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Supplementary Figure 1 - PDF file 44K, Supplementary Figure 1A-D. Median hormone levels at baseline, C2 and EOS

Purpose: Breast cancer is a heterogeneous disease, and markers for disease subtypes and therapy response remain poorly defined. For that reason, we employed a prospective neoadjuvant study in locally advanced breast cancer to identify molecular signatures of gene expression correlating with known prognostic clinical phenotypes, such as inflammatory breast cancer or the presence of hypoxia. In addition, we defined molecular signatures that correlate with response to neoadjuvant chemotherapy.Experimental Design: Tissue was collected under ultrasound guidance from patients with stage IIB/III breast cancer before four cycles of neoadjuvant liposomal doxorubicin paclitaxel chemotherapy combined with local whole breast hyperthermia. Gene expression analysis was done using Affymetrix U133 Plus 2.0 GeneChip arrays.Results: Gene expression patterns were identified that defined the phenotypes of inflammatory breast cancer as well as tumor hypoxia. In addition, molecular signatures were identified that predicted the persistence of malignancy in the axillary lymph nodes after neoadjuvant chemotherapy. This persistent lymph node signature significantly correlated with disease-free survival in two separate large populations of breast cancer patients.Conclusions: Gene expression signatures have the capacity to identify clinically significant features of breast cancer and can predict which individual patients are likely to be resistant to neoadjuvant therapy, thus providing the opportunity to guide treatment decisions.

CYTOF Panel

PDF file - 797K, Figure S1. Barrier phenotype of human brain microvascular endothelial cells and astrocytes in monocultures and co-cultures. Figure S2. alpha/beta-crystallin does not affect cell viability of TNBC cells in standard monolayer culture. Figure S3. alpha/beta-crystallin overexpression increases liver metastases in an orthotopic model of TNBC. Figure S4. Silencing alpha/beta-crystallin inhibits liver metastases in an orthotopic model of TNBC. Figure S5. alpha/beta-crystallin does not affect proliferation or apoptosis in primary mammary tumors and brain metastatic lesions determined at autopsy. Table S1. Patient characteristics Table S2. alpha/beta-crystallin expression in paired tumors. Table S3. Association of alpha/beta-crystallin expression with breast cancer subtype. Table S4. Incidence of metastases in mice in orthotopic models of TNBC .

Supplementary Figures S1, S2, S3 and Tables S1, S2

SPADE tree generated by CYTOF analysis

Supplementary Figures S1-S3; Tables S1, S2 from Gene Expression Profiles of Multiple Breast Cancer Phenotypes and Response to Neoadjuvant Chemotherapy

Purpose: The use of tumor-derived proteins as cancer vaccines is complicated by tolerance to these self-antigens. Tolerance may be broken by immunization with activated, autologous, ex vivo generated and antigen-loaded, antigen-presenting cells (APC); however, targeting tumor antigen directly to APC in vivo would be a less complicated strategy. We wished to test whether targeted delivery of an otherwise poorly immunogenic, soluble antigen to APC through their mannose receptors (MR) would induce clinically relevant immunity.Experimental Design: Two phase I studies were conducted with CDX-1307, a vaccine composed of human chorionic gonadotropin beta-chain (hCG-β) fused to an MR-specific monoclonal antibody, administered either locally (intradermally) or systemically (intravenously) in patients with advanced epithelial malignancies. An initial dose escalation of single-agent CDX-1307 was followed by additional cohorts of CDX-1307 combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) and the Toll-like receptor (TLR) 3 agonist polyinosinic-polycytidylic acid (poly-ICLC) and TLR7/8 agonist resiquimod to activate the APC.Results: CDX-1307 induced consistent humoral and T-cell responses to hCG-β when coadministered with TLR agonists. Greater immune responses and clinical benefit, including the longest duration of stable disease, were observed with immunization combined with local TLR agonists. Immune responses were induced equally efficiently in patients with elevated and nonelevated levels of serum hCG-β. Antibodies within the serum of vaccinated participants had tumor suppressive function in vitro. Toxicity consisted chiefly of mild injection site reactions.Conclusions: APC targeting and activation induce adaptive immunity against poorly immunogenic self-antigens which has implications for enhancing the efficacy of cancer immunotherapy. Clin Cancer Res; 17(14); 4844–53. ©2011 AACR.

