Mark Ayers

Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes

Gastric cancer, a leading cause of cancer-related deaths, is a heterogeneous disease. We aim to establish clinically relevant molecular subtypes that would encompass this heterogeneity and provide useful clinical information. We use gene expression data to describe four molecular subtypes linked to distinct patterns of molecular alterations, disease progression and prognosis. The mesenchymal-like type includes diffuse-subtype tumors with the worst prognosis, the tendency to occur at an earlier age and the highest recurrence frequency (63%) of the four subtypes. Microsatellite-unstable tumors are hyper-mutated intestinal-subtype tumors occurring in the antrum; these have the best overall prognosis and the lowest frequency of recurrence (22%) of the four subtypes. The tumor protein 53 (TP53)-active and TP53-inactive types include patients with intermediate prognosis and recurrence rates (with respect to the other two subtypes), with the TP53-active group showing better prognosis. We describe key molecular alterations in each of the four subtypes using targeted sequencing and genome-wide copy number microarrays. We validate these subtypes in independent cohorts in order to provide a consistent and unified framework for further clinical and preclinical translational research.

IFN-γ–related mRNA profile predicts clinical response to PD-1 blockade

Programmed death-1–directed (PD-1–directed) immune checkpoint blockade results in durable antitumor activity in many advanced malignancies. Recent studies suggest that IFN-&ggr; is a critical driver of programmed death ligand-1 (PD-L1) expression in cancer and host cells, and baseline intratumoral T cell infiltration may improve response likelihood to anti–PD-1 therapies, including pembrolizumab. However, whether quantifying T cell–inflamed microenvironment is a useful pan-tumor determinant of PD-1–directed therapy response has not been rigorously evaluated. Here, we analyzed gene expression profiles (GEPs) using RNA from baseline tumor samples of pembrolizumab-treated patients. We identified immune-related signatures correlating with clinical benefit using a learn-and-confirm paradigm based on data from different clinical studies of pembrolizumab, starting with a small pilot of 19 melanoma patients and eventually defining a pan-tumor T cell–inflamed GEP in 220 patients with 9 cancers. Predictive value was independently confirmed and compared with that of PD-L1 immunohistochemistry in 96 patients with head and neck squamous cell carcinoma. The T cell–inflamed GEP contained IFN-&ggr;–responsive genes related to antigen presentation, chemokine expression, cytotoxic activity, and adaptive immune resistance, and these features were necessary, but not always sufficient, for clinical benefit. The T cell–inflamed GEP has been developed into a clinical-grade assay that is currently being evaluated in ongoing pembrolizumab trials.

A Randomized Phase II/III Study of Dalotuzumab in Combination With Cetuximab and Irinotecan in Chemorefractory, KRAS Wild-Type, Metastatic Colorectal Cancer

BACKGROUND Insulin-like growth factor type 1 receptor (IGF-1R) mediates resistance to epidermal growth factor receptor (EGFR) inhibition and may represent a therapeutic target. We conducted a multicenter, randomized, double blind, phase II/III trial of dalotuzumab, an anti-IGF-1R monoclonal antibody, with standard therapy in chemo-refractory, KRAS wild-type metastatic colorectal cancer. METHODS Eligible patients were randomly assigned to dalotuzumab 10mg/kg weekly (arm A), dalotuzumab 7.5mg/kg every alternate week (arm B), or placebo (arm C) in combination with cetuximab and irinotecan. Primary endpoints were progression-free survival (PFS) and overall survival (OS). Secondary endpoints included exploratory biomarker analyses. All statistical tests were two-sided. RESULTS The trial was prematurely discontinued for futility after 344 eligible KRAS wild-type patients were included in the primary efficacy population (arm A = 116, arm B = 117, arm C = 111). Median PFS was 3.9 months in arm A (hazard ratio [HR] = 1.33, 95% confidence interval [CI] = 0.98 to 1.83, P = .07) and 5.4 months in arm B (HR = 1.13, 95% CI = 0.83 to 1.55, P = .44) compared with 5.6 months in arm C. Median OS was 10.8 months in arm A (HR = 1.41, 95% CI = 0.99 to 2.00, P = .06) and 11.6 months in arm B (HR = 1.26, 95% CI = 0.89 to 1.79, P = .18) compared with 14.0 months in arm C. Grade 3 or higher asthenia and hyperglycaemia occurred more frequently with dalotuzumab compared with placebo. In exploratory biomarker analyses, patients with high IGF-1 mRNA tumors in arm A had numerically better PFS (5.6 vs 3.6 months, HR = 0.59, 95% CI = 0.28 to 1.23, P = .16) and OS (17.9 vs 9.4 months, HR = 0.67, 95% CI = 0.31 to 1.45, P = .31) compared with those with high IGF-1 mRNA tumors in arm C. In contrast, in arm C high IGF-1 mRNA expression predicted lower response rate (17.6% vs 37.3%, P = .04), shorter PFS (3.6 vs 6.6 months, HR = 2.15, 95% CI = 1.15 to 4.02, P = .02), and shorter OS (9.4 vs 15.5 months, HR = 2.42, 95% CI = 1.21 to 4.82, P = .01). CONCLUSIONS Adding dalotuzumab to irinotecan and cetuximab was feasible but did not improve survival outcome. IGF-1R ligands are promising biomarkers for differential response to anti-EGFR and anti-IGF-1R therapies.

