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Purpose: Increasing tumor-infiltrating lymphocytes (TIL) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. Experimental Design: We obtained pretreatment core-needle biopsies from 105 patients with stage I–III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from Oct 22, 2015 through July 24, 2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, programmed death-ligand 1 (PD-L1) IHC, multiplex immunofluorescence, and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathologic response to NAT. Results: The pCR rate was 40% (42/105). Higher TCR clonality (median = 0.2 vs. 0.1, P = 0.03), PD-L1 positivity (OR: 2.91, P = 0.020), higher CD3+:CD68+ ratio (median = 14.70 vs. 8.20, P = 0.0128), and closer spatial proximity of T cells to tumor cells (median = 19.26 vs. 21.94 μm, P = 0.0169) were associated with pCR. In a multivariable model, closer spatial proximity of T cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage. Conclusions: In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

BACKGROUND: Current treatment for early stage triple negative breast cancer (TNBC) includes neoadjuvant systemic chemotherapy (NAST), which is used to assess disease biology and the need for adjuvant treatment in case of residual disease at the time of surgery, also known as residual cancer burden (RCB). Patients with TNBC who experience RCB-0 (pathologic complete response [pCR]) or RCB-I after NAST have an excellent prognosis whereas patients with significant residual disease (RCB-II or RCB-III) are at a high risk of relapse. Standard NAST for TNBC achieves pCR in 30-50% of cases. NAST typically consists of anthracycline-based chemotherapy followed or preceded by a taxane +/- carboplatin. Disease progression (PD) is uncommon in TNBC patients receiving NAST and little is known regarding outcomes in patients who have PD during the initial phase of NAST. METHODS: Total 316 TNBC patients were evaluated from two prospectively accrued clinical trials of NAST (NCT02276443 and NCT01334021). The ARTEMIS trial (NCT02276443) aims to improve pCR rates by adding targeted therapy to chemotherapy as the second phase of NAST for those patients who do not experience at least a 70% volumetric reduction after 4 cycles of doxorubicin/cyclophosphamide (AC). Unique histopathologic features including % stromal tumor-infiltrating lymphocytes (sTIL), presence of mesenchymal histology (high vimentin expression by IHC), and androgen receptor expression are used to guide second phase therapy. RESULTS: 31 TNBC patients had PD while receiving AC as the first phase of NAST (10%; 95% CI= 6.69-13.31%). 9 of 31 patients proceeded to standard chemotherapy and all had RCB II/III disease. 22 of 31 patients were enrolled to targeted therapy trials. 6 were treated with the EGFR inhibitor panitumumab + carboplatin/paclitaxel, 9 with atezolizumab + nab-paclitaxel, and 7 with everolimus, bevacizumab, and liposomal doxorubicin (DAE). Of these 22 patients, 3 (13.6%) had pCR/RCB-0, 1 (4.5%) RCB-I and 18 (81.8%) had RCB II/III. All 4 patients who experienced RCB-0/I had T2N0 disease at diagnosis. 2 had sTIL < 5% and 2 patients had 70% sTIL. CONCLUSION: PD is uncommon while receiving NAST. Patients with TNBC and progression on initial NAST with AC are unlikely to achieve pCR or RCB-I status despite subsequent standard chemotherapy. Combination chemotherapy with targeted therapy on clinical trial resulted in a numerically higher rate of pCR+RCB-I (18%) as salvage therapy, but this was not statistically significant and requires confirmation in larger trials. Citation Format: Haven Garber, Gaiane Rauch, Beatriz Adrada, Rosalind Candelaria, Elizabeth Mittendorf, Alastair Thompson, Jennifer Litton, Senthil Damodaran, Bora Lim, Banu Arun, Naoto Ueno, Vicente Valero, Nuhad Ibrahim, Rashmi Murthy, Debu Tripathy, Helen Piwnica-Worms, Fraser Symmans, Lei Huo, Stacy Moulder. Residual cancer burden in patients with early stage triple negative breast cancer who progress on anthracycline-based neoadjuvant chemotherapy in an ongoing clinical trial (ARTEMIS) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-16-09.

