Absence of PD-L1 expression on tumor cells in the context of an activated immune infiltrate may indicate impaired IFNγ signaling in non-small cell lung cancer.

BACKGROUND: In non-small cell lung cancer (NSCLC), PD-L1 expression on either tumor cells (TC) or both TC and tumor-infiltrating immune cells (IC) is currently the most used biomarker in cancer immunotherapy. However, the mechanisms involved in PD-L1 regulation are not fully understood. To provide better insight in these mechanisms, a multiangular analysis approach was used to combine protein and mRNA expression with several clinicopathological characteristics. PATIENTS AND METHODS: Archival tissues from 640 early stage, resected NSCLC patients were analyzed with immunohistochemistry for expression of PD-L1 and CD8 infiltration. In addition, mutational status and expression of a selection of immune genes involved in the PD-L1/PD-1 axis and T-cell response was determined. RESULTS: Tumors with high PD-L1 expression on TC or on IC represent two subsets of NSCLC with minimal overlap. We observed that PD-L1 expression on IC irrespective of expression on TC is a good marker for inflammation within tumors. In the tumors with the highest IC expression and absent TC expression an association with reduced IFNγ downstream signaling in tumor cells was observed. CONCLUSIONS: These results show that PD-L1 expression on TC and IC are both independent hallmarks of the inflamed phenotype in NSCLC, and TC-negative/IC-high tumors can also be categorized as inflamed. The lack of correlation between PD-L1 TC and IC expression in this subgroup may be caused by impaired IFNγ signaling in tumor cells. These findings may bring a better understanding of the tumor-immune system interaction and the clinical relevance of PD-L1 expression on IC irrespective of PD-L1 expression on TC.

Differential regulation of PD-L1 expression by immune and tumor cells in NSCLC and the response to treatment with atezolizumab (anti-PD-L1).

Programmed death-ligand 1 (PD-L1) expression on tumor cells (TCs) by immunohistochemistry is rapidly gaining importance as a diagnostic for the selection or stratification of patients with non-small cell lung cancer (NSCLC) most likely to respond to single-agent checkpoint inhibitors. However, at least two distinct patterns of PD-L1 expression have been observed with potential biological and clinical relevance in NSCLC: expression on TC or on tumor-infiltrating immune cells (ICs). We investigated the molecular and cellular characteristics associated with PD-L1 expression in these distinct cell compartments in 4,549 cases of NSCLC. PD-L1 expression on IC was more prevalent and likely reflected IFN-γ-induced adaptive regulation accompanied by increased tumor-infiltrating lymphocytes and effector T cells. High PD-L1 expression on TC, however, reflected an epigenetic dysregulation of the PD-L1 gene and was associated with a distinct histology described by poor immune infiltration, sclerotic/desmoplastic stroma, and mesenchymal molecular features. Importantly, durable clinical responses to atezolizumab (anti-PD-L1) were observed in patients with tumors expressing high PD-L1 levels on either TC alone [40% objective response rate (ORR)] or IC alone (22% ORR). Thus, PD-L1 expression on TC or IC can independently attenuate anticancer immunity and emphasizes the functional importance of IC in regulating the antitumor T cell response.

Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab.

Although programmed death-ligand 1-programmed death 1 (PD-L1-PD-1) inhibitors are broadly efficacious, improved outcomes have been observed in patients with high PD-L1 expression or high tumor mutational burden (TMB). PD-L1 testing is required for checkpoint inhibitor monotherapy in front-line non-small-cell lung cancer (NSCLC). However, obtaining adequate tumor tissue for molecular testing in patients with advanced disease can be challenging. Thus, an unmet medical need exists for diagnostic approaches that do not require tissue to identify patients who may benefit from immunotherapy. Here, we describe a novel, technically robust, blood-based assay to measure TMB in plasma (bTMB) that is distinct from tissue-based approaches. Using a retrospective analysis of two large randomized trials as test and validation studies, we show that bTMB reproducibly identifies patients who derive clinically significant improvements in progression-free survival from atezolizumab (an anti-PD-L1) in second-line and higher NSCLC. Collectively, our data show that high bTMB is a clinically actionable biomarker for atezolizumab in NSCLC.

