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Recently MHC-II protein expression levels in breast cancer tumor cells were shown to predict response to the PD-1 inhibitor pembrolizumab on a neoadjuvant clinical trial for ER+ and TNBC (ISPY-2). To better understand this biomarker and avoid post-market issues, we aimed to investigate possible pitfalls of MHC-II/HLA-DR quantification, prevalence of HLA-DR expression in breast cancer (BC) cells and its association with clinical data in a cohort of 372 BC patents. We assessed tumor and non-tumor HLA-DR expression by multiplex fluorescence immunohistochemistry on 8 TMAs containing 372 surgical BC samples, comprised of 239 ER+ and 123 TNBCs. Patient characteristics are shown in table 1. PanCK was used to define the tumor area and HLA-DR quantification was performed by training an object classifier on QuPath. Upon visual examination, 23 samples showed patchy panCK expression. We excluded normal breast epithelium and in situ components, which showed varying degrees of HLA-DR. 44.4% of breast cancers expressed HLA-DR, with 8.3% showing high tumor expression (HLA-DRHI; ≥20% of tumor cells). TNBC showed higher prevalence of HLA-DRHI cases (35% HLADR+, 14.6% HLA-DRHI, mean tumor HLA-DR expression (% of tumor cells) 10.3%, range 0-84%) than ER+ BC (51% HLADR+, 5.4% HLA-DRHI, mean 7.4%, range 0-66.5%). In some cases, HLA-DR and panCK expression were mutually exclusive. In other cases the lack of panCK expression highlighted the presence of stromal cells within the tumor area that were not evident on H&E. Tumoral MHC-II expression correlated with the presence of TILs for ER+ BC (r=0.18, p=0.0158) and TNBC (r=0.20, p= 0.0450), specifically CD3 (r=0.49 p Table 1: Patient characteristicsTNBCER+Total number of cases239123Median age48,563Treatmenttreatment naïve26%45%neoadjuvant chemotherapy74%4%pre-surgical letrozole-51%Positive lymph node58%14%StageI27%82%II20%27%III67%8%IV0%1% Citation Format: Paula Ines Gonzalez Ericsson, Justin M Balko, Violeta Sanchez, Melinda Sanders. Evaluation of tumor-specific MHC-II expression as a biomarker [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-19.

Purpose: The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME). Experimental Design: We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood. Results: In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden. Conclusions: We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Purpose: Preclinical data demonstrating androgen receptor (AR)–positive (AR+) triple-negative breast cancer (TNBC) cells are sensitive to AR antagonists, and PI3K inhibition catalyzed an investigator-initiated, multi-institutional phase Ib/II study TBCRC032. The trial investigated the safety and efficacy of the AR-antagonist enzalutamide alone or in combination with the PI3K inhibitor taselisib in patients with metastatic AR+ (≥10%) breast cancer. Patients and Methods: Phase Ib patients [estrogen receptor positive (ER+) or TNBC] with AR+ breast cancer received 160 mg enzalutamide in combination with taselisib to determine dose-limiting toxicities and the maximum tolerated dose (MTD). Phase II TNBC patients were randomized to receive either enzalutamide alone or in combination with 4 mg taselisib until disease progression. Primary endpoint was clinical benefit rate (CBR) at 16 weeks. Results: The combination was tolerated, and the MTD was not reached. The adverse events were hyperglycemia and skin rash. Overall, CBR for evaluable patients receiving the combination was 35.7%, and median progression-free survival (PFS) was 3.4 months. Luminal AR (LAR) TNBC subtype patients trended toward better response compared with non-LAR (75.0% vs. 12.5%, P = 0.06), and increased PFS (4.6 vs. 2.0 months, P = 0.082). Genomic analyses revealed subtype-specific treatment response, and novel FGFR2 fusions and AR splice variants. Conclusions: The combination of enzalutamide and taselisib increased CBR in TNBC patients with AR+ tumors. Correlative analyses suggest AR protein expression alone is insufficient for identifying patients with AR-dependent tumors and knowledge of tumor LAR subtype and AR splice variants may identify patients more or less likely to benefit from AR antagonists.

The recent approval of anti-PD-L1 immunotherapy in combination with nAB-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor-immune microenvironment (TIME). Patients with TNBC are routinely treated with neoadjuvant chemotherapy (NAC). Stromal tumor-infiltrating lymphocytes (sTILs) in the pre-treatment diagnostic biopsy are predictive of pathologic complete response (pCR). In patients with residual disease (RD) at surgery, sTILs confer good prognosis. However, the effect of chemotherapy on sTILs and how it influences the TIME are poorly understood. We examined immune-gene expression patterns before and after NAC in a series of 83 breast tumors, including 44 TNBCs, from patients with RD. sTILs were enumerated by standardized guidelines. Gene expression patterns were tested for association with recurrence-free (RFS) and overall survival (OS). T cell receptor sequencing (TCRseq) was performed on a subset (n=15) of tumors. In 4 patients undergoing NAC, PD-1-high and -negative CD8+ peripheral blood mononuclear cells (PBMCs) were profiled using single-cell RNAseq and multiplexed cytokine secretion assays. Post-NAC sTILs (≥30%) were only predictive of outcome (RFS p=0.019; OS p=0.05) in TNBC patients, but not in non-TNBC patients (RFS p=0.28; OS p=0.78) confirming that the prognostic capacity of sTILs is confined to TNBC. Pre-NAC sTILs were not predictive of outcome in either group, likely due to exclusion of patients experiencing pCR. The change in sTILs during NAC did not prognosticate outcome in TNBC, suggesting that in the post-NAC setting, only the most proximal measurement of sTILs is meaningful. However, these results did suggest that NAC alters the TIME. To examine the interplay among NAC, the TIME, and clinical outcomes, we tested the change in expression of 770 immune-related genes during NAC in univariate cox-proportional hazards models. In non-TNBC, no change in expression of any single gene was associated with RFS or OS at a false-discovery rate (FDR) of 10%. In TNBC, individual changes in 12 genes and 204 genes were identified as associated with RFS and OS, respectively (FDR Citation Format: Justin M Balko, Mellissa Nixon, Paula I Gonzalez-Ericsson, Mark A Pilkinton, Wyatt J McDonnell, Violeta Sanchez, Susan R Opalenik, Sherene Loi, Brent Rexer, Vandana Abramson, Valerie Jansen, Simon Mallal, Jonathan D Marotti, Kevin Shee, Todd W Miller, Melinda E Sanders, Ingrid A Mayer, Roberto Salgado. Immunologic correlates of long-term outcome in the residual disease of triple-negative breast cancer after neoadjuvant chemotherapy [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 P3-08-15.

Purpose: Immunotherapies targeting PD-1/L1 enhance pathologic complete response (pCR) rates when added to standard neoadjuvant chemotherapy (NAC) regimens in early-stage triple-negative, and possibly high-risk estrogen receptor–positive breast cancer. However, immunotherapy has been associated with significant toxicity, and most patients treated with NAC do not require immunotherapy to achieve pCR. Biomarkers discerning patients benefitting from the addition of immunotherapy from those who would achieve pCR to NAC alone are clearly needed. In this study, we tested the ability of MHC-II expression on tumor cells, to predict immunotherapy-specific benefit in the neoadjuvant breast cancer setting. Patients and Methods: This was a retrospective tissue-based analysis of 3 cohorts of patients with breast cancer: (i) primary nonimmunotherapy-treated breast cancers (n = 381), (ii) triple-negative breast cancers (TNBC) treated with durvalumab and standard NAC (n = 48), and (iii) HER2-negative patients treated with standard NAC (n = 87) or NAC and pembrolizumab (n = 66). Results: HLA-DR positivity on ≥5% of tumor cells, defined a priori, was observed in 10% and 15% of primary non-immunotherapy–treated hormone receptor–positive and triple-negative breast cancers, respectively. Quantitative assessment of MHC-II on tumor cells was predictive of durvalumab + NAC and pembrolizumab + NAC (ROC AUC, 0.71; P = 0.01 and AUC, 0.73; P = 0.001, respectively), but not NAC alone (AUC, 0.5; P = 0.99). Conclusions: Tumor-specific MHC-II has a strong candidacy as a specific biomarker of anti–PD-1/L1 immunotherapy benefit when added to standard NAC in HER2-negative breast cancer. Combined with previous studies in melanoma, MHC-II has the potential to be a pan-cancer biomarker. Validation is warranted in existing and future phase II/III clinical trials in this setting.

