Liu, MC

Abstract Background: Late recurrence is characteristic of ER+ breast cancers. Despite an apparently effective adjuvant endocrine therapy, many breast cancers recur years after their initial endocrine treatment. Why some tumors recur early (<3 years) and some recur later (>5 years) is poorly understood. If systemic endocrine therapies killed all cells, recurrence would reflect only the appearance of new disease. Thus, we hypothesized that cells that survive and lie dormant may be driven, in part, by altered wiring of their cell death signaling. We, therefore, studied how cell death signaling is differentially wired in primary tumors that will recur early versus those that will recur later. Method: Genes involved in apoptosis, autophagy, ferroptosis, necrosis, and pyroptosis were identified from KEGG to initiate network feature analysis of gene expression data from public and our first in-house gene expression dataset. Data were collected from ER+ breast cancer pre-endocrine treatment samples with up to 20 years follow-up. Publicly available datasets used were GSE6532, GSE2034, GSE7390, GSE17705, GSE12093, and TCGA. We applied our Knowledge-fused Differential Dependency Network (KDDN) analysis tool to the public datasets; KDDN has provided powerful new insights into signaling in breast and other cancers. Common gene-gene interactions (edges) predicted in at least two different datasets were extracted from all KDDN analyses results. To strengthen the relevance of these features, predicted network edges that represent known protein-protein interactions (PPI) were identified from the STRING database, and these edges were noted in the signaling graphs. Final network graphs were constructed using the common edges from all overlaid networks. We conducted IPA analysis on all nodes in the final network and selected those incorporating network hubs. We took a similar approach to our second in-house dataset, which we used for independent testing. Here, patients were included if their tumor exhibited an initial reduction in volume of at least 40% by four months in response to neo-adjuvant Letrozole. Patients were then classified into two groups during follow-up of up to 3.7 years: i) initial tumor size reduction followed by continued response (expected to recur late); ii) initial reduction followed by tumor regrowth (expected to recur early). KDDN analysis was performed on pretreatment samples from these two groups and a network created annotated with PPI information. Results: MAPK8 and CYCS (Molecular Mechanisms of Cancer, p=1.58E-52), TNFRSF1A Neuroinflammation Signaling Pathway, p=1.26E-54), RELA, and NFKB1 (Colorectal Cancer Metastasis Signaling, p=7.94E-35), were identified as hubs. Hubs may be critical signaling components driving the differences between tumors that will become dormant and recur late. Connections between SLC25A6 and SQSTM1 (p = 0.008), BIRC2 and GABARAP (p = 0.021) in the early group, and AKT3 and IRS2 (p = 0.014) in the late group, were shared between the two final networks. With longer follow-up time on the second in-house dataset, we will better define the two groups and identify additional common phenotype specific gene-gene interactions. Citation Format: Clarke R, Dixon M, Jin L, Pearce D, Turnbull A, Selli C, Hu R, Zwart A, Wang Y, Xuan J, Sengupta S, Sims A, Liu MC. Local network topology differences between early and late recurrence in ER+ breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-17.