Cathepsin G is broadly expressed in acute myeloid leukemia and is an effective immunotherapeutic target

Background aims PR1 is an HLA-A2 restricted leukemia-associated antigen derived from neutrophil elastase and proteinase 3, both of which are normally stored in the azurophil granules of myeloid cells but overexpressed in myeloid leukemic cells. PR1-specific cytotoxic lymphocytes (PR1-CTLs) have activity against primary myeloid leukemia in vitro and in vivo and thus could have great potential in the setting of adoptive cellular therapy (ACT). Adult peripheral blood–derived PR1-CTLs are infrequent but preferentially lyse myeloid leukemia cells. We sought to examine PR1-CTLs in umbilical cord blood (UCB) because UCB units provide a rapidly available cell source and a lower risk of graft-versus-host disease, even in the setting of mismatched human leukocyte antigen (HLA) loci. Methods We first determined the frequency of PR1-CTLs in HLA-A2 + UCB units and then successfully expanded them ex vivo using repeated stimulation with PR1 peptide-pulsed antigen-presenting cells (APCs). After expansion, we assessed the PR1-CTL phenotype (naive, effector, memory) and function against PR1-expressing target cells. Results PR1-CTLs are detected at an average frequency of 0.14% within the CD8 + population of fresh UCB units, which is 45 times higher than in healthy adult peripheral blood. UCB PR1-CTLs are phenotypically naive, consistent with the UCB CD8 + population as a whole. In addition, the cells can be expanded by stimulation with PR1 peptide-pulsed APCs. Expansion results in an increased frequency of PR1-CTLs, up to 4.56%, with an average 20-fold increase in total number. After expansion, UCB PR1-CTLs express markers consistent with effector memory T cells. Expanded UCB PR1-CTLs are functional in vitro as they are able to produce cytokines and lyse PR1-expressing leukemia cell lines. Conclusions This study is the first report to show that T cells specific for a leukemia-associated antigen are found at a significantly higher frequency in UCB than adult blood. Our results also demonstrate specific cytotoxicity of expanded UCB-derived PR1-CTLs against PR1-expressing targets. Together, our data suggest that UCB PR1-CTLs could be useful to prevent or treat leukemia relapse in myeloid leukemia patients.

Chemotherapy/ targeted therapy are both known to trigger evolution of treatment resistant clones that can lead to relapse. Allogeneic stem cell transplant (alloSCT) for refractory Chronic Lymphocytic Leukemia (CLL) patients is associated with better outcomes. We hypothesized that allogeneic T-cell immunotherapies, including alloSCT and donor lymphocyte infusion (DLI) would impact tumor evolution through the application of selective immunologic pressure with reciprocal changes in the T-cell compartment. Here, we tested a cohort of 24 heavily pre-treated CLL patients treated. Treatments consisted of alloSCT alone, or with follow-up DLI, which are two established mediators of effective Graft versus Leukemia (GVL). Our cohort included 11 patients who relapsed (denoted as non-responder, NR) after alloSCT and 13 patients who had complete response (CR) after alloSCT, with 11/13 patients showing durable CR with a median post-transplant overall survival (OS) of 9.8 years. We mapped the evolutionary trajectories of tumor cells by whole exome sequencing (WES) of sort purified CLL in post-transplant relapsed patients. To investigate changes in immune repertoire and gene expression post-transplant, CD3 positive T-cells from peripheral blood and bone marrows of CLL patients at complete donor chimerism were analyzed both at bulk and at the single cell level. We found evidence of subclonal leukemic evolution in the majority of our CLL patient cohort after nonmyeloablative HLA-matched alloSCT. Different patterns of CLL evolution were observed, and these changes included putative CLL drivers in every case. In all of the 11 patients with longitudinal post-alloSCT samples available, we observed branched CLL evolution in 4 patients, linear evolution in 4 patients, and no evolution in 3 patients. These data suggest that differential sensitivity of leukemic subclones to allogeneic T cell killing may underlie the branched and linear evolution that we observed, and therefore can shape leukemic subclonal architecture after transplant. Of note, we found that clonal CLL was more responsive to alloSCT in comparison to CLL with subclonal disease architecture.To identify T-cells with GVL potential, we first cataloged potential neoantigens by screening mutated regions in CLL with in silico HLA binding prediction models. Neoantigen specific T-cells were then sorted from longitudinal peripheral blood samples using tetramers, followed by identification of GVL specific TCR in both bulk and single cell setting. We were able to identify T-cells that coevolved with specific tumorigenic lesions in a subset of CLL patients. Taken together, our results suggest that donor-derived antigen-specific T-cells mediate clonal selection of CLL with concurrent changes in allogeneic T-cells, and that these changes can be monitored in longitudinal patient samples. Citation Format: Celine Kerros, John P. Miller, Xizeng Mao, Haven R. Garber, Hannah C. Beird, Jianhua Zhang, Jason Roszik, Paul Leonard, Li Zhao, Sahil Seth, Pei Lin, Huandong Sun, William G. Wierda, Issa F. Khouri, Karen Clise-Dwyer, Andrew Futreal, Shoudan Liang, Koppikar Priya, Jeffrey Molldrem. Deep profiling of T-cell repertoire and tumor heterogeneity in chronic lymphocytic leukemia patients following allogeneic T-cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1516.