A transcriptional MAPK Pathway Activity Score (MPAS) is a clinically relevant biomarker in multiple cancer types.

KRAS- and BRAF-mutant tumors are often dependent on MAPK signaling for proliferation and survival and thus sensitive to MAPK pathway inhibitors. However, clinical studies have shown that MEK inhibitors are not uniformly effective in these cancers indicating that mutational status of these oncogenes does not accurately capture MAPK pathway activity. A number of transcripts are regulated by this pathway and are recurrently identified in genome-based MAPK transcriptional signatures. To test whether the transcriptional output of only 10 of these targets could quantify MAPK pathway activity with potential predictive or prognostic clinical utility, we created a MAPK Pathway Activity Score (MPAS) derived from aggregated gene expression. In vitro, MPAS predicted sensitivity to MAPK inhibitors in multiple cell lines, comparable to or better than larger genome-based statistical models. Bridging in vitro studies and clinical samples, median MPAS from a given tumor type correlated with cobimetinib (MEK inhibitor) sensitivity of cancer cell lines originating from the same tissue type. Retrospective analyses of clinical datasets showed that MPAS was associated with the sensitivity of melanomas to vemurafenib (HR: 0.596) and negatively prognostic of overall or progression-free survival in both adjuvant and metastatic CRC (HR: 1.5 and 1.4), adrenal cancer (HR: 1.7), and HER2+ breast cancer (HR: 1.6). MPAS thus demonstrates potential clinical utility that warrants further exploration.

Gene Expression Profiling in BRAF-Mutated Melanoma Reveals Patient Subgroups with Poor Outcomes to Vemurafenib That May Be Overcome by Cobimetinib Plus Vemurafenib.

Purpose: The association of tumor gene expression profiles with progression-free survival (PFS) outcomes in patients with BRAFV600-mutated melanoma treated with vemurafenib or cobimetinib combined with vemurafenib was evaluated.Experimental Design: Gene expression of archival tumor samples from patients in four trials (BRIM-2, BRIM-3, BRIM-7, and coBRIM) was evaluated. Genes significantly associated with PFS (P < 0.05) were identified by univariate Cox proportional hazards modeling, then subjected to unsupervised hierarchical clustering, principal component analysis, and recursive partitioning to develop optimized gene signatures.Results: Forty-six genes were identified as significantly associated with PFS in both BRIM-2 (n = 63) and the vemurafenib arm of BRIM-3 (n = 160). Two distinct signatures were identified: cell cycle and immune. Among vemurafenib-treated patients, the cell-cycle signature was associated with shortened PFS compared with the immune signature in the BRIM-2/BRIM-3 training set [hazard ratio (HR) 1.8; 95% confidence interval (CI), 1.3-2.6, P = 0.0001] and in the coBRIM validation set (n = 101; HR, 1.6; 95% CI, 1.0-2.5; P = 0.08). The adverse impact of the cell-cycle signature on PFS was not observed in patients treated with cobimetinib combined with vemurafenib (n = 99; HR, 1.1; 95% CI, 0.7-1.8; P = 0.66).Conclusions: In vemurafenib-treated patients, the cell-cycle gene signature was associated with shorter PFS. However, in cobimetinib combined with vemurafenib-treated patients, both cell cycle and immune signature subgroups had comparable PFS. Cobimetinib combined with vemurafenib may abrogate the adverse impact of the cell-cycle signature. Clin Cancer Res; 23(17); 5238-45. ©2017 AACR.

Characterization of PD-L1 expression and immune cell infiltration in nasopharyngeal cancer.

