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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.

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

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: 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 like the genomic amplification of HER2. We constructed a vaccine based on a novel alphaviral vector encoding a portion of HER2 (VRP-HER2). METHODS: In preclinical studies, mice were immunized with VRP-HER2 before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and anti-tumor 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 three 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 one partial response and two 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 anti-tumor 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.

PURPOSE: Based on promising predinical 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 HER2(mut) nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2(mut) detection. EXPERIMENTAL DