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Background: Although immune checkpoint inhibitors (ICI) are most widely used in PD-L1 positive, metastatic triple-negative breast cancer (TNBC), the clinical utility of ICI in other tumor subtypes and patient populations is not well defined. In this study, we measured infiltration of multiple immune cell populations in primary and metastatic HR+ tumors to infer potential benefit from ICI. We investigated how risk stratifying features such as pathologic grade and Oncotype Dx scores affect immune cell infiltration across subtypes. Lastly, we compared immune cell infiltration in breast tumors from younger versus older women, as our pre-clinical studies demonstrated differential enrichment of immune response pathways by age and association with improved response to ICI. Methods: ImmunoProfile, a multiplexed immunofluorescence (IF) assay, was performed on FFPE tissue from 167 archived breast cancer tumors from the Dana-Farber Cancer Institute (HR+/HER2- n=124, HER2+ n=21, TNBC n=22). Unmatched samples were obtained from either primary breast (HR+/HER2- n=104, HER2+ n=13, TNBC n=15) or metastatic core biopsies. IF staining signal was measured for T cell markers CD8 and co-localized CD8/PD-1, T regulatory cell marker FOXP3, PD-L1 and PD1 expression on tumor cells as well as PD-L1 expression on inflammatory cells. Inflammatory, tumoral (TPS) and combined inflammatory and tumor (CPS) pathologic PD-L1 scores were calculated. PD-L1 staining was also assessed by IHC (E1L3N) staining and compared to multiplexed IF stains. IF staining signal (Vectra analyzed, log2+1 transformed) was analyzed against clinical and pathologic variables, including tumor subtype, primary versus metastatic status, pathologic grade, and Oncotype Dx score, using non-parametric statistical comparisons. Given the decline in adaptive immunity that occurs with normal physiological aging and our own group’s previous reports, we also explored whether immune cell staining signal associated with age. Results: TPS, CPS, and inflammatory PD-L1 scores strongly correlated between IHC and IF staining methods (Spearman r = 0.660, 0.683, 0.638 respectively, p < 2.2e-16). When HR+/HER2- (n=124), HER2+ (n=21) and TNBC (n=22) cases were analyzed separately, there was no statistical difference between unmatched primary and metastatic cases in any of the analyzed immune cell population stains for any subtype. When all primary cases were analyzed, TNBC showed higher CD8, CD8/PD1, PD1 (all p < 0.05), and FOXP3 staining as well as IF CPS, TPS and inflammatory PD-L1 scores (all p < 0.005) than HR+ cases. HR+/HER2- cases from women < age 65 (n=56) showed higher intra-tumoral CD8 (p=0.039), PD1 (p=0.043), and CD8/PD1 (p=0.022) than cases from women > age 65 (n=38). There was a statistically significant step-wise increase in IF staining of all immune cell populations with increasing pathologic grade (I, II, III) as well as Oncotype Dx scores (< 11, 11-25, >25) among primary HR+/HER2- cases (n=74). Conclusions: Strong correlations between PD-L1 scores based on IF and IHC methods suggest clinical utility for multiplexed IF immune cell staining in therapeutic planning. ImmunoProfile demonstrated the previously known pattern that primary TNBC contains higher immune cell infiltration than HR+ or HER2+ tumors. Women < age 65 with HR+ cancer showed higher levels of CD8 and PD1 stained populations, suggesting there may be greater utility of ICI in younger patients. Lastly, the association of higher pathologic grade and Oncotype Dx score with increasing immune cell infiltration supports exploration of ICI in biologically unfavorable primary HR+ cancers. Citation Format: Tess A. O'Meara, Tanya E. Keenan, Adrienne G. Waks, Kristen D. Felt, Bijaya Sharma, Scott Rodig, Melissa Hughes, Nancy U. Lin, Judith Agudo, Jennifer L. Guerriero, Sandra S. McAllister, Elizabeth Mittendorf, Sara Tolaney. Multiplexed immunofluorescence staining of intra-tumoral immune cell populations and associations with immunohistochemical, clinical, and pathologic variables in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-05.