Concurrent HER2 therapies for each patient for whom CYTOF analysis was done

Patient cohort schematic

Mutation of HER2 to obscure binding of Trastuzumab and Pertuzumab

Supplementary Legend - PDF file 22K, Supplementary Legend

Purpose: Based on promising preclinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2mut detection.Experimental Design: Tumor tissue positive for HER2mut was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2mut).Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2mut cases were identified locally. Twenty-one of these 22 HER2mut cases were estrogen receptor positive. Sixteen patients [median age 58 (31–74) years and three (2–10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI), 13%–55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8–31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2mut in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%–94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%–100%). In addition, ctDNA HER2mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression.Conclusions: Neratinib is active in HER2mut, nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2mut cancers for clinical trial participation. Clin Cancer Res; 23(19); 5687–95. ©2017 AACR.

Supplementary Figure 2 - PDF file 275K, Supplementary Figure 2A-D. Changes in hormone levels over time per patient; responders (stable disease >6 months) highlighted in red

Purpose: Basal-like breast tumors are typically (ER/PR/HER2) triple-negative and are associated with a high incidence of brain metastases and poor clinical outcomes. The molecular chaperone αB-crystallin is predominantly expressed in triple-negative breast cancer (TNBC) and contributes to an aggressive tumor phenotype in preclinical models. We investigated the potential role of αB-crystallin in brain metastasis in TNBCs.Experimental Design: αB-crystallin expression in primary breast carcinomas and brain metastases was analyzed by immunohistochemistry among patients with breast cancer with brain metastases. αB-crystallin was overexpressed or silenced in two different TNBC cell lines. The effects on cell adhesion to human brain microvascular endothelial cells (HBMEC) or extracellular matrix proteins, transendothelial migration, and transmigration across a HBMEC/astrocyte coculture blood–brain barrier (BBB) model were examined. In addition, the effects of overexpressing or silencing αB-crystallin on brain metastasis in vivo were investigated using orthotopic TNBC models.Results: In a cohort of women with breast cancer brain metastasis, αB-crystallin expression in primary breast carcinomas was associated with poor overall survival and poor survival after brain metastasis, even among patients with TNBC. Stable overexpression of αB-crystallin in TNBC cells enhanced adhesion to HBMECs, transendothelial migration, and BBB transmigration in vitro, whereas silencing αB-crystallin inhibited these events. αB-crystallin promoted adhesion of TNBC cells to HBMECs, at least in part, through an α3β1 integrin–dependent mechanism. αB-crystallin overexpression promoted brain metastasis, whereas silencing αB-crystallin inhibited brain metastasis in orthotopic TNBC models.Conclusion: αB-crystallin is a novel regulator of brain metastasis in TNBC and represents a potential biomarker and drug target for this aggressive disease. Clin Cancer Res; 20(1); 56–67. ©2013 AACR.

Supplementary Table 1 from Phase I Study Utilizing a Novel Antigen-Presenting Cell–Targeted Vaccine with Toll-like Receptor Stimulation to Induce Immunity to Self-antigens in Cancer Patients

Individual histograms of perforin expression by memory CD8 T cell cluster pre- and post-vaccination

