Naveen Dakappagari

Phase I Study of BIIB028, a Selective Heat Shock Protein 90 Inhibitor, in Patients with Refractory Metastatic or Locally Advanced Solid Tumors

Purpose: Heat shot protein 90 (Hsp90) is a ubiquitous molecular chaperone involved in protein folding, activation, and assembly, including key mediators of signal transduction, cell-cycle control, and transcriptional regulation. We conducted a phase I dose-finding and pharmacokinetic/pharmacodynamic study of BIIB028, a prodrug designed to inhibit Hsp90 activity. Experimental Design: Patients with advanced solid tumors were enrolled and received escalating doses of BIIB028 intravenously twice a week in 21-day cycles (3 + 3 design). Response was evaluated after two cycles. Results: Forty-one patients received doses of 6 to 192 mg/m2. The maximum tolerated dose was 144 mg/m2. Dose-limiting toxicities were syncope (n = 1) and fatigue (n = 1). Common toxicities at least possibly related to drug were grades 1 to 2, including fatigue (46%), diarrhea (44%), nausea (44%), vomiting (29%), hot flushes (29%), and abnormal dreams (17%). The concentration–time curves for day 1 and day 18 for both prodrug and active metabolite (CF2772) showed a negligible difference. There was a dose-dependent increase in plasma exposure for BIIB028 (CF3647) and CF2772 with plasma half-life of 0.5 and 2.1 hours, respectively. Pharmacodynamic analyses showed significant increases in Hsp70 in peripheral blood mononuclear cells and significantly decreased circulating human EGF receptor-2 extracellular domain in all patients who received BIIB028 at dose levels of 48 mg/m2 or more. Stable disease for at least eight cycles (24 weeks) was achieved in 5 (12%) patients (for durations of 6, 6, 8, 12.5, and 19 months). Conclusion: BIIB028 is a well-tolerated molecule that showed target impact and was associated with prolonged stable disease in two patients. Clin Cancer Res; 19(17); 4824–31. ©2013 AACR.

Deep exploration of the immune infiltrate and outcome prediction in testicular cancer by quantitative multiplexed immunohistochemistry and gene expression profiling

ABSTRACT Platinum-based chemotherapy is usually curative for patients with testicular germ cell tumors (TGCT), but a subset of patients experience disease progression and poor clinical outcomes. Here, we tested whether immune profiling of TGCT could identify novel prognostic markers and therapeutic targets for this patient cohort. We obtained primary and metastatic TGCT samples from one center. We performed immune profiling using multiplexed fluorescence immunohistochemistry (FIHC) for T-cell subsets and immune checkpoints, and targeted gene expression profiling (Nanostring nCounter Immune panel). Publically available data sets were used to validate primary sample analyses. Nearly all samples had some degree of T-cell infiltration and immune checkpoint expression. Seminomas were associated with increased CD3+ T-cell infiltration, decreased Regulatory T-cells, increased PD-L1, and increased PD-1/PD-L1 spatial interaction compared with non-seminomas using FIHC. Gene expression profiling confirmed these findings and also demonstrated increased expression of T-cell markers (e.g., IFNγ, and LAG3) and cancer/testis antigens (e.g., PRAME) in seminomas, whereas non-seminomas demonstrated high neutrophil and macrophage gene signatures. Irrespective of histology, advanced TGCT stage was associated with decreased T-cell and NK-cell signatures, while Treg, neutrophil, mast cell and macrophage signatures increased with advanced stage. Importantly, cancer/testis antigen, neutrophil, and CD8+/regulatory T-cell signatures correlated with recurrence free survival. Thus, deep immune characterization of TGCT using IHC and gene expression profiling identified activated T-cell infiltration which correlated with seminoma histology and good prognosis. These results may provide a rationale for testing of anti-PD-1/PD-L1 agents and suggest prognostic markers.