Background: Immunological differences between HER2+ breast tumors that are HR+ versus HR- have not been widely explored. DAPHNe was a single-arm prospective clinical trial enrolling patients with clinical anatomic stage II-III HER2+ breast cancer to neoadjuvant treatment with THP. Here, we examine differential expression of immune-related proteins between HR+/HER2+ and HR-/HER2+ tumors in pre-treatment biopsies from trial participants, and analyze correlates of pathologic response to THP, overall and by HR status. Methods: A baseline research biopsy was required in all participants prior to initiation of neoadjuvant therapy. All patients received 12 weeks of neoadjuvant THP; 5 patients also received neoadjuvant AC given incomplete clinical response to THP, and are excluded from residual cancer burden (RCB) analyses. The trial primary endpoint, feasibility of de-escalation to antibody doublet therapy only (HP) following pCR, was previously reported. Protein expression profiling of baseline research biopsies was performed on the NanoString GeoMx® platform using a 58-protein immune cell profiling panel. Regions of interest (ROI; up to 12 per slide) were identified by a pathologist based on tumor cell, immune cell, and/or T cell presence, and all ROI were segmented into tumor vs stroma. Protein data quality controls were normalized by isotypes. Linear mixed effect models were used for differential expression comparisons; p-values were estimated using Satterthwaite’s method. Results: 97 pre-treatment breast tumor specimens were analyzed: 64 patients had HR+ tumors, 32 HR-, 1 unknown; 67 patients had favorable (RCB 0/1) response to preop therapy, 30 patients had unfavorable response (RCB 2/3). Multiple significant differences were observed between protein expression in HR+ versus HR- tumors (Table 1). Multiple immune cell types, as well as checkpoint proteins PD-L1 and IDO1, were higher in HR- patients, whereas checkpoint proteins B7-H3 and TIM3 were higher in HR+ patients. In the overall population, higher HER2 and PD-L1 expression were significant predictors of favorable RCB response, while higher ER alpha and Bcl-2 expression were significant predictors of unfavorable RCB response (Table 2). Conclusions: As anticipated, the strength of HER2 expression and ER expression were the most significant predictors of favorable and unfavorable RCB response to neoadjuvant THP, respectively. This highly multiplexed protein expression analysis demonstrated significant differences between the immune microenvironment of HR+ and HR- HER2+ breast tumors, implying that distinct immunological targets should be explored in the treatment of HER2+ breast cancer according to HR status. Table 1.Differences between HR+/HER2+ and HR-/HER2+ patientsStroma compartmentTumor compartmentHigher in HR+ER alpha* (fold-change 2.25)ER alpha* (8.94)Fibronectin (1.68)Bcl-2* (1.98)B7-H3 (1.44)Fibronectin* (1.90)PR (1.69)TIM-3 (1.37)Higher in HR-CD27* (fold-change 1.36)IDO1 (1.50)IDO1 (1.77)S100B (1.44)CD45 (1.45)MART1 (1.33)CD3 (1.35)CD20 (1.34)STING (1.33)CD4 (1.30)ICOS (1.30)CD45RO (1.28)CD40 (1.28)CD127 (1.27)VISTA (1.25)PD-L1 (1.24)4-1BB (1.21)*FDR p-value < 0.05 (otherwise, p-value < 0.05). Table 2: Predictors of RCB response Overall populationHigher in RCB 0/1HER2* (fold-change 3.75)PD-L1 (1.25)Higher in RCB 2/3ER alpha* (2.44)Bcl-2 (1.46)HR+/HER2+ patientsStroma compartmentTumor compartmentHigher in RCB 0/1HER2* (fold-change 4.28)HER2* (4.21)CTLA4 (1.64)Higher in RCB 2/3Bcl-2* (2.18)*FDR p-value < 0.05 (otherwise, p-value < 0.05).HR-/HER2+ patients are not shown in Table 2 as only 2 HR- patients had unfavorable RCB response. Citation Format: Adrienne G. Waks, Ying Huang, Tyler Hether, Esther R Ogayo, Sapana Kadel, Jillian Alberti, Sara M Tolaney, Ian E Krop, Prajan Divakar, Otto Metzger, Madison L O'Donnell, Sarah Church, Eric P Winer, Jennifer L. Guerriero, Elizabeth A Mittendorf. Correlation of immune-related protein expression with hormone receptor (HR) status and pathologic response to neoadjuvant paclitaxel/trastuzumab/pertuzumab (THP) among patients with early-stage HER2+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-03.

Background: The optimal systemic therapy strategy, neoadjuvant vs adjuvant therapy, for patients with small clinically node negative HER2+ breast cancers is uncertain. The goal of this study was to evaluate the incidence of pathologic nodal disease in cT1-2 N0 HER2+ patients, treated with upfront surgery or neoadjuvant chemotherapy (NAC), to better define selection criteria for initial treatment approach. Methods: Our prospectively maintained database was queried for cT1-2 N0 HER2+ breast cancer patients diagnosed 2/12/2015 - 10/13/2020 (after publication of the Adjuvant Paclitaxel and Trastuzumab trial). Patients without axillary surgery were excluded (n =23). HER2 positivity was defined per ASCO/CAP guidelines. Pre-NAC and/or pre-operative axillary ultrasound was not routinely performed. Patient characteristics and rates of pN+ disease were compared by treatment strategy using Chi square and Wilcoxon rank-sum tests. Nodes with isolated tumor cells were considered negative (pN0) in the upfront surgery setting but positive (pN+) after NAC. Logistic regression determined factors associated with pN+ disease. Results: A total of 579 eligible patients were identified; 368 (63.6%) were treated with upfront surgery and 211 (36.4%) with NAC. Upfront surgery patients were older (median 55 years [range 23-88] vs 49 [24-79], p Table 1.Pathologic nodal stage among upfront surgery and NAC patientsUpfront Surgery (N=368)pN+pN0P valuecT category< 0.001T1mi (N=48)6 (10.4%)42 (89.6%)T1a (N=26)3 (11.5%)23 (88.5%)T1b (N=87)7 (8.0%)80 (92.0%)T1c (N=154)38 (24.7%)116 (75.3%)T2 (N=53)19 (35.8%)34 (64.2%)NAC (N=211)ypN+ypN0P valuecT category0.719T1b (N=7)1 (14.3%)6 (85.7%)T1c (N=30)5 (16.7%)25 (83.3%)T2 (N=174)20 (11.5%)154 (88.5%) Citation Format: Anna Weiss, Adrienne G. Waks, Alison Laws, Sara M. Tolaney, Eric P. Winer, Elizabeth A. Mittendorf, Ann H. Partridge, Tari A. King. Pathologic nodal staging and systemic therapy among patients with cT1-2N0 HER2+ breast cancer: A prospective single institution cohort analysis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-02.

