Chumsri, Saranya

Abstract Background: Previous failed trials of anti-EGFR therapy (TBCRC001) in breast cancer have not directly assessed EGFR amplification instead relying on pathway activation surrogates. Previous studies have examined EGFR amplification primarily retrospectively for research purposes without being part of a standard diagnostic evaluation. We evaluated 5,606 clincally advanced breast cases for which comprehensive genomic profiling was performed in the course of clinical care to identify EGFR altered mBC cases. Methods: DNA was extracted from 40 microns of FFPE sections from 5,605 mBC. Comprehensive genomic profiling (CGP) was performed using a hybrid-capture, adaptor ligation based next generation sequencing assay of up to 236 or 315 genes to a mean coverage depth of >600X. The results were analyzed for base substitutions, short insertions and deletions, selected rearrangements, and copy number changes. Results: 155 (2.7%) of 5,605 mBC featured EGFR alterations. 126 (10.6%) featured EGFR amplification, and 27 (2.2%) featured ERBB2 subs/indels, with no cases harboring both amplification and another GA of EGFR. These patient (pts) had a median age of 55 years (range 30 to 78). For EGFR altered cases, specimens utilized for CGP included 59 (38%) from the patient's primary BC and 93 (60%) from metastatic sites including liver (14%), lymph node (14%), lung (8%), soft tissue (6%), skin (5%) and with information not available for 3 cases. 75 (48%) mBC were submitted as IDC, 69 (45%) as breast carcinoma NOS, 5 (3 %) as metaplastic BC, and 7 (5%) as other mBC. For the 126 EGFR amplified breast patients, quantitative estimation of EGFR copy number ranged from 6 to 380 copies, with a median of 12 copies. A patient with heavily pretreated case and whose TNBC harbored 32 copies of EGFR was begun on erlotinib and has experienced a 20 months ongoing complete response to erlotinib, and will be presented as will other outcomes and biomarker characterization of these cases. Conclusions: Given anecdotal evidence that mBC is responsive to anti-EGFR targeted therapies, prediction of benefit from such therapies may be linked to sensitive and specific detection of EGFR alteration in mBC cases. This study demonstrates a 2.7% EGFR alteration rate of mBC, with a striking median copy number of 12 for the subset of EGFR amplified cases. Given these findings along with anecdotal report of patient benefit, utilization of CGP in the course of clinical care or for clinical trials for mBC may help optimize treatement with targeted therapies for these patients including the direction of anti-EGFR monotherapy to EGFR altered patients. Citation Format: Ali SM, Wang K, Johnson A, Rodriguez AA, Elvin JA, Vergilio J-A, Suh JH, Chumsri S, Morosini D, Yelensky R, Lipson D, Chmielecki J, Miller VA, Ross JS, Chang J, Stephens PJ. EGFR genomic alterations in 5,605 cases of refractory and metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-02.

Abstract Background: Entinostat, a class I HDAC inhibitor (HDACi), has shown promising activity in ENCORE 301, a randomized, placebo-controlled, phase II trial of entinostat + exemestane (EE) vs. exemestane + placebo (EP) in advanced hormone receptor-positive breast cancer progressed on nonsteroidal aromatase inhibitors. ENCORE 301 met the primary progression free survival endpoint and showed a median 8.3-month improvement in the overall survival (OS) exploratory endpoint for the EE arm. Emerging preclinical work suggests that entinostat has immunomodulatory effects and can eradicate modestly immunogenic mouse tumors in combination with immune checkpoint blockade agents via reduction of circulating myeloid-derived suppressor cells (MDSC). Based on these data, we conducted an analysis of immune subsets in blood samples from ENCORE 301 breast cancer patients. Method: Blood was collected from a subset of 49 patients (27 EE and 22 EP) representative of the 130 patients enrolled in ENCORE 301 on cycle 1 day 1 (C1D1; pre-treatment), C1D2, C1D8, and C1D15 for biomarker analysis. Of these, 34 patient samples (20 EE and 14 EP) were analyzed for circulating immune subsets. The percent change in subsets at C1D15 vs. baseline was assessed based on the following surface markers: Lin-MDSC (lin; CD3, CD19, CD56)-HLA-DR-CD11b+CD33+), granulocytic MDSC (CD14-CD11b+CD33+), monocytic MDSC (Lin-HLA-DR-CD11b+CD33+CD14+), immature MDSC (Lin-HLA-DR-CD11b+CD33+CD14-), CD8+ T-cells (CD4-CD8+), Foxp3-CD4+ T-cells (CD8-CD4+Foxp3-), and Tregs (CD4+CD8-CD25hiFoxp3+). Monocytes were analyzed for three populations: CD14+, CD14+HLA-DRhi, and CD14+HLA-DRlow/negative. In addition, PD-1, CTLA-4, and TIM-3 were measured on T-cell subsets, and CD40 was measured on MDSCs. Results: In line with preclinical data, we observed a significant reduction in granulocytic MDSC (-14.67% vs. +20.56%, p 0.029) and monocytic MDSC (-62.3% vs. +1.97%, p 0.002) in EE. Entinostat did not alter immature MDSC levels (-20.9% vs. -15.0%, p 0.93) suggesting a downstream effect of entinostat on MDSC subsets. Interestingly, CD40, a costimulatory receptor required for MDSC-mediated immune suppression was significantly down-regulated in all MDSC subsets except granulocytic MDSC where a downward trend was observed. Entinostat did not significantly impact the ratio of CD8+ T-cells per CD4+ T-cells or per Tregs or alter expression of CTLA4, PD-1, or TIM3 on T-cell subsets. Reduced expression of HLA-DR on monocytes has been associated with poor prognosis in cancer. Consistent with entinostat-mediated immunomodulatory effects, a significant increase in the number of HLA-DR+ monocytes (34.1% vs. -11.38%, p 0.0004) and level of HLA-DR expression on monocytes (16.3% vs. -4.7%; p 0.015) was observed. Conclusion: Data with entinostat combined with exemestane in ENCORE 301 provide the first evidence of HDACi-mediated reduction of immunosuppressive MDSCs and increased immunocompetent CD14+HLA-DRhi monocytes in patients. These findings may explain the improved OS seen with EE in ENCORE 301 and provide strong rationale for planned combination studies of entinostat with immune checkpoint blockade agents. Citation Format: Chumsri S, Lee M-J, Tomita Y, Lee S, Tomita S, Cruickshank S, Ordentlich P, Trepel JB. Epigenetic immune modulation by entinostat in breast cancer: Correlative analysis of ENCORE 301 trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-11-10.