Abstract Background: The live attenuated non-pathogenic Edmonston MV vaccine strain has advantages as an oncolytic platform given its tumor specificity, potent bystander effect, and ability to be engineered and retargeted. MV-NIS expresses the human thyroidal sodium-iodide symporter (NIS) and is selectively oncolytic, entering tumor cells through CD46 (overexpressed on many cancers, including breast cancer of all subtypes) and Nectin-4. NIS expression in MV-NIS infected cells permits noninvasive monitoring of virus spread by SPECT-CT imaging of Tc-99m pertechnetate or I-123 uptake. Methods: NCT01846091 is a standard 3+3 phase I trial of a single IT administration of MV-NIS in pts with recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN) or metastatic breast cancer (MBC). Primary objectives are (a) safety and tolerability and (b) maximally tolerated single dose. The secondary clinical objective is to preliminarily assess antitumor efficacy at and away from the MV injection site. Key eligibility criteria were: absence of standard therapy with life prolonging intent; at least one lesion >1 cm amenable to percutaneous injection; and no impending visceral crisis. MV-NIS was administered on D1 with mandatory SPECT-CT at baseline (BL) and on D3&D8; repeat SPECT-CT on D15&D21 if the prior result was positive; mandatory tumor biopsies on D3&D21; optional tumor biopsies on D8&D15; assessments for viremia and viral shedding at BL and on D3,D8,D15,D21; and standard imaging for restaging at BL,D21,W6,W12. Results: Accrual completed with 12 evaluable pts (6 SCCHN and 6 MBC) at 3 dose levels (108, 3x108, 109 TCID50). The MBC group included 5 HR+/HER2- pts and 1 pt with mixed HR+/HER2- and HR+/HER2+ disease. 5 pts had evidence of disease progression prior to study participation. No dose limiting toxicities were observed among the MBC pts; AEs possibly related to MV-NIS in this group were gr2 fatigue, gr1 flu-like illness, gr2 lymphopenia (all n=1). No SCCHN responses were observed. Best response for the MBC pts was: stable disease (SD) >6 wks, n=4; clinical response, n=1; progression, n=1. One MBC pt with SD for 12 wks had positive SPECT/CT imaging at and away from the injection site on D3&D8 and was the only pt seronegative for measles IgG antibodies prior to MV-NIS exposure. The MBC pt who responded after initial MV-NIS exposure was the only pt with low viral RNA in blood (D3); she received additional doses at W9&W13 without toxicity through an expanded access protocol exemption and had disease progression by W19. No viral shedding was detected from mouth rinse or urine in any pt. MV was detected in tumor samples from all pts treated at the highest dose level. Additional blood and tissue analyses are in progress. Conclusion: These results demonstrate the safety of IT MV-NIS administration, provide early evidence of biologic activity in MBC, and support the possibility of viral replication in tumors remote from the IT injection site. A MV strain encoding the immunomodulatory neutrophil activating protein transgene has been constructed (MV-s-NAP) with preclinical evidence of improved antitumor activity and immunogenicity. The phase I MV-s-NAP trial will start recruitment in Fall 2018. Citation Format: Liu MC, Peng K-W, Federspiel MJ, Russell SJ, Brunton BA, Zhou Y, Packiriswamy N, Hubbard JM, Loprinzi CL, Peethambaram PP, Ruddy KJ, Allred JB, Galanis E, Okuno SH. Phase I trial of intratumoral (IT) administration of a NIS-expressing derivative manufactured from a genetically engineered strain of measles virus (MV) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-21-03.

Abstract Cyclin dependent kinase 4 and 6 inhibitors (CDK4/6i) are standard first line (1L) and second line (2L) treatments of ER+ MBC. However, the optimal treatment strategy after progression on a CDK 4/6i is unknown. Given concern for rapid disease progression after discontinuation of CDK4/6is (Bashour, J Cancer 2017), further data on responses to subsequent lines of therapy post CDK4/6i are needed. We performed a single institution retrospective review of patients (pts) with ER+ MBC who received 1L or 2L CDK4/6i to examine the prescribing patterns and clinical responses to post-CDK4/6i treatment. Methods: We identified 136 ER+ MBC pts prescribed a CDK4/6i at Mayo Clinic Rochester (from 12/2014 to 2/2018) on 1L or 2L who received at least 30 days of therapy and ≥ two clinic visits