OBJECTIVES: Locally recurrent or metastatic nasopharyngeal cancer (NPC) remains an important challenge, with more effective and durable therapeutic options needed. Cancer immunotherapy, and in particular therapies that target the PD-L1/PD-1 immune checkpoint pathway, may provide new options to treat NPC patients. This study evaluated PD-L1 and CD8 expression levels and the respective associations with clinical and histopathological characteristics of patients with NPC. MATERIALS AND METHODS: Diagnostic tumour biopsies were obtained before radical radiotherapy with or without chemotherapy from 161 patients with NPC. These biopsies were analysed for PD-L1 expression levels on tumour cells (TC) and tumour-infiltrating immune cells (IC), and for CD8 T-cell infiltration. Results were correlated with baseline characteristics and clinical outcomes with standard-of-care treatment regimens. Additionally, pre- and post-treatment-paired tumour samples were analysed (n=146). RESULTS: 75% of tumours expressed PD-L1 on IC and 24% on TC. Baseline clinical characteristics of stage, sex and age did not correlate with PD-L1 expression. Additionally, overall survival and progression-free survival of standard-of-care treatment did not correlate with baseline PD-L1 expression. CD8 levels did correlate with clinical outcomes; however, results were confounded by other baseline characteristics. After treatment, PD-L1 expression dropped a median of 1.5% on IC and a median of 2.75% on TC. Median CD8 expression dropped 1.9%. CONCLUSIONS: Majority of NPC biopsy samples demonstrated PD-L1 expression on ⩾1% of IC, with fewer expressing PD-L1 on TC. In contrast to previous smaller studies, no prognostic value was observed for PD-L1 expression levels in patients with NPC.

Biomarker analysis of the NeoSphere study: pertuzumab, trastuzumab, and docetaxel versus trastuzumab plus docetaxel, pertuzumab plus trastuzumab, or pertuzumab plus docetaxel for the neoadjuvant treatment of HER2-positive breast cancer.

BACKGROUND: NeoSphere showed significantly higher pathologic complete response (pCR) with neoadjuvant pertuzumab, trastuzumab, and docetaxel compared with trastuzumab plus docetaxel, pertuzumab plus trastuzumab, or pertuzumab plus docetaxel. We assessed associations between human epidermal growth factor receptor 2 (HER2) pathway-related biomarkers and clinical outcome in response to these regimens. METHODS: Tumor, serum, and whole blood samples were collected at baseline and post neoadjuvant treatment before surgery. Associations between biomarkers and pCR, and between biomarkers and clinical variables were assessed in the overall and estrogen receptor (ER)-positive and ER-negative populations. Changes in serum marker levels between baseline and post-neoadjuvant treatment were examined. RESULTS: No markers were associated with pCR across all groups; however, significant associations were observed for two markers in individual groups. High HER2 was significantly associated with higher pCR rates (P = 0.001) and a significant treatment interaction (P = 0.0236) with pertuzumab, trastuzumab, and docetaxel (odds ratio 2.07, P = 0.01). Low serum transforming growth factor alpha (TGFα) was associated with higher pCR rates with pertuzumab plus trastuzumab (P = 0.04) without a significant treatment interaction. Presence of truncated HER2 did not affect pCR. A non-significant decreased pCR benefit was observed consistently across groups in patients with mutated PIK3CA while the treatment benefit from pertuzumab was maintained when comparing the trastuzumab plus docetaxel and pertuzumab, trastuzumab, and docetaxel groups. Notably, PIK3CA exon 9 mutations were associated with residual disease (pooled groups), which was not found for exon 20 mutations. Serum HER2 extracellular domain levels were significantly increased between baseline and post-neoadjuvant treatment in the non-trastuzumab-treated group, and decreased in the trastuzumab-containing groups (likely due to trastuzumab's mechanism of action). Differences in biomarker profiles according to ER status were observed. CONCLUSIONS: The observed associations of HER2 protein levels with sensitivity to pertuzumab, and of PIK3CA exon 9 mutation to lack of sensitivity to HER2-targeted monoclonal antibody treatment, warrant further investigation. Previously reported findings of truncated forms of HER2 as resistance markers to HER2-targeted treatment could not be confirmed in NeoSphere. Conventional HER2 assessment should continue and HER2 remains the only biomarker suitable for patient selection in this population. TRIAL REGISTRATION: Clinicaltrials.gov, NCT00545688 . Registered on 16 October 2007.

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.

Heterogeneity and clinical significance of ESR1 mutations in ER-positive metastatic breast cancer patients receiving fulvestrant.