Background: Somatic alterations in the FGFR pathway (mainly FGFR1, amplified in about 15% of ER+ BC) have been implicated in resistance to endocrine therapy (ET), and more recently, to CDK4/6i as well. Based on our preclinical data showing that the FGFR pan-inhibitor erdafitinib when added to fulvestrant/palbociclib resulted in marked PDX regressions, we initiated a phase Ib trial combining erdafitinib with fulvestrant/palbociclib in patients with ER+/HER2-/FGFR-amplified MBC (NCT03238196) to determine safety, tolerability and anti-tumor activity of this combination. Methods: Patients with evaluable ER+/HER2- MBC with FGFR1-4 amplification (detected on tumor next generation sequencing or plasma ctDNA) exposed to at least one ET regimen (but no more than 2 lines of chemotherapy) in the metastatic setting, were treated with fulvestrant, palbociclib (standard of care dosing/ schedule) and oral erdafitinib, tested in 4 doses ranging from 4 mg to 8 mg daily. Once MTD reached, we planned to enroll 20 patients in the expansion portion of the trial. Tumor blocks were collected for FGFR1 FISH amplification analysis, and plasma ctDNA was collected at baseline, 4 weeks and at treatment discontinuation. Tumor assessments were performed every 8 weeks. Results: Since August 2017, 26 eligible patients with evaluable ER+/HER2-/FGFR-amplified MBC were enrolled across 4 institutions. Patient characteristics are summarized in Table 1. Main grade 1 and 2 adverse events (AE) were consistent with on-target toxicities of erdafitinib and/or palbociclib: mucositis (67%), hyperphosphatemia (61%), dysgeusia (52%), diarrhea (48%), fatigue (48%), neutropenia (47%), hand-foot syndrome (38%), anemia (29%), and onycholysis (14%). Febrile neutropenia occurred in 5% patients, no cases of central serous retinopathy were seen. Serious AE were rare: one grade 4 elevation of transaminases (DLT; attributed to fulvestrant), one grade 3 colitis (attributed to erdafitinib), and one thromboembolic event (attributed to palbociclib). In combination with fulvestrant/ palbociclib, the MTD of erdafitinib was 6 mg. No drug-drug interaction was seen. 8 patients were deemed non-evaluable for anti-tumor effect as treatment discontinuation (mainly due to AE) occurred prior to first tumor assessment. Of the 18 evaluable patients: 7 had disease progression, 8 had stable disease (4 of which discontinued treatment due to AE), 3 have not completed their first tumor assessment, 4 are still on treatment; median PFS was 3 months and CBR at 6 months was 28%. However, higher PFS (6 months) was seen in 6/8 patients with high levels of FGFR1 amplification (FISH FGFR1:CEP8 ratio >5; gene copy number >10) and in both patients with FGFR3 amplification. Conclusion: To our knowledge, this is the first time an FGFR inhibitor has been tested in combination with ET and CDK4/6i in patients with MBC harboring FGFR alterations. Erdafitinib-related side effects appeared to be on target, leading to treatment discontinuation in several patients despite optimal medical treatment. Clinical activity was seen in heavily pre-treated patients with molecular evidence of high FGFR amplification despite 100% prior exposure to ET and CDK4/6i. Full clinical and correlative work will be presented at the meeting, and a future phase II trial is being planned. Table 126 / 35 patients accrued13 in escalation, 13 in expansion(ongoing)Median age53 (35 - 75)Race/ ethnicityWhite 22Black 1Asian 2Hispanic 1Median number of lines of treatment in the metastatic setting4 (1 - 5)Prior lines of treatment in the metastatic settingEndocrine therapy 100%Fulvestrant 28%CDK4/6i 100%PI3K pathway inhibitor 80%1 line chemo 65%2 lines chemo 45%FGFR1 amplification23FGFR3 amplification2FGFR4 amplification1 Citation Format: Ingrid A. Mayer, Barbara B. Haley, Vandana G. Abramson, Adam Brufsky, Brent Rexer, Erica Stringer-Reasor, Komal L. Jhaveri, Melinda Sanders, Paula I. Ericsson-Gonzalez, Fei Ye, Carlos L. Arteaga. A phase Ib trial of fulvestrant + CDK4/6 inhibitor (CDK4/6i) palbociclib + pan-FGFR tyrosine kinase inhibitor (TKI) erdafitinib in FGFR-amplified/ ER+/ HER2-negative metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD1-03.

Background: Although neoadjuvant chemotherapy (NAC) induces complete response in 30-40% of triple-negative breast cancers (TNBC), patients with residual disease at surgery have poor prognosis and limited treatment options until recurrence. Tumor-infiltrating lymphocytes (TILs) in the residual disease are a positive prognostic factor, but how specific immune composition of the tumor guides outcome is unclear, resulting in a lack of understanding of how to employ immunotherapies in the adjuvant setting. Methods: We assessed multiple immunologic biomarkers in a series of 99 residual TNBCs after NAC. Immune markers were assessed by multiple methods, including H&E-based TILs analysis, standard immunohistochemistry (IHC; CD8, CD20, CD56, FOXP3, LAG-3, B7-H4, HLA-A) and multiplexed immunofluorescence (mIF; HLA-DR, GZMB, CD4, CD8, PD-L1, pan-CK, PD1, CD3). Association among parameters were assessed (up to n=98) including clinical outcome after surgery (n=94). Results: As previously demonstrated, TILs in residual tumors after NAC predicted both RFS and OS (p=0.0093 and p=0.0376, respectively). H&E-scored TILs were highly correlated to CD3, CD4 and CD8 positive T cells (Spearman r range 0.34-0.4850; all p Conclusions: We have found a paradoxical association of GZMB+ CD8+ T cells with a negative prognosis in NAC-treated TNBC. We hypothesize that while these T cells are poised for cytotoxic activity, they remain restricted through sub-localization outside the tumor core, downregulation of HLA-A on tumor cells preventing interaction with the T cell receptor, and upregulation of B7H4 expression, which has been shown to inhibit cytotoxic T cell activity. Patients with high CD8+GZMB+ tumors in the post-NAC setting may benefit from adjuvant immunotherapy, particularly in combination with therapies that enhance MHC-I (e.g. HLA-A) antigen presentation. Citation Format: Paula I Gonzalez-Ericsson, Violeta Sanchez, Roberto Salgado, Jennifer Bordeaux, Ju Y Kim, Christine Vaupel, Henry Gomez, Melinda E Sanders, Justin M Balko. The immune landscape of residual triple-negative breast cancers after neoadjuvant chemotherapy [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 PD5-07.

Background Papillary thyroid carcinoma (PTC) in children has a distinctive set of clinicopathologic features and molecular signature compared to their adult counterparts. The recent recommendation to reclassify encapsulated follicular variant of papillary thyroid carcinoma (EFVPTC) without invasion as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is based on evidence derived almost exclusively from studies in adults. Clinicopathologic studies restricted to pediatric NIFTP are limited. Methods We retrospectively analyzed all pediatric PTC and NIFTP diagnosed and treated in our institution from 1999 to 2016 (n = 31). Results Using recently published consensus diagnostic criteria, we identified 3 NIFTP and 2 infiltrative follicular variants of papillary thyroid carcinoma (FVPTC) among 31 cases. Two of the NIFTP cases were initially diagnosed as EFVPTC. All 3 patients with NIFTP had unifocal tumors of lower American Joint Committee on Cancer (AJCC) classification (T2 or lower) and were free of lymph node or distant metastasis. Total (n = 1) or completion (n = 2) thyroidectomy was performed in all cases, and only 1 NIFTP patient received subsequent radioablative therapy. No residual or recurrent disease has been observed during follow-up (15–138 months) in patients with NIFTP. Conclusions Our experience with NIFTP in children is similar to outcomes reported in adult studies, suggesting that pediatric NIFTP behave indolently as evidenced by the absence of local recurrence in our cohort.

Checkpoint inhibitors produce durable responses in numerous metastatic cancers, but immune-related adverse events (irAEs) complicate and limit their benefit. IrAEs can affect organ systems idiosyncratically; presentations range from mild and self-limited to fulminant and fatal. The molecular mechanisms underlying irAEs are poorly understood. Here, we report a fatal case of encephalitis arising during anti-programmed cell death receptor 1 therapy in a patient with metastatic melanoma. Histologic analyses revealed robust T cell infiltration and prominent programmed death ligand 1 expression. We identified 209 reported cases in global pharmacovigilance databases (across multiple cancer types) of encephalitis associated with checkpoint inhibitor regimens, with a 19% fatality rate. We performed further analyses from the index case and two additional cases to shed light on this recurrent and fulminant irAE. Spatial and multi-omic analyses pinpointed activated memory CD4+ T cells as highly enriched in the inflamed, affected region. We identified a highly oligoclonal T cell receptor repertoire, which we localized to activated memory cytotoxic (CD45RO+GZMB+Ki67+) CD4 cells. We also identified Epstein-Barr virus-specific T cell receptors and EBV+ lymphocytes in the affected region, which we speculate contributed to neural inflammation in the index case. Collectively, the three cases studied here identify CD4+ and CD8+ T cells as culprits of checkpoint inhibitor-associated immune encephalitis.

Background Misidentifying parathyroid glands (PGs) during thyroidectomies or parathyroidectomies could significantly increase postoperative morbidity. Imaging systems based on near infrared autofluorescence (NIRAF) detection can localize PGs with high accuracy. These devices, however, depict NIRAF images on remote display monitors, where images lack spatial context and comparability with actual surgical field of view. In this study, we designed an overlay tissue imaging system (OTIS) that detects tissue NIRAF and back-projects the collected signal as a visible image directly onto the surgical field of view instead of a display monitor, and tested its ability for enhancing parathyroid visualization. Study Design The OTIS was first calibrated with a fluorescent ink grid and initially tested with parathyroid, thyroid, and lymph node tissues ex vivo. For in vivo measurements, the surgeon's opinion on tissue of interest was first ascertained. After the surgeon looked away, the OTIS back-projected visible green light directly onto the tissue of interest, only if the device detected relatively high NIRAF as observed in PGs. System accuracy was determined by correlating NIRAF projection with surgeon's visual confirmation for in situ PGs or histopathology report for excised PGs. Results The OTIS yielded 100% accuracy when tested ex vivo with parathyroid, thyroid, and lymph node specimens. Subsequently, the device was evaluated in 30 patients who underwent thyroidectomy and/or parathyroidectomy. Ninety-seven percent of exposed tissue of interest was visualized correctly as PGs by the OTIS, without requiring display monitors or contrast agents. Conclusions Although OTIS holds novel potential for enhancing label-free parathyroid visualization directly within the surgical field of view, additional device optimization is required for eventual clinical use.

Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists., Era+ breast cancer patients often develop resistance to endocrine therapy. Here, the authors show that FGFR1 amplification is a resistance mechanism to CDK4/6 inhibitor and endocrine therapy and that combined treatment with FGFR, CDK4/6, and anti-estrogens is a potential therapeutic strategy in Era+ breast cancer tumors.

Purpose:The phase III ExteNET trial showed improved invasive disease-free survival in patients with HER2+ breast cancer treated with neratinib versus placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in patients with ER+/HER2+ tumors. We thus sought to discover mechanisms that may explain the benefit from extended adjuvant therapy with neratinib.Experimental Design: Mice with established ER+/HER2+ MDA-MB-361 tumors were treated with paclitaxel plus trastuzumab ± pertuzumab for 4 weeks, and then randomized to fulvestrant ± neratinib treatment. The benefit from neratinib was evaluated by performing gene expression analysis for 196 ER targets, ER transcriptional reporter assays, and cell-cycle analyses.Results:Mice receiving “extended adjuvant” therapy with fulvestrant/neratinib maintained a complete response, whereas those treated with fulvestrant relapsed rapidly. In three ER+/HER2+ cell lines (MDA-MB-361, BT-474, UACC-893) but not in ER+/HER2− MCF7 cells, treatment with neratinib induced ER reporter transcriptional activity, whereas treatment with fulvestrant resulted in increased HER2 and EGFR phosphorylation, suggesting compensatory reciprocal crosstalk between the ER and ERBB RTK pathways. ER transcriptional reporter assays, gene expression, and immunoblot analyses showed that treatment with neratinib/fulvestrant, but not fulvestrant, potently inhibited growth and downregulated ER reporter activity, P-AKT, P-ERK, and cyclin D1 levels. Finally, similar to neratinib, genetic and pharmacologic inactivation of cyclin D1 enhanced fulvestrant action against ER+/HER2+ breast cancer cells.Conclusions:These data suggest that ER blockade leads to reactivation of ERBB RTKs and thus extended ERBB blockade is necessary to achieve durable clinical outcomes in patients with ER+/HER2+ breast cancer.