Relationship between immune gene signatures and clinical response to PD-1 blockade with pembrolizumab (MK-3475) in patients with advanced solid tumors

Meeting abstracts Immune checkpoint inhibition with anti–PD-1 monoclonal antibodies such as pembrolizumab has demonstrated robust, durable anti-tumor activity against many advanced malignancies. We analyzed immune-related gene expression profiles in pembrolizumab-treated patients with advanced

MCL1 and BCL-xL Levels in Solid Tumors Are Predictive of Dinaciclib-Induced Apoptosis

Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclibs antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.

A phase II study of clinical activity of SCH 717454 (robatumumab) in patients with relapsed osteosarcoma and Ewing sarcoma

Robatumumab (19D12; MK‐7454 otherwise known as SCH717454) is a fully human antibody that binds to and inhibits insulin‐like growth factor receptor‐1 (IGF‐1R). This multiinstitutional study (P04720) determined the safety and clinical efficacy of robatumumab in three separate patient groups with resectable osteosarcoma metastases (Group 1), unresectable osteosarcoma metastases (Group 2), and Ewing sarcoma metastases (Group 3).

Dalotuzumab in chemorefractory KRAS exon 2 mutant colorectal cancer: Results from a randomised phase II/III trial.

Limited data are available on the efficacy of anti‐IGF‐1R agents in KRAS mutant colorectal cancer (CRC). We analysed the outcome of 69 chemorefractory, KRAS exon 2 mutant CRC patients who were enrolled in a double‐blind, randomised, phase II/III study of irinotecan and cetuximab plus dalotuzumab 10 mg/kg once weekly (arm A), dalotuzumab 7.5 mg/kg every second week (arm B) or placebo (arm C). Objective response rate (5.6% vs. 3.1% vs. 4.8%), median progression‐free survival (2.7 vs. 2.6 vs. 1.4 months) and overall survival (7.8 vs. 10.3 vs. 7.8 months) were not statistically significantly different between treatment groups. Most common grade ≥3 treatment‐related toxicities included neutropenia, diarrhoea, hyperglycaemia, fatigue and dermatitis acneiform. Expression of IGF‐1R, IGF‐1, IGF‐2 and EREG by quantitative real‐time polymerase chain reaction was assessed in 351 patients from the same study with available data on KRAS exon 2 mutational status. Median cycle threshold values for all biomarkers were significantly lower (i.e., higher expression, p < 0.05) among patients with KRAS wild‐type compared to those with KRAS exon 2 mutant tumours. No significant changes were found according to location of the primary tumour with only a trend towards lower expression of IGF‐1 in colon compared to rectal cancers (p = 0.06). Albeit limited by the small sample size, this study does not appear to support a potential role for anti‐IGF‐1R agents in KRAS exon 2 mutant CRC. Data on IGF‐1R, IGF‐1 and IGF‐2 expression here reported may be useful for patient stratification in future trials with inhibitors of the IGF pathway.