1096 Background: Previous reports suggest the incidence of brain metastasis is higher in patients with hereditary BRCA1 mutations compared to BRCA1 noncarriers among breast cancer patients who develop recurrent disease. PARP inhibitors are now standard therapies for metastatic breast cancer patients with germline BRCA1 or BRCA2 mutations ( gBRCA1/2) based on their efficacy in treating systemic disease. However, as management of systemic disease improves, a concern is that patients with hereditary BRCA mutations may experience higher rates of disease progression in the CNS. We aimed to estimate the incidence of brain metastasis in breast cancer patients with gBRCA1/2 using a prospectively maintained gBRCA database and to assess the impact of brain metastasis on survival. Methods: To determine incidence, we queried a prospectively maintained electronic database that included patients referred to the MDACC genetics department and who underwent gBRCA1/2 testing. We identified patients with stage I-III invasive breast cancer who were treated between 2000-2017 and assessed for disease recurrence and brain metastasis. To expand our cohort for descriptive characteristics (separate from the incidence analysis), we queried the Breast Medical Oncology database for patients with brain metastasis who had undergone BRCA1/2 testing outside the genetics department or at outside institutions. Results: Of 474 patients with Stage I-III breast cancer and gBRCA1, 77 (16.2%) developed distant metastasis (median f/u: 9.1 years). Of these patients, 34/77 (44.2%) developed brain metastasis. In comparison, 42 of 318 (13.2%) of gBRCA2 patients with Stage I-III breast cancer developed distant recurrence (median f/u: 8.4 years), and 7/42 (16.7%) experienced brain metastasis. In gBRCA1 patients with brain metastasis, 45/48 (83.8%) had triple negative disease, and the median time from diagnosis to brain metastasis was 2.45 years. The brain was among the initial sites of disease recurrence in 24/48 (50%) of gBRCA1 patients. For gBRCA1 patients with distantly recurrent disease, median OS from diagnosis was 3.19 years for patients with brain metastasis vs. 5.37 years for patients without brain mets (HR 0.54; 95% CI 0.34 to 0.85; P = 0.0082). Conclusions: Brain metastasis is frequent among breast cancer patients with recurrent disease and hereditary BRCA1 mutations. Development and testing of agents with intracranial activity is critical for improving long-term outcomes in gBRCA1 patients with metastatic breast cancer.