Identifying parathyroid glands can be challenging for surgeons during head and neck operations. For the surgeon, it becomes essential to preserve healthy parathyroid glands, while successfully removing diseased parathyroid glands. This study presents PTeye – a commercial fiber probe-based device that was designed for intraoperative parathyroid identification by detecting near infrared autofluorescence. The results of this study demonstrate that PTeye had 95% accuracy in identifying parathyroid glands in 78 patients. The device had an overall detection rate of 97% for healthy and diseased parathyroid glands. PTeye could also quantify parathyroid vascularity in real-time by using indocyanine green during neck surgeries.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that does not respond to endocrine therapy or human epidermal growth factor receptor 2 (HER2)-targeted therapies. Individuals with TNBC experience higher rates of relapse and shorter overall survival compared to patients with receptor-positive breast cancer subtypes. Preclinical discoveries are needed to identify, develop, and advance new drug targets to improve outcomes for patients with TNBC. Herein, we report that MYCN, an oncogene typically overexpressed in tumors of the nervous system or with neuroendocrine features, is heterogeneously expressed within a substantial fraction of primary and recurrent TNBC and is expressed in an even higher fraction of TNBCs that do not display a pathological complete response after neoadjuvant chemotherapy. We performed high-throughput chemical screens on TNBC cell lines with varying amounts of MYCN expression and determined that cells with higher expression of MYCN were more sensitive to bromodomain and extra-terminal motif (BET) inhibitors. Combined BET and MEK inhibition resulted in a synergistic decrease in viability, both in vitro and in vivo, using cell lines and patient-derived xenograft (PDX) models. Our preclinical data provide a rationale to advance a combination of BET and MEK inhibitors to clinical investigation for patients with advanced MYCN-expressing TNBC.

This study presents PTeye – a fiber probe-based device – that (i) yielded 95% accuracy in label-free identification of parathyroid glands in 88 patients and (ii) could quantitatively assess parathyroid vascularity using indocyanine green during neck surgeries.

Background: Neratinib is a potent, irreversible pan-HER tyrosine kinase inhibitor. The phase III trial ExteNET showed improved disease-free survival in patients (pts) with HER2+ breast cancer treated with neratinib vs placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in pts with ER+ tumors. Thus, we sought to elucidate mechanisms that may explain the benefit from extended adjuvant therapy with neratinib in pts with ER+/HER2+ breast cancer using a human-in-mouse model that simulates the clinical outcomes seen in ExteNET. Results: Mice with established ER+/HER2 amplified MDA-361 tumors were treated with trastuzumab (tz) + paclitaxel (pac) for 4 weeks, and then randomized to fulvestrant (fulv) ± neratinib for 4 weeks. All MDA-361 tumors exhibited a prompt and marked reduction in volume after tz/pac treatment; 10 mice achieved a complete response (CR) before receiving 'extended adjuvant' therapy with fulv (n=5) or neratinib/fulv (n=5). A CR was maintained with neratinib/fulv following tz/pac. However, mice treated with fulv alone, relapsed rapidly (p Conclusions: Neratinib/fulv but not fulv alone maintained complete responses of ER+/HER+ tumors following treatment with tz/pac or pertuzumab/tz/pac, reminiscent of the results in ExteNET. Neratinib treatment promoted ER transcriptional activity whereas ER downregulation with fulv was associated with increased HER2 signaling. In ER+/HER2+ breast cancer cells and tumors, neratinib/fulv synergistically inhibited growth, cyclin D1 expression, and AKT and MAPK activation, thus providing a plausible mechanism to explain the results in the ExteNET trial. Citation Format: Sudhan DR, Schwarz LJ, Guerrero-Zotano AL, Nixon M, Formisano L, Croessmann S, Gonzalez Ericsson PI, Sanders ME, Balko JM, Avogadri-Connors F, Cutler RE, Lalani AS, Bryce R, Auerbach A, Arteaga CL. Extended adjuvant neratinib/fulvestrant blocks ER/HER2 crosstalk and maintains complete responses of ER+/HER2+ tumors following treatment with chemotherapy and anti-HER2 therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-13-08.

Background: FGFR1 amplification (amp) occurs in ˜15% of breast cancers (BC) and associates with poor prognosis and resistance to endocrine therapy. CCND1 regulates cell cycle progression and is amplified in 15-20% of BC. Co-amp of FGFR1 occurs in 30-40% of CCND1amp tumors, suggesting the possibility of oncogene cooperativity. CDK4/6 inhibitors, which block the action of cyclin D1/CDK4 complexes at the G1-to-S transition, are approved for treatment of ER+ BC and FGFR inhibitors are in early phase clinical trials. Results: Between 11/2013 and 03/2017, 191 BCs from 188 patients with metastatic (M) or refractory locoregional recurrent (RLRR) BC at Vanderbilt (VICC) were profiled by targeted next gen sequencing (Foundation OneTM). These are included within the 2131 publicly available BC sequencing results in GENIE (Foundation OneTM and MSK-IMPACT). Among the GENIE cohort, rates were: FGFR1amp 7% (n=156), CCND1amp 12% (n=261) and CCND1/FGFR1co-amp 3% (n=58). Additional cases showed FGFR1 missense mutations (n=16) and deep deletions (n=5). When the analysis was limited to the VICC cohort allowing restriction to ER+ BC, FGFR1amp (16%) and CCND1amp (23%) rates are similar to rates in primary BC in TCGA (13% FGFR1 [p = 0.44] and 19% CCND1 [p = 0.24]). In GENIE, the most frequent co-mutations in FGFR1amp tumors were TP53 (31%), PIK3CA (21%), GATA3 (13%), CDH1 (11%) and MAP3K1 (10%). However, TP53 and PIK3CA mutations were less common among FGFR1amp tumors than FGFR1non-amp cases (p 0.016). Histopathologic correlation on tumors from our institution show a majority of FGFR1 and/or CCND1 amp BC (64%) were ER+/HER2–; 33% of ER+/FGFR1amp tumors were PR–. Distinctive histologic features associated with FGFR1 and/or CCND1 amp were lobular histology (17%) and neuroendocrine differentiation (14%), 0-10%TILs (94%) and high proliferative rate (46%). Conclusion: FGFR1amp and CCND1amp rates in TCGA are similar to those seen in MBC/LRRBC (GENIE) suggesting FGFR1 can function as both a driver mutation and de novo mechanism of endocrine resistance early in tumorigenesis. Frequent co-amp with CCND1 and lower rates of TP53 and PIK3CAmut also support a driver role for FGFR1amp and FGFR1/CCND1co-amp. The observation of neuroendocrine features in a subset of these tumors suggests lineage plasticity. This may be a consequence of genomic alterations promoting anti-estrogen resistance and is consistent with recently published BC outcome data associating neuroendocrine differentiation with higher grade ER+ tumors, frequent 8p amp, which includes FGFR1, and worse disease-free and overall survival. The frequency of FGFR1amp suggests genotype specific trials with FGFR inhibitors would be highly feasible. Whether FGFR1/CCND1 co-amplified tumors are candidates for treatment with a combination of FGFR and CDK4/6 inhibitors requires further investigation. Citation Format: Gonzalez-Ericsson PI, Estrada MV, Formisano L, Jansen VM, Mayer IA, Rexer BN, Abramson VG, Levy M, Balko JM, Stricker TP, Arteaga CL, Sanders ME. Genenomic landscape of breast cancers with FGFR1 amplification and FGFR1/CCND1 co-amplification revealed by targeted capture next generation sequencing [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-07.

Background: We have previously shown that some breast cancers express major histocompatibility complex II (MHC-II), correlating with enhanced immune infiltration. In other tumor types, we have shown that MHC-II expression on tumor cells predicts clinical response to checkpoint inhibition. We sought to determine the direct effects of MHC-II on anti-tumor immunity and characterize mechanisms of immune escape in this breast cancer subset. Methods: To determine the functional effects of MHC-II on tumor cells, we generated isogenic mouse breast tumor cells with enforced MHC-II expression and determined their ability to generate tumors in syngeneic mice, the impact on immunity, and their response to checkpoint inhibition. In a series of molecularly-characterized HER2+ (n=8) and triple-negative breast cancers (TNBC; n=103), we performed immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) for Lag-3, PD-L1, CD4, CD8, FCRL6, and granzyme B. Results: Following injection in syngeneic immunocompetent mice, MHC-II+ mouse breast tumors were more frequently rejected (p=0.04) and recruited greater numbers of CD4+ TILs. When MHC-II+ tumors escaped rejection, they expressed higher degrees of PD-1 and Lag-3 in the tumor and in the draining lymph node. Since Lag-3 is a checkpoint that specifically targets MHC-II, we hypothesized that MHC-II+ breast cancers escape anti-tumor immunity through suppressing MHC-II-mediated antigen presentation. Combinations of anti-Lag-3 and anti-Pd-1 antibodies inhibited growth of MHC-II+ tumors. These findings led us to also explore Fc receptor-like 6 (FCRL6), a previously reported MHC-II receptor expressed on NK and cytotoxic T cells. Residual MHC-II+ TNBC post-neoadjuvant chemotherapy (NAC) recruited greater numbers of CD4+ and CD8+ TILs (p=0.0001 and p=0.0002), suggesting enhanced immune recognition. However, MHC-II+ TNBCs also demonstrated a greater frequency of Lag-3+ and FCRL6+ TILs (p Conclusions: These data suggest that MHC-II+ breast tumors are immunologically active and circumvent anti-tumor immunity by targeting MHC-II antigen presentation through recruitment of Lag-3+ and FCRL6+ TILs. We describe herein FCRL6 as a novel bona fide immune checkpoint which targets MHC-II, which may impact a variety of cancers. MHC-II expression status may be a useful biomarker for patient stratification on anti-PD-1/anti-Lag-3 combination, and eventually, anti-PD-1/anti-FCRL6 combinations in patients with breast cancer. Citation Format: Balko JM, Johnson DB, Ericsson-Gonzalez P, Nixon MJ, Salgado R, Sanchez V, Shreeder DM, Rimm DL, Loi S, Kim JY, Bordeaux J, Sanders ME, Davis RS. Breast tumor-specific MHC-II expression drives a unique pattern of adaptive resistance to antitumor immunity through MHC-II receptor checkpoint engagement [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-08-02.