during treatment. For the 1L and 2L cohorts we assessed the time to treatment failure during (TTF-1) and after CDK4/6i therapy (TTF-2). TTF was defined as time from start of therapy to discontinuation for any cause. Additionally, we assessed overall survival (OS) post CDK4/6i discontinuation (defined as the date of TTF-1 to death or last follow up). Results: The study cohort included 81 and 55 pts treated with 1L and 2L CDK4/6is, respectively. In the 1L cohort, palbociclib/letrozole (82.3%) and palbociclib/fulvestrant (13.6%) were most commonly prescribed. Treatment was discontinued in 39/81 pts due to: progression (84.6%), intolerability (7.7%), and other (7.7%). The median TTF-1 was 19.7 mo (95% CI 11.2 – 25.4 mo). Subsequent treatment and response data were available in 37/39 pts that progressed on 1L CDK4/6i. The most commonly prescribed regimens included single-agent hormonal therapy (29.7%), everolimus/exemestane (27.0%), and single agent chemotherapy (21.7%). The overall median TTF-2 were as follows: everolimus/exemestane 13.2 mo [95%CI 0.33 mo – not reached (NR)]; single agent hormonal therapy 3.1 mo (95%CI 1.4 – 5.4 mo); and single agent chemotherapy 4.1 mo (95%CI 1.4 - 5.4 mo). With a median post-CDK4/6i follow up of 11.2 mo, we observed 8 deaths and the median OS-post CDK4/6i was NR. In the 2L cohort, common regimens were palbociclib/letrozole (63.6%) and palbociclib/fulvestrant (27.3%). Treatment was discontinued in 30/55 pts due to: progression (73.3%), intolerability (13.3%), and other (13.3%). The median TTF-1 in the 2L cohort was 8.3 mo (95%CI 4.6 – 12.7 mo). Subsequent treatment data were available in 24/30 pts and included single-agent hormonal therapy [n=7 (29.2%); median TTF-2 4.7 mo; 95%CI 1.9 – 14.0 mo], everolimus/exemestane [n=6 (25.0%); median TTF-2 3.2 mo, 95% CI 0.8-10.1 mo)] and single agent chemotherapy [n=6 (25.0%); median TTF-2 2.6 mo; 95%CI 1.7 mo – NR)]. With a median post-CDK4/6i follow up 18.7 mo in the 2L cohort, we observed 11 deaths, and the median OS-post CDK4/6i was 11.8 mo (95%CI 11.8 mo – NR). Conclusions: Single and combination-based endocrine strategies are commonly administered post-CDK4/6i without clinical evidence for rapid deterioration. The observation of a median treatment duration lasting >12 months in patients receiving everolimus/exemestane post-CDK4/6i supports ongoing studies evaluating drugs that target PI3K/mTOR pathway in combination and following progression on a CDK4/6i. Citation Format: Giridhar KV, Choong GM, Leon-Ferre RA, O'Sullivan CC, Ruddy KJ, Haddad TC, Hobday TJ, Peethambaram PP, Moynihan TJ, Loprinzi CL, Liu MC, Suman VJ, Goetz MP. Clinical management of metastatic breast cancer (MBC) after CDK 4/6 inhibitors: A retrospective single-institution study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-09.

Abstract Background: The LAR subtype is a genomically distinct subset of TNBC. Using a large cohort of non-metastatic TNBC patients (pts) with long term follow-up, we sought to further characterize the clinicopathologic features and outcomes of LAR vs non-LAR TNBC. Methods: From a cohort of 9982 women with surgically-treated non-metastatic breast cancer, 605 met criteria for TNBC (ER/PR<1% and HER2-negative) by central pathology. RNA extracted from 304 FFPE tumor specimens using the HighPure RNA extraction kit was subjected to TruSeq RNA Access library preparation and sequencing on a HiSeq2500. Adequate RNA was available for 283 pts. Tumors were classified as LAR or non-LAR using a shrunken centroid model, CABAL (Clustering Among BAsal and Luminal androgen receptor). In addition to previously described analyses [Leon-Ferre et al, Breast Cancer Res Treat 2017], immunohistochemical (IHC) androgen receptor (AR) staining was performed and the impact of various parameters on invasive disease-free survival (IDFS) and overall survival (OS) was assessed using Cox proportional hazards models. Results: 58 (20%) tumors were classified as LAR and 225 (80%) as non-LAR. Compared to non-LAR, LAR pts were older (mean age 65 vs 54) and more often postmenopausal (79%vs53%), both p=0.01. Apocrine histology was more common among LAR tumors (21%vs0%), which were also lower grade (grade3: 69%vs95%) and had lower Ki-67 (Ki-67>15%: 64%vs82%), all p<0.01. Additionally, LAR tumors had lower median stromal tumor infiltrating lymphocytes (TILs, 20%vs25%) and were less frequently lymphocyte-predominant [≥50% stromal or intratumoral TILs (19%vs32%)], although neither reached statistical significance. AR IHC was available for 223 of 283 tumors. Median AR IHC score in LAR was 65% (range 0-100%) vs 0% (range 0-90%) in non-LAR. T/N stage, surgery type, and receipt of adjuvant chemotherapy (AdjCT) or radiotherapy were similar between LAR and non-LAR. LAR pts had shorter IDFS and OS compared to non-LAR (5.6 vs 11.8 yrs and 10.8 vs 20.8 yrs, respectively), although this did not reach statistical significance. Test of proportional hazard assumption was not significant for IDFS or OS (p = 0.30 and 0.09). IDFS estimates were numerically higher in LAR vs non-LAR (80.2%vs70.5%,p = 0.92) at 3yrs post-diagnosis; whereas the opposite was true (40.9%vs55.6%,p = 0.07) after 10yrs. OS estimates at 3 and 5yrs were similar between LAR and non-LAR, but at 10yrs OS was inferior in LAR (40.9%vs66.4%,p = 0.24). In a univariate analysis including both LAR and non-LAR, older age, higher N stage, lower TILs and absence of AdjCT were associated with poorer IDFS and OS. In a multivariate analysis, higher N stage and absence of AdjCT remained associated with both poorer IDFS and OS; while lower stromal TILs were associated with poorer IDFS (p=0.01), and with a trend towards poorer OS (p=0.07). Conclusions: LAR TNBCs occurred in older women, were lower grade, and had lower TIL density than nonLAR tumors. While significant differences in IDFS or OS were not demonstrated, LAR pts exhibited a numerically lower risk of a disease event at 3yrs, but higher risk by 10yrs compared to nonLAR pts. In the entire cohort, higher N stage, absence of AdjCT and lower TILs were independently associated with poorer outcomes. Citation Format: Leon-Ferre RA, Polley M-Y, Liu H, Kalari KR, Boughey JC, Liu MC, Cafourek V, Negron V, Ingle JN, Thompson KJ, Tang X, Barman P, Carlson E, Visscher DW, Carter JC, Couch FJ, Goetz MP. Characteristics, outcomes and prognostic factors of luminal androgen receptor (LAR) triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-08-01.

Abstract Background: In women with breast cancer receiving neoadjuvant chemotherapy, residual cancer burden (RCB) predicts distant recurrence and survival. In those with high risk tumors, locoregional recurrence (LRR) remains a concern, and has been associated with type of local therapy received. We evaluated the impact of local therapy on LRR in the ISPY-2 TRIAL. Methods: Data were analyzed in Stata 14.2, using Chi2 test, log rank test, and a Cox proportional hazards model. RCB was considered a categorical variable (0/1 versus 2/3), as described in prior publications. Breast surgery categories were lumpectomy +/- radiotherapy, or mastectomy +/- radiotherapy. Axillary surgery was defined as sentinel lymph node (SLN) surgery (≤6 nodes removed) or axillary dissection (>6 nodes). Results: Follow up data from the I-SPY2 TRIAL were available for 630 patients (median follow up 2.76 yrs, range 0.4-7.2). Type of local therapy was significantly associated with clinical stage at presentation, with stage III patients most frequently undergoing mastectomy + radiation (p<0.001). Women with higher RCB were more likely to undergo mastectomy than those with lower RCB (61.3% vs 48.8% mastectomy rate, p=0.002), and more likely to receive adjuvant radiotherapy (62.0% vs 53.9%, p=0.048). There was no association between clinical stage, type of surgery, or radiotherapy and LRR (Table). Higher RCB was significantly associated with LRR, with 3 year locoregional recurrence free rate of 95.1% in RCB 0/1 versus 89.9% in RCB 2/3 (p=0.003). In a Cox model adjusting for clinical stage, tumor subtype, surgical therapy, RCB status, nodal radiation, and age, significant predictors for LRR were tumor subtype and RCB status. Hazard ratio (HR) for LRR in those with RCB 0/1 was 0.39 compared to those with RCB 2/3 (95% CI 0.17-0.87, p=0.021). There was no difference in LRR between breast conservation and mastectomy; within the breast conservation group, those who had lumpectomy alone had higher hazard of LRR compared to those having lumpectomy + radiation (HR 3.1, 95% CI 1.1-9.2, p=0.043). Conclusions: Extent of surgical therapy was not associated with local tumor control, regardless of advanced tumor stage at presentation. Rather, tumor biology and response to therapy were the best predictors of LRR. These data highlight the opportunity to minimize the morbidity of