509 Background: In TNBC patients (pts) receiving NAST, increasing tumor infiltrating lymphocytes (TILs) is associated with higher pathologic complete response (pCR) rates. However, since the presence of TIL do not consistently predict pCR, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. Methods: T cell receptor (TCR) sequencing, PD-L1 immunohistochemistry and multiplex immunofluorescence were performed on prospectively collected pre-NAST tumor samples from 98 pts with stage I-III TNBC enrolled in ARTEMIS (NCT: 02276443). TCR clonality was calculated using Shannon’s entropy. PD-L1+ was defined as ≥1% immune cell staining. Response to NAST was defined using the residual cancer burden (RCB) index. Associations between TCR clonality, immune phenotype, and response were examined with the Wilcoxon rank sum test, Spearman’s rank correlation and multivariable logistic regression using stepwise elimination (threshold p > 0.2), as appropriate. Results: The pCR rate was 39% (38/98). pCR was associated with higher TCR clonality (median = 0.2 [in pts with pCR] vs 0.1 [in pts with residual disease], p = 0.05). Notably, the association between pCR and higher TCR clonality was observed in pts with ≥5% TIL (n = 61; p = 0.05) but not in pts with < 5% TIL (n = 37; p = 0.87). Among pts with ≥5% TIL, TCR clonality emerged as the only independent predictor of response in a multivariable model of tumor immune characteristics (odds ratio/0.1 increase in TCR clonality: 3.0, p = 0.021). PD-L1+ status was associated with higher TCR clonality (median = 0.2 [in PD-L1+] vs 0.1 [in PD-L1-], p = 0.004). Higher TCR clonality was associated with higher CD3+ (rho = 0.32, p = 0.0018) and CD3+CD8+ (rho = 0.33, p = 0.0013) infiltration but lower expression of PD-1 on CD3+ (rho = -0.24, p = 0.021) and CD3+CD8+ cells (rho = -0.21, p = 0.037). Conclusions: In TNBC, a more clonal T cell population is associated with an immunologically active microenvironment (higher CD3+ and CD3/8+ T cell; lower PD-1+CD3+ and PD-1+CD3/8+ T cell; PD-L1+) and favorable response to NAST, especially in pts with ≥5% TIL, suggesting a role for deep immune phenotyping in further refining the predictive value of TILs.

It is now well established that the host’s adaptive immune system plays an important role in identifying and eliminating cancer cells in much the same way that intracellular pathogens are cleared during an adaptive immune response to infection. From a therapeutic standpoint, the adaptive immune system is unique in that it can co-evolve alongside a developing tumor. However, tumor acquisition of immune evasive phenotypes, such as class-I MHC downregulation, remains a major limitation of successful T-cell immunotherapy. We propose a stochastic model that couples tumor and CD8+ T-cell adaptive immune compartments. This framework accurately models relevant empirical findings, including the growth-threshold conjecture of immune activation, and predicts experimental observations including “sneak-through,” wherein intermediate-growth threats are penalized relative to their slower- and faster-growing counterparts. We demonstrate that predicted optimal growth strategies depend on whether or not the threat may acquire an immune-evasive phenotype as well as the mode of immune detection. The model is able to accurately characterize age-dependent cancer incidence data as a function of decreasing T-cell turnover and repertoire diversity. Lastly, we quantify therapeutic efficacy of immunotherapy in the setting of an immune-evasive threat. Our model serves as a first attempt at modeling stochastic cancer evolution sculpted by an adaptive immune compartment. Citation Format: Jason T. George, Haven R. Garber, Jeffrey J. Molldrem, Herbert Levine. Co-evolution between tumor cells and immune system in the setting of T-cell immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B59.

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies. Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples. Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivo. Conclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386–96. ©2018 AACR.

Appreciation for genomic and immune heterogeneity in cancer has grown though the relationship of these factors to treatment response has not been thoroughly elucidated. To better understand this, we studied a large cohort of melanoma patients treated with targeted therapy or immune checkpoint blockade (n = 60). Heterogeneity in therapeutic responses via radiologic assessment was observed in the majority of patients. Synchronous melanoma metastases were analyzed via deep genomic and immune profiling, and revealed substantial genomic and immune heterogeneity in all patients studied, with considerable diversity in T cell frequency, and few shared T cell clones (, Melanoma: Tumor differences within a patient may explain heterogeneous responses Patients with metastatic melanoma display molecular and immune differences across tumor sites associated with differential drug responses. A team led by Jennifer Wargo from the University of Texas MD Anderson Cancer Center, Houston, USA, studied the radiological responses of 60 patients with metastatic melanoma, half of whom received targeted drug therapy and half of whom received an immune checkpoint inhibitor. The majority (83%) showed differences in responses across metastases. The group then profiled tumors in a subset, and found molecular and immune heterogeneity in different tumors within the same patient. Heterogeneity in mutational and immune profiles within tumors from individual patients could explain differences in treatment response. Knowing this, the authors emphasize the importance of acquiring biopsies from more than one tumor site in order to best tailor therapies to the features of metastatic cancer.