BackgroundDespite the broad success of immune checkpoint inhibition (ICI e.g. anti-Programmed Death Ligand-1 [PD-L1]) in cancer treatment, tumor-intrinsic factors leading to intrinsic and acquired resistance are poorly understood. Tumor specific MHC-I expression is indispensable for anti-PD-1/L1 response as complete loss of MHC-I via B2M deletion results in inability of CD8+ T cells to recognize tumor-associated antigens. However, MHC-I is heterogeneously downregulated or lost in many tumor types. Tumor cell destruction can also occur through non-synaptic mechanisms, in a so-called ‘field effect’. Therefore, we modeled heterogeneous loss of MHC-I expression in breast cancer and experimentally evaluated how heterogeneous MHC-I loss affects response to anti-PD-L1 therapy.MethodsWe performed quantitative immunofluorescence for MHC-I and Pan-CK on breast cancer tumors (n=410). To determine the functional effect of MHC-I heterogeneity on anti-PD-L1 response, we used an immunocompetent EMT6 orthotopic mammary tumor model which ubiquitously expresses MHC-I at baseline. Using CRISPR/Cas9, we engineered EMT6 cells with B2m loci excision resulting in complete knockout of MHC-I on the cell surface. We then orthotopically implanted B2m-comtetent and B2m-KO cells at varying inoculum ratios (100:0, 90:10, 50:50, 10:90, 0:100) into syngeneic Balb/C mice and assessed immune responsiveness and efficacy of checkpoint inhibition. Additionally, to look at how loss of MHC-I affects the tumor microenvironment we will use the PanCancer Immune NanoString panel (n=770 genes) to evaluate gene expression patterns in tumor cells and infiltrating immune cells.ResultsIn patient samples, we identified high diversity in MHC-I expression across all clinical subtypes, with triple negative breast cancer (TNBC) having the highest MHC-I expression. Chemotherapy-treated tumors had higher MHC-I levels than untreated tumors. In mice when 10% of cells were B2m-KO, we observed a 50% reduction in complete eradication of EMT6 tumors with aPD-L1 treatment and reduced disease-stabilization and no complete responses when a 50% mixture of MHC-I deficient cells. An increasing percentage of B2m KO leads to worse outcomes overall and a decrease in infiltrating T cells.ConclusionsOur work suggests that there is an ICI-responsive phenotype that is driven by heterogeneity in MHC-I expression levels. As little as 10% of tumor specific MHC-I loss can lead to therapeutic resistance and a decrease in complete responders. This represents that early TNBC may be less responsive to single-agent PD-L1 due to specific percentages of MHC-I loss. MHC-I expression can influence therapy outcomes and potentially lead to novel observations of how to overcome lack of, or limited, MHC-I expression.

BackgroundImmune checkpoint inhibitors (ICI) have improved patient survival in some cancer types but yielded limited success in breast cancer. Combinations of ICI (αPD-L1/PD-1) and chemotherapy have been FDA-approved for metastatic TNBC patients, and potentially in the early breast cancer setting, but many patients remain non-responsive to ICI. B7-H4 is a B7 family ligand with proposed immunosuppressive functions being explored as a cancer immunotherapy target and may be associated with resistance to αPD-L1. We confirmed an inverse expression pattern between B7-H4 and PD-L1 in breast tumor cells, which has previously been noted by others. B7-H4 was expressed in immune-excluded tumors, while PD-L1 was expressed in immune-infiltrated tumors. Based on these findings, we hypothesized ectopic B7-H4 expression would induce αPD-L1 resistance through immune cell suppression in vivo.MethodsUsing an immunocompetent and αPD-L1-sensitive EMT6 orthotopic mammary cancer model, we induced ectopic expression of B7-H4 and performed animal survival studies to assess therapy response, and RNA analysis to assess changes to cell signaling among tumor infiltrating immune cells. Finally, we performed transcriptomic correlation analyses from the cancer cell line encyclopedia dataset to identify potential regulators of B7-H4 in breast cancer.ResultsIn the αPD-L1-sensitive EMT6 mammary cancer model, tumors with cell-surface B7-H4 expression were more resistant to immunotherapy. Additionally, tumor infiltrating immune cells had reduced immune activation signaling based on transcriptomic analysis. We also observed strong correlation with B7-H4 mRNA and epithelial cell markers, in contrast to gene expression markers of mesenchymal cells.ConclusionsOur data support the hypothesis that B7-H4 induces tumor resistance to αPD-L1 ICI through an immunosuppressive function. Additionally, the strong correlation of B7-H4 to epithelial cell markers suggests a potential regulatory mechanism of B7-H4 expression independent of PD-L1 regulation.

BackgroundImmune checkpoint inhibitors (ICI) improve patient survival in some cancer types but yield limited success in breast cancer. Phase-III clinical trials in triple-negative breast cancer (TNBC) patients, who harbor extensive tumor-infiltrating lymphocytes, demonstrate increased progression-free survival (IMpassion130) and pathologic complete response (KEYNOTE-522). Consequently, combinations of ICI and chemotherapy have been FDA-approved for metastatic TNBC patients. However, the therapeutic benefit of ICI alone and the most efficacious chemotherapy combinations are poorly characterized. We sought to model ICI response in vivo to elucidate the mechanisms of immunotherapy efficacy in breast cancer and ascertain the therapeutic benefits of different chemotherapeutic combinations with ICI.MethodsUsing an immunocompetent EMT6 orthotopic mammary tumor model, we investigated the efficacy of single-agent immunotherapy and in combination with standard-of-care chemotherapy (paclitaxel [PAC] or doxorubicin [DOX]). We used single-cell RNA sequencing and bulk RNA and T-cell receptor (TCR) sequencingto assess the cellular landscape of the primary tumor in response to combinatorial therapeutic strategies and identify systemic genetic alterations and T-cell expansion, respectively.ResultsSingle-agent anti-PD-L1 robustly suppressed primary tumor growth (p =0.0046) and extended survival (pConclusionsWe identify a heterogeneously ICI-responsive in vivo model that emulates TNBC patient response to combinatorial ICI approaches. We describe single-agent ICI efficacy in upregulating cytotoxic immune cell infiltration and expansion within the primary tumor that diminishes tumor growth and enhances survival. Moreover, this study describes differential responses in a genetically similar host, which reflects heterogeneous patient response to ICI. Further characterization may identify systemic biomarkers and tumor antigen-specific T cell clones to accurately predict immunotherapy response in patients and uncover mechanisms for sensitizing refractory tumors to ICI

We describe a 44-year-old female with triple-negative breast cancer who developed skin erythaema, sclerosis and contracture of her entire right breast 15 months after completion of post-lumpectomy chemotherapy and radiotherapy, consistent with post-irradiation morphoea (PIM). PIM is a rare complication of breast irradiation that impairs a patient's quality of life. PIM is located usually at the radiation port or in the surrounding tissue. Clinically, PIM is misdiagnosed commonly as lymphoedema and cellulitis in the early inflammatory phase, and recurrent breast cancer, chronic radiodermatitis (CRD), radiation-induced fibrosis (RIF), post-irradiation pseudosclerodermatous panniculitis (PIPP), atypical vascular lesions (AVL) or angiosarcoma (AS) in the late burnout phase. Arriving at the correct diagnosis typically requires a multidisciplinary approach, including a skin biopsy for confirmation. To date, satisfactory treatment of this condition has been challenging. and the clinical outcome after therapy is often unsatisfactory.

Breast conserving surgery is the preferred treatment for women diagnosed with early stage invasive breast cancer. To ensure successful breast conserving surgeries, efficient tumour margin resection is required for minimizing tumour recurrence. Currently surgeons rely on touch preparation cytology or frozen section analysis to assess tumour margin status intraoperatively. These techniques have suboptimal accuracy and are time-consuming. Tumour margin status is eventually confirmed using postoperative histopathology that takes several days. Thus, there is a need for a real-time, accurate, automated guidance tool that can be used during tumour resection intraoperatively to assure complete tumour removal in a single procedure. In this paper, we evaluate feasibility of a 3-dimensional scanner that relies on Raman Spectroscopy to assess the entire margins of a resected specimen within clinically feasible time. We initially tested this device on a phantom sample that simulated positive tumour margins. This device first scans the margins of the sample and then depicts the margin status in relation to an automatically reconstructed image of the phantom sample. The device was further investigated on breast tissues excised from prophylactic mastectomy specimens. Our findings demonstrate immense potential of this device for automated breast tumour margin assessment to minimise repeat invasive surgeries.

Background: Lymphocyte-activation gene 3 (LAG-3) is a T-cell checkpoint regulator and a current target in immunotherapy trials. LAG-3's main ligand is MHC class II (MHC-II), to which it binds with higher affinity than CD4. Binding of LAG3 to MHC-II antigen-presenting cells negatively regulates cellular proliferation, activation, and homeostasis of T cells, similarly to CTLA-4 and PD-1, suggesting that antibodies targeting LAG-3 may demonstrate similar anti-tumor immune effects. Hypothesis: We recently reported an association of MHC-II on tumor cells and its involvement in mediating sensitivity to PD-1/PD-L1 monoclonal antibodies. MHC-II demonstrates a strong bimodal expression pattern on tumor cells from a variety of tissues, including those of the breast. In breast cancer patients, tumor-specific MHC-II expression on TNBCs is correlated with a 'hot' immune environment. We hypothesized that 1) MHC-II expression may drive potent anti-tumor immune responses and 2) MHC-II-positive tumors that generate immunotolerance may develop a specific immune checkpoint dependency on LAG-3, since LAG-3 is the inhibitory receptor for MHC-II-mediated antigen presentation. Methods: To determine the functionality of MHC-II in driving anti-tumor immune responses, we constitutively expressed the MHC-II master regulator CIITA in MMTV-neu mouse tumor cells and determined their ability to form tumors in immunocompetent syngeneic hosts. To evaluate the association of MHC-II+ tumors with LAG-3 expression, we evaluated LAG-3-positivity by immunohistochemistry (IHC) in lymphocytic infiltrates in a series of 111 post-NAC TNBC specimens from patients with residual disease remaining after presurgical chemotherapy. Tumor-infiltrating lymphocytes (TILs) were scored by H&E, PD-L1 and MHC-II (HLA-DR) were scored in the stroma and tumor compartments using automated quantitative immunofluorescence (AQUA). Results: Enforced expression of MHC-II via constitutive expression of CIITA caused rejection in 60% of mice, while only 11% of mice rejected MMTV-neu tumors expressing the vector control (Fisher's exact p=0.04). All rejecting mice were immune to rechallenge with parental (non-CIITA-expressing) MMTV-neu cells, suggesting a memory effector response. Clinically, 11/102 patients (10.8%) had LAG-3+ immune cells in their tumor. LAG-3+ tumors were strongly correlated with MHC-II positivity in tumor cells (p20%). When this subset was analyzed, LAG-3 positivity retained its association with tumor MHC-II expression (p=0.0001), while the association of LAG-3 with stromal PD-L1 was reduced below the level of significance (p=0.052). Conclusions: MHC-II expression causes increased immune activation in breast cancers, consistent with our previous findings. MHC-II positivity in breast tumors may identify a population with preferential dependence on the LAG-3 checkpoint, which may be important for future immunotherapy trials. Citation Format: Balko JM, Loi S, Giltnane JM, Combs S, Estrada MV, Sanchez V, Rimm D, Sanders ME, Salgado R, Gomez H, Johnson DB. MHC-II positive breast tumors are more immunogenic and may preferentially select for LAG-3-positive tumor immune infiltrates [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-10-02.