Purpose:Immune-based therapy for metastatic breast cancer has had limited success, particularly in molecular subtypes with low somatic mutations rates. Strategies to augment T-cell infiltration of tumors include vaccines targeting established oncogenic drivers such as the genomic amplification of HER2. We constructed a vaccine based on a novel alphaviral vector encoding a portion of HER2 (VRP-HER2).Patients and Methods:In preclinical studies, mice were immunized with VRP-HER2 before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and antitumor function were evaluated. We tested VRP-HER2 in a phase I clinical trial where subjects with advanced HER2-overexpressing malignancies in cohort 1 received VRP-HER2 every 2 weeks for a total of 3 doses. In cohort 2, subjects received the same schedule concurrently with a HER2-targeted therapy.Results:Vaccination in preclinical models with VRP-HER2 induced HER2-specific T cells and antibodies while inhibiting tumor growth. VRP-HER2 was well tolerated in patients and vaccination induced HER2-specific T cells and antibodies. Although a phase I study, there was 1 partial response and 2 patients with continued stable disease. Median OS was 50.2 months in cohort 1 (n = 4) and 32.7 months in cohort 2 (n = 18). Perforin expression by memory CD8 T cells post-vaccination significantly correlated with improved PFS.Conclusions:VRP-HER2 increased HER2-specific memory CD8 T cells and had antitumor effects in preclinical and clinical studies. The expansion of HER2-specific memory CD8 T cells in vaccinated patients was significantly correlated with increased PFS. Subsequent studies will seek to enhance T-cell activity by combining with anti-PD-1.

Supplementary Table 1 - PDF file 4K, Supplementary Table 1. Median hormone levels at baseline, start of cycle 2 (C2), and end of study (EOS) and the percent change in levels from baseline to C2 and C2 to EOS

Table S3

Background Poly (ADP-ribose) polymerase inhibitors (PARPi) have demonstrated efficacy in treating solid tumors with Homologous Recombination Deficiency (HRD), the inability to repair DNA double-stranded breaks through the Homologous Recombination Repair (HRR) pathway. Specific genetic and epigenetic alterations result in defective HRR function. Bi-allelic loss of BRCA1 or BRCA2 principally drives HRR deficiency. While BRCA1/2 are instrumental to HRR, multiple genes, including PALB2, impact the HRR pathway. PALB2 mutations occur in an estimated 0.97% to 3.66% of solid tumors [AACR GENIE PALB2] and are associated with susceptibility to various cancers. No clinically approved therapies specifically targeting PALB2 currently exist. Emerging evidence suggests that patients with germline or somatic PALB2 mutations may benefit from PARPi treatment, which has potential to be a new tumor agnostic therapy option across a wide range of solid tumors. Methods PAVO is a pan-tumor, single-arm, multicenter Phase-II study assessing the safety and efficacy of niraparib (a PARPi) in patients who harbor a confirmed PALB2 mutation. The study plans to enroll up to 110 adult subjects. Eligible participants must have: locally advanced or metastatic solid tumor(s); confirmed pathogenic or likely pathogenic somatic or germline PALB2 mutation; received all standard of care (SOC) therapy for their tumor type, or are unlikely to derive benefit from SOC therapy in the opinion of the treating physician; ECOG performance status of 0 or 1; life expectancy of ≥ 12 weeks with adequate organ/bone marrow function. Exclusion criteria include a confirmed somatic or germline BRCA1/2 mutation, prior treatment with any PARPi, ovarian or prostate cancer, or rapid progression while on platinum-based therapy in the metastatic setting. Niraparib will be administered in 28 day cycles with daily dosing, as outlined in the protocol. Participants will continue study treatment until documented radiographic progression, unacceptable toxicity, death, or consent withdrawal. The primary study endpoint is objective response rate (ORR), defined as the proportion of participants who have partial or complete response to therapy as assessed by Independent Central Review. Secondary endpoints include DOR, PFS, and CBR. PAVO is sponsored by Tempus with support from GSK (GlaxoSmithKline). The trial opened in March 2022. Tempus molecular data tracking (integrated NGS and EMR data) and the TIME Trial program (rapid match of patients to Just in TIME sites for clinical trials), enable patient identification and prescreening. Enrollment occurs through a combination of TIME and prospective clinical sites, where individualized prescreening models are in development. New site identification and referral of molecularly eligible patients to enrolling centers are ongoing. Clinical Trial Registry: NCT05169437 Citation Format: Tian Zhang, Thomas Weart, Matthias Weiss, Drew Murray, Minaxi Jhawer, Edward Huynh, Shumei Kato, Amy Cummings, Lydia Usha, Arvinder Bhinder, Rajiv Desai, Brad Johnson, Anjali Avadhani, Cecile Rose T. Vibat, Lauren Lopez, Brynna Driscoll, Annajane Ward, Christie K. Rice, Blathnaid Donovan, Scott Sherrin, Mykel Robble, Stephanie O'Leary, Kimberly Blackwell, Amine Aziez, Stephanie Petrone, Kathleen Harnden, Kimberly Strickland, Sonya Reid, Mark Robson, Andrew S. Paulson, Afshin Dowlati. PAVO: A phase-II, open label, single arm study of niraparib in patients with locally advanced/metastatic PALB2 mutated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT055.