Mutations in ESR1 have been associated with resistance to aromatase inhibitor (AI) therapy in patients with ER+ metastatic breast cancer. Little is known of the impact of these mutations in patients receiving selective oestrogen receptor degrader (SERD) therapy. In this study, hotspot mutations in ESR1 and PIK3CA from ctDNA were assayed in clinical trial samples from ER+ metastatic breast cancer patients randomized either to the SERD fulvestrant or fulvestrant plus a pan-PI3K inhibitor. ESR1 mutations are present in 37% of baseline samples and are enriched in patients with luminal A and PIK3CA-mutated tumours. ESR1 mutations are often polyclonal and longitudinal analysis shows distinct clones exhibiting divergent behaviour over time. ESR1 mutation allele frequency does not show a consistent pattern of increases during fulvestrant treatment, and progression-free survival is not different in patients with ESR1 mutations compared with wild-type patients. ESR1 mutations are not associated with clinical resistance to fulvestrant in this study.

PTEN loss in circulating tumour cells correlates with PTEN loss in fresh tumour tissue from castration-resistant prostate cancer patients.

BACKGROUND: PTEN gene loss occurs frequently in castration-resistant prostate cancer (CRPC) and may drive progression through activation of the PI3K/AKT pathway. Here, we developed a novel CTC-based assay to determine PTEN status and examined the correlation between PTEN status in CTCs and matched tumour tissue samples. METHODS: PTEN gene status in CTCs was evaluated on an enrichment-free platform (Epic Sciences) by fluorescence in situ hybridisation (FISH). PTEN status in archival and fresh tumour tissue was evaluated by FISH and immunohistochemistry. RESULTS: Peripheral blood was collected from 76 patients. Matched archival and fresh cancer tissue was available for 48 patients. PTEN gene status detected in CTCs was concordant with PTEN status in matched fresh tissues and archival tissue in 32 of 38 patients (84%) and 24 of 39 patients (62%), respectively. CTC counts were prognostic (continuous, P=0.001). PTEN loss in CTCs associated with worse survival in univariate analysis (HR 2.05; 95% CI 1.17-3.62; P=0.01) and with high lactate dehydrogenase (LDH) in metastatic CRPC patients. CONCLUSIONS: Our results illustrate the potential use of CTCs as a non-invasive, real-time liquid biopsy to determine PTEN gene status. The prognostic and predictive value of PTEN in CTCs warrants investigation in CRPC clinical trials of PI3K/AKT-targeted therapies.

Safety and Pharmacokinetics/Pharmacodynamics of the First-in-Class Dual Action HER3/EGFR Antibody MEHD7945A in Locally Advanced or Metastatic Epithelial Tumors.

PURPOSE: The novel dual-action humanized IgG1 antibody MEHD7945A targeting HER3 and EGFR inhibits ligand-dependent HER dimer signaling. This phase I study evaluated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of MEHD7945A. EXPERIMENTAL DESIGN: Patients with locally advanced or metastatic epithelial tumors received escalating doses of MEHD7945A (1-30 mg/kg) every 2 weeks (q2w) until disease progression or intolerable toxicity. An expansion cohort was enrolled at the recommended phase II dose (14 mg/kg, q2w). Plasma samples, tumor biopsies, FDG-PET were obtained for assessment of pharmacokinetics, and pharmacodynamic modulation downstream of EGFR and HER3. RESULTS: No dose-limiting toxicities or MEHD7945A-related grade ≥ 4 adverse events (AE) were reported in dose-escalation (n = 30) or expansion (n = 36) cohorts. Related grade 3 AEs were limited to diarrhea and nausea in the same patient (30 mg/kg). Related AEs in ≥20% of patients ≤24 hours after the first infusion included grade 1/2 headache, fever, and chills, which were managed with premedication and/or symptomatic treatment. Pharmacodynamic data indicated target inhibition in 25% of evaluable patients. Best response by RECIST included 2 confirmed partial responses in squamous cell carcinomas of head and neck (SCCHN) patients with high tumor tissue levels of the HER3 ligand heregulin; 14 patients had stable disease ≥8 weeks, including SCCHN (n = 3), colorectal cancer (n = 6), and non-small cell lung cancer (n = 3). CONCLUSIONS: MEHD7945A was well-tolerated as single agent with evidence of tumor pharmacodynamic modulation and antitumor activity in SCCHN. Phase II studies were initiated with flat (nonweight-based) dosing at 1,100 mg q2w in SCCHN and colorectal cancer.