The presence of predicted neo-antigens in any given tumor is highly correlated with total somatic mutational burden of the same tumor genome. However, many potential neo-antigens are never transcribed, translated, or presented as antigens, partially because they lie in regions of the genome that are transcriptionally-repressed by cytosine methylation of promoter-CpG islands. Treatment with DNA methyltransferase inhibitors (DNMTi) can hypothetically lead to re-expression of many potential neo-antigens. Furthermore, the presence of neo-antigens have been linked to immunotherapy outcomes in patients. Our hypothesis is that tumors with reduced mutational burden can be maximized for neo-antigen presentation by activating transcription and translation of these sequences through DNMTi treatment. Our preliminary results showed that the DNMTi guadecitabine (SGI-110), a second-generation hypomethylating agent, treatment decreases methyl-cytosine in genomic DNA both in vitro and in vivo. In addition, SGI-110 treatment enhances MHC-II expression on murine mammary carcinoma MMTV-Neu cells upon IFN-γ stimulation in vitro and increases T-cell infiltration in vivo. Currently, we are performing whole-exome-sequencing and RNA-sequencing to track somatic mutations from DNA to RNA. We will further track somatic mutations from RNA→ MHC-presented peptide sequences using MHC immunoprecipitation followed by mass spectroscopy analysis. In addition, we will explore the role of guadecitabine therapeutic priming on response to αPD-L1 immunotherapy. These studies will provide a pre-clinical data to evaluate the potential for combined epigenetic and immune-therapy in a clinical trial for breast cancer. Citation Format: Luo N, Estrada VM, Sanders ME, Balko JM. Improving immunotherapy response by epigenetic modulation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-04-02.

Background: Amplification/overexpression of ERBB receptors and/or ligands has been associated with resistance to anti-HER2 therapies. Pan-HER is a mixture of six antibodies targeting each of the ERBB receptors, EGFR, HER2 and HER3, with synergistic pairs of antibodies. Each pair of antibodies simultaneously blocks ligand binding and/or induces target degradation, thus preventing compensatory mechanisms to anti-ERBB therapies. We examined the antitumor activity of Pan-HER against drug-sensitive and -resistant HER2+ breast cancer cells and xenografts. Results: Pan-HER exhibited potent growth inhibitory activity against a panel of HER2+ breast cancer cells (BT474, MDA-453, MDA-361, SUM190, HCC1954, UACC893 and SKBR3). Growth inhibition was associated with internalization and degradation of EGFR, HER2 and HER3. Pan-HER was superior to the combination of trastuzumab/pertuzumab (TP) against HER2+/PIK3CA mutant MDA-361, HCC1954, UACC893 and MDA-453 cells. We next compared the effect of Pan-HER against BT474, HCC1954 and MDA-361 xenografts established in nude mice to that of trastuzumab/lapatinib (TL), TP and T-DM1. All treatments were effective across the panel of xenografts. In mice with MDA-361 tumors, Pan-HER and TP were superior to TL. Immunoblot analysis showed significant downregulation of EGFR, HER2 and HER3 only in tumors treated with Pan-HER. After a complete response, treatment was discontinued. Among mice with BT474 xenografts treated with TP, TL and T-DM1, 25-50% of mice exhibited a tumor recurrence within 50 weeks of follow-up, while no recurrences were registered in mice treated with Pan-HER. Tumors recurring after TP and T-DM1 expressed significantly higher HER3 and P-HER3 protein levels and NRG1 mRNA levels. HCC1954 xenografts recurring after T-DM1 also overexpressed NRG1 mRNA compared to tumors before therapy. We next examined the effect of Pan-HER against trastuzumab-resistant HR6 (BT474) cells (Ritter et al. CCR 2007) and HCC1954 and UACC893 cells with acquired resistance to T-DM1 (TDR; IC50 >5-, >6- and 600-fold in HR6, UACC893-TDR and HCC1954-TDR cells, respectively, vs. parental cells). All T-DM1-resistant cells expressed significantly higher HER3 and P-HER3 protein levels and NRG1 mRNA and protein levels. Treatment with the HER3 neutralizing antibody LJM716 resensitized HR6 and HCC1954-TDR cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Mice with HR6 tumors were treated with Pan-HER, TL, TP and T-DM1. Only Pan-HER arrested HR6 tumor growth and downregulated EGFR, HER2, HER3, P-HER3 and P-AKT. Finally, HCC1954-TDR tumors rapidly grew in vivo despite treatment with T-DM1. Administration of Pan-HER to mice bearing HCC1954-TDR xenografts growing in the presence of T-DM1, induced rapid tumor regressions. Conclusions: These data suggest that multitarget therapeutic interventions, such as Pan-HER, which simultaneously remove and/or block all ERBB receptors and ligands, are a feasible and effective approach against HER2-overexpressing cancers both sensitive and resistant to anti-HER2 therapies. Citation Format: Schwarz LJ, Hutchinson KE, Estrada MV, Sanders ME, Dugger TC, Formisano L, Guerrero AL, Red-Brewer M, Young CD, Lantto J, Pedersen MW, Kragh M, Horak ID, Arteaga CL. Pan-HER, an antibody mixture with antitumor activity against drug-resistant HER2-overexpressing breast cancers with high ERBB ligand expression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-09.

Clinical trials have demonstrated the efficacy of combining phosphoinositide 3-kinase (PI3K) inhibitors with endocrine therapies in hormone therapy-refractory breast cancer. However, biomarkers of PI3K pathway dependence in ER+ breast cancer have not been fully established. Hotspot mutations in the alpha isoform of PI3K (PIK3CA) are frequent in ER+ disease and may identify tumors that respond to PI3K inhibitors. It is unclear whether PIK3CA mutations are the only biomarker to suggest pathway dependence and response to therapy. We performed correlative molecular characterization of primary and metastatic tissue from patients enrolled in a phase Ib study combining buparlisib (NVP-BKM-120), a pan-PI3K inhibitor, with letrozole in ER+, human epidermal growth factor-2 (HER2)-negative, metastatic breast cancer. Activating mutations in PIK3CA and inactivating MAP3K1 mutations marked tumors from patients with clinical benefit (≥6 months of stable disease). Patients harboring mutations in both genes exhibited the greatest likelihood of clinical benefit. In ER+ breast cancer cell lines, siRNA-mediated knockdown of MAP3K1 did not affect the response to buparlisib. In a subset of patients treated with buparlisib or the PI3Kα inhibitor alpelisib each with letrozole where PAM50 analysis was performed, nearly all tumors from patients with clinical benefit had a luminal A subtype. Mutations in MAP3K1 in ER+ breast cancer may be associated with clinical benefit from combined inhibition of PI3K and ER, but we could not ascribe direct biological function therein, suggesting they may be a surrogate for luminal A status. We posit that luminal A tumors may be a target population for this therapeutic combination.

Acquired resistance to CDK4/6 small molecule inhibitors in breast cancer arises through mechanisms that are yet uncharacterized. In this study, we used a kinome-wide siRNA screen to identify kinases which when downregulated yields sensitivity to the CDK4/6 inhibitor ribociclib. In this manner, we identified PDK1 as a key modifier of ribociclib sensitivity in estrogen receptor-positive MCF-7 breast cancer cells. Pharmacological inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, another CDK4/6 inhibitor, synergistically inhibited proliferation and increased apoptosis in a panel of ER+ breast cancer cell lines. Ribociclib-resistant breast cancer cells selected by chronic drug exposure displayed a relative increase levels of PDK1 and activation of the AKT pathway. Analysis of these cells revealed that CDK4/6 inhibition failed to induce cell cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E and D1, activated phospho-CDK2 and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib in combination with GSK2334470 or the PI3Kα inhibitor alpelisib decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K-PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors.

Purpose: Neoadjuvant chemotherapy (NAC), the standard of care for a subset of breast cancer patients, is known to have immunologic effects. With emerging data showing improved response rates with anti-PD-1/PD-L1 immunotherapy in combination with chemotherapy, the effects of NAC on systemic and local anti-tumor immunity require further study. Biomarkers of anti-tumor immunity are needed to identify which patients are most likely to respond to immunotherapy. Our previous work has shown that changes in the peripheral blood can be observed over the course of NAC for breast cancer. Peripheral blood biomarkers are attractive because of the relative ease of sampling compared to site of disease. Residual cancer burden (RCB) is a useful surrogate marker of long-term prognosis, as patients who experience a pathologic complete response (pCR) have better outcomes than those with residual disease (RD). Methods: We previously identified an 8 gene signature of cytotoxicity, derived from single cell RNA sequencing of PD-1Hi CD8+ T cells, which are enriched for tumor-reactive T cells. Using a custom NanoString panel, we tested expression of this gene signature in whole blood collected prior to definitive surgery in 88 breast cancer patients (TNBC, n=21; HER2+, n=17; ER+, n= 54; PR+, n=53) across two cohorts (VUMC, n=58; DFCI, n=30), 64 of whom had received NAC (pCR, n=11; RD, n=53). We further investigated peripheral blood gene expression using RNA sequencing (n=58; 34 post-NAC, 24 untreated). Results: In two cohorts of breast cancer patients, expression of the 8 gene signature (FGFBP2 + GNLY + GZMB + GZMH + NKG7 + LAG3 + PDCD1 - HLA-G) was highest in patients with RD who experienced a recurrence within three years compared to those with pCR (p Citation Format: Margaret L Axelrod, Paula I Gonzalez-Ericsson, Xiaopeng Sun, Riley E Bergman, Joshua Donaldson, Sara M Tolaney, Ian E Krop, Ana C Garrido-Castro, Melinda E. Sanders, Ingrid A Mayer, Justin M Balko. Peripheral blood gene signatures predict response to neoadjuvant chemotherapy in breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-06.