Background: De-escalation of adjuvant therapy following pathologic complete response (pCR) to an abbreviated neoadjuvant regimen in HER2-positive (HER2+) breast cancer is the focus of a recently initiated national trial. However the feasibility of this approach, and its appeal to patients (pts) and providers, has not been formally investigated. Methods: We conducted a single arm pilot trial to determine the feasibility of de-escalated adjuvant therapy (HP-only) in patients with pCR following neoadjuvant THP. Eligible pts had clinical anatomic stage II-III treatment-naïve HER2+ breast cancer. All pts received weekly paclitaxel x12 doses, and HP every 3 weeks x4 doses (up to 6 doses allowed in case of surgical delay). The primary objective was to assess adherence to protocol-specified antibody doublet therapy (HP-only, without cytotoxic chemotherapy) in the adjuvant setting among pts with pCR (ypT0/isN0) following neoadjuvant THP; the primary endpoint was receipt of adjuvant cytotoxic chemotherapy, assessed 3 months post-operatively. Trastuzumab emtansine (T-DM1) was not considered cytotoxic chemotherapy. Among pts with pCR to THP, de-escalation would be deemed infeasible if the true rate of adherence to HP-only were 90% power to reject the null if the true rate of adherence were >95% (Binomial exact test; one-sided alpha=0.05). Questionnaires were administered and records were reviewed to assess pts’ and physicians’ decision-making about adjuvant systemic therapy following pCR and non-pCR. Results: 98 pts received at least one dose of THP on study. Median age was 50 yrs (range 24-78), pts were 99% female, 86% had stage II/14% stage III tumors, 32% ER/PR negative. No pts progressed during neoadjuvant THP. Five pts had incomplete clinical response following THP and received AC prior to surgery. They were classified as non-pCR and censored from further analyses. At the time of analysis, 93 pts were evaluable for response to neoadjuvant therapy (1 pt withdrew from study early; 4 pts had not reached surgery by the data cutoff). Overall pCR rate was 55% (51/93 pts); 10%, 28%, and 2% of pts had RCB I, II, and III responses, respectively (this excluded patients who received AC preoperatively). Of 51 pts who had pCR to THP, 40 had verified data available regarding adjuvant chemotherapy receipt at data cutoff. 39/40 pts (97.5%, 95% CI 86.8-99.9%) who had pCR did not receive adjuvant cytotoxic chemotherapy, meeting the trial’s prespecified threshold for declaring this a feasible approach (primary endpoint), though data remain pending for 11 pts with pCR and will be available at presentation. Of 30 pts who did not experience pCR to THP and had adjuvant chemotherapy status verified at data cutoff, 14/30 pts received adjuvant cytotoxic chemotherapy, and 16/30 pts did not receive adjuvant chemotherapy. The most common reasons cited by pts for non-receipt of adjuvant cytotoxic chemotherapy, despite residual disease at surgery, were (N=21): plan for adjuvant T-DM1 alone (67% of pts), good response to preop chemo (38% of pts), and plan for adjuvant hormonal therapy (24% of pts). The most common reason cited by treating physicians for non-administration of adjuvant chemotherapy, despite residual disease at surgery, was plan for adjuvant T-DM1 (17/21 (81%) physicians). With brief follow-up (median 10.2 mos), there were no breast cancer recurrences. Conclusions: De-escalation of adjuvant cytotoxic chemotherapy among pts who experience pCR in early stage HER2+ breast cancer appears to be an acceptable approach for both pts and physicians, though data are not yet complete and will be updated at the time of presentation. The long-term efficacy of this approach will be determined in the ongoing national CompassHER2-pCR trial. Citation Format: Adrienne G. Waks, Neelam V. Desai, Tianyu Li, Philip D. Poorvu, Ann H. Partridge, Natalie Sinclair, Laura M. Spring, Meredith Faggen, Michael Constantine, Jillian C. Alberti, Julia Deane, Shoshana M. Rosenberg, Sara M. Tolaney, Ian E. Krop, Nadine M. Tung, Nabihah Tayob, Tari A. King, Elizabeth A. Mittendorf, Eric P. Winer. The DAPHNE trial: A feasibility study of chemotherapy de-escalation based on response to neoadjuvant paclitaxel-HP (THP) in HER2-positive 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 PD3-05.

This abstract was withdrawn by the authors. Citation Format: Metzger Filho O, Janiszewska M, Guo H, Yardley D, Mayer I, Spring L, Arteaga C, Wrabel E, DeMeo M, Freedman R, Tolaney S, Waks A, Bardia A, Parsons H, Partridge A, Mayer E, King T, Polyak K, Viale G, Winer E, Krop I. Withdrawn [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 P1-15-01.