Upregulation of Periostin and Reactive Stroma Is Associated with Primary Chemoresistance and Predicts Clinical Outcomes in Epithelial Ovarian Cancer.

PURPOSE: Up to one third of ovarian cancer patients are intrinsically resistant to platinum-based treatment. However, predictive and therapeutic strategies are lacking due to a poor understanding of the underlying molecular mechanisms. This study aimed to identify key molecular characteristics that are associated with primary chemoresistance in epithelial ovarian cancers. EXPERIMENTAL DESIGN: Gene expression profiling was performed on a discovery set of 85 ovarian tumors with clinically well-defined response to chemotherapies as well as on an independent validation dataset containing 138 ovarian patients from the chemotreatment arm of the ICON7 trial. RESULTS: We identified a distinct "reactive stroma" gene signature that is specifically associated with primary chemoresistant tumors and was further upregulated in posttreatment recurrent tumors. Immunohistochemistry (IHC) and RNA in situ hybridization (RNA ISH) analyses on three of the highest-ranked signature genes (POSTN, LOX, and FAP) confirmed that modulation of the reactive stroma signature genes within the peritumoral stromal compartments was specifically associated with the clinical chemoresistance. Consistent with these findings, chemosensitive ovarian cells grown in the presence of recombinant POSTN promoted resistance to carboplatin and paclitaxel treatment in vitro. Finally, we validated the reactive stroma signature in an independent dataset and demonstrated that a high POSTN expression level predicts shorter progression-free survival following first-line chemotherapy. CONCLUSIONS: Our findings highlight the important interplay between cancer and the tumor microenvironment in ovarian cancer biology and treatment. The identified reactive stromal components in this study provide a molecular basis to the further development of novel diagnostic and therapeutic strategies for overcoming chemoresistance in ovarian cancer.

Relationship between HER2 expression and efficacy with first-line trastuzumab emtansine compared with trastuzumab plus docetaxel in TDM4450g: a randomized phase II study of patients with previously untreated HER2-positive metastatic breast cancer.

INTRODUCTION: The purpose of this study was to retrospectively explore the relationship between human epidermal growth factor receptor 2 (HER2) messenger RNA (mRNA) expression and efficacy in patients receiving trastuzumab plus docetaxel (HT) or trastuzumab emtansine (T-DM1). METHODS: Patients with HER2-positive, locally advanced or metastatic breast cancer (MBC) were randomly assigned to HT (n=70) or T-DM1 (n=67). HER2 status was assessed locally using immunohistochemistry or fluorescence in situ hybridization and confirmed retrospectively by central testing. HER2 mRNA expression was assessed using quantitative reverse transcriptase polymerase chain reaction. RESULTS: HER2 mRNA levels were obtained for 116/137 patients (HT=61; T-DM1=55). Median pretreatment HER2 mRNA was 8.9. The risk of disease progression in the overall population was lower with T-DM1 than with HT (hazard ratio (HR)=0.59; 95% confidence interval (CI) 0.36 to 0.97). This effect was more pronounced in patients with HER2 mRNA≥median (HR=0.39; 95% CI 0.18 to 0.85) versus

Biomarker analyses from a placebo-controlled phase II study evaluating erlotinib±onartuzumab in advanced non-small cell lung cancer: MET expression levels are predictive of patient benefit.

