Teiko Sumiyoshi

Membrane-Proximal Epitope Facilitates Efficient T Cell Synapse Formation by Anti-FcRH5/CD3 and Is a Requirement for Myeloma Cell Killing

Summary The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.

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.

Myeloid cell biology and inhibition of anti-tumor immune responses by MPDL3280A in urothelial bladder cancer

Treatment options for metastatic urothelial bladder cancer (UBC) are limited. Mutational complexity is known to be high in UBC and may correlate with increased immunogenicity. MPDL3280A, a human PD-L1 monoclonal antibody containing an engineered Fc-domain designed to promote a Th1-driven response, has demonstrated a RECIST response rate of 43% in diagnostically selected, pretreated patients with UBC. A total of 68 patients (67 with efficacy evaluable) were enrolled in the UBC cohort of the Phase I study; 45% were PD-L1 IHC diagnostic positive as defined by expression of PD-L1 on ≥ 5% of tumor-infiltrating immune cells. In the prescreened UBC population, the prevalence of PD-L1-positive patients was 27%. Comprehensive gene expression analyses of UBC tumors were conducted to interrogate the tumor immune microenvironment in PD-L1-positive tumors and to identify potential mechanisms associated with response or resistance to MPDL3280A. In this study, PD-L1-positive tumors exhibited a high prevalence of gene expression markers associated with T-effector cells (Teff), including perforin, IFNγ, CD8A, granzyme B, granzyme A and EOMES. Additionally, a low baseline signature of genes associated with myeloid cell markers, including IL1B and IL8, appeared to be statistically significantly associated (P<0.01) with MPDL3280A response, suggesting a potential role for myeloid biology in resistance to MPDL3280A treatment in UBC. Tumor burden markers, including CA-125, CA19-9 and human chorionic gonadotropin (HCG), have been associated with chemotherapy response markers in UBC. A marked decrease in these markers, including CEA, CA19-9, CA-125 and HCG, was observed with MPDL3280A response after 1 treatment cycle, potentially enabling an on-treatment monitoring alternative for response to therapy. Similarly, evaluation of cytokines on treatment identified markers, including IL-6 and IL-10, elevated as early as Cycle 2 only in patients without response to MPDL3280A. These circulating cytokines and tumor-associated gene signatures suggest potential mechanisms associated with resistance and response to MPDL3280A in UBC and provide a rationale for informed combination strategies to further improve treatment benefit in this indication.

Abstract POSTER-THER-1441: Biomarker evaluation of phase 1 clinical trials of antibody-drug conjugates (ADCs) in platinum resistant ovarian cancer

Purpose: DNIB0600A and DMUC5754A are two ADCs that conjugate the anti-mitotic agent MMAE with anti-NaPi2b and anti-MUC16 monoclonal antibodies, respectively. Both ADCs have shown promising anti-tumor activity in patients with platinum resistant ovarian cancer. Here we report biomarker analysis in patient samples collected from these phase 1 studies. The main goal of this study is to evaluate tissue-based biomarkers that can predict response or resistance to these ADCs. We also explored the utility of serum protein biomarkers and circulating tumor cells (CTCs) as potential surrogates for monitoring treatment response to ADCs and disease progression. Methods: Biomarker analysis was done on 55 ovarian cancer patients treated with clinically relevant doses (1.8-3.2mg/kg) from DNIB0600A and DMUC5754A Phase 1 studies. Protein and mRNA expression levels of NaPi2b and MUC16 targets were assessed in archival tumor specimen by immunohistochemistry (IHC) and qRT-PCR respectively. Serum collected at baseline and post-treatment were analyzed by CA125 and HE4 ELISA assays as well as by the OLINK 96-plex PEA protein biomarker panel. CTCs at baseline and post-treatment were analyzed using the Veridex CellSearch System. Results: Target expression in tumor tissues for both NaPi2b and MUC16 measured by IHC and qRT-PCR are concordant. High NaPi2b or MUC16 expression (IHC 2+/3+) was identified in all responders by RECIST criteria (11 from DNIB0600A and 5 from DMUC5754A) for respective target, while no patient from either study with IHC 0 showed RECIST response. In patients treated with DNIB0600A, longitudinal changes in serum CA125 level correlated with RECIST response. Additionally, CTC was detected in 60% of patients at baseline in the DNIB0600A trial, and decreased CTC counts was observed after 1-2 cycles of treatment for two-third of patients. In patients treated with DMUC5754A, circulating CA125 (i.e. extra-cellular domain of MUC16 shed in circulation) is cleared after initial dosing; therefore other ovarian cancer biomarkers including HE4 were assessed. Baseline serum HE4 level correlates well with the tumor burden at pre-treatment in DMUC5754A trial, and showed excellent correlation with RECIST response post-treatment. Conclusions: Target expression in archival tumor tissues is predictive to clinical response to ADCs. CTC enumeration as well as serum HE4 could be used as potential surrogate biomarkers for monitoring treatment response in ovarian cancer. Further validation of these findings is required. Citation Format: Yulei Wang, Ron Firestein, Lisa Ryner, Walter Darbonne, Yinghui Guan, Shan Lu, YJ Choi, Yuanyuan Xiao, Paul Polakis, Becky Suttmann, Rupal Desai, Ling Fu, Ola Saad, Kirsten Achilles Poon, Mitch Denker, Vincent Leveque, Teiko Sumiyoshi, Mark Lackner, David Shames, Eric Humke, Daniel Mayslar. Biomarker evaluation of phase 1 clinical trials of antibody-drug conjugates (ADCs) in platinum resistant ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1441.