extensive surgical therapy for patients with excellent response to systemic therapy. LRR rates by clinical features and treatment status FrequencyLRR RateP valueClinical Stage 0.5I240 (47.5%)5.8% II185 (36.6%)8.7% III80 (15.8%)6.3% Tumor Subtype 0.014ER+PR+Her2-161 (26.4%)3.1% ER+PR-Her2-56 (9.2%)3.6% Her2+176 (28.9%)6.3% Triple negative216 (35.5%)11.1% Local therapy 0.169Lumpectomy85 (13.5%)11.8% Lumpectomy with radiation198 (31.4%)5.6% Mastectomy173 (27.5%)5.2% Mastectomy with radiation174 (27.6%)8.6% Axillary surgery 0.23None5 (0.8%)20% SLN329 (52.2%)5.8% ALND296 (47%)8.5% Axillary radiation 0.535Yes42 (6.7%)9.5% No588 (93.3%)7.0% Axillary management 0.2No surgery or radiation5 (0.8%)20.0% SLN312 (50%)5.3% SLN+Axillary radiation17 (2.7%)8.3% ALND271 (43%)10.3% ALND+Axillary radiation25 (4%)5.4% RCB 0.0020/1293 (50.1%)3.8% 2/3292 (49.9%)10.3% Citation Format: Silverstein J, Suleiman L, Yau C, Price ER, Singhrao R, Yee D, DeMichele A, Isaacs C, Albain KS, Chien AJ, Forero-Torres A, Wallace AM, Pusztai L, Ellis ED, Elias AD, Lang JE, Lu J, Han HS, Clark AS, Korde L, Nanda R, Northfelt DW, Khan QJ, Viscusi RK, Euhus DM, Edmiston KK, Chui SY, Kemmer K, Wood WC, Park JW, Liu MC, Olopade O, Leyland-Jones B, Tripathy D, Moulder SL, Rugo HS, Schwab R, Lo S, Helsten T, Beckwith H, I-SPY 2 TRIAL Consortium, Berry DA, Asare SM, Esserman LJ, Boughey JC, Mukhtar RA. The impact of local therapy on locoregional recurrence in women with high risk breast cancer in the neoadjuvant I-SPY2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-14-01.

Abstract Background: Thirty percent of patients with breast cancer may experience chest wall recurrence, which is associated with a higher risk of developing distant metastases and a poor prognosis. Cancer cells may evade immune rejection through the programmed cell death 1 (PD-1) pathway. Pembrolizumab, an anti-PD-1 antibody, binds PD-1 and inhibits its interaction with the programmed death ligand 1 (PD-L1) to facilitate tumor immune rejection. We hypothesize that pembrolizumab may be an effective therapy in chest wall recurrence, given the inflammatory nature, and the high expression of PD-1 in tumors with lymphovascular invasion. Platinum agents may enhance anti-tumor immunity in a synergistic manner, and the combination of pembrolizumab and carboplatin has demonstrated efficacy in advanced lung cancer. In this study, the combination of pembrolizumab and carboplatin is being evaluated in breast cancer patients with chest wall disease. Methods: This is a randomized phase II study of breast cancer patients with advanced, unresectable breast cancer involving the chest wall, being conducted through the Translational Breast Cancer Research Consortium (TBCRC). Patients may have hormone resistant disease (at least 2 prior lines of hormone therapy), triple negative breast cancer, or refractory HER2+ disease for enrollment. They may have other sites of distant metastases, have received any number of prior lines of therapy, have had prior surgery, but prior chest wall radiation is not necessary. Eighty-four patients at 7 TBCRC sites will be randomized 2:1 to treatment with pembrolizumab 200 mg IV and carboplatin AUC 5 IV every 3 weeks followed by pembrolizumab 200 mg IV alone every 3 weeks (Arm A, n=56) or carboplatin AUC 5 IV every 3 weeks (Arm B, n=28), with an option for patients in Arm B to cross-over to single agent pembrolizumab 200 mg IV every 3 weeks (arm Bx) on progression. Patients will undergo imaging with CT chest, abdomen, and pelvis at baseline and every 2 cycles of treatment for response evaluation. The primary endpoint is the disease control rate in the chest wall and distant sites at 18 weeks of treatment, and this study is powered to determine a 20% difference in disease control rates between arms A and B (hazard ratio of 0.52, α= 0.10, β= 0.20). After 18 patients are enrolled into Arm B, an interim analysis for futility will be conducted to enable early closure of that arm for lack of efficacy. Secondary endpoints in the study are toxicity, progression free survival, and response based on PD-L1 expression and irRECIST. Exploratory endpoints, which will be studied using peripheral blood testing and chest wall tumor biopsies at baseline and after 2 cycles of treatment, include determining associations of response with changes in tumor and peripheral blood immune composition, soluble PD-L1 expression, circulating tumor cells, cell free DNA, and tumor PD-L1 and MYC genomic expression. Ultimately this study promises to improve our understanding of checkpoint inhibition and chemotherapy for chest wall disease, and the underlying mechanism of action. This study is open for enrollment and 2 patients are currently enrolled. (NCT03095352). Citation Format: Vidula N, Goga A, Hwang J, Liu MC, Park BH, Nanda R, Pohlmann PR, Storniolo AM, Brufsky A, Abramson V, Rugo HS. A randomized phase II study of pembrolizumab in combination with carboplatin versus carboplatin alone in breast cancer patients with chest wall disease, with immunologic and genomic correlative studies [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT3-04-04.

Abstract Background: The explosion in new treatment options targeting immune checkpoints, HER signaling, DNA repair deficiency, AKT, and other pathways calls for updated breast cancer subtypes beyond HR and HER2 status to predict which patients will respond to which treatments. Here we leverage the I-SPY 2 TRIAL biomarker program over the past 8 years across 10 treatment arms to elucidate a minimal set of biomarkers that may improve response prediction in a modern treatment context, and to investigate which new patient phenotypes are identified by these response-predictive biomarkers. Methods: 986 patients were considered in this analysis. Treatments included paclitaxel alone (or with trastuzumab (H) in HER2+) or combined with investigational agents: veliparib/carboplatin (VC); neratinib; MK2206; ganitumab; ganetespib; AMG386; TDM1/pertuzumab (P); H/P; and pembrolizumab (Pembro). 24 prospectively defined, mechanism-of-action and pathway-based expression and phospho-protein signatures/biomarkers assayed from pre-treatment biopsies were previously found to be predictive in a particular agent/arm in pre-specified analysis. Here we evaluate these biomarkers in all patients. We assessed association between each biomarker and response in the population as a whole and within each arm and HR/HER2 subtype using a logistic model. To identify optimal dichotomizing thresholds for select biomarkers, 2-fold cross-validation was repeated 500 times. Our analysis is exploratory and does not adjust for multiplicities. Results: Our initial set of 24 predictive biomarkers reflects DNA repair deficiency (n=2), immune activation (n=7), ER signaling (n=2), HER2 signaling (n=4), proliferation (n=2), phospho-activation of AKT/mTOR (n=2), and ANG/TIE2 (n=1) pathways, among others. Biomarkers reflecting similar biology are correlated and cluster together. We make use of this correlation structure to reduce the dimensionality of the biomarker set to five predictive signals: proliferation, DNA repair deficiency (DRD), immune-engaged (Immune+), luminal/ER (lum), and HER2-activated. These biomarkers, when dichotomized, identify patient groups with differential predicted sensitivities to I-SPY 2 agents and are present at different proportions within receptor subtypes. For instance, in the HER2- subset, Immune+/DRD+ patients are predicted sensitive to both VC and Pembro, and account for 39% of TN, but only 12% of HR+HER2-. On the other end of the spectrum, only 17% of TN are Immune-/DRD-, compared to the majority (56%) of HR+HER2-. There are also subsets of patients positive for only one marker. For the HER2+ subset, 67% are HER2-activated+, and 25% lum+; of these HER2-activated+ patients are more likely to be Immune+ (44%), vs 23% in lum+. HER2-activated+/Immune+ patients have higher predicted sensitivity to HER2-targeted agents than lum+ or Immune- patients. In all, these molecular phenotypes predict sensitivity to one or more I-SPY 2 investigational agents for 75% of the ˜ 1000 patients. Conclusion: Molecular phenotypes reflecting proliferation, immune engagement, HER2-activation, luminal/ER-signaling, and DNA repair deficiency may provide a roadmap to guide treatment prioritization for emerging therapeutics. Citation Format: Wolf DM, Yau C, Wulfkuhle J, Petricoin E, Campbell M, Brown-Swigart L, Hirst G, Asare S, Zhu Z, Lee EP, Delson A, Pohlmann P, I-SPY 2 TRIAL Consortium, Hylton N, Liu MC, Symmans F, DeMichele A, Yee D, Berry D, Esserman L, van 't Veer L. Identifying breast cancer molecular phenotypes to predict response in a modern treatment landscape: Lessons from ˜1000 patients across 10 arms of the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-02.

Abstract Background: Neoadjuvant chemotherapy is administered to patients with operable breast cancer to downstage the tumor to allow for less extensive surgery and to provide prognostic information about drug efficacy and risk of disease recurrence. Patients who achieve a pathological complete response (pCR) following neoadjuvant treatment have a more favorable outcome than patients with residual invasive disease. Single-agent poly(ADP-ribose) polymerase (PARP) inhibitors have clinical efficacy in BRCA-mutated breast cancer. Niraparib, a potent and selective PARP1/2 inhibitor, is approved for maintenance treatment of patients with recurrent ovarian cancer and has demonstrated strong antitumor activity in in vivo studies with BRCA1-mutant breast cancer. The objective of this study is to evaluate the antitumor activity of single-agent niraparib in the neoadjuvant treatment of patients with localized, human epidermal growth factor receptor 2 (HER2)-negative, BRCAmut breast cancer. Trial Design: This is an open-label, single-arm pilot study with a target enrollment of 20 evaluable patients. Eligible patients are those ≥18 years old with histologically-confirmed HER2-negative localized breast cancer and either a BRCA1 or BRCA2 mutation (germline or somatic) and no prior anti-cancer therapies for the current malignancy. Patients will receive 200 mg of oral niraparib once daily for 2 months, after which they may either proceed directly to surgery or receive chemotherapy at the discretion of the physician. The primary endpoint is tumor response rate based on the change in tumor volume as measured by breast MRI after 2 months of treatment with niraparib; a response is defined as ≥30% reduction of tumor volume from baseline. Secondary endpoints include pCR rate, tumor response rate based on the change in tumor volume as measured by breast ultrasound, and safety and tolerability. Data will be summarized in a descriptive nature by frequency distributions (number and percentage of patients) for categorical variables and by the mean, median, and standard deviation for continuous variables. Tumor response rate will be tabulated together with its 95% binomial exact confidence interval. Funding: TESARO, Inc., Waltham, MA, USA sponsored the study. Citation Format: Han H, Hamilton E, Irie H, Isakoff S, Jelovac D, Liem A, Liu MC, Milillo A, Nangia J, Page D, Reeves J, Santa-Maria C, Duncan M, Graham JR, Chen J, Dezube BJ, Spring L. Open-label, single-arm study evaluating the antitumor activity and safety of niraparib as neoadjuvant treatment in patients with localized, HER2-negative, BRCA-mutant breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT3-03-01.