PURPOSE: In a recent phase II study of onartuzumab (MetMAb), patients whose non-small cell lung cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit with onartuzumab plus erlotinib (O+E) versus erlotinib. We describe development and validation of a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers as predictors of patient benefit. EXPERIMENTAL DESIGN: Biomarkers related to MET and/or EGF receptor (EGFR) signaling were measured by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA. RESULTS: A positive correlation between IHC, Western blotting, and MET mRNA expression was observed in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement with O+E in patients with high MET copy number (mean≥5 copies/cell by FISH); however, benefit was maintained in "MET IHC-positive"/MET FISH-negative patients (HR, 0.37; P=0.01). MET, EGFR, amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant benefit with onartuzumab; a nonsignificant OS improvement was observed in patients with high tumor MET mRNA levels (HR, 0.59; P=0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519 (P=0.09) in favor of onartuzumab treatment. CONCLUSIONS: MET IHC remains the most robust predictor of OS and progression-free survival benefit from O+E relative to all examined exploratory markers.

Discovery and development of DNA methylation-based biomarkers for lung cancer.

Lung cancer remains the primary cause of cancer-related deaths worldwide. Improved tools for early detection and therapeutic stratification would be expected to increase the survival rate for this disease. Alterations in the molecular pathways that drive lung cancer, which include epigenetic modifications, may provide biomarkers to help address this major unmet clinical need. Epigenetic changes, which are defined as heritable changes in gene expression that do not alter the primary DNA sequence, are one of the hallmarks of cancer, and prevalent in all types of cancer. These modifications represent a rich source of biomarkers that have the potential to be implemented in clinical practice. This perspective describes recent advances in the discovery of epigenetic biomarkers in lung cancer, specifically those that result in the methylation of DNA at CpG sites. We discuss one approach for methylation-based biomarker assay development that describes the discovery at a genome-scale level, which addresses some of the practical considerations for design of assays that can be implemented in the clinic. We emphasize that an integrated technological approach will enable the development of clinically useful DNA methylation-based biomarker assays. While this article focuses on current literature and primary research findings in lung cancer, the principles we describe here apply to the discovery and development of epigenetic biomarkers for other types of cancer.

High-throughput detection of clinically relevant mutations in archived tumor samples by multiplexed PCR and next-generation sequencing.

PURPOSE: Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false-positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high-content genetic profiling in archived clinical samples. EXPERIMENTAL DESIGN: We first validated the technical performance of MMP-seq in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin-embedded (FFPE) clinical samples using matched fresh-frozen tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 patients with endometrial cancer. RESULTS: We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false-positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K (phosphoinositide 3-kinase) and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA. CONCLUSIONS: MMP-seq provides a robust solution for comprehensive, reliable, and high-throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice.

High heregulin expression is associated with activated HER3 and may define an actionable biomarker in patients with squamous cell carcinomas of the head and neck.

PURPOSE: Tumors with oncogenic dependencies on the HER family of receptor tyrosine kinases (RTKs) often respond well to targeted inhibition. Our previous work suggested that many cell lines derived from squamous cell carcinomas of the head and neck (SCCHNs) depend on autocrine signaling driven by HER2/3 dimerization and high-level co-expression of HRG. Additionally, results from a Phase I trial of MEHD7495A, a dual-action antibody that blocks ligand binding to EGFR and HER3, suggest that high-level HRG expression was associated with clinical response in SCCHN patients. Here we explore the hypothesis that high-level HRG expression defines a subpopulation of SCCHNs with activated HER3. EXPERIMENTAL DESIGN: qRT-PCR expression profiling was performed on >750 tumors of diverse origin, including >150 therapy-naïve, primary, and recurrent SCCHNs. Activated HER3, defined by immunoprecipitation of phospho-HER3, was compared to HRG expression in SCCHN samples. Paracrine versus autocrine expression was evaluated using RNA-in situ hybridization. RESULTS: SCCHN tumors express the highest levels of HRG compared to a diverse collection of other tumor types. We show that high HRG expression is associated with activated HER3, whereas low HRG expression is associated with low HER3 activation in SCCHN tumors. Furthermore, HRG expression is higher in recurrent SCCHN compared to patient-matched therapy naïve specimens. CONCLUSIONS: HRG expression levels define a biologically distinct subset of SCCHN patients. We propose that high-level expression of HRG is associated with constitutive activation of HER3 in SCCHN and thus defines an actionable biomarker for interventions targeting HER3.