CDK4/6 inhibitors (CDK4/6i) in combination with antiestrogens have revolutionized the treatment of ER+ metastatic breast cancer (MBC), significantly prolonging survival. However, this combination is not curative, and tumors eventually acquire resistance. Following progression on this combination, patients are left with limited treatment options. A diverse array of mechanisms of resistance to CDK4/6i + antiestrogens have been described. However, laboratory models that capture this heterogeneity of resistance mechanisms are lacking. Patient-derived organoids (PDOs) provide a rapid, robust and reliable platform that recapitulates intra-tumor heterogeneity, partially mimics the cancer microenvironment, and accurately predicts drug response. We aspired to generate a platform of CDK4/6i-resistant breast cancer PDOs to serve as models for understanding acquired resistance to CDK4/6i + antiestrogens and identifying therapies to overcome resistance. We successfully established 16 PDOs out of 32 biopsies (50% efficiency) of metastates from patients with ER+ MBC progressing on CDK4/6i (palbociclib or abemaciclib) + antiestrogens (letrozole or fulvestrant; median response to combination = 9 months). Our collection includes PDOs derived from lobular (n=3) and inflammatory (n=2) breast cancers and reflects racial/ethnic diversity (50% white/not Hispanic; 18.8% Hispanic; 12.5% Black; 12.5% other/unknown). Next-gen sequencing reports were available for 10 patients from which organoids were established, revealing alterations associated with CDK4/6i and/or antiestrogen resistance, including ESR1 (n=2), HER2/ERBB2 (n=2), PTEN (n=2), CCNE1 (n=1), NF1 (n=1), and ARID1A (n=1). Furthermore, one biopsy and its derived organoid lost ER expression, and 5 harbored PIK3CA activating mutations. Thus far, we have performed targeted DNA-sequencing on 7 PDOs and found 13/15 (86.7%) concordance with driver mutations from tumor NGS reports. PDOs established from CDK4/6i-resistant biopsies maintained resistance to palbociclib or abemaciclib ± fulvestrant (500 nM each) in 3D cell viability assays (6 days of treatment). In contrast, control PDOs established from primary ER+ breast cancer surgical samples (n=2) were sensitive to each CDK4/6i ± fulvestrant (median viability for combination=25.6-31.5% for control vs 65.2-80.5% for resistant). GSEA analysis of RNA-seq data from control (n=2) and CDK4/6i-resistant (n=6) PDOs cultured in estrogen-depleted media ± 200 nM palbociclib revealed that palbociclib treatment resulted in downregulation of E2F target and G2M checkpoint signatures in control but not resistant PDOs. Next, we performed a high-throughput screen of 1,000 compounds in 3 resistant PDOs. One PDO showed exquisite sensitivity to G2/M cell cycle checkpoint components, including CDK1, PLK1, Aurora kinase, ATR, Chk1, and Wee1 inhibitors. Finally, treatment of 10 resistant PDOs with the CDK2/4/6 inhibitor PF-06873600 revealed that the CCNE1 (cyclin E1)-amplified PDO was highly sensitive (IC50=130 nM vs >1000 nM), supporting that CCNE1-amplified tumors are vulnerable to CDK2 inhibition. Conclusions: PDOs can be successfully established from ER+ MBC biopsies, maintain the resistant phenotype in culture, retain driver alterations found in tumors from which they were derived, and fail to suppress E2F targets following treatment with CDK4/6i. Therefore, these PDOs represent valuable models to understand and explore diverse mechanisms of CDK4/6i resistance and therapeutic vulnerabilities. Citation Format: Ariella B. Hanker, Sumanta Chatterjee, Yunguan Wang, Dan Ye, Dhivya R. Sudhan, Brian M. Larsen, Lauren C. Smith, Yilin Zhang, Vishal Kandagatla, Kuntal Majmudar, Ezequiel Renzulli, Saurabh Mendiratta, Kimberly Blackwell, Alana L. Welm, Sunati Sahoo, Nisha Unni, Cheryl M. Lewis, Tao Wang, Ameen A. Salahudeen, Carlos L. Arteaga. A platform of CDK4/6 inhibitor-resistant patient-derived breast cancer organoids illuminates mechanisms of resistance and therapeutic vulnerabilities [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 PD2-01.