Background: Hormone receptor-positive (HR+) tumors have fewer tumor-infiltrating lymphocytes (TILs) and lower response rates to immune checkpoint inhibitors (ICI), either as single agents or in combination with chemotherapy, than triple negative cancers. However, some HR+ cancers do respond to ICI and biomarkers that accurately reflect the immune microenvironment may help guide the use of ICI therapy. Prior evidence suggests that macrophage-related immune pathways may be relevant to the pathophysiology of HR+ BC. Methods: HR+/HER2- patients were identified from a prospective trial of preoperative bevacizumab (preop bev) followed by bev with adriamycin/cyclophosphamide/paclitaxel dose-dense chemotherapy (chemo). Tumor samples were collected at diagnosis and surgery (pre-tx and post-tx), and PD-L1 expression (by immunohistochemistry), TILs, and Nanostring PanCancer Immune Profiling Panel were evaluated on both pre-tx and post-tx specimens. Pre-tx whole transcriptome sequencing was performed. Pathologic response at surgery was centrally assessed by Miller-Payne (MP) and residual cancer burden (RCB) scores. An immune score was calculated for each pre-tx specimen by integrating 10 published immune signatures. Immune cell subsets were inferred from bulk transcriptional data using CIBERSORT and immune cell-specific signatures from MSigDB. Results: 55 patients who received trial therapy and had at least 1 evaluable specimen were included for analysis. Pre-tx TILs and tumor PD-L1 (tPD-L1) scores are shown in the table. 18% of pre-tx tumors had “high” (≥10%) TILs and “high” TILs were associated with significantly higher immune signature score (p=0.004). Immune score correlated highly with proportion of CIBERSORT anti-tumor M1 macrophages as well as CD8 T-cell signatures (r>0.65 and p5%) in TILs or tPD-L1 from pre-tx to post-tx were rare: 2 pts each had large changes in TIL or tPD-L1 score (N=38/N=31 pairs, respectively). Conclusions: High levels of tumor-lymphocyte interaction were seen in only a minority of untreated HR+ breast tumors, and did not typically change with chemo plus bev. An immune score derived from bulk RNAseq correlated with histological observations in these specimens. Nonetheless, TILs, tPD-L1, and signature-derived immune score were significantly associated with pathologic response to preop treatment in HR+ disease. Early data suggest that the role of M1 macrophages in HR+ tumors warrants further investigation. ScoreTILs (N=50 evaluable)Tumor PD-L1 (N=51)0%0 pts (0%)28 pts (55%)>0-5% (low)19 (38%)18 (35%)>5-10% (intermediate)22 (44%)3 (6%)>10% (high)9 (18%)2 (4%) Citation Format: Waks AG, Stover DG, Barry W, Dillon D, Gjini E, Rodig SJ, Brock J, Baltay M, Savoie J, Winer EP, Krop I, Tolaney SM. The immune microenvironment in hormone receptor-positive breast cancer patients and relationship to treatment outcome following preoperative chemotherapy plus bevacizumab [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 PD6-09.

Background: Proliferation is the strongest predictor of response to neoadjuvant chemotherapy in estrogen receptor-positive (ER+) breast cancer. Evidence of immune activation has also been associated with improved response to neoadjuvant chemotherapy in breast cancer. We hypothesized that immune signatures may be associated with response independent of proliferation in ER+ breast cancers. Approach: We compiled microarray expression data from breast cancer biopsies obtained prior to neoadjuvant chemotherapy on 465 ER-positive/HER2-negative patients by reported pathologic receptor status. We evaluated the association of 118 published gene expression signatures with response to neoadjuvant chemotherapy, based on study-defined pathologic complete response (pCR) versus residual disease (RD). Results: Overall, 42 of 118 signatures were significantly associated with response to neoadjuvant chemotherapy in ER+ breast cancer (FDR-corrected p0.30, p0.4, p Conclusions: Gene expression signatures associated with "immune activation" identify a subset of ER+ breast cancers with higher rates of pCR to neoadjuvant chemotherapy. These "immune activation" signatures appear to be proliferation-independent and may provide additional predictive information to existing gene expression-based approaches for ER+ breast cancer. Citation Format: Stover DG, Waks AG, Erica ML, Brugge JS, Winer EP, Selfors LM. Immune activation signatures identify a subset of ER+ breast cancers with increased pathologic complete response to neoadjuvant chemotherapy. [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 P3-07-31.

While recent studies have begun characterizing the metastatic breast cancer (MBC) genomics, our understanding of mechanisms of acquired endocrine-resistance, their induced cell-state, and their altered drug-response profile, remains lacking. We collected biopsies from patients with MBC with detailed clinicopathologic features. To date we profiled 520 exomes, and 291 transcriptomes, with 126 patients having multiple biopsy exomes. Curated endocrine-relapse set include 60 patients with pre-treatment and post-relapse exomes. Characterization of candidate mechanisms of resistance (MOR) included 909 RNA-seq profiles of T47D cells with introduced MOR under various drugs. In the acquired endocrine resistance cohort, as expected, we found frequent ESR1 acquired mutations (13 pt, 22%). Additionally, we identified acquired activating SNVs and amplifications in oncogenic receptor tyrosine kinases (RTKs) in 18/60 (30%), including EGF family - HER2 (n=7), ERRB3 (R525Q), and EGFR (S116F), and FGF family - FGFR1 (n=5), FGFR2 (n=4), and FGF3 amplicon (n=4). RNA-seq of T47D cells overexpressing HER2 activating mutations revealed a distinct cell-state (HER2-ACT). Similarly, FGFR activation revealed a district FGFR-ACT state. The transcriptional signatures of these HER2 and FGFR states were remarkably similar (odd-ratio= 91, p= 2.64E-94). To characterize the cell-state common to HER and FGFR, we defined RTK-ACT with 358 overlapping marker genes (see table). Canonical (estradiol) ER signaling is slightly elevated in RTK-ACT, however this state is strongly associated with growth-factor driven ER signaling, suggesting reprogramming of ER from AF2, to AF1 signaling. Consistent with this, RTK-ACT had significantly higher MAPK activation. Additionally, RTK-ACT Induced stronger similarity to Basal-state, enrichment in motility/migration, mesenchymal, and stem-like features, with top genes including CDH3, MBP7, and S100, ETV, DUSP, SPRY families (see table). To study RTK-driven state in-vivo, we analyzed RNA-seq from our MBC biopsies, and compared tumors with activating RTK mutations (n=38) with WT (n=118), and inferred activated RTK in-vivo (RTK.ACT.iv). Our in-vitro and in-vivo states show significant overlap (OR=4.71, p=9.42E-20). Furthermore, characterization of RTK.ACT.iv, recapitulated all the cell-state features observed in RTK.ACT - including higher growth-factors ER signaling, MAPK, and basal-like state (see table). We further studied the viability and transcription of these RTKs under various drugs (12 treatments), including fulvestrant, palbociclib, and specific tyrosine kinase inhibitors (TKIs) - Neratinib (pan-HER inhibitor) and FIIN-3 (FGFR-i). We found that HER2-ACT and FGFR-ACT signatures remain robust when treated with fulvestrant, palbociclib, and their combinations, as compared to TKIs suppression (see table). in-line with our viability results - suggesting intrinsic resistance to CDK4/6i and sensitivity to TKIs. This study demonstrated that activating RTKs constitute of prevalent modality of acquired resistance to endocrine therapies, inducing a distinct state with clinical implications - suggesting the potential benefit of combination therapies with specific TKIs over CDK4/6i. The common MAPK activity and our preliminary results - suggests the potential of convergence-node targeting strategy with added MEK or SHP2 inhibition. TableGene set AGene set BOdds-ratioP-value (two-sided)Fisher''s Exact test matHER2-ACT -FGFR-ACT -912.64E-9469, 131, 131, 22704HER2 S653C, L755S, V777L, L869R vs. GFP, under DMSO, rank genes based on the logFC (top 200)FGFR1, FGFR2 (WT, N550K, M538I, K660N) vs. GFP, Parental, Under DMSO, rank genes based on the logFC (top 200)HER2-ACT_1000 -FGFR-ACT_1000 -18.98.28E-248358, 642, 642, 21758HER2 S653C, L755S, V777L, L869R vs. GFP, under DMSO, rank genes based on the logFC- top 1000FGFR1, FGFR2 (WT, N550K, M538I, K660N) vs. GFP, Parental, Under DMSO, rank genes based on the logFC - top 1000RTK-ACTHALLMARK_ESTROGEN_RESPONSE_EARLY, MSigDB3.030.004229, 191, 349, 22474RTK-ACTER driven by Growth Factors, PMID: 208897186.771.74E-059, 86, 349, 22585RTK-ACTMEK_UP.V1_UP (MAPK), MSigDB10.95.72E-1827, 169, 331, 22496RTK-ACTRAS ONCOGENE (MAPK), PMID: 162730928.445.77E-1220, 158, 338, 22553RTK-ACTWU_CELL_MIGRATION, PMID 187243907.648.96E-1119, 165, 339, 22502RTK-ACTHUPER_BREAST_BASAL_VS_LUMINAL_UP, PMID 17409405131.38E-079, 45, 349, 22621RTK-ACTHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, MSigDB3.770.00031411, 189, 347, 22477RTK-ACTLIM_MAMMARY_STEM_CELL_UP, PMID 203461513.119.56E-0622, 467, 336, 22205RTK-ACTRTK-ACT.iv -4.719.42E-2060, 940, 298, 21992In MBC biopsies, compare RTK mutations with WT, rank genes based on the logFC - top 1000RTK-ACT.iv (in-vivo)HALLMARK_ESTROGEN_RESPONSE_EARLY, MSigDB1.950.020316, 184, 984, 22088RTK-ACT.ivER driven by Growth Factors, PMID: 208897182.640.0076310, 85, 990, 22193RTK-ACT.ivMEK_UP.V1_UP (MAPK), MSigDB2.863.91E-0522, 174, 978, 22098RTK-ACT.ivRAS ONCOGENE (MAPK), PMID: 162730921.620.13212, 166, 988, 22152RTK-ACT.ivWU_CELL_MIGRATION, PMID 187243903.573.60E-0725, 159, 975, 22115RTK-ACT.ivHUPER_BREAST_BASAL_VS_LUMINAL_UP, PMID 174094059.515.43E-1016, 38, 984, 22235RTK-ACT.ivHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, MSigDB2.090.0073817, 183, 983, 22090RTK-ACT.ivLIM_MAMMARY_STEM_CELL_UP, PMID 203461511.420.089229, 460, 971, 21819HER2-ACTHER2-ACT - Fulv3006.53E-183109, 91, 91, 22750HER2-ACTHER2-ACT - Palbo2331.46E-163101, 99, 99, 22749HER2-ACTHER2.ACT - Fulv + Palbo2197.64E-15999, 101, 101, 22742HER2-ACTHER2.ACT - Neratinib63.19.78E-7257, 143, 143, 22707HER2-ACTHER2.ACT - Fulv + Neratinib59.33.53E-6855, 145, 145, 22724FGFR-ACTFGFR.ACT - Palbo19003.2e-319157, 43, 43, 22778FGFR-ACTFGFR.ACT - Fulv + Palbo12501.38E-290148, 52, 52, 22767FGFR-ACTFGFR.ACT - Fulv8661.72E-266140, 60, 60, 22764FGFR-ACTFGFR.ACT - Palbo + FIIN.31062.88E-10474, 126, 126, 22710FGFR-ACTFGFR.ACT - Fulv + Palbo + FIIN.378.41.14E-8464, 136, 136, 22705FGFR-ACTFGFR.ACT - FIIN.367.42.16E-7559, 141, 141, 22730FGFR-ACTFGFR.ACT - Fulv + FIIN.336.89.47E-4541, 159, 159, 22733 Citation Format: Ofir Cohen, Pingping Mao, Utthara Nayar, Jorge E Buendia-Bue, Dewey Kim, Esha Jain, Karla Helvie, Daniel Abravanel, Kailey J Kowalski, Christian Kapstad, Samuel Freeman, Victor Adalsteinsson, Seth A Wander, Adrienne G Waks, Gad Getz, Aviv Regev, Eric Winer P Winer, Nancy U Nancy U. Lin, Nikhil Wagle. Acquired activating mutations in RTKs confer endocrine resistance in ER+ metastatic breast cancer through ER-reprogramming, MAPK signaling, and an induced stem-like cell state [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 GS2-02.