The breast cancer microenvironment comprises a complex stroma including tumor-infiltrating lymphocytes (TILs), which can either stimulate tumor progression or promote anti-tumor immunity in response to tumor-derived cues. In general, of all clinical subtypes, triple-negative breast cancer (TNBC) is characterized by the most extensive infiltration of TILs within tumor stroma, which is consistent with the observation that TNBC seems to clinically respond to immunotherapies at the highest rates. Immune checkpoint blockade (ICB), an immunotherapy that promotes prolonged activation of cytotoxic immune cells to mount robust anti-tumorigenic responses, has yielded limited success in treating breast cancer. IMpassion130 was the first clinical trial to indicate that combining anti-PD-L1 with standard-of-care chemotherapy (nab-paclitaxel) to treat TNBC increases progression-free survival in patients exclusively those with PD-L1 positive tumors. Furthermore, the KEYSTONE-522 trial showed that administering anti-PD-1 in addition to various neoadjuvant chemotherapies increased the pathologic complete response in early stage TNBC patients. Despite promising evidence for immunotherapy success, both clinical trials lacked an experimental ICB-only group, and thus cannot address the therapeutic benefit of ICB alone, or which chemotherapy combination would maximize this benefit. Finally, mechanisms of resistance to ICB in breast cancer remain unexplored. We sought to model ICB response in vivo to elucidate the mechanisms responsible for immunotherapy efficacy in breast cancer, explore the synergistic effects of ICB with chemotherapies, and model ICB resistance.In this study, we investigated the efficacy of anti-PD-L1 as single-agent or in combination with paclitaxel or doxorubicin in the EMT6 (Balb/c) orthotopic mammary tumor model. In this model, single-agent immunotherapy was efficacious in reducing primary tumor growth compared to combination treatment, with a small proportion of complete responses, whereas modest benefit was observed with either chemotherapy alone. Following two rounds of treatment, we analyzed the tumor-immune microenvironment by flow cytometry and gene expression analysis. Anti-PD-L1 alone or in combination with either chemotherapy enhanced infiltration of cytotoxic and effector T cell as well as natural killer cells into the tumor microenvironment. Using gene expression analysis, we observed elevated expression of myeloid recruitment and activation markers in combination-treated tumors, supporting a known role of chemotherapy-induced cell death in myeloid recruitment; however as chemotherapy did not add benefit to tumor response or survival, it is unclear if this effect is detrimental or supportive. Interestingly, completely responsive anti-PD-L1 treated tumors that eventually recurred retained resistance to ICB upon re-implantation in naïve recipient mice, suggesting that tumor-intrinsic factors may contribute to resistance.Herein, we explore an in vivo model that corroborates clinical response to combinatorial immunotherapy approaches in breast cancer patients. We report the immunogenic efficacy of single-agent ICB that upregulates tumoricidal immune cell infiltration into the primary tumor, thereby controlling tumor growth, albeit without achieving complete response in all mice. Additionally, post-therapy recurrent tumors retain resistance upon transplantation, indicating tumor-specific adaptive resistance. This study has potentially significant clinical implications for re-evaluating the contributions of chemotherapy in combination with ICB in TNBC patients. Citation Format: Ann Hanna, Paula I. Gonzalez-Ericsson, Violeta Sanchez, Melinda E. Sanders, Justin M. Balko. Evaluating the efficacy of immunotherapy in triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-14.

BACKGROUND Women with atypical hyperplasia (AH) on breast biopsy have a substantially increased risk of breast cancer (BC). Here the BC risk for the extent and subtype of AH is reported for 2 separate cohorts. METHODS All samples containing AH were included from 2 cohorts of women with benign breast disease (Mayo Clinic and Nashville). Histology review quantified the number of foci of atypical ductal hyperplasia (ADH) and atypical lobular hyperplasia (ALH). The BC risk was stratified for the number of AH foci within AH subtypes. RESULTS The study included 708 Mayo AH subjects and 466 Nashville AH subjects. In the Mayo cohort, an increasing number of foci of AH was associated with a significant increase in the risk of BC both for ADH (relative risks of 2.61, 5.21, and 6.36 for 1, 2, and ≥3 foci, respectively; P for linear trend = .006) and for ALH (relative risks of 2.56, 3.50, and 6.79 for 1, 2, and ≥3 foci, respectively; P for linear trend = .001). In the Nashville cohort, the relative risks of BC for ADH were 2.70, 5.17, and 15.06 for 1, 2, and ≥3 foci, respectively (P for linear trend < .001); for ALH, the relative risks also increased but not significantly (2.61, 3.48, and 4.02, respectively; P = .148). When the Mayo and Nashville samples were combined, the risk increased significantly for 1, 2, and ≥3 foci: the relative risks were 2.65, 5.19, and 8.94, respectively, for ADH (P < .001) and 2.58, 3.49, and 4.97, respectively, for ALH (P = .001). CONCLUSIONS In 2 independent cohort studies of benign breast disease, the extent of atypia stratified the long-term BC risk for ADH and ALH. Cancer 2016. © 2016 American Cancer Society.

Background: Activation of the phosphoinositide-3-kinase (PI3K) pathway has been associated with resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer. Recently, we reported a Stand Up 2 Cancer (SU2C) Phase Ib trial of buparlisib, an oral, reversible, pan-PI3K inhibitor in combination with the aromatase inhibitor letrozole in patients with metastatic ER+/HER2– breast cancer (n=51) who had previously progressed on endocrine therapy. In this study, 31% of patients demonstrated clinical benefit (CR, PR and SD ≥6 months; Mayer et al. JCO 2014). Clinical activity was observed in patients with PIK3CA-wild type (PIK3CA-WT) and PIK3CA-mutant (PIK3CA-MUT) tumors. We performed targeted next-generation sequencing (tNGS) to identify somatic alterations associated with clinical benefit to the combination therapy. Methods: tNGS was performed using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) in DNA extracted from tumors of 33 study patients (22 samples from archived primary/untreated and 11 from metastatic biopsies). Detected alterations were tested for association with clinical benefit (Fisher's exact test) and progression-free survival (PFS; log-rank test). For PFS analysis, patients were censored if they discontinued buparlisib for toxicity. Results: The most commonly detected alterations occurred in PIK3CA (36%), TP53 (30%), MAP3K1 (27%), GATA3 (24%), CCND1 (24%), CDH1 (21%) and PTEN (21%). Additional alterations of note included FGFR1 amplification (15%), MYC amplification (12%), ESR1 mutations (6%) and ERBB2 mutations (6%). Probable inactivating mutations occurring in MAP3K1 (MAP3K1-MUT) were significantly associated with improved clinical benefit, regardless of other mutations (6/9 patients, 67%, P=0.044). PIK3CA-MUT tumors trended toward greater clinical benefit (7/12, 58%, P=0.067). Patients with coexisting PIK3CA-MUT and MAP3K1-MUT tumors derived the largest clinical benefit (5/7, 70% P=0.07) compared to patients with only PIK3CA-MUT (2/5; 40%, P=1.0) or only MAP3K1-MUT tumors (1/2; 50%, P=1.0). Only 2/19 (11%) patients with PIK3CA-WT/MAP3K1-WT cancers benefitted from treatment. Both MAP3K1 and PIK3CA alterations were each also associated with increased PFS (p=0.03 and p=0.009, respectively). Three of 5 (60%) patients with tumors with FGFR1 amplification achieved clinical benefit (including a MAP3K1-MUT tumor and a PIK3CA-MUT/MAP3K1-MUT tumor), suggesting that FGFR1 may preferentially signal via PI3K and/or FGFR1 amplifications are not associated with resistance to the combination of aromatase inhibitors and PI3K inhibitors. Conclusions: Both MAP3K1 and PIK3CA are mutated at higher frequencies in luminal A breast cancer, suggesting that this alteration pattern (MAP3K1-MUT + PIK3CA-MUT) is a surrogate for low grade ER+ breast cancers with PI3K dependence. It is also possible that MAP3K1 mutations may predispose tumor cells to sensitivity to PI3K inhibition, but this speculation requires further investigation. Finally, patients with ER+/FGFR1-amplified cancers appeared to derive clinical benefit from combined therapy with letrozole and buparlisib. Citation Format: Balko JM, Hicks M, Berger MF, Solit DB, Bouvier N, Sanders ME, Estrada MV, Won H, Cantley LC, Mayer IA, Arteaga CL. Genomic alterations indicative of a luminal A subtype associate with clinical benefit to buparlisib and letrozole in endocrine-resistant ER+/HER2– 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 P3-07-41.

Background: Neoadjuvant chemotherapy (NAC) is frequently used in triple negative breast cancers (TNBC), and patients who achieve pathological complete response (pCR) following NAC have improved outcomes over those with residual disease (non-pCR). Unfortunately only 30% of TNBC patients achieve pCR, with no good treatment options for other 70% with non-pCR. Tumor infiltrating lymphocytes (TILs) in TNBC are predictive of pCR to NAC, and TILs found in the residual disease further prognosticate patients with residual disease into an improved prognosis subset. These data suggest there is an immune component to TNBC that might be affected by chemotherapy. Given the advent of immunotherapy trials in breast cancer, specifically in combination with NAC, there is an unmet need to gain a better understanding of the immune microenvironment in TNBC. Objective: We investigated the role of NAC on the immune microenvironment by examining breast cancer samples before (diagnostic biopsy) and after (residual disease) NAC, as these data are pertinent to the design and analysis of ongoing clinical trials in breast cancer combining immunotherapy with concurrent chemotherapy. Methods: RNA was extracted from 51 paired (pre- and post-NAC) breast cancer specimens with extensive residual disease, and analyzed by nCounter analysis for expression of >750 immune-related genes. Functional immune signature scores were generated, compared between matched pre- and post-NAC, and stratified by breast cancer molecular subtype (luminal (n=4), Her2-enriched (n=5), basal-like (n=17)). DNA extracted from a subset of these samples was utilized for T cell receptor sequencing to explore changes in T cell clonality following NAC in each subtype. Results: Across all subtypes of breast cancer, immune scores decreased after NAC consistent with a broad decrease in TILs observed histologically. When samples were stratified by molecular subtype using the PAM50 analysis, luminal A/B and basal-like patients demonstrated a decrease in immune signatures after NAC, while Her2-enriched patients exhibited a global increase in immune scores. Importantly, basal-like patients had the greatest immune signature changes, including decreases in T cell functions and CD8+ T cell, T helper and T regulatory signatures (p>0.05). Conversely, these signatures were increased after NAC in the Her2-enriched molecular subtype. Analyses of T-cell clonality are ongoing, but should yield insight into the effect of NAC on the landscape of effector T-cells in the micro-environment. Conclusions: Our work suggests that NAC decreases immune infiltrate signatures, specifically related to effector T cells in patients who do not achieve pCR. However, we observed an increase after NAC in the same signatures in Her2-enriched patients, suggesting that these patients' tumors may respond differently to chemotherapy on the immune-molecular level. As clinical trials progress in TNBC with the combination of chemotherapy and immune therapy, more work is needed to understand who might benefit from these therapy regimens. Citation Format: Hicks MJ, Estrada MV, Sanders ME, Salgado R, Cook RS, Arteaga CL, Balko JM. Differential effect of chemotherapy on immune gene expression signatures based on molecular subtype of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-03.