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

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.Biomarker: Gene signature predicts drug responses and patient outcomesA clinical score based on the activity of genes that regulate cell signaling can predict drug sensitivity and patient outcomes across a range of cancer types. Marie-Claire Wagle, Daniel Kirouac and their colleagues at Genentech in South San Francisco, California, USA developed an index that aggregates expression levels of 10 genes involved in modulating the mitogen-activated protein kinase (MAPK) pathway. This “MAPK Pathway Activity Score”, or MPAS, performed as well or better than other more complicated, genome-based tools at predicting whether drugs that inhibit MAPK-related enzymes were active against tumor cell lines. Retrospective analyses of clinical datasets also showed that MPAS correlated with survival outcomes in patients with melanoma, colon cancer, and breast cancer. The authors suggest that MPAS should be evaluated more broadly and perhaps implemented as a clinically informative biomarker.

Abstract 4257: Gene expression and genomic drift comparative analysis between patient-derived conditionally reprogrammed cells and original tumors

First two authors contributed equally Last two authors contributed equally Recent studies have shown that ex vivo propagation of normal tissues or patient-derived tumor cells in presence of irradiated fibroblast feeder cells and ROCK inhibitor can rapidly establish conditionally reprogramed cells (CRCs). In case of normal tissues, the induction of CRCs was reversible when the ROCK inhibitor and the feeder cells were removed, resulting in CRCs differentiating to its tissue origin (Liu et al.2012). Previous publications suggested that the establishment of such cell models provides new strategies to understand acquired resistance during treatment (Crystal et al 2015) and to predict treatment response (Liu et al. 2014). However, gene expression modulations and genomic drifting during the establishment of CRC propagation have not been thoroughly studied. The primary goal of this study is to molecularly characterize alterations between the original tumor tissues and the derived models growing with or without ROCK inhibitor. Understanding in-depth molecular fluctuations in this patient-derived ex vivo system will facilitate its appropriate use for tumor biology experimentation. Herein, tumors from prostate cancer and breast cancer patients were surgical removed and cryopreserved at the clinical site then processed and cultured as previously described (Liu et al. 2012). Gene expression profiling and next-generation sequencing were carried out on the original tumor tissues and cellular models passaged during the CRC propagation in the presence or absence of ROCK inhibitor. Gene expression analysis of the prostate cancer cells and the breast cancer cells were carried out using a 93-gene prostate cancer-focused Fluidigm panel and a 800 gene NanoString breast cancer-focused panel, respectively. Cancer hotspot mutations were analyzed using the Ion Torrent Cancer Hotspot v2 NGS assay. Through aforementioned genomic and transcriptomic interrogations, we demonstrated the extent of indication-relevant gene expression modulation during establishment and propagation of these cells. We also characterize cancer hotspot mutations in the primary tumor cells and the stability of those mutations during ex vivo propagation. These results should begin to inform the appropriate use of the CRC model for tumor biology experimentation. Citation Format: Jessica Li, Bonnie Liu, Rin Nakamura, Heidi Savage, Shoji Ikeda, Timothy Wilson, Teiko Sumiyoshi, Garret Hampton, Lukas Amler, Mark Lackner, Shih-Min A. Huang, Walter C. Darbonne. Gene expression and genomic drift comparative analysis between patient-derived conditionally reprogrammed cells and original tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4257.

Abstract 2409: Ultrasensitive detection of genomic alterations in cell-free DNA by Droplet Digital PCR

As cancer cells die, they release small pieces of DNA into the bloodstream. These molecules of DNA are called circulating tumor DNA (ctDNA). ctDNA carry somatic aberrations that are found in the originating tumor tissue and can serve as excellent biomarkers. They can be extremely valuable as tools for disease monitoring, and samples for predictive and prognostic biomarkers. However, limited quality and quantity of formalin-fixed paraffin-embedded (FFPE) tissues and circulating cell-free DNA (cfDNA) samples available from patients pose a big challenge to performing DNA quantification, mutation profiling and copy number variation analysis. We explored the feasibility of using Droplet Digital PCR (ddPCR) technology to address this challenge. Using ddPCR, we studied the ability to perform high-sensitivity mutation analysis and accurate quantification of allele frequencies, and compared this approach to alternate methods such as next-generation sequencing (NGS). For mutation profiling, we examined a total of 22 mutation detection ddPCR assays across 7 oncogenes - KRAS, PIK3CA, EGFR, BRAF, NRAS, cKIT and AKT1. The assay sensitivity for the majority of mutation detection assays was found to be approximately 0.03%. We have identified/reconfirmed various mutations using matching FFPE/plasma samples from patients in various cancer types. Moreover, we have adapted the previously published HER2 CNV ddPCR assay to detect HER2 copy number variation in cfDNA. We will present data showing validation of this platform using commercially available DNA control, archival FFPE tissue and cfDNA samples and demonstrate improved sensitivity and specificity. The platform is being routinely used in our labs to analyze samples from multiple clinical trials. These efforts enable the development of a non-invasive method to overcome existing challenges to provide molecular understanding of patient9s tumor evolution, and aid in the development of personalized therapies for cancer patients. Citation Format: Nga Wan Rachel Tam, Teiko Sumiyoshi, Rajesh Patel, Sundari Sarma, Astrid Kiermaier, Rajiv Raja. Ultrasensitive detection of genomic alterations in cell-free DNA by Droplet Digital PCR. [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 2409. doi:10.1158/1538-7445.AM2015-2409

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.