Abstract Background: Targeting immune checkpoint programmed death receptor 1 (PD-1)/PD-L1 pathway has shown promising clinical activity with some preliminary association of clinical benefit with PD-L1 expression on tumors. Recent preclinical and clinical studies highlight the beneficial immunomodulatory potential of epigenetic therapy. Entinostat is a class I specific histone deacetylase inhibitor (HDACi). A promising preclinical study showed that entinostat in combination with immune checkpoint blockade agent can eradicate modestly immunogenic breast tumors in mice via reduction in immunosuppressive myeloid-derived suppressor cells. In this study, we investigated the effects of entinostat on expression of immune-related genes in breast cancer cells to further explore the potential mechanism of its combined activity. Method: Gene expression was assessed on Nanostring platform using the nCounter GX Human ImmunologyV2 panel comprised of 594 immune-related and 15 reference genes. Gene expression was normalized to the internal positive controls and reference genes using nSolver2.0 software. Hormone receptor-positive (HR+) breast cancer (MCF-7 and T47D) and triple negative breast cancer (TNBC) cell lines (MDA-MB-231 and Hs578T) were used for the analysis. Gene expression analysis was performed on control and after 24-hour treatment of entinostat at clinically relevant 125 and 500 nM concentrations. Results: Overall, a greater number of immune-related genes were induced > 2 fold with entinostat at 125 and 500 nM in TNBC compared to HR+: 77 and 118 genes in MDA-MB-231, 80 and 147 genes in Hs578T, 20 and 64 genes in MCF-7, and 73 and 72 genes in T47D, respectively. In particular, MHC class I (HLA-A, HLA-B, HLA-C) and II (HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA, and HLA-DRB1) genes were induced by entinostat in a dose dependent manner (range 1.5-22.44 fold). These inductions were observed in both HR+ and TNBC cell lines. Interestingly, we found higher baseline expression and a several fold increase in PD-L1 expression in TNBC. PD-L1 mRNA expression increased by 1.74 and 2.14 fold in MDA-MB-231 and 3 and 9.6 fold in Hs578T with 125 and 500 nM treatment, respectively. Corresponding increase in PD-L1 protein expression after entinostat treatment was also observed. In contrast, there appeared to be no significant changes in PD-L1 expression after entinostat treatment in MCF-7 and T47D. Furthermore, we also identified 21 genes that were differentially induced by entinostat in TNBC but not in HR+. These genes include PTPN22, ARG2, CISH, IL17A, ICAM2, KIR3DL1, CXCR3, TLR2, CFD, CCR5, IL13, LILRA3, IL8, TNFRSF9, DPP4, MR1, SELPLG, PTGS2, IL1B, CD3D, and MBL2. No significant change in PDL2 expression was observed in any of the cell lines. Conclusion: Our data suggest that entinostat induces immune-related genes involved in antigen presentation in both ER+ and TNBC cells, potentially increasing the immunogenicity of these tumors. Given the significant induction of PD-L1 expression with entinostat in TNBC, our preclinical data provides support for further investigation of entinostat in combination with anti-PD1 or anti-PD-L1 in this subtype of breast cancer. Citation Format: Chumsri S, Necela BM, Ordentlich P, Advani P, Moreno-Aspitia A, McLaughlin SA, Geiger X, McDonough M, Vallow LA, Perez EA, Thompson EA. Immunomodulatory effects of entinostat on PD-L1 and MHC class I and II in different subtypes of breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-04-02.