Abstract 853: Novel quantitative multiplexed PD-1/PD-L1 immunohistochemistry test provides superior prediction of treatment response in melanoma patients

While PD-1/L1 axis-targeted therapies have shown promising clinical responses, their use in combination therapies is associated with both benefits and safety concerns. Response rates for single-agent anti-PD-1 therapies are significantly higher in biomarker positive patients, therefore there is a need to utilize predictive diagnostics to enhance benefit-risk profiles and guide treatment decisions. To address this, we developed a novel quantitative multiplexed immunohistochemistry assay that provides objective quantitation of PD-L1 positive cells, but more importantly assesses interactions with immune cells (PD-1+ or CD8+) in formalin-fixed paraffin embedded (FFPE) clinical specimens. The clinical validity of this assay was verified in a small series of melanoma patients treated with anti-PD1 targeted therapies/agents. FFPE melanoma tissues from patients who received anti-PD-1 therapy were fluorescently stained with a combination of anti-PD-1, PD-L1, and S100 antibodies plus DAPI or a combination of anti-CD8, PD-L1, and S100 antibodies plus DAPI. Each slide was then imaged on Vectra platform and the frequencies of biomarker positive cells (PD-L1, PD-1, and CD8) and their interaction scores were objectively evaluated using proprietary Automated Quantitative Analysis (AQUA®) algorithms. Analytical sensitivity, precision, and accuracy were established using standardized PD-L1 and PD-1 tissue control arrays composed of cell lines and lymphoid organs, while range of biomarker expression was verified on archived melanoma clinical specimens (n = 30), including samples taken from melanoma patients prior to anti-PD-1 therapies (n = 21). Frequencies of PD-L1 positive cells could be accurately quantified within 1% to 100% range in predefined control cell line mixtures. PD-L1 and PD-1 measurements were highly reproducible (R2 = 0.98 and 0.97, respectively). A broad range of PD-L1 and PD-1 expression and interaction scores were observed in archival clinical specimens (n = 53). In a cohort of 21 advanced melanoma patients treated with nivolumab (n = 5) or pembrolizumab (n = 16), the PD-1/L1 interaction score was found to reliably distinguish responders from non-responders (p = 0.03) while PD-L1 alone (p = 0.15) or CD-8 alone (p = 0.23) did not. Additionally, patients exhibiting higher PD-1/L1 interaction scores had superior response rates (78% vs. 17%, p = 0.03). Responders experienced significantly longer median progression-free survival (177 vs. 85 days, p = 0.014), and fewer deaths (22% vs 58%) compared with patients having lower PD-1/L1 interaction scores. In terms of diagnostic utility, the PD-1/L1 multiplex test showed superior predictive power (78% Positive Predictive Value, 83% Negative Predictive Value) compared with PD-L1 expression alone. Additional studies are underway to fully establish diagnostic utility and aid in treatment guidance. Citation Format: Jennifer Bordeaux, Douglas Johnson, Jeffrey Sosman, Ju Young Kim, Christine Vaupel, Bashar Dabbas, Justin Cates, Jeff Hall, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. Novel quantitative multiplexed PD-1/PD-L1 immunohistochemistry test provides superior prediction of treatment response in melanoma patients. [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 853.

Abstract 1302: High complexity flow cytometry panels to monitor target expression, T-cell activation and suppresssion by novel immunotherapies in hematomalignancy clinical trials

The renaissance of cancer immunotherapies and the positive clinical responses observed with chimeric antigen receptor modified T cells in the hematomalignancy setting has stimulated substantial interest in monitoring immune cell activation and suppression to determine efficacy, prognosis and safety in new agent investigational trials. However, immunophenotyping capacity has been limited and required multi-test tube panels. To address the growing needs of clinical trials, we have developed three fit-for-purpose high complexity (10 or more markers) T-cell phenotyping flow cytometry panels on a qualified LSR FortessaTM platform that enables detection of up to 15 markers in a single test tube. The first panel allows identification of multiple phenotypes along the T-cell differentiation pathway, namely, T-naive (TN), T-stem cell memory (TSCM), T-effector memory (TEM) and T-central memory (TCM) and T-effector memory RA+ (TEMRA). The second panel identifies the most common helper T-cell phenotypes such as Th1, Th2, Treg, and Th17. A third panel determines the functional status of T-cells (e.g., activation vs. suppression) but also enables quantitation of important checkpoint inhibitor expression (e.g., PD-1) on T-cells of interest. These high complexity flow cytometry panels can serve as powerful tools for comprehensive examination of T-cells in a small volume of patient specimen. We believe these new flow cytometry panels will have a substantial impact on the determination of efficacy and safety correlates of immunomodulating agents administered alone or in combination to patients with leukemia. Citation Format: Ghanashyam Sarikonda, Devika Ashok, Anil Pahuja, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. High complexity flow cytometry panels to monitor target expression, T-cell activation and suppresssion by novel immunotherapies in hematomalignancy clinical trials. [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 1302. doi:10.1158/1538-7445.AM2015-1302