Background: The heterogeneity of TNBC includes a subtype that is androgen dependent and whose growth is inhibited by antiandrogen therapy. We conducted a multicenter phase II trial which established the activity of bicalutamide for the treatment of patients (pts) with metastatic ER/PR-negative breast cancer TBCRC011 (Gucalp CCR 2013). We now report results of whole genome, next generation sequencing of the 26 evaluable pts to molecularly classify the study population and to identify potential biomarkers of response to bicalutamide. We hypothesized that those pts whose tumors express increased AR output are most likely to benefit from anti-androgen therapy. Methods: Archival formalin-fixed, paraffin-embedded (FFPE) samples were collected from either primary or metastatic site and RNA was isolated using standard techniques. Whole transcriptome sequencing was performed using the Illumina HiSeq 2000 platform. This data set was compared to that of TCGA, the PAM50 and the Lehmann TN subtypes for molecular classification. Bioinformatic analysis defined a model of AR transcriptional output based on androgen stimulated AR+ ER/PR- breast cancer cell lines (HCC202, SUM185, MDA453) that was applied to the TBCRC011 data set as a predictor of response. Gene set analysis was performed to test secondary hypotheses. Results: 21/26 pts provided adequate gene expression estimates for further analysis. Principal component analysis in comparison to whole transcriptomes from breast cancers in the TCGA show that TBCRC011 tumors associate with the basal like (BL) intrinsic subtype, and this was confirmed by PAM50 classification. Relative AR expression of TBCRC011 cases was similar to that of BLBC in the TCGA. There is a weak but statistically significant positive correlation between AR expression by IHC and transcriptome (r=0.395, p=0.034). An expression-based model of AR activity was derived from AR+ ER/PR- cell lines exposed to androgen. TBCRC011 samples segregate with the TCGA samples predicted to be AR responsive by this model. However, the AR output signature did not predict clinical benefit (CR+PR+SD>24 weeks) as defined in TBCRC011. Unexpectedly, by the Lehmann TNBC subtype criteria TBCRC011 tumors were not consistently molecularly classified as luminal AR (BL1-1, BL2-1, Immunomodulatory-5, Luminal AR-4, Mesenchymal-2, Mesenchymal Stem Like-2, Unclassified-6). None of the 4 pts with clinical benefit were classified as BL by the Lehmann model. Gene set analysis revealed that estrogen signal pathways were associated with the number weeks on therapy, and that samples with high scoring AR by IHC exhibited expression patterns of medullary breast cancer. Conclusions: The majority of samples demonstrated expression variation consistent with TCGA basal like breast cancer. Although cell line models predict enrichment of AR activity in both the TCGA basal like and TBCRC011 cohorts, this AR output signature did not predict clinical benefit of bicalutamide. Molecular classification found clinical benefit beyond the luminal AR subtype but exclusive of basal like 1 and 2. Whether IHC or molecular subtype is the optimal biomarker for pt selection for antiandrogen therapy remains uncertain. Citation Format: Tiffany A Traina, Ayca Gucalp, William Polkinghorn, Steven Isakoff, Sara Tolaney, Lisa Carey, James Ingle, Lisle Nabell, Andres Forero, Hope Rugo, Kimberly Blackwell, Minetta Liu, Matthew Soloway, Lisle Mose, Dilip Giri, Agnes Viale, Clifford Hudis, Charles Sawyers, Joel Parker. Whole transcriptome analysis of AR+ ER/PR- metastatic breast cancers treated with bicalutamide on TBCRC011 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-01.