Background: Patients undergoing thyroidectomy may have inadvertent damage or removal of the parathyroid gland(s) due to difficulty in real-time parathyroid identification. Near-infrared autofluorescence (NIRAF) has been demonstrated as a label-free modality for intraoperative parathyroid identification with high accuracy. This study presents the translation of that approach into a user-friendly clinical prototype for rapid intraoperative guidance in parathyroid identification. Methods: A laboratory (lab)-built spectroscopy system that measures NIRAF in tissue was evaluated for identifying parathyroid glands in vivo across 162 patients undergoing thyroidectomy and/or parathyroidectomy. Based on these results, a clinical prototype called PTeye was designed with a user-friendly interface and subsequently investigated in 35 patients. The performance of the lab-built system and the clinical prototype were concurrently compared side by side by a single user with 20 patients in each group. The influence of (i) intrapatient and interpatient variability of NIRAF in thyroid and parathyroid glands and (ii) thyroid and parathyroid pathology on intraoperative parathyroid identification were investigated. The effect of blood on NIRAF intensity of parathyroid and thyroid was tested ex vivo with the PTeye system to assess if a hemorrhagic surgical field would affect parathyroid identification. Accuracy of both systems were determined by correlating the acquired data with either visual confirmation by a surgeon for unexcised parathyroid glands or histology reports for excised parathyroid glands. Results: The overall accuracy of the lab-built system in guiding parathyroid identification was 92.5%, while the PTeye system achieved an accuracy of 96.1%. Unlike the lab-built system, the PTeye could guide parathyroid identification even as the operating room lights remained on and required only 25% of the laser power used by the lab-built setup. Parathyroid glands had elevated NIRAF intensity compared to thyroid and other neck tissues, regardless of thyroid or parathyroid pathology. Blood did not seem to affect tissue NIRAF measurements obtained with both systems. Conclusion: In this study, the clinical prototype PTeye demonstrated high accuracy for label-free intraoperative parathyroid identification. The intuitive interface of the PTeye that can guide in identifying parathyroid tissue in the presence of ambient room lights suggests that it is a reliable and easy-to-use tool for surgical personnel.

Background Difficulty in identifying the parathyroid gland during neck operations can lead to accidental parathyroid gland excisions and postsurgical hypocalcemia. A clinical prototype called as PTeye was developed to guide parathyroid gland identification using a fiber-optic probe that detects near-infrared autofluorescence from parathyroid glands as operating room lights remain on. An Overlay Tissue Imaging System was designed concurrently to detect near-infrared autofluorescence and project visible light precisely onto parathyroid gland location. Methods The PTeye and the Overlay Tissue Imaging System were tested in 20 and 15 patients, respectively, and a modified near-infrared imaging system was investigated in 6 patients. All 41 patients underwent thyroidectomy or parathyroidectomy. System accuracy was ascertained with surgeon's visual confirmation for in situ parathyroid glands and histology for excised parathyroid glands. Results There was no observable difference between near-infrared autofluorescence of healthy and diseased parathyroid glands. The PTeye identified 98% of the parathyroid gland, whereas the near-infrared imaging system and the Overlay Tissue Imaging System identified 100% and 97% of the parathyroid glands, respectively. Conclusion The PTeye can guide in real-time parathyroid gland identification even with ambient operating room lights. The near-infrared imaging system performs parathyroid gland imaging with high sensitivity, whereas the Overlay Tissue Imaging System enhances parathyroid gland visualization directly within the surgical field without requiring display monitors. These label-free technologies can be valuable adjuncts for identifying parathyroid glands intraoperatively.

Potentiating anti-tumor immunity by inducing tumor inflammation and T cell-mediated responses are a promising area of cancer therapy. Immunomodulatory agents that promote these effects function via a wide variety of mechanisms, including upregulation of antigen presentation pathways. Here, we show that major histocompatibility class-I (MHC-I) genes are methylated in human breast cancers, suppressing their expression. Treatment of breast cancer cell lines with a next-generation hypomethylating agent, guadecitabine, upregulates MHC-I expression in response to interferon-γ. In murine tumor models of breast cancer, guadecitabine upregulates MHC-I in tumor cells promoting recruitment of CD8+ T cells to the microenvironment. Finally, we show that MHC-I genes are upregulated in breast cancer patients treated with hypomethylating agents. Thus, the immunomodulatory effects of hypomethylating agents likely involve upregulation of class-I antigen presentation to potentiate CD8+ T cell responses. These strategies may be useful to potentiate anti-tumor immunity and responses to checkpoint inhibition in immune-refractory breast cancers., Immunotherapy often fails as a single option treatment in cancer. Here, the authors show that targeting of DNA methyltransferases, such as DNMT1, can potentiate anti-tumor immunity and response to checkpoint inhibition by increasing MHC gene expression and the recruitment of CD8+ T cells.

Background: Current evidence indicates that patients with Human Epidermal Growth Factor Receptor 2 (HER2)-positive Breast Cancer (BC) benefit from HER2-targeted therapies. Accurate determination of HER2 status is critical to ensure that the correct patients are offered targeted treatment while patients with HER2-negative tumors–who are unlikely to benefit from anti-HER2 therapy– are spared from the adverse effects of these costly drugs. Guidelines for performance and reporting of HER2 testing, first published in 2007 by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP), were updated in October 2013. The new reporting criteria are based on a combination of HER2:CEP17 ratio and average HER2 copy number. Methods: We retrospectively reviewed HER2 dual probe FISH test results sequentially performed by the Cytogenetics Laboratory at Vanderbilt University from January to May 2014 since implementation of the updated guidelines. The cases were then rescored using the 2007 guidelines. The clinicopathologic features of cases with a change in Her2 status after scoring with the 2013 guidelines were examined for statistical significance. Results: If the 266 performed HER2 FISH tests had been scored using the 2007 guidelines the results would have been the following: amplified (n = 44, 16%), equivocal (n = 28, 10%) and not amplified (n = 194, 70%). However, using the 2013 guidelines the cases were actually reported as follows: amplified (n=57, 21%), equivocal (n=22, 8.3%) and not amplified (n=187, 68%). Additionally, 18 cases demonstrated genetic heterogeneity in >25% of cells. The updated guidelines resulted in a change in Her2 status in 12% of tests (32/266; p less than 0.0001); 13 changed from equivocal to amplified, 13 cases changed from not amplified to equivocal and 6 cases changed from equivocal to not amplified. Cases with a change in Her2 status following implementation of the new guidelines were more likely to demonstrate genetic heterogeneity (28% [9/32] vs. 4% [9/234]; p less than 0.0001). Furthermore, hormone receptor (HR)-negative tumors scored as not amplified by the 2007 guidelines were more likely to undergo an upgrade in HER2 status under the 2013 guidelines than HR-positive tumors (26% [11/41] vs. 7% [11/153], p less than 0.0001). Conclusions: The 2013 reporting criteria, based on a combination of HER2:CEP17 ratio and average HER2 copy number, increase the number of patients eligible for HER2-targeted therapies while decreasing equivocal results. Tumors that demonstrate genetic heterogeneity for Her2 or are HR-negative account for a significant percentage of these newly eligible cases. Correlation with clinical response is required to confirm the proposed improved analytical validity of the updated guidelines. Clinical trials to evaluate the benefit of anti-Her2 therapy in patients with genetic heterogeneity are in the planning phase. Citation Format: Monica V Estrada, Jena M Giltnane, Ferrin C Wheeler, Ashwini Yenamandra, Vandana Abramson, Ingrid A Mayer, Julie Means, Brent Rexer, Ingrid M Meszoely, Melinda E Sanders. Genetic heterogeneity for Her2 accounts for a significant percentage of breast cancers changing Her2 status following implementation of the 2013 CAP/ASCO HER2 reporting guidelines [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-32.