e14023 Background: Despite recent progress, robust treatment strategies that lead to durable remission are still lacking for many cancer types. This disease is difficult to treat owing in part to the complexity introduced by a heterogeneous population of cancer cells capable of evolving mechanisms of resistance to traditional therapy. Nonetheless, the discovery and continued optimization of T-cell immunotherapy has revolutionized the treatment of many cancers. This treatment strategy stands out from other approaches in its unique ability to co-evolve alongside an evading tumor. While promising, such therapies are also complex. For example, allogeneic stem cell transplantation leverages a donor-derived T-cell repertoire to treat patients with refractory hematologic malignancies and relies upon a delicate balance between desirable anti-tumor effects and potentially life-threatening graft-versus-host-disease. Currently, the decision to utilize this therapy and others like it is largely influenced by prior empirical evidence. Thus, there is great need for quantitative models of the cancer-immune interaction to generate testable predictions of treatment outcome, which could then be validated prior to T-cell immunotherapy administration. Methods: We develop a foundational mathematical model to investigate the properties of stochastic tumor-immune co-evolution using applied stochastic process theory and probabilistic analysis. We use this model to predict the effects of reduced immunity, T-cell diversity, and thymic turnover rates on cancer incidence, and compare model simulations to cancer evolutionary data. Results: We predict that changes in T-cell diversity, and to a lesser degree thymic turnover, increase the chance of tumor progression. When applied to experimental data, we demonstrate that the observations are consistent with co-evolution between an indolent cancer population and the adaptive immune system prior to clinical disease. Conclusions: Our results provide a fundamental framework for analyzing the interaction dynamics of an evolving threat like cancer and the adaptive immune system in order to better understand and predict immunotherapeutic efficacy.

585 Background: We previously reported a median tumor volume loss of 88% (range 30-98%) in 13 patients with early stage BRCA1/2 mutant breast cancer treated on a neoadjuvant trial of the PARP inhibitor talazoparib. The effects of PARP inhibition on immune aspects of the TiME in early-stage breast cancer has not been well described. The goal of this study was to evaluate the TiME in pre and post-treatment core biopsies from enrolled patients. Methods: Eleven paired core biopsies were available for examination. Tumor infiltrating lymphocytes (TILs) were quantified by H&E stained slides by a central pathologist. Specimens were assessed by multiplex immunofluorescence (mIF) using a panel of 6 biomarkers (PD-1, PD-L1, CD3, CD8, CD68 and CK) with the Opal 7-color Kit in LEICA BOND auto stainer, Vectra automated quantitative pathology imaging system and inForm software (PerkinElmer). Results: In the analyzed core biopsies, there was an increase in TILs evaluated by H&E in post-treatment compared to baseline (mean 36 vs 11%). By mIF there was an increase in CD3+ T cell and CD3+CD8+ cytotoxic T cell density in post-treatment samples compared to baseline, summarized in table. PD-L1 expression in tumor cells was rare in the cohort. There was no difference in CD3+PD-1+ or CD3+CD8+ PD-1+ lymphocytes in pre and post-treatment specimens. There was also no differences in macrophages (CD68+). Evaluation of immune phenotype and imaging response will be presented in the final analysis. Conclusions: This is the first study phenotyping the immune response to neoadjuvant talazoparib in BRCA-mutant breast cancer patients. In this small cohort, intratumoral and stromal CD3+ T cells and CD3+CD8+ cytotoxic T cells increased after two months of talazoparib. Clinical trial information: NCT02282345. [Table: see text]

