HR+ cancers. Utilizing a discovery approach, we identified genes associated with a high risk of recurrence in HR+ patients, which were validated in an independent data set. Molecular classification based on PAM50 and TNBC subtyping stratified clinical high-risk patients into distinct prognostic subsets. Patients with high expression of immune-related genes showed superior DFS in both HR+ and TNBC. These results may inform patient management and drug development in early breast cancer.

Molecular, Pathological, Radiological, and Immune Profiling of Non-brainstem Pediatric High-Grade Glioma from the HERBY Phase II Randomized Trial

Summary The HERBY trial was a phase II open-label, randomized, multicenter trial evaluating bevacizumab (BEV) in addition to temozolomide/radiotherapy in patients with newly diagnosed non-brainstem high-grade glioma (HGG) between the ages of 3 and 18 years. We carried out comprehensive molecular analysis integrated with pathology, radiology, and immune profiling. In post-hoc subgroup analysis, hypermutator tumors (mismatch repair deficiency and somatic POLE/POLD1 mutations) and those biologically resembling pleomorphic xanthoastrocytoma ([PXA]-like, driven by BRAF_V600E or NF1 mutation) had significantly more CD8+ tumor-infiltrating lymphocytes, and longer survival with the addition of BEV. Histone H3 subgroups (hemispheric G34R/V and midline K27M) had a worse outcome and were immune cold. Future clinical trials will need to take into account the diversity represented by the term “HGG” in the pediatric population.

Abstract 1553: Biomarker evaluation in a randomized phase 2 study of MEHD7945A (MEHD) versus cetuximab (Cet) in ≥2 line recurrent/metastatic (R/M) squamous cell carcinomas of the head and neck (SCCHN) [MEHGAN]

Background MEHD is a novel dual-action humanized IgG1 antibody that blocks ligand binding to EGFR and HER3, inhibiting all major ligand-dependent HER complex signaling. Preclinical and Phase 1a clinical data suggested ligand-driven HER3 signaling as a promising target for therapy in a subset of patients with SCCHN. Results from the MEHGAN study showed comparable objective response rates and PFS for MEHD and Cet (Fayette et al, ESMO 2014). Here we report the results of comprehensive and comparative biomarker analyses from that study. Methods Archival and fresh (as available) tumor tissues were evaluated to characterize the biology of anti-HER therapy in SCCHN, and to identify potential predictive biomarkers for improved outcomes with MEHD compared to Cet, with particular attention to the HER3 ligand NRG1. NRG1 and ERBB3 RNA expression was measured by both ISH (data analysis are ongoing) and qRT-PCR. Additionally, extensive gene expression analyses and HPV detection were performed by qRT-PCR. Results Of 121 randomized patients in MEHGAN, 107 had archival tissues with sufficient tumor content and quality for biomarker analyses. Key findings include: 1) Most patients with CT RECIST responses on either treatment arm had higher (≥ median) tumor expression levels of NRG1 as measured by qRT-PCR 2) EGFR ligands such as amphiregulin were co-expressed with NRG1, consistent with preclinical analysis in an independent panel of SCCHN tumor samples (Genentech data on file). 3) 24 HPV (+) patients (20%) were identified, consistent with published prevalence reports. 4) Higher EGFR and HER3 ligand expression was observed in HPV (-) samples relative to HPV (+) samples and, moreover, no responses were seen in HPV (+) patients. These results are consistent with prior correlative ligand observations and may point to differential roles for HER signaling biology in HPV (-) versus HPV (+) SCCHN. Conclusions NRG1 expression did not predict enhanced responsiveness to MEHD versus Cet or, conversely, resistance to Cet. NRG1 and EGFR ligands appear to have similar expression patterns in SCCHN. Higher levels of NRG1 and EGFR ligands were associated with greater activity for both MEHD and Cet, and were consistently observed in HPV (-) SCCHN versus HPV (+) SCCHN. These data suggest distinct HER signaling biology in these 2 patient groups and warrant further evaluation to potentially inform treatment approaches in SCCHN. * We would like acknowledge and thank all of the MEHGAN study investigators and patients. Citation Format: Elicia Penuel, Amy V. Kapp, An Do, Rachel Tam, Teiko Sumiyoshi, Chaitra Marathe, Susan Sa, Franklin Peale, Mark Lackner, Scott Holden, Tanguy Seiwert, Andrea Pirzkall. Biomarker evaluation in a randomized phase 2 study of MEHD7945A (MEHD) versus cetuximab (Cet) in ≥2 line recurrent/metastatic (R/M) squamous cell carcinomas of the head and neck (SCCHN) [MEHGAN]. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1553. doi:10.1158/1538-7445.AM2015-1553

Abstract 2708: A custom gene expression panel for consensus molecular subtype classification of archival primary and metastatic colorectal cancers