Background: The CDK4/6 inhibitor palbociclib (P) combined with endocrine therapy (ET) prolongs progression-free survival in previously untreated and treated hormone receptor positive/HER2 negative (HR+/HER2-) metastatic breast cancer (MBC). The most common toxicity with P is neutropenia, typically non-cumulative and uncomplicated. Given observed benefits of P in metastatic BC, this single arm phase II trial was designed to determine the feasibility and toxicity of combination adjuvant P and ET for HR+/HER2- early BC (EBC). Methods: Eligible patients (pts) had HR+/HER2- stage II (not T2N0)-III EBC, with prior completion of 3-24 mo of ET (either AI or tamoxifen) without significant adverse events (AE). Pts received P at 125 mg daily, 3 wk on/1 wk off in a 28d cycle, plus continuous ET, for planned duration 2 yrs. Pts were removed from study for toxicity, non-adherence, or other events related to tolerability; pts who recurred or completed 2 yrs of therapy were censored for the primary endpoint. The primary objective was to evaluate the treatment discontinuation rate at 2 yrs; a rate of >50%, would indicated a non-feasible treatment duration (null hypothesis). Discontinuation rates at 2 yrs are estimated by Kaplan Meier with 95% confidence bands. A sample size of 160 pts provided 92% power to reject the null hypothesis using a one-sided alpha = 0.025 if the true rate of discontinuation is Results: Between 3/2014 and 11/2015, 162 pts initiated P; the majority had stage III EBC (52%) and received prior chemotherapy (63%). As of 05/2017, 120 (74%) have completed at least 1 yr of P + ET, and 50 (31%) have completed 2 yrs of P + ET. Early discontinuation of protocol treatment was reported for 59 pts (36%), including 49 events (30%) related to protocol-mandated (9%) and non-mandated (21%) tolerability. The cumulative rate of all discontinuations was 15.1% at 6 mos, 20.9% at 12 mos and 27.8% at 18 mos. Half of all non-mandated discontinuations occurred within the first 6 mos of initiation of therapy, and the rate decreased with greater provider and pt education. Median duration of pts still on treatment is 20 mos (inter-quartile range: 18 to 21 mos). The rate of grade 3/4 neutropenia was 77%, with 0 cases of febrile neutropenia. Other common all-grade P-related AE > 20% included fatigue 65%, alopecia 25%, mucositis 24%, and anemia 24%. 32% of pts required one dose reduction, 16% required two. There have been 2 BC recurrence events and 1 chemotherapy-related AML. Updated data for the primary analysis of feasibility and tolerability, as well as pharmacogenomics, QOL, and adherence, will be presented. Conclusions: In this single arm phase II trial, the majority of pts have completed at least 1 year of adjuvant P + ET therapy, with no new toxicity signals. Non-protocol discontinuations have decreased with education. Updated results for the primary analysis will be presented. As in the MBC setting, extended duration palbociclib appears feasible and tolerable for most pts. The efficacy of 2 years of P and ET will be addressed by the phase III PALLAS trial (NCT NCT02513394). Citation Format: Mayer EL, DeMichele AM, Guo H, Miller KD, Rugo HS, Schneider B, Waks AG, Come SE, Mulvey T, Huang Bartlett C, Koehler M, Barry W, Winer EP, Burstein HJ. Adjuvant palbociclib plus endocrine therapy for hormone receptor positive/HER2 negative breast cancer: A phase II feasibility study [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 PD5-06.

In estrogen-receptor positive (ER+) breast cancer, therapies that target the ER have led to reductions in recurrence and mortality. However, in the metastatic setting, resistance to ER-targeted therapies is near universal. Various resistance mechanisms have been proposed, including ESR1 activating mutations and upregulation of alternative signal transduction pathways, yet clinically relevant resistance mechanisms have not been clearly defined. We conducted a genome-scale gain-of-function screen in ER+ breast cancer cell lines, spanning 17,255 overexpressed open reading frames (ORFs), to investigate genes whose overexpression is sufficient to confer resistance to selective estrogen receptor degraders (SERDs). Among several validated resistance genes, the fibroblast growth factor receptor (FGF/FGFR) pathway was among the top pathways, with several FGF ligands scoring as top resistance genes in this screen. In parallel, we performed whole exome sequencing (WES) in paired pre-treatment and post-resistance biopsies from 60 patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-targeted therapy. The FGFR pathway was altered in 40% (24 of 60) of the post-resistance biopsies; alterations included FGFR1 amplifications (9 patients), FGFR2 amplifications and activating mutations (4 patients), and FGF3 amplifications (17 patients). In 12 out of the 24 cases (5 FGFR1, 4 FGFR2, 3 FGF3), the FGF/FGFR pathway alterations were present in the resistant tumors and not detected in the pre-treatment tumors, suggesting that these alterations were acquired under the selective pressure of ER-directed therapy. Overexpression of wildtype FGFR1, FGFR2, FGF3 in ER+ breast cancer cells (T47D and MCF7) led to resistance to fulvestrant as well as the combination of fulvestrant and the CDK4/6 inhibitor palbociclib. This resistance phenotype was reversed by the FGFR inhibitor PD173074, and, to a lesser extent, the MEK inhibitor trametinib and mTOR inhibitor everolimus, suggesting several potential treatment strategies. The three FGFR2 mutations (M538I, N550K and K660N) identified in these patients induced hyperactive FGFR signaling and conferred resistance to fulvestrant, which was abrogated by the irreversible FGFR inhibitors FIIN-2 and FIIN-3. Together, these results offer new insights into endocrine resistance and suggest new clinical approaches to overcome or preempt therapeutic resistance. Citation Format: Pingping Mao, Ofir Cohen, Kailey Kowalski, Justin Kusiel, Jorge Buendia, Pedro Exman, Seth A. Wander, Adrienne G. Waks, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired FGF and FGFR alterations confer resistance to estrogen receptor (ER) targeted therapy in metastatic ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 314.