Pretransplant conditioning regimens critically determine outcomes in the setting of allogeneic stem cell transplantation (allo-SCT). The use of nucleoside analogs such as fludarabine (Flu) in combination with i.v. busulfan (Bu) has been shown to be highly effective as a pretransplant conditioning regimen in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS). Because leukemia relapse remains the leading cause of death after allo-SCT, we studied whether clofarabine (Clo), a nucleoside analog with potent antileukemia activity, can be used to complement Flu. In a preliminary report, we previously showed the safety and efficacy of Clo ± Flu with i.v. Bu in 51 patients with high-risk AML, CML, and MDS. The study has now been completed, and we present long-term follow-up data on the entire 70-patient population, which included 49 (70%), 8 (11%), and 13 (19%) patients with AML, MDS, and CML, respectively. Thirteen patients (19%) were in complete remission, and 41 patients (59%) received matched unrelated donor grafts. Engraftment was achieved in all patients. Sixty-three patients (90%) achieved complete remission. There were no deaths reported at day +30, and the 100-day nonrelapse mortality rate was 4% (n = 3). Thirty-one percent of patients (n = 22) developed grades II to IV acute graft-versus-host disease, and the median overall survival and progression-free survival times were 2.4 years and .9 years, respectively. Our results confirm the safety and overall and progression-free survival advantage of the arms with higher Clo doses and lower Flu doses, which was most prominent in the AML/MDS group.

There have been significant advances in the treatment of metastatic melanoma through targeted and immunotherapy, however a significant proportion of patients still progress on these regimens with many experiencing mixed responses. Intense research efforts to better understand resistance are underway, and multiple molecular resistance mechanisms to targeted therapy have been identified. The appreciation of genetic heterogeneity as a contributor to resistance to therapy has grown, though immune heterogeneity has been poorly characterized. The goal of the present study is to better understand the molecular and immune heterogeneity in synchronous melanoma metastases at the time of disease progression. In this study, we prospectively evaluated 32 tumors from 15 patients who were treatment-naïve (n = 4), or had received prior targeted (n = 4) or immunotherapy (n = 7). Whole exome sequencing demonstrated between 4-41% of non-synonymous exonic mutations (NSEM) were restricted to individual tumors within a patient. Deep profiling of infiltrating immune cell subsets by flow cytometry and immunohistochemistry analyses confirmed the immune infiltrate between synchronous metastases to be highly heterogeneous, specifically in regards to CD4 and CD8 T lymphocytes. In aggregate, 92% of these T cell clones were unique to distinct tumors within the same patient, with limited overlap with clones detected in the blood. NetMHC3.4 neoantigen prediction demonstrated a large fraction of predicted neoantigens were restricted to individual tumors, with over 10% of these presenting extremely high predicted affinity. Importantly, analysis of RECIST measurements of individual lesions within the same patient suggested this molecular and immune heterogeneity could contribute to differential tumor growth and response to therapy within the same patient. This has important clinical implications, and suggests a single tumor biopsy may not be sufficiently representative of the molecular and immune landscape of multiple tumors within the same individual. Citation Format: Alexandre Reuben, Christine N. Spencer, Peter A. Prieto, John P. Miller, Xizeng Mao, Wei-Shen Chen, Hannah Cheung, Hong Jiang, Cara Haymaker, Mariana Petaccia De Macedo, Haven R. Garber, Pei-Ling Chen, Vancheswaran Gopalakrishnan, Jacob Austin-Breneman, Courtney W. Hudgens, Jason Roszik, Patrick Hwu, Scott E. Woodman, Lynda Chin, Michael A. Davies, Rodabe N. Amaria, Sapna P. Patel, Alexander J. Lazar, Michael T. Tetzlaff, Karen C. Dwyer, Ignacio I. Wistuba, Padmanee Sharma, James P. Allison, Jianhua Zhang, Andrew Futreal, Zachary A. Cooper, Jennifer A. Wargo. Genomic and immune heterogeneity in synchronous melanoma metastases is associated with differential tumor growth and response to therapy. [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 2392.

