Supplemental Table 1. Biomarker Associations with Outcome

Legend for Supplementary Figures 1-2

Supplemental Figure 1. Number of cycles received

Supplemental Figure 2. Representative biomarker staining

Abstract Background Up to 30% of breast cancer patients will eventually relapse with metastatic disease. With an increasing array of therapeutic options, there is an ongoing need for predictive biomarkers to help guide treatment strategies including sequencing of therapies in the metastatic setting. We sought to evaluate the prognostic and predictive potential of a panel-specific tumor mutational burden (TMB) in metastatic breast cancer patients. Methods METAMORPH is a prospective, longitudinal cohort study. Eligible patients (pts) had newly diagnosed or progressive metastatic breast cancer and enrolled prior to starting a new line of therapy (physician’s choice) at the University of Pennsylvania. Pts underwent tissue biopsy of a suspected metastatic site. Tumor samples were analyzed for mutations and copy number alterations (CNA’s) using our institution’s CLIA-certified Center for Personalized Diagnostics (CPD) targeted gene panel, which evolved over the course of the study from 20 genes to 152 genes. TMB-high (TMB-H) was defined as ≥3 mutations and/or copy-number gains (CNG) among 18 genes shared across all panel versions. Pts were followed for time to progression (TTP), progression-free survival (PFS), and overall survival (OS). The frequency of rapid progressors and rapid death (defined as having progressed or died within 3 months of enrollment, respectively) was assessed. Results Three hundred pts enrolled from 2013-2020, of whom 200 pts had CPD reports generated. Of these, 12 pts were excluded due to either no treatment change on enrollment (n=11) or different primary cancer on biopsy (n=1). Thus 188 pts were included in this analysis. The median age was 55 years (range 28-79). 77% of pts identified as white, 18% as Black or African American, and 3.2% as Asian. Pts had a median of 1 line (range 0-12) of prior systemic therapy in the metastatic setting. 46.8% had no prior therapies for MBC, while 31% had ≥3 prior lines of therapy. 74.4% were HR+, 22.8% TNBC, and 2.7% HR-/HER2+. 6.9% of the cohort were classified as TMB-H. The average mutation/CNG rate was 2.2/sample, and 22.5% had no mutations or CNA’s. The most common mutations were TP53 (35%) and PIK3CA (26%). While TMB-H patients showed a statistically non-significant trend towards shorter median TTP and PFS compared with TMB-L, they comprised a significantly greater proportion of rapid progressors (54.5% vs 24.1%, p=0.027), with an odds ratio for rapid progression of 3.8 (95% CI 1.08-13.2). In a multivariate logistic regression analysis, TMB-H remained independently associated with rapid progression when adjusted for receptor subtype and next line of therapy. Receptor subtype analysis revealed that ER- (including ER-/PR+) patients with TMB-H had a shorter median TTP compared to ER- TMB-L (147 vs 68 days, p=0.03). TMB-H was also associated with significantly shorter OS compared with TMB-L (587 vs 648 days, p=0.02; HR 2.2 [95% CI 1.11-4.41]). 44.4% of TMB-H pts died within 3 months of enrollment, as compared to 11.0% of TMB-L pts (p=0.005), with an odds ratio for rapid death, adjusted for number of previous lines of therapy and receptor subtype, of 6.7 (95% CI 1.5-31.0). Conclusion MBC pts who are TMB-H represent a population who are highly resistant to standard therapies, progress rapidly, and have significantly shorter overall survival with more rapid time to death. Our data support further studies investigating the utility of TMB as a predictive biomarker in directing patients away from standard treatment options and towards novel approaches e.g. immunotherapy. Citation Format: Igor Makhlin, Amy S Clark, Paul Wileyto, Noah Goodman, John Ndicu, Shannon DeLuca, Candace Clark, S. William Stavropoulos, Natalie Shih, Michael D Feldman, Susan M Domchek, Jennifer M Matro, Payal D Shah, Hayley M Knollman, Kevin R Fox, Kara N Maxwell, Lewis A Chodosh, Angela DeMichele. Investigating the clinical utility of tumor mutational burden in predicting rapid progression and death in patients with metastatic 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 PD9-10.

PURPOSEWomen with breast cancer have a 4%-16% lifetime risk of a second primary cancer. Whether mutations in genes other than BRCA1/2 are enriched in patients with breast and another primary cancer over those with a single breast cancer (S-BC) is unknown.PATIENTS AND METHODSWe identified pathogenic germline mutations in 17 cancer susceptibility genes in patients with BRCA1/2-negative breast cancer in 2 different cohorts: cohort 1, high-risk breast cancer program (multiple primary breast cancer [MP-BC], n = 551; S-BC, n = 449) and cohort 2, familial breast cancer research study (MP-BC, n = 340; S-BC, n = 1,464). Mutation rates in these 2 cohorts were compared with a control data set (Exome Aggregation Consortium [ExAC]).RESULTSOverall, pathogenic mutation rates for autosomal, dominantly inherited genes were higher in patients with MP-BC versus S-BC in both cohorts (8.5% v 4.9% [P = .02] and 7.1% v 4.2% [P = .03]). There were differences in individual gene mutation rates between cohorts. In both cohorts, younger age at first breast cancer was associated with higher mutation rates; the age of non-breast cancers was unrelated to mutation rate. TP53 and MSH6 mutations were significantly enriched in patients with MP-BC but not S-BC, whereas ATM and PALB2 mutations were significantly enriched in both groups compared with ExAC.CONCLUSIONMutation rates are at least 7% in all patients with BRCA1/2 mutation-negative MP-BC, regardless of age at diagnosis of breast cancer, with mutation rates up to 25% in patients with a first breast cancer diagnosed at age < 30 years. Our results suggest that all patients with breast cancer with a second primary cancer, regardless of age of onset, should undergo multigene panel testing.

Background : While several comprehensive genomic sequencing tests are clinically available for breast cancer(BC), little is known about the spectrum of findings reported in the general population and clinical utility of findings for patients(pts). Here we report tumor sequencing from the METAMORPH study, a comprehensive genomic testing approach in pts with metastatic(met) BC. Methods : Pts with either known or suspected BC mets consented to and clinically underwent concurrent diagnostic and research tumor biopsies(bx). FFPE specimens were profiled via Illumina TruSeq Cancer Panel next generation sequencing platform covering 212 amplicons in 47 cancer genes. Pathology, treatment and outcome data were prospectively collected and tracked. Aside from Her2-directed treatment, therapy was not mutation (mut)-matched. Results : 64 pts enrolled between 11/2013 – 05/2015. Of these, 48 had bx successfully sequenced (75%). Of those without sequencing, 5 had negative/insufficient tissue, 2 had insufficient DNA, remainder no bx/pending. Median age of those sequenced was 56 (range 31-78); 81% Caucasian, 17% African American. 25% (12 pts) presented with de novo stage IV disease. Of those with recurrence (n=36), 83% had prior adjuvant chemotherapy; 81% hormone receptor positive(HR+) had prior endocrine therapy. Median # prior lines of therapy for met disease was 2 (IQR 0 – 8). Tumor characteristics, including mut analyses, are shown in Table 1. # muts did not differ significantly by subtype(p=0.22). Frequency of TP53 and PIK3CA hotspot muts was nearly identical to TCGA. Median # muts was 1 for pts with both de novo mets and recurrence(p=0.79). # of muts was not associated with time to recurrence(p=0.80). Excluding pts found to have TP53 mut only or ERBB2 alterations in known Her2+ disease, 42% of pts were identified as having at least one potentially actionable alteration ( PIK3CA mut, AKT1 mut or EGFR amplification). Median time to treatment failure(TTF) on subsequent therapy was 4.1 months for overall group, and 4.1, 6.2, and 1.6 months for HR+/Her2-, any Her2+ and TN, respectively, adjusted for line of therapy(p=0.03). After adjustment for # lines of prior met therapy, TTF was 4.7 vs. 4.1 months for pts with any mut vs. none(p=0.89); 5.7 vs 4.1 months for PIK3CA + vs. not (p=0.94); 3.3 vs. 6.5 months for TP53 + vs. not (p=0.03). Conclusion : Pts with met BC have frequent and potentially actionable muts.While overall # of muts did not affect response, tumors with TP53 muts had shorter response to subsequent therapy in this cohort. Additional data are needed to determine the clinical utility of mut testing in met BC, for both standard and mut-matched therapy. Citation Format: Soucier-Ernst D, Colameco C, Troxel AB, Clark C, Shih N, Maxwell KN, Morrissette J, Lieberman D, Feldman M, Goodman N, Bradbury A, Clark A, Domchek S, Fox K, Glick J, Matro J, Nathanson K, Chodosh L, DeMichele A. Mutational spectrum and tumor response in metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-05.

IMPORTANCE There is a paucity of immune signatures identifying patients with human epidermal growth factor receptor 2 (HER2)-positive invasive breast cancer (IBC) at risk for treatment failure following trastuzumab and chemotherapy.OBJECTIVE To determine whether circulating anti-HER2 CD4-positive (CD4(+)) T-helper type 1 (Th1) immunity correlates with recurrence in patients with completely treated HER2-positive IBC.DESIGN, SETTING, AND PARTICIPANTS Hypothesis-generating exploratory translational analysis at a tertiary care referral center of patients with completely treated HER2-positive IBC with median (interquartile range) follow-up of 44 (31) months. Anti-HER2 Th1 responses were examined using peripheral blood mononuclear cells pulsed with 6 HER2-derived class II-promiscuous peptides via interferon-gamma (IFN-gamma) enzyme-linked immunospot assay.MAIN OUTCOMES AND MEASURES T-helper type 1 response metrics were anti-HER2 responsivity, repertoire (number of reactive peptides), and cumulative response across 6 peptides (spot-forming cells [SFCs]/10(6) cells). Anti-HER2 Th1 responses in treatment-naive patients (used as an immunologic baseline) were compared with those in patients completing trastuzumab and chemotherapy; in the latter group, analyses were stratified by recurrence status. Recurrence was defined as any locoregional or distant breast event, or both. Cox regression analysis estimated the instantaneous hazard of recurrence (ie, disease-free survival [DFS]) stratified by anti-HER2 Th1 responsivity.RESULTS In 95 women with HER2-positive IBC (median [range] age, 49 [24-85] years; 22 treatment-naive, 73 treated with trastuzumab and chemotherapy), depressed anti-HER2 Th1 responsivity (recurrence, 2 of 25 [8%], vs nonrecurrence, 40 of 48 [83%]; P<.001), mean (SD) repertoire (0.1 [0.1] vs 1.5 [0.2]; P<.001), and mean (SD) cumulative response (14.8 [2.0] vs 80.2 [11.0] SFCs/106 cells; P<.001) were observed in patients incurring recurrence (n = 25) compared with patients without recurrence (n = 48). After controlling for confounding, anti-HER2 Th1 responsivity remained independently associated with recurrence (P<.001). This immune disparity was mediated by anti-HER2 CD4(+) T-bet(+) IFN-gamma(+) (Th1)-not CD4(+)GATA-3(+) IFN-gamma(+) (Th2) or CD4(+) CD25(+) FoxP3(+) (T-reg)-phenotypes, and not attributable to immune incompetence. When stratifying trastuzumab plus chemotherapy-treated patients by Th1 responsivity, Th1-nonresponsive patients demonstrated a worse DFS (median, 47 vs 113 months; P<.001) compared with Th1-responsive patients (hazard ratio, 16.9 [95% CI, 3.9-71.4]; P <.001).CONCLUSIONS AND RELEVANCE Depressed anti-HER2 Th1 response is a novel immune correlate to recurrence in patients with completely treated HER2-positive IBC. These data underscore a role for immune monitoring in patients with HER2-positive IBC to identify vulnerable populations at risk of treatment failure.

Abstract BACKGROUND: We have previously demonstrated a loss of anti-HER2 CD4 T-helper type 1 responses (Th1resp) in HER2+ invasive breast cancer (IBC) pts relative to healthy donors. Compared with pathologic complete response (pCR) after neoadjuvant trastuzumab/chemotherapy (T/C), residual disease at surgery ( METHOD: Th1resp were generated from PBMCs pulsed with 6 HER2 class II peptides by measuring IFN-γ production via ELISPOT, and compared between HER2+ IBC groups. Th1resp metrics were anti-HER2 responsivity, no. of reactive peptides (repertoire), and cumulative response across 6 peptides (spots/10^6 cells). Th1resp of RESULTS: The study comprised 85 pts. The diminished anti-HER2 Th1resp in treatment-naïve HER2+ IBC pts (n = 21) - assessed by responsivity, repertoire, or cumulative response - did not improve globally in T/C-treated IBC pts (n = 64). Within this T/C-treated cohort, neoadjuvant T/C receipt (60.9%) was associated with higher repertoire (1.5 vs 0.7; p = 0.04), but not responsivity or cumulative response, compared with adjuvant T/C. CONCLUSION: Beyond the known association between ER status and pathologic response following neoadjuvant T/C in HER2+ breast cancer, we identify a novel correlation between Note: This abstract was not presented at the meeting. Citation Format: Jashodeep Datta, Erik Berk, Shuwen Xu, Elizabeth Fitzpatrick, Lea Lowenfeld, Noah Goodman, David Lewis, Robert E. Roses, Angela DeMichele, Brian J. Czerniecki. Depressed anti-HER2 CD4 Th1 responses correlate with residual disease following neoadjuvant therapy in HER2+ breast cancer patients and can be restored by dendritic cell vaccination. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1308. doi:10.1158/1538-7445.AM2015-1308

Abstract Purpose: The G1–S checkpoint of the cell cycle is frequently dysregulated in breast cancer. Palbociclib (PD0332991) is an oral inhibitor of CDK4/6. Based upon preclinical/phase I activity, we performed a phase II, single-arm trial of palbociclib in advanced breast cancer. Experimental Design: Eligible patients had histologically confirmed, metastatic breast cancer positive for retinoblastoma (Rb) protein and measureable disease. Palbociclib was given at 125 mg orally on days 1 to 21 of a 28-day cycle. Primary objectives were tumor response and tolerability. Secondary objectives included progression-free survival (PFS) and assessment of Rb expression/localization, KI-67, p16 loss, and CCND1 amplification. Results: Thirty-seven patients were enrolled; 84% hormone-receptor (HR)+/Her2−, 5% HR+/Her2+, and 11% HR−/Her2−, with a median of 2 prior cytotoxic regimens. Two patients had partial response (PR) and 5 had stable disease ≥ 6 months for a clinical benefit rate (CBR = PR + 6moSD) of 19% overall, 21% in HR+, and 29% in HR+/Her2− who had progressed through ≥2 prior lines of hormonal therapy. Median PFS overall was 3.7 months [95% confidence interval (CI), 1.9–5.1], but significantly longer for those with HR+ versus HR− disease (P = 0.03) and those who had previously progressed through endocrine therapy for advanced disease (P = 0.02). Grade 3/4 toxicities included neutropenia (51%), anemia (5%), and thrombocytopenia (22%). Twenty-four percent had treatment interruption and 51% had dose reduction, all for cytopenias. No biomarker identified a sensitive tumor population. Conclusions: Single-agent palbociclib is well tolerated and active in patients with endocrine-resistant, HR+, Rb-positive breast cancer. Cytopenias were uncomplicated and easily managed with dose reduction. Clin Cancer Res; 21(5); 995–1001. ©2014 AACR.

1511 Background: Multiplex panel testing studies evaluating cancer susceptibility in breast cancer (BC) patients suggest that between 4-11% of BRCA1/2 negative individuals have a deleterious mutation in a high or moderate penetrance gene. The range in mutation positivity is likely due to heterogeneity of the studied patient populations. Methods: We performed targeted, massively parallel sequencing of 795 BRCA1/2 negative BC patients in a research laboratory and analyzed 18 cancer susceptibility genes.Three high risk groups were studied: multiple primary cancers (MP, n = 315), early onset breast cancer (EOBC, n = 323), and familial breast cancer (FBC, n = 415). MP was defined as at least one BC and at least one other primary malignancy excluding non-melanoma skin cancer, EOBC was defined as BC under age 40, and FBC was defined as at least three first to third degree relatives with BC under age 75. Results: Overall, 78 patients (10%) were found to have a deleterious mutation. Mutations were found most commonly in CHEK2 (n = 32, 4.0%), ATM (n = 14, 1.8%), TP53 (n = 10, 1.3%), and MSH6 (n = 4, 0.5%). One to two patients each had deleterious mutations in BARD1, BRIP1, CDKN2A, MRE11A, MSH2, NBN, PALB2, PTEN, PMS2, and RAD50; no mutations were identified in CDH1, MLH1, or STK11. Deleterious mutations were more common in MP patients versus FBC patients without MP (12% versus 7%, p = 0.04) but not EOBC patients without MP (10%, p = NS). CHEK2 and MSH6 mutations were more common in MP versus non-MP patients (6% vs 3% and 1.3% vs none, respectively, p = 0.05) whereas ATM mutations were more common in non-MP patients (0.6% vs 2.5%, p = 0.05). The most common cancers in mutation carriers were a second primary breast cancer, sarcoma, melanoma, hematological malignancies, ovarian, thyroid and uterine cancer. There was a similar distribution of malignancies in MP mutation carriers versus non-mutation carriers. Sequencing is ongoing for 276 additional MP patients to validate these findings. Conclusions: Our results indicate that multiplex panel testing may find a higher rate of mutations in patients with MP malignancies as compared to patients with FBC alone; however, the type of second primary malignancy may not be predictive of mutation status.

INTRODUCTION:A progressive loss of circulating anti-human epidermal growth factor receptor-2/neu (HER2) CD4(+) T-helper type 1 (Th1) immune responses is observed in HER2(pos)-invasive breast cancer (IBC) patients relative to healthy controls. Pathologic complete response (pCR) following neoadjuvant trastuzumab and chemotherapy (T + C) is associated with decreased recurrence and improved prognosis. We examined differences in anti-HER2 Th1 responses between pCR and non-pCR patients to identify modifiable immune correlates to pathologic response following neoadjuvant T + C.METHODS:Anti-HER2 Th1 responses in 87 HER2(pos)-IBC patients were examined using peripheral blood mononuclear cells pulsed with 6 HER2-derived class II peptides via IFN-γ ELISPOT. Th1 response metrics were anti-HER2 responsivity, repertoire (number of reactive peptides), and cumulative response across 6 peptides (spot-forming cells [SFC]/10(6) cells). Anti-HER2 Th1 responses of non-pCR patients (n = 4) receiving adjuvant HER2-pulsed type 1-polarized dendritic cell (DC1) vaccination were analyzed pre- and post-immunization.RESULTS:Depressed anti-HER2 Th1 responses observed in treatment-naïve HER2(pos)-IBC patients (n = 22) did not improve globally in T + C-treated HER2(pos)-IBC patients (n = 65). Compared with adjuvant T + C receipt, neoadjuvant T + C - utilized in 61.5 % - was associated with higher anti-HER2 Th1 repertoire (p = 0.048). While pCR (n = 16) and non-pCR (n = 24) patients did not differ substantially in demographic/clinical characteristics, pCR patients demonstrated dramatically higher anti-HER2 Th1 responsivity (94 % vs. 33 %, p = 0.0002), repertoire (3.3 vs. 0.3 peptides, p < 0.0001), and cumulative response (148.2 vs. 22.4 SFC/10(6), p < 0.0001) versus non-pCR patients. After controlling for potential confounders, anti-HER2 Th1 responsivity remained independently associated with pathologic response (odds ratio 8.82, p = 0.016). This IFN-γ(+) immune disparity was mediated by anti-HER2 CD4(+)T-bet(+)IFN-γ(+) (i.e., Th1) - not CD4(+)GATA-3(+)IFN-γ(+) (i.e., Th2) - phenotypes, and not attributable to non-pCR patients' immune incompetence, host-level T-cell anergy, or increased immunosuppressive populations. In recruited non-pCR patients, anti-HER2 Th1 repertoire (3.7 vs. 0.5, p = 0.014) and cumulative response (192.3 vs. 33.9 SFC/10(6), p = 0.014) improved significantly following HER2-pulsed DC1 vaccination.CONCLUSIONS:Anti-HER2 CD4(+) Th1 response is a novel immune correlate to pathologic response following neoadjuvant T + C. In non-pCR patients, depressed Th1 responses are not immunologically "fixed" and can be restored with HER2-directed Th1 immune interventions. In such high-risk patients, combining HER2-targeted therapies with strategies to boost anti-HER2 Th1 immunity may improve outcomes and mitigate recurrence.

We report a case of asbestosis in a 63-year-old man, diagnosed 45 years after exposure to asbestos. The case is unusual in the finding of hemorrhagic pleural effusion without associated malignancy. We report a case of asbestosis in a 63-year-old man, diagnosed 45 years after exposure to asbestos. The case is unusual in the finding of hemorrhagic pleural effusion without associated malignancy.