Abstract Background: Combining cyclin dependent 4/6 kinase inhibitors (CDK4/6i) with endocrine therapy (ET) has resulted in clinically significant improvements in progression-free survival (PFS) in persons with hormone-receptor (HR)-positive metastatic breast cancer (MBC). However, nearly all patients will progress on CDK4/6i and ET and the mechanisms associated with primary and secondary resistance are mostly unknown. The identification of biomarkers associated with response to CDK4/6i is therefore a major research priority. PROMISE is a multicenter prospective cohort study designed to perform a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome to identify alterations in molecular or cellular features associated with primary endocrine resistance (e.g. disease progression ≤ 12 months on treatment) and acquired resistance to CDK4/6i. Additionally, patient derived xenografts (PDX) and organoids are generated to test new drug strategies designed to reverse resistance to CDK4/6i and ET. Methods: Eligible participants include women for whom palbociclib is recommended in combination with either letrozole (first-line) or fulvestrant (second-line) therapy for HR-positive MBC. As of July 2018, 14 patients have been enrolled; the target accrual is 250 patients, who will be followed over the course of 36 months and followed for at least 12 months after the close of enrollment. Blood and tumor biopsies are obtained at baseline, at the end of cycle 2, and at progression. Whole exome sequencing (germline and tumor) and RNAseq (whole transcriptome) are performed on tumor samples in a CAP/CLIA lab (TEMPUS) and results will be provided back to all patients for later use. Additionally, PDX and organoids are generated from tumor biopsies at baseline and during progression. Blood, stool, and urine samples are collected for additional correlative studies. The primary objective of this trial is to identify novel genomic variants and pathways associated with early progression (≤ 12 months) among women with advanced HR-positive breast cancer treated with palbociclib and ET. Secondary objectives include identification of the biomolecular features within the gut (stool) microbiome and exploration of the metabolomics and proteomics of ER-positive MBC. Results from the interrogation of these biospecimens will be critical to gain insights into treatment resistance. Funding is provided by Mayo Clinic Center for Individualized Medicine, TEMPUS, and an ASCO Career Development Award (COS). ClinicalTrials.gov Identifier: NCT03281902. Citation Format: O'Sullivan CC, Suman VJ, Kalari KR, Moyer A, Sung J, Moreno-Aspitia A, Northfelt DW, Liu MC, Haddad TC, Chumsri S, Couch FJ, Weinshilboum RM, Wang L, Goetz MP. Genetic analysis in blood, urine, stool and tumor samples from patients with advanced or metastatic estrogen receptor positive and HER2 negative breast cancer receiving palbociclib and endocrine therapy (PROMISE) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT3-02-08.

Abstract Background. The presence and number of circulating tumor cells (CTCs) are prognostic for breast cancer treatment outcome. Direct imaging assays traditionally employ four markers to identify canonical epithelial CTCs: nucleus, exclusion (CD45), and inclusion (EpCAM and cytokeratin). There is intense interest in the ability to phenotype CTCs in order to provide a noninvasive means by which to predict treatment benefit from endocrine therapy and/or HER2-directed therapy in breast cancer. To address this, a 6-parameter assay for detection of ER and HER2 expression on CTCs was developed. We applied this assay to four well characterized breast cancer cell lines representative of various ER and HER2 phenotypes. Methods. BT474, MCF-7, SKBR3, or MDA-MB-231 cells were spiked into peripheral blood from healthy donors and processed using the AccuCyte® sample preparation system; nucleated cells, including CTCs, are captured onto glass slides (8 slides per 7.5 mL blood sample) for subsequent immunofluorescent staining. Slides were stained using the combined epithelial marker and ER/HER2 CTC assay and then analyzed with the CyteFinder® imaging system. CTCs were identified as nucleated cells with positive EpCAM and/or cytokeratin staining, and negative CD45 staining. ER and HER2 expression were assessed as present or absent. Results. All cell lines expressed both cytokeratin and EpCAM, except for MDA-MB-231 which was EpCAM-negative. The ER / HER2 expression patterns observed were consistent with reported phenotype: BT474 (+/+), MCF-7 (+/–), SKBR3 (–/+), and MDA-MB-231 (–/–). Conclusions. Identification of epithelial CTCs and phenotypic characterization of ER and HER2 status are feasible in a combined assay applied to a single blood sample. This approach has implications for efficiency and cost effectiveness, which are of particular importance given the interest in longitudinal testing. Assay evaluation is currently underway using blood samples from breast cancer patients with known receptor status, treatment history, and clinical outcomes. Results will be available for presentation at the meeting. Citation Format: Liu MC, Sun Y, Ramirez A, Campton D, George T, Haselkorn KE, Clein A, Gadi V, Sabath D, Kaldjian E. A novel six-parameter assay for comprehensive phenotyping of circulating tumor cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-11.