Stratification of Colorectal Cancer (CRC) into actionable molecular subtypes has tremendous clinical value. Recently, a consolidated classifier identified four molecularly distinct CRC subtypes (CMS1-4) that were associated with unique biology and clinical outcomes based on global transcriptional analysis of frozen tissues. Here, we developed and applied a novel CRC panel that is ideally suited for transcriptional classification of archival clinical samples. Findings from in silico analysis demonstrated that the 800 genes on our panel could accurately classify CRC samples from external public datasets into the correct CMS subtypes. We applied our panel in the analysis of a novel cohort of 312 formalin-fixed paraffin-embedded (FFPE) tissues from 205 patients, and were able to detect all 4 CMS subtypes in primary CRCs and in metastases. When we examined the CMS subtypes of primary tumors and matched metastases from 50 patients we found 70% of cases to be concordant, as were key biologies, such as WNT/MYC pathway activation in CMS2 and EMT features in tumors of the CMS4 subtypes. This was confirmed by in situ hybridization (ISH) using the markers ASCL2 for CMS2 and SPARC for CMS4, respectively. Discordance in the CMS subtypes between primary tumors and matched metastases were observed in 30% of cases and may reflect tumor heterogeneity. Our findings suggest that our CRC-focused panel many have clinical utility for CMS classification of FFPE samples, and point to potential risks of using CMS subtypes of primary tumors to inform clinical decision-making at the metastatic stage in a subset of patients. Citation Format: Ling-Yuh Huw, Robert Piskol, Felipe de Sosa e Melo, Doris Kim, Xueping Qu, Hartmut Koeppen, Mark Lackner, Garret Hampton, Omar Kabbarah, Rachel Tam. A custom gene expression panel for consensus molecular subtype classification of archival primary and metastatic colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2708. doi:10.1158/1538-7445.AM2017-2708

Development and Application of a Microfluidics-Based Panel in the Basal/Luminal Transcriptional Characterization of Archival Bladder Cancers.

In the age of personalized medicine stratifying tumors into molecularly defined subtypes associated with distinctive clinical behaviors and predictable responses to therapies holds tremendous value. Towards this end, we developed a custom microfluidics-based bladder cancer gene expression panel for characterization of archival clinical samples. In silico analysis indicated that the content of our panel was capable of accurately segregating bladder cancers from several public datasets into the clinically relevant basal and luminal subtypes. On a technical level, our bladder cancer panel yielded robust and reproducible results when analyzing formalin-fixed, paraffin-embedded (FFPE) tissues. We applied our panel in the analysis of a novel set of 204 FFPE samples that included non-muscle invasive bladder cancers (NMIBCs), muscle invasive disease (MIBCs), and bladder cancer metastases (METs). We found NMIBCs to be mostly luminal-like, MIBCs to include both luminal- and basal-like types, and METs to be predominantly of a basal-like transcriptional profile. Mutational analysis confirmed the expected enrichment of FGFR3 mutations in luminal samples, and, consistently, FGFR3 IHC showed high protein expression levels of the receptor in these tumors. Our bladder cancer panel enables basal/luminal characterization of FFPE tissues and with further development could be used for stratification of bladder cancer samples in the clinic.

Abstract B024: Comprehensive biomarker profiling of matched primary and metastatic estrogen receptor positive breast cancers

Patients with newly diagnosed, early stage estrogen receptor positive (ER+) breast cancer often show disease free survival in excess of five years following surgery and systemic adjuvant therapy. As such, an important question is whether diagnostic tumor tissue from the primary lesion offers an accurate molecular portrait of the cancer post recurrence and thus may be used for predictive diagnostic purposes for patients with relapsed, metastatic disease. To address this question, we performed detailed biomarker analyses on matched, asynchronous primary and metastatic tumors from 77 patients with ER+ breast cancer. The class I phosphatidylinositol 39 kinase (PI3K) is thought to be an important driver in ER+ breast cancer and has been linked to acquired resistance to hormonal therapy. We thus examined whether mutations in PIK3CA and loss of PTEN showed differences in primary and metastatic samples. We also sought to look more broadly at markers reflective of proliferation, molecular subtype, and key receptors and signaling pathways. To accomplish this, we developed an analysis platform using the Fluidigm BioMark™ microfluidics system to measure the relative expression of 90 breast cancer related genes in formalin-fixed paraffin-embedded (FFPE) tissue. Application of this panel of assays to matched tumor pairs showed a very high concordance between primary and metastatic tissue, with generally few changes in mutation status, proliferative markers, or gene expression between matched samples. Thus, archival primary tumor tissue may still provide an accurate portrait of biomarker status in patients with disease recurrence. Citation Format: Jill M. Spoerke, Erica B. Schleifman, Rupal Desai, Yuanyuan Xiao, Cheryl Wong, Ilma Abbas, Carol O9Brien, Rajesh Patel, Teiko Sumiyoshi, Ling Fu, Rachel Tam, Hartmut Koeppen, Timothy Wilson, Rajiv Raja, Garret M. Hampton, Mark R. Lackner. Comprehensive biomarker profiling of matched primary and metastatic estrogen receptor positive breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B024.