Abstract 2840: Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA With the advent of multiple HER-2 targeting agents, the need for a diagnostic test that accurately predicts the levels of HER2 in newly diagnosed breast cancer patients has never been greater. The goal of this study was to develop an immunofluorescence based assay to objectively and reproducibly quantify HER-2 protein level in Formalin-Fixed Paraffin-Embedded (FFPE) specimens using AQUA Technology and to provide a binary readout, thus eliminating reflex testing and enabling quick treatment decisions. Methods: Three well known HER-2 antibody clones, A0485, CB11, and SP3 were evaluated across a three-log dilution series under four different antigen retrieval conditions on a tissue microarray (TMA) containing 80 breast cancer cases with known central HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) amplification status. The top six immunofluorescence assay combinations out of 108 conditions were quantified by AQUA scoring based on the their ability to accurately separate negative and positive cases using receiver operating characteristic (ROC) analysis. These top six assay conditions were then assessed on an independent TMA containing 94 breast cancer cases and their AQUA scores were compared to HER2 protein levels determined by reverse phase protein microarray (RPMA®), HER2 mRNA levels assessed by RNAscope® as well as to the current “gold standard” IHC and FISH amplification. Results: Top three conditions clearly segregating HER2 positive and HER2 negative breast cancer cases were selected and analyzed on an independent TMA. Conditions associated with both SP3 and A0485 antibodies demonstrated high correlations with both HER2 protein levels determined by RPMA analysis and HER2 mRNA levels assessed by RNAscope (spearmans rho >0.85). Conclusion: Assessment of HER2 status using AQUA Technology was confirmed using two expression platforms, including both protein (RPMA) and RNA (RNAscope) supporting the utilization of the AQUA methodology for diagnostic test development. Citation Format: Jennifer Bordeaux, Krupa Chandrasekaran, Sue Beruti, Mike Nerenberg, Corinne Ramos, David Rimm, Jelveh Lameh, Naveen Dakappagari. Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2840. doi:10.1158/1538-7445.AM2014-2840

Abstract 2843: Development of a binary diagnostic immunofluorescence assay by AQUA® technology for accurate detection of HER-2 levels in breast cancer specimens

HER2 is a prognostic and predictive marker for breast cancer patients and its expression is routinely evaluated by immunochemistry (IHC). Scoring of IHC slides is prone to operator subjectivity and equivocal results make selection of appropriate therapy difficult, ultimately affecting patient outcomes. We describe development and validation of a reproducible quantitative immunofluorescence assay to accurately assess HER-2 levels in Formalin-Fixed Paraffin-Embedded (FFPE) breast cancer specimens by AQUA Technology that avoids the ambiguity of equivocal results and enables critical treatment decisions. Methods: A tissue microarray (n=80) composed of breast cancer cases with known IHC and fluorescence in situ hybridization (FISH) status was used to characterize and optimize assay performance for three HER-2 antibody clones; A0485, CB11, and SP3. Eight dilutions of each antibody were tested with four different antigen retrieval conditions. A total of 108 assay conditions were evaluated by receiver operator characteristic (ROC) analysis for sensitivity and specificity. Equal weight was given to sensitivity and specificity to select the most robust assay. The top six assay conditions were then assessed using 45 whole tissue breast cancer specimens to identify one condition with highest sensitivity and specificity. The selected assay condition was evaluated on an additional 90 breast cancer specimens (training set) annotated for IHC and FISH scores to determine the binary cut point for the HER-2 AQUA assay®, the cut point was then confirmed on a validation set composed of 90 independent breast cancer specimens. The final assay was analytically validated in accordance with College of American Pathologists (CAP) guidelines utilizing over 120 independent breast cancer specimens. Results: Based on an evaluation of over 400 breast cancer specimens with nearly equal distribution of 0, 1+, 2+ and 3+ cases, HER-2 antibody clone, SP3, which recognizes the extracellular domain of the receptor, clearly segregated HER2 positive specimens from HER2 negative breast cancer cases. Conclusion: The availability of a highly reproducible quantitative binary test facilitates rapid treatment decisions with appropriate HER-2 targeting biologics on the market. Citation Format: Lakshmi Krupa Chandrasekaran, Jennifer Bordeaux, Sue Beruti, Naveen Dakappagari, Mike Nerenberg, Jelveh Lameh, Armin Graber, David Rimm, Bruce Robbins, Nagesh Rao. Development of a binary diagnostic immunofluorescence assay by AQUA® technology for accurate detection of HER-2 levels in breast cancer specimens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2843. doi:10.1158/1538-7445.AM2014-2843