Preclinical to clinical translation of anti-PD-1 blockade

Meeting abstracts Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. Immunotherapy with anti–PD-1 monoclonal antibodies such as pembrolizumab shows robust,

Abstract 1307: Assessment of gene expression in peripheral blood from patients with advanced melanoma using RNA-seq before and after treatment with anti-PD-1 therapy with pembrolizumab (MK-3475)

Pembrolizumab (MK-3475) is a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1) that is currently being studied in clinical trials across more than 30 types of cancer. Gene expression profiling was used to explore a less invasive method for gaining new insights into the pembrolizumab mechanism of action and to assess the potential for blood-based predictive biomarkers. RNA-seq data were obtained from the whole blood of 44 patients with advanced melanoma enrolled in the phase 1 KEYNOTE-001 clinical study before and after the first cycle of treatment with pembrolizumab. Objective response rates (ORR) were assessed per RECIST v1.1 by independent central review. The pembrolizumab dose and treatment schedule varied among the patients studied and included 10 mg/kg given once every 3 weeks (Q3W) (n = 21), 10 mg/kg Q2W (n = 12), and 2 mg/kg Q3W (n = 11). Among the 44 patients with melanoma included in the analysis, 75% received previous ipilimumab treatment. The ORR was 32%. Significant posttreatment changes in gene expression were confirmed for the target, PD-1, and its key ligand, programmed death ligand 1 (PD-L1). Changes in PD-1 and PD-L1 gene expression did not, however, show a significant association with ORR. Statistically significant posttreatment changes for an interferon-gamma (IFNG)-related 10-gene signature were observed (P Citation Format: Mark D. Ayers, Michael Nebozhyn, Heather A. Hirsch, Razvan Cristescu, Erin E. Murphy, S. Peter Kang, Scot W. Ebbinghaus, Terrill K. McClanahan, Andrey Loboda, Jared K. Lunceford. Assessment of gene expression in peripheral blood from patients with advanced melanoma using RNA-seq before and after treatment with anti-PD-1 therapy with pembrolizumab (MK-3475). [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 1307. doi:10.1158/1538-7445.AM2015-1307

Abstract 1328: Molecular characterization of mouse syngeneic tumor models in response to treatment with anti-PD-1 immunotherapy

Although there are a variety of immune cells localized in the tumor microenvironment, they are generally present in a quiescent state. The goal of cancer immunotherapy is to activate immune effector cells in the tumor microenvironment to recognize tumor cells and elicit an immune response that results in elimination of the tumor. Checkpoint inhibitors and activators that target T-cell receptors provide attractive targets for cancer immunotherapies by either enhancing T-cell activation pathways or inhibiting T-cell checkpoint/anergy pathways. In 2014, the anti-programmed death receptor 1 (PD-1) antibody pembrolizumab (MK-3475) was approved for use in patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. Pembrolizumab is currently being explored in multiple clinical trials covering ∼30 different cancer types. A more complete understanding of the mechanism of action and biology associated with both response and resistance to pembrolizumab is critical to better inform future clinical development and to provide insight into the development of additional immuno-oncology therapies. Preclinical mouse syngeneic tumor models have been used extensively to support the clinical development of drugs such as pembrolizumab and to help identify novel targets that have the potential to synergize with anti-PD-1 therapies. We employed a panel of mouse syngeneic tumor models that have been used to assess the efficacy of muDX400, a fully murinized surrogate antibody of pembrolizumab. Tumors from these models were extensively characterized at the molecular and cellular levels at baseline and after muDX400 treatment. Data obtained from multiple experimental sources, including RNA expression, DNA mutation and copy number, fluorescence-activated cell sorting, and immunohistochemistry, have been integrated to inform pembrolizumab mechanisms of action and resistance, pharmacodynamic biomarkers, responder identification, and indication selection. These findings are also being compared to data being obtained from ongoing clinical trials of pembrolizumab to help better understand whether these preclinical models can be translated to the clinic. These preclinical syngeneic tumor models also provide the opportunity to formulate and test specific hypotheses to more completely understand the biology behind the clinical successes currently observed with novel cancer immunotherapies, including anti-PD-1 antibodies such as pembrolizumab. The hypotheses under evaluation include mutational burden, immune cell activation and migration, interferon signaling, antigen presentation (MHC class I and II), and expression of novel targets that will help lead to the development of the next generation of cancer immunotherapies. Citation Format: Heather A. Hirsch, Elaine M. Pinheiro, Mingmei Cai, Yanhong Ma, Manjiri Sathe, Mark Ayers, Terrill K. McClanahan. Molecular characterization of mouse syngeneic tumor models in response to treatment with anti-PD-1 immunotherapy. [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 1328. doi:10.1158/1538-7445.AM2015-1328