Abstract Background: Entinostat, a class I HDAC inhibitor (HDACi), has shown promising activity in ENCORE 301, a randomized, placebo-controlled, phase II trial of entinostat + exemestane (EE) vs. exemestane + placebo (EP) in advanced hormone receptor-positive breast cancer progressed on nonsteroidal aromatase inhibitors. ENCORE 301 met the primary progression free survival endpoint and showed a median 8.3-month improvement in the overall survival (OS) exploratory endpoint for the EE arm. Emerging preclinical work suggests that entinostat has immunomodulatory effects and can eradicate modestly immunogenic mouse tumors in combination with immune checkpoint blockade agents via reduction of circulating myeloid-derived suppressor cells (MDSC). Based on these data, we conducted an analysis of immune subsets in blood samples from ENCORE 301 breast cancer patients. Method: Blood was collected from a subset of 49 patients (27 EE and 22 EP) representative of the 130 patients enrolled in ENCORE 301 on cycle 1 day 1 (C1D1; pre-treatment), C1D2, C1D8, and C1D15 for biomarker analysis. Of these, 34 patient samples (20 EE and 14 EP) were analyzed for circulating immune subsets. The percent change in subsets at C1D15 vs. baseline was assessed based on the following surface markers: Lin-MDSC (lin; CD3, CD19, CD56)-HLA-DR-CD11b+CD33+), granulocytic MDSC (CD14-CD11b+CD33+), monocytic MDSC (Lin-HLA-DR-CD11b+CD33+CD14+), immature MDSC (Lin-HLA-DR-CD11b+CD33+CD14-), CD8+ T-cells (CD4-CD8+), Foxp3-CD4+ T-cells (CD8-CD4+Foxp3-), and Tregs (CD4+CD8-CD25hiFoxp3+). Monocytes were analyzed for three populations: CD14+, CD14+HLA-DRhi, and CD14+HLA-DRlow/negative. In addition, PD-1, CTLA-4, and TIM-3 were measured on T-cell subsets, and CD40 was measured on MDSCs. Results: In line with preclinical data, we observed a significant reduction in granulocytic MDSC (-14.67% vs. +20.56%, p 0.029) and monocytic MDSC (-62.3% vs. +1.97%, p 0.002) in EE. Entinostat did not alter immature MDSC levels (-20.9% vs. -15.0%, p 0.93) suggesting a downstream effect of entinostat on MDSC subsets. Interestingly, CD40, a costimulatory receptor required for MDSC-mediated immune suppression was significantly down-regulated in all MDSC subsets except granulocytic MDSC where a downward trend was observed. Entinostat did not significantly impact the ratio of CD8+ T-cells per CD4+ T-cells or per Tregs or alter expression of CTLA4, PD-1, or TIM3 on T-cell subsets. Reduced expression of HLA-DR on monocytes has been associated with poor prognosis in cancer. Consistent with entinostat-mediated immunomodulatory effects, a significant increase in the number of HLA-DR+ monocytes (34.1% vs. -11.38%, p 0.0004) and level of HLA-DR expression on monocytes (16.3% vs. -4.7%; p 0.015) was observed. Conclusion: Data with entinostat combined with exemestane in ENCORE 301 provide the first evidence of HDACi-mediated reduction of immunosuppressive MDSCs and increased immunocompetent CD14+HLA-DRhi monocytes in patients. These findings may explain the improved OS seen with EE in ENCORE 301 and provide strong rationale for planned combination studies of entinostat with immune checkpoint blockade agents. Citation Format: Peter Ordentlich, Min-Jung Lee, Yusuke Tomita, Sunmin Lee, Saori Tomita, Scott Cruickshank, Saranya Chumsri, Jane B. Trepel. Epigenetic immune modulation by entinostat in breast cancer: Correlative analysis of ENCORE 301 trial. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A030.