Background: While great strides have been made in the treatment of ER+ MBC, therapeutic resistance is nearly universal. The genomic evolution of ER+ breast cancer in the metastatic setting under the selective pressure of multiple lines of therapies is not well understood. To address this, we analyzed the clonal dynamics of serial metastatic samples (mets) to evaluate how tumors evolve and to identify acquired resistance mechanisms. Methods: We performed WES on 462 clinically annotated samples from 208 patients (pts) with ER+ MBC, including 67 primary tumor biopsies, 229 metastatic biopsies and 160 blood samples (cfDNA). Pts with multiple mets included cases with temporally concordant metastatic tumor and blood samples (48 pts) and cases with serial mets obtained over the course of treatment in the metastatic setting (69 pts). Treatments given between the serial mets included CDK4/6 inhibitors (23 pts), and selective estrogen receptor degraders (19 pts), among others. Results: In the temporally-concordant mets, we found that cfDNA mutations (muts) largely overlap with muts found in tumor biopsies, capturing >85% of clonal tumor muts. However, we observed a higher level of heterogeneity in cfDNA compared to biopsies (p.value< 1.05e-19, Welch test) and a subset of high-confidence muts that were only detected in cfDNA, including in clinically important genes such as ESR1, PIK3CA, KRAS, and ERBB2. Analysis of serial mets was used to elucidate the evolutionary dynamics within the metastatic setting under the selective pressure of treatment. The median duration between mets was 112 days and the median number of inter-biopsy unique treatments was two. Most tumors continued to evolve within the metastatic setting, with 50 out of 69 pts (72%) acquiring a meaningful sub-clone (50% increase in relative cancer cell fraction) and 31 out of 69 (45%) acquiring muts in known cancer genes, including a subset acquiring a plausible resistance alteration such as alterations that dysregulate ER (5 out of 69 pts, 7%; ESR1 mut, FOXA1 amplification (amp), NCOR1 bi-allelic deletion (del)), ERBB (4%; ERBB2 amp, ERBB3 mut), RAS (4%; KRAS mut, NRAS amp, NF1 del), FGF/FGFR (12%; FGFR2 mut, FGFR1/2 amp, FGF3 amp), and cell cycle (13%; RB1 del, CDK4 amp, AURKA amp, CDKN2A del). Finally, in pts who had multiple mets, we observed several cases of evolutionary convergence toward equivalent resistance mechanisms including convergent RB1 loss as a mechanism of resistance to a CDK4/6 inhibitor and convergent BRCA2 reversion following resistance to a PARP inhibitor. Conclusions: This study demonstrates that ER+ MBC continues to evolve under the selective pressure of treatments in the metastatic setting. These findings elucidate the challenge of studying high complexity and heavily treated tumors, while also highlighting some commonalities in the evolutionary trajectories selected by these treatments. The multiplicity of clinically relevant genomic alterations acquired in these advanced stages highlights the need for serial biopsies and the potential to inform post-progression therapeutic choices through targeting the acquired dependencies in post-progression tumors. Citation Format: Cohen O, Buendia-Buendia J, Wander S, Nayar U, Mao P, Waks A, Kim D, Freeman S, Adalsteinsson V, Helvie K, Livitz D, Rosebrock D, Leshchiner I, Dellostritto L, Garrido-Castro A, Jain E, Periyasamy S, Mackichan C, Lloyd M, Marini L, Krop I, Garraway L, Getz G, Winer E, Lin N, Wagle N. Evolutionary analysis of 462 serial metastatic biopsies from 208 patients with estrogen receptor-positive (ER+) metastatic breast cancer (MBC) using whole exome sequencing (WES) [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 PD9-02.

Background: Hormone receptor-positive (HR+) breast cancers (BC) have fewer tumor-infiltrating lymphocytes (TILs) and lower response rates to immune checkpoint inhibitors in early phase studies than other breast cancer subtypes. Immune biomarkers that accurately reflect the immune microenvironment have important clinical implications in HR+ BC patients. Prior evidence suggests that macrophage-related immune pathways may be relevant to the pathophysiology of HR+ BC. Methods: Patients identified from a prospective trial of preoperative bevacizumab (preop bev) followed by bev with adriamycin/cyclophosphamide/paclitaxel dose-dense chemotherapy (chemo). Tumor samples were collected at diagnosis and surgery (pre-tx and post-tx). TILs and immunohistochemical staining for PD-L1, CD8, and CD68 were scored. Whole transcriptome sequencing (RNAseq) and Nanostring PanCancer Immune Profiling Panel were performed. Pathologic response at surgery was assessed by Miller-Payne (MP) and residual cancer burden (RCB) scores. An immune score was calculated for each pre-tx specimen by integrating 10 published immune signatures. Immune cell subsets were inferred from bulk transcriptional data using CIBERSORT. Results: 55 patients had at least 1 evaluable specimen and were included for analysis. 18% of pre-tx tumors had ‘high' (≥10%) TILs and ‘high' TILs were associated with significantly higher immune signature score (p=0.004). Immune score correlated highly with proportion of CIBERSORT anti-tumor M1 macrophage and CD8 T-cell signatures (r>0.65 and p Conclusions: Most HR+/HER2- breast tumors demonstrate low levels of anti-tumor immune activity; however, those with higher levels have a more favorable response to chemo plus bev. Assessment of immune activity based on RNA signatures is consistent with histology and immune-related protein expression. T-cell- and checkpoint-related biomarkers tend to decrease following preoperative chemo plus bev in HR+/HER2- breast cancer. Following treatment with chemotherapy/bevacizumab, we observe increased expression of chemokines and complement pathway genes. Citation Format: Adrienne G. Waks, Daniel G. Stover, William T. Barry, Deborah A. Dillon, Evisa Gjini, Scott J. Rodig, Jane E. Brock, Michele Baltay, Jennifer Savoie, Eric P. Winer, Ian E. Krop, Sara M. Tolaney. The immune microenvironment in hormone receptor-positive breast cancer and treatment outcome following preoperative chemotherapy plus bevacizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4564.