Dual targeting of EGFR and HER3 with MEHD7945A overcomes acquired resistance to EGFR inhibitors and radiation.

EGF receptor (EGFR) inhibition is efficacious in cancer therapy, but initially sensitive tumors often develop resistance. In this study, we investigated the potential to overcome acquired resistance to EGFR inhibitors with MEHD7945A, a monoclonal antibody that dually targets EGFR and HER3 (ErbB3). In cancer cells resistant to cetuximab and erlotinib, we found that MEHD7945A, but not single target EGFR inhibitors, could inhibit tumor growth and cell-cycle progression in parallel with EGFR/HER3 signaling pathway modulation. MEHD7945A was more effective than a combination of cetuximab and anti-HER3 antibody at inhibiting both EGFR/HER3 signaling and tumor growth. In human tumor xenograft models, we confirmed the greater antitumor potency of MEHD7945A than cetuximab or erlotinib. MEHD7945A retained potent activity in tumors refractory to EGFR inhibitor alone. Furthermore, MEHD7945A also limited cross-resistance to radiation in EGFR inhibitor-resistant cells by modulating cell-cycle progression and repair processes that control apoptotic cell death. Taken together, our findings confirm an important role of compensatory HER3 signaling in the development of acquired resistance to EGFR inhibitors and offer preclinical proof-of-concept that MEHD7945A can effectively overcome EGFR inhibitor resistance.

Phosphoinositide 3-kinase (PI3K) pathway alterations are associated with histologic subtypes and are predictive of sensitivity to PI3K inhibitors in lung cancer preclinical models.

PURPOSE: Class 1 phosphatidylinositol 3-kinase (PI3K) plays a major role in cell proliferation and survival in a wide variety of human cancers. Here, we investigated biomarker strategies for PI3K pathway inhibitors in non-small-cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Molecular profiling for candidate PI3K predictive biomarkers was conducted on a collection of NSCLC tumor samples. Assays included comparative genomic hybridization, reverse-transcription polymerase chain reaction gene expression, mutation detection for PIK3CA and other oncogenes, PTEN immunohistochemistry, and FISH for PIK3CA copy number. In addition, a panel of NSCLC cell lines characterized for alterations in the PI3K pathway was screened with PI3K and dual PI3K/mTOR inhibitors to assess the preclinical predictive value of candidate biomarkers. RESULTS: PIK3CA amplification was detected in 37% of squamous tumors and 5% of adenocarcinomas, whereas PIK3CA mutations were found in 9% of squamous and 0% of adenocarcinomas. Total loss of PTEN immunostaining was found in 21% of squamous tumors and 4% of adenocarcinomas. Cell lines harboring pathway alterations (receptor tyrosine kinase activation, PI3K mutation or amplification, and PTEN loss) were exquisitely sensitive to the PI3K inhibitor GDC-0941. A dual PI3K/mTOR inhibitor had broader activity across the cell line panel and in tumor xenografts. The combination of GDC-0941 with paclitaxel, erlotinib, or a mitogen-activated protein-extracellular signal-regulated kinase inhibitor had greater effects on cell viability than PI3K inhibition alone. CONCLUSIONS: Candidate biomarkers for PI3K inhibitors have predictive value in preclinical models and show histology-specific alterations in primary tumors, suggesting that distinct biomarker strategies may be required in squamous compared with nonsquamous NSCLC patient populations.

Phase Ib study of drozitumab combined with first-line mFOLFOX6 plus bevacizumab in patients with metastatic colorectal cancer.

In this multicenter phase Ib study, drozitumab was given in combination with the mFOLFOX6 regimen and bevacizumab in patients with previously untreated, locally advanced recurrent or metastatic colorectal cancer on day 1 of every 14-day cycle. Nine patients were treated at 2 different cohort dose levels of drozitumab. No dose-limiting toxicities occurred at either dose level and the maximum tolerated dose was not reached. Two patients had a partial response of 4.93 and 4.96 months duration. Cohort 2 dose level is the recommended starting dose level for future trials.