Pancreatic cancer is the fourth-leading cause of death in the United States and one of the most aggressive known malignancies. New and innovative advances in treatment are desperately needed. One promising area of investigational treatment for pancreatic cancer involves the use of immunotherapy. The development of immunotherapy for pancreatic cancer has been hampered by difficulty in generating tumor-reactive lymphocytes from resected specimens and by a lack of appropriate target antigens expressed on tumor cells. Innovative strategies have been developed with the use of peripheral blood lymphocytes that are genetically engineered to express T-cell receptors targeting common tumor antigens, including cancer-testis antigens, such as the MAGE-A3 antigen. Cancer-testis antigens pose excellent targets for immunotherapy because they are expressed in cancer and in the testis, an immune-privileged site, but have limited expression in normal tissue. An additional advantage in targeting cancer-testis antigens for immunotherapy is that their expression can be selectively up-regulated in tumor cells via epigenetic regulation with chromatin remodeling agents. Current interest in targeting cancer-testis antigens in pancreatic cancer is well-founded because cancer-testis antigens have been shown to be expressed in pancreatic cancer as potential targets for therapy. In our studies, we validated the expression pattern of cancer-testis antigens in resected specimens of pancreatic cancer and tested the hypothesis that treatment of pancreatic cancer cells with chromatin remodeling agents would render them more sensitive to antigen-specific T lymphocytes. We focused predominately on the MAGE-A3 antigen because it is highly expressed in pancreatic cancer, and several immunotherapeutic strategies are in clinical trials targeting this specific antigen. The results of these studies have important translational implications and provide the rationale for combined treatment with chromatin remodeling agents and immunotherapeutic approaches for pancreatic cancer.

Targeted therapy against oncogenic BRAF (BRAFV600E) is a promising new therapeutic approach for the treatment of melanoma with up to 80% of patients with stage IV melanoma responding to treatment. Despite these results, the median duration of response is only 8.5 months. Investigators propose combining this therapy with other targeted agents to address redundancy and signaling through different oncogenic pathways in hopes to improve the durability of response. An alternate approach is to combine BRAF-targeted agents with immunotherapy based on evidence that oncogenic BRAF contributes to immune escape. We recently reported data supporting this hypothesis, showing that treatment of melanoma cell lines with MEK or selective BRAFV600E inhibition results in increased expression of melanocyte differentiation antigens (MDAs) which is associated with enhanced recognition by antigen-specific T cells. The purpose of these studies was to validate these findings by assaying expression of MDAs in patients with metastatic melanoma treated with BRAFV600E inhibition. We also sought to test immune cell infiltrate in tumors to analyze host immune response. Patients with metastatic melanoma undergoing treatment with a selective BRAFV600E inhibitor were consented on a tissue procurement protocol approved through the IRB at the Massachusetts General Hospital. Patients underwent treatment with the BRAFV600E inhibitor (PLX4032) per protocol (960 mg given twice daily). Tumor biopsies and blood draws were performed pre-treatment and 10-14 days after initiation of treatment. RNA was harvested and expression of MDAs was assayed by quantitative RT-PCR. The level of MITF, the master transcriptional regulator of melanocyte which controls MDA expression, was also assayed. Immunohistochemistry was performed on formalin-fixed, paraffin embedded tissue for MDAs, as well as CD4 and CD8. The effect of BRAFV600E inhibition on T lymphocyte function was analyzed by assaying fold-expansion and viability in blood samples collected before and during treatment. Treatment with a selective inhibitor of BRAFV600E resulted in significantly increased levels of MDAs (up to 95-fold) which was associated with higher levels of MITF. In addition, CD8 T cell infiltrate also increased after treatment. Of note, T cell function was preserved after treatment with a specific inhibitor of BRAFV600E. These data provide in vivo correlates to our in vitro findings that selective BRAFV600E inhibition enhances MDA expression in melanoma and suggest a possible improved host immune response. The studies provide further support for potential synergy between BRAF-targeted therapy and immunotherapy in the treatment of melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 958. doi:10.1158/1538-7445.AM2011-958

Meeting abstracts Despite recent advances in the treatment of metastatic melanoma through targeted and immunotherapy, the majority of patients do not achieve a durable response. Research efforts to better understand responses are underway, and numerous molecular mechanisms of resistance to targeted