Backgound: Tumor-infiltrating lymphocytes (TILs) are associated with improved prognosis in TNBCs, with several retrospective analyses demonstrating that TNBCs with high baseline TILs have higher rates of pathologic complete response (pCR) to NAC. Moreover, the TIL burden in the RD of patients who do not achieve pCR to NAC is also correlated with prognosis. However, insight into the molecular pathways in TNBC which modulate heterogeneity in host anti-tumor immune responses is lacking. To address this gap in knowledge, we analyzed TILs retrospectively in a cohort of clinically and molecularly characterized TNBCs with RD after NAC. Methods: TILs were scored in H&E stained slides by expert pathologists in the post-treatment tumors of 92 NAC-treated TNBC patients with RD at the time of resection and in 44 matched baseline diagnostic biopsies. Genomic alterations in the RD were assayed using targeted next-generation sequencing (tNGS) while selected transcriptional signatures were evaluated by NanoString as previously published (Balko et al, Cancer Discovery 2014). Differences in pre- and post-NAC TILs were compared between tumors harboring alterations in cell cycle, PI3K/mTOR, growth factor receptors, Ras/MAPK and DNA repair pathways. Associations of TILs with transcriptional signatures were also tested. Results: A strong positive association of TILs in NAC-treated specimens was observed with RFS (coxPH p=0.0001, relative risk reduction of 3.4% for each % of TILs) and OS (p=0.0016; relative risk reduction of 2.8% for each % of TILs). In multivariate analysis with stage, age, node status and RD tumor cellularity, TILs in the post-NAC disease remained a significant predictor of RFS and OS (p=0.0008 and p=0.007, respectively). TILs tended to decrease with NAC in paired samples, although this decrease was not statistically significant (p=0.07). Genetic alterations in the Ras/MAPK (amplifications in KRAS, BRAF, RAF1 and truncations in NF1) and cell cycle pathway (alterations in CCND1-3, CDK4, CDK6, CCNE1, RB, AURKA and CDKN2A) were associated with lower TILs in RD (p=0.005 and p=0.05, respectively). A significant inverse linear correlation was detected between a transcriptional signature of Ras/MAPK activation (Pratilas et al, PNAS 2009) and TILs in the RD (Spearman’s r=-0.42; p=0.00028). Total number of alterations of likely functional significance detected by tNGS showed no association with TILs, suggesting that the association of Ras/MAPK deregulation and cell cycle alterations with TILs may be a pathway-specific effect. In TNBC cell lines, chemical inhibition of MEK transcriptionally up-regulated MHC-I and MHC-II molecules, while simultaneously down-regulating mRNA expression of the immune checkpoint inhibitor PD-L1 (MDA-231 p=0.00002, BT549 p=0.0003, and SUM159PT p=0.009). In vivo experiments confirming these associations are underway. Conclusions: Our data suggest a strong correlation of Ras/MAPK pathway activation with immune-evasion and outcome in TNBC. With additional mechanistic understanding, rational design of clinical trials combining MEK inhibitors with PD-L1 antibodies in TNBC may be warranted. Citation Format: Justin M Balko, Carsten Denkert, Roberto Salgado, Martin O'Hely, Peter Savas, Paul A Beavis, Phil K Darcy, Susan Combs, David L Rimm, Jennifer M Giltnane, Monica V Estrada, Melinda E Sanders, Rebecca S Cook, Kai Wang, Vincent A Miller, Phillip J Stephens, Roman Yelensky, Joseph A Pinto, Franco Doimi, Henry Gomez, Carlos L Arteaga, Sherene Loi. Reduced tumor lymphocytic infiltration in the residual disease (RD) of post-neoadjuvant chemotherapy (NAC) triple-negative breast cancers (TNBC) is associated with Ras/MAPK activation and poorer survival [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S1-08.

The standard of care for breast cancer patients includes treatment with chemotherapy, antiestrogens, and targeted inhibitors. There are more than 100 drugs approved to treat breast cancers with more in the pipeline of discovery and approval; however, few biomarkers have been identified for individualized, patient-specific treatment planning. Currently, drug regimens are chosen based on the presence or absence of specific receptors, including progesterone receptor (PR), estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER2) and patient response is assessed by changes in tumor size weeks after treatment. Unfortunately, many patients do not initially respond to therapy or develop a resistance, and face a greater risk of recurrence and death. Herein we present a novel, light-based technology that can accurately predict individual tumor response to anti-cancer drugs based on drug-induced changes in metabolism. Cellular metabolism is a powerful indicator response to treatment, because the oncogenic drivers targeted by therapeutic agents often regulate cellular metabolism. In this study, we demonstrate the sensitivity of optical metabolic imaging to predict therapeutic response in xenografts in vivo, and in primary tumor derived organoids. Optical metabolic imaging utilizes mulitphoton fluorescence intensity and fluorescence lifetime imaging to probe the concentrations and protein-binding dynamics of cellular NADH and FAD, two coenzymes of metabolism. A composite endpoint, the OMI index, provides a robust, dynamic readout of cellular metabolism. First, we probed metabolic changes within ER+/HER2+ tumors treated with trastuzumab, an anti-HER2 antibody. After 48h of a single injection of trastuzumab (HER2 antibody), the OMI index of responsive tumors (ER+/HER2+) decreased (p Citation Format: Alex J Walsh, Rebecca S Cook, Melinda E Sanders, Carlos L Arteaga, Melissa C Skala. Optical metabolic imaging predicts therapeutic response in breast cancer tumors and organoids [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-38.

Objectives To determine if combined measurements from dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and diffusion weighted MRI (DW-MRI), obtained before and after the first cycle of neoadjuvant chemotherapy (NAC), are superior to single parameter measurements for predicting pathological complete response (pCR) in breast cancer patients.

In previous work, we demonstrated the presence of hydroxyapetite (type II microcalcification), HAP, in triple negative MDA-MB-231 breast cancer cells. We used (18)F-NaF to detect these types of cancers in mouse models as the free fluorine, (18)F(-), binds to HAP similar to bone uptake. In this work, we investigate other bone targeting agents and techniques including (99m)Tc-MDP SPECT and Osteosense 750EX FMT imaging as alternatives for breast cancer diagnosis via targeting HAP within the tumor microenvironment.Thirteen mice were injected subcutaneously in the right flank with 10(6) MDA-MB-231 cells. When the tumor size reached ~0.6 cm(3), mice (n=9) were injected with ~37 MBq of (99m)Tc-MDP intravenously and then imaged one hour later in a NanoSPECT/CT or injected intravenously with 4 nmol/g of Osetosense 750EX and imaged 24 hours later in an FMT (n=4). The imaging probe concentration in the tumor was compared to that of muscle. Following SPECT imaging, the tumors were harvested, sectioned into 10 μm slices, and underwent autoradiography or von Kossa staining to correlate (99m)Tc-MDP binding with HAP distribution within the tumor. The SPECT images were normalized to the injected dose and regions-of-interest (ROIs) were drawn around bone, tumor, and muscle to obtain the radiotracer concentration in these regions in units of percent injected dose per unit volume. ROIs were drawn around bone and tumor in the FMT images as no FMT signal was observed in normal muscle.Uptake of (99m)Tc-MDP was observed in the bone and tumor with little or no uptake in the muscle with concentrations of 11.34±1.46 (mean±SD), 2.22±0.95, and 0.05±0.04%ID/cc, respectively. Uptake of Osteosense 750EX was also observed in the bone and tumor with concentrations of 0.35±0.07 (mean±SD) and 0.04±0.01picomoles, respectively. No FMT signal was observed in the normal muscle. There was no significant difference in the bone-to-tumor ratio between the two modalities (5.1±2.3 for SPECT and 8.8±2.2 for FMT) indicating that there is little difference in tumor uptake between these two agents.This study provides evidence of the accessibility of HAP within the breast tumor microenvironment as an in vivo imaging target for bone-seeking agents. SPECT imaging using (99m)Tc-MDP can be rapidly translated to the clinic. FMT imaging using Osteosense 750EX is not currently approved for clinical use and is limited to animal research.

Purpose: To develop and evaluate a new method for detecting calcium deposits using their characteristic magnetic susceptibility effects on magnetic resonance (MR) images at high fields and demonstrate its potential in practice for detecting breast microcalcifications. Methods: Characteristic dipole signatures of calcium deposits were detected in magnetic resonance phase images by computing the cross-correlation between the acquired data and a library of templates containing simulated phase patterns of spherical deposits. The influence of signal-to-noise ratio and various other MR parameters on the results were assessed using simulations and validated experimentally. The method was tested experimentally for detection of calcium fragments within gel phantoms and calcium-like inhomogeneities within chicken tissue at 7 T with optimized MR acquisition parameters. The method was also evaluated for detection of simulated microcalcifications, modeled from biopsy samples of malignant breast cancer, inserted in silico into breast magnetic resonance imaging (MRIs) of healthy subjects at 7 T. For both assessments of calcium fragments in phantoms and biopsy-based simulated microcalcifications in breast MRIs, receiver operator characteristic curve analyses were performed to determine the cross-correlation index cutoff, for achieving optimal sensitivity and specificity, and the area under the curve (AUC), for measuring the method’s performance. Results: The method detected calcium fragments with sizes of 0.14–0.79 mm, 1 mm calcium-like deposits, and simulated microcalcifications with sizes of 0.4–1.0 mm in images with voxel sizes between (0.2 mm)3 and (0.6 mm)3. In images acquired at 7 T with voxel sizes of (0.2 mm)3–(0.4 mm)3, calcium fragments (size 0.3–0.4 mm) were detected with a sensitivity, specificity, and AUC of 78%–90%, 51%–68%, and 0.77%–0.88%, respectively. In images acquired with a human 7 T scanner, acquisition times below 12 min, and voxel sizes of (0.4 mm)3–(0.6 mm)3, simulated microcalcifications with sizes of 0.6–1.0 mm were detected with a sensitivity, specificity, and AUC of 75%–87%, 54%–87%, and 0.76%–0.90%, respectively. However, different microcalcification shapes were indistinguishable. Conclusions: The new method is promising for detecting relatively large microcalcifications (i.e., 0.6–0.9 mm) within the breast at 7 T in reasonable times. Detection of smaller deposits at high field may be possible with higher spatial resolution, but such images require relatively long scan times. Although mammography can detect and distinguish the shape of smaller microcalcifications with superior sensitivity and specificity, this alternative method does not expose tissue to ionizing radiation, is not affected by breast density, and can be combined with other MRI methods (e.g., dynamic contrast-enhanced MRI and diffusion weighted MRI), to potentially improve diagnostic performance.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecological system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these micro-environments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases.

Fibroepithelial lesions with cellular stroma are frequently termed cellular fibroadenomas although criteria for distinguishing them from a phyllodes tumor are vague and subjective. However, the clinical implications and surgical management for these 2 lesions may be different. We randomly selected 21 cases of fibroepithelial lesions sent in consultation to the senior author that were challenging to classify as cellular fibroadenoma or phyllodes tumor. One to 2 representative slides of each case along with patient age were sent to 10 pathologists who specialize in breast pathology. The World Health Organization criteria for phyllodes tumors and a diagnosis form were included with the study set. For the purposes of data reporting, fibroadenoma and cellular fibroadenoma are considered together. In only 2 cases was there uniform agreement as to whether the tumor represented a fibroadenoma or phyllodes tumor. Of the remaining 19 cases, if the diagnoses of fibroadenoma and benign phyllodes tumor were combined and separated from borderline and malignant phyllodes tumors, there was 100% agreement in 53% of cases and 90% agreement in 79% of cases. This study highlights the difficulty that exists in distinguishing some cellular fibroadenomas from phyllodes tumors even for pathologists who specialize in breast pathology. However, there appears to be considerable agreement when cellular fibroadenomas and benign phyllodes tumors are distinguished from borderline and malignant phyllodes tumors. Further studies are needed to determine if there is a clinically significant difference between cellular fibroadenomas and benign phyllodes tumors and how to better distinguish them from borderline and malignant phyllodes tumors.