Abstract Background: Blood-based testing can be used as a noninvasive method to recover and analyze circulating tumor-derived cells for clinical use. Circulating cancer-associated macrophage-like cells (CAML) are specialized myeloid cells found in peripheral blood and associated with the presence of solid malignancies. We measured CAMLs prospectively in peripheral blood to ascertain their prevalence, specificity, and sensitivity in relation to breast disease status at clinical presentation. Methods: We report on two related but separate studies: 1) CellSieve microfilters were used to isolate CAMLs from blood samples of patients with known malignant disease (n = 41). Prevalence and specificity was compared against healthy volunteers (n = 16). 2) A follow-up double-blind pilot study was conducted on women (n = 41) undergoing core-needle biopsy to diagnose suspicious breast masses. Results: CAMLs were found in 93% of known malignant patients (n = 38/41), averaging 19.4 cells per sample, but none in the healthy controls. In subjects undergoing core biopsy for initial diagnosis, CAMLs were found in 88% of subjects with invasive carcinoma (n = 15/17) and 26% with benign breast conditions (n = 5/19). Conclusion: These preliminary pilot studies suggest that the presence of CAMLs may differentiate patients with malignant disease, benign breast conditions, and healthy individuals. Impact: We supply evidence that this previously unidentified circulating stromal cell may have utility as a screening tool to detect breast cancer in various malignancies, irrespective of disease stage. Cancer Epidemiol Biomarkers Prev; 25(7); 1037–42. ©2016 AACR.

An all-in-one Circulating Tumor Cell (CTC) isolation platform with streamlined multiplex integration of proteomic and genomic clinical testing.

Abstract Background: Blood-based biopsies can be used as a non-invasive method to recover both Circulating Tumor Cells (CTCs) and Circulating Cancer Associated Macrophage-Like cells (CAMLs) from the blood of cancer patients. CAMLs are a newly-defined circulating immune cell type, described as a subtype of circulating stromal cells, known to engulf tumor cells/debris. CAMLs are identified by their large size, 30-300 microns, and aberrant nuclear structure. We studied the peripheral blood of cancer patients to ascertain the prevalence, specificity and sensitivity of CAMLs in relation to their disease status at presentation, including a variety of benign and malignant diseases. We supply evidence that this previously unidentified circulating cell can be used as a screening tool to detect solid tumors in numerous malignancy subtypes in all disease stages. Methods: CellSieveTM microfilters were used to isolate CAMLs from 7.5 mL of whole peripheral blood. The pore size of CellSieveTM is 7 microns, capable of isolating both CTCs and CAMLs based on size. Collected cells were fixed, permeabilized, and stained with DAPI and antibodies against cytokeratin 8, 18 and 19, CD14, and CD45. CAMLs were defined as enlarged, multinuclear cells with diffuse cytoplasmic cytokeratin staining, and/or CD14+, and/or CD45+. CTCs were defined as filamentous cytokeratin cells that are CD45-. Study 1: Blinded peripheral blood samples from 61 cancer patients were tested, including Stage I-IV breast, pancreatic, lung and prostate cancers, and newly diagnosed, untreated patients. Non-blinded blood samples from 30 healthy volunteers served as controls. Study 2: Double blinded peripheral blood samples from 20 patients with abnormal mammogram results were drawn at time of tissue biopsy and screened. After analysis, unblinding showed a distribution of malignant breast disease (n = 10), benign (n = 8), or atypical hyperplasia (AH) (n = 2). Results: Study 1: CAMLs were found in 94% patients with Stage III/IV cancers, 87% with Stage I/II cancers, and 89% of all patients, regardless of cancer type. Specifically, CAMLs were found in 77% of prostate, 100% of pancreatic, 80% of lung, and 100% of breast patient samples. CAMLs were not found in healthy control samples (sensitivity: 84-96%, Specificity: 100%, PPV: 100%, NPV: 88-97%). Study 2: CAMLs were found in 8 patients with malignant disease, 2 with benign disease, and 2 with AH (sensitivity: 80%, Specificity: 75%, PPV: 80%, NPV: 75%) Conclusions: We present evidence that CAMLs can be used as a non-invasive blood based biopsy, to detect the anatomical presence of solid malignancies and potentially pre-malignant lesions. The presence of CAMLs in newly diagnosed patient samples suggests their use as a blood biomarker for early stage cancer screening in a broad population. Preliminary data from a double blind breast cancer study suggests that CAMLs may differentiate between benign and malignant breast diseases. Citation Format: Daniel Adams, Katherine Alpaugh, Massimo Cristofanilli, Stuart Martin, Saranya Chumsri, Raymond C. Bergen, Susan Tsai, Martin Edelman, Peixuan Zhu, Shuhong Li, Olga V. Makarova, Platte T. Amstutz, Cha-Mei Tang, Jeffrey R. Marks. Cancer associated macrophage-like cells as a blood-based biomarker for the screening of solid tumors. [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 5165. doi:10.1158/1538-7445.AM2015-5165

AbstractRecent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch® CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45−/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45− cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45− cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45−/DAPI+ cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using non‐EpCAM dependent isolation. © 2014 The Authors. Published by Wiley Periodicals, Inc.