Despite the reduction in cancer recurrence and mortality provided by therapies that target the estrogen receptor (ER), resistant ER+ breast cancer remains the most common cause of breast cancer death. Beyond mutations in ER in 25-30% of patients treated with aromatase inhibitors, which deplete circulating estrogen, our understanding of clinical mechanisms of resistance to agents that target the ER remains incomplete. We used whole exome sequencing (WES) of metastatic biopsies to identify mechanisms of resistance in patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-directed agents, including aromatase inhibitors, tamoxifen, and fulvestrant. We noted mutations in human epidermal growth factor receptor 2 (HER2) in metastatic biopsies from 12 patients out of 168. Of these, eight mutations had previously been described as activating. Examination of the treatment-naïve primary tumors in the five patients with activating mutants where WES from the matched treatment-naïve primary tumor was available revealed no evidence of pre-existing mutations in four of the five patients, suggesting that these four mutations were acquired under the selective pressure of ER-directed therapy. These acquired HER2 mutations were mutually exclusive with ER mutations, suggesting a distinct mechanism of resistance to ER-directed therapies. In vitro analysis through expression in ER+/HER2- cell lines confirmed that these mutations conferred estrogen independence. In addition, and in contrast to ER mutations, these HER2 mutations resulted in resistance to tamoxifen, fulvestrant, and the CDK4/6 inhibitor palbociclib. The mutants result in increased AKT and ERK phosphorylation in ER+ cell lines, while simultaneously repressing ER levels and ER-dependent transcriptional targets. One mutation observed in two patients, S653C, occurring in the transmembrane domain and not previously observed in breast cancer, was shown to likely function through constitutive dimerization, a mechanism of activation not previously described for this mutation. Resistance caused by all four mutations was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib, suggesting a novel effective treatment strategy in these patients. Thus, we have shown that acquired activating HER2 mutations can confer endocrine resistance in ER+/HER2- breast cancer, and that such resistance can be effectively reversed by combination therapies that include an anti-HER2 inhibitor. Citation Format: Utthara Nayar, Ofir Cohen, Christian Kapstad, Adrienne Waks, Seth A. Wander, Corrie Painter, Samuel Freeman, Priyanka Ram, Nicole Persky, Lori Marini, Karla Helvie, Nelly Oliver, Cynthia X. Ma, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired HER2 mutations in ER+ metastatic breast cancer confer resistance to ER-directed therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4952.

Background: While great strides have been made in the treatment of estrogen receptor-positive (ER+) metastatic breast cancer (MBC), therapeutic resistance invariably occurs. A better understanding of the underlying resistance mechanisms is critical to enable durable control of this disease. Methods: We performed whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) on metastatic tumor biopsies from 88 patients with ER+ MBC who had developed resistance to one or more ER-directed therapies. For 27 of these patients, we sequenced the treatment-naïve primary tumors for comparison to the resistant specimens. Tumors were analyzed for point mutations, insertions/deletions, copy number alterations, translocations, and gene expression. Detailed clinicopathologic data was collected for each patient and linked to the genomic information. Results: WES of all metastatic samples demonstrated several recurrently altered genes whose incidence differed significantly from primary, treatment-naïve ER+ breast cancers sequenced in the TCGA study (TCGA). These include ESR1 mutations (n=17, 19.3%; 32.86 fold enrichment, q.value Comparing to matched primary samples from the same patient, many alterations were found to be acquired in several cases, including for ESR1, ERBB2, PIK3CA, PTEN, RB1, AKT1, and others. Initial analysis of RNA-seq data from metastatic samples (n=59) allowed classification of individual resistance mechanisms into broader resistance modes based on the observed transcriptional state. Conclusions: We present a genomic landscape of resistant ER+ MBC using WES and RNA-seq. Multiple genes were recurrently altered in these tumors at significantly higher rates than in ER+ primary breast cancer. When compared with matched primary tumors from the same patient, alterations in these and other genes were often found to be acquired after treatment, suggesting a role in resistance to ER-directed therapies and/or metastasis. Potential resistance mechanisms appear to fall into several categories; integrating RNA-seq data may enhance the ability to identify these categories even when genomic alterations are not identified. Multiple clinically relevant genomic and molecular alterations are identified in metastatic biopsies– with implications for choice of next therapy, clinical trial eligibility, and novel drug targets. Citation Format: Cohen O, Kim D, Oh C, Waks A, Oliver N, Helvie K, Marini L, Rotem A, Lloyd M, Stover D, Adalsteinsson V, Freeman S, Ha G, Cibulskis C, Anderka K, Tamayo P, Johannessen C, Krop I, Garraway L, Winer E, Lin N, Wagle N. Whole exome and transcriptome sequencing of resistant ER+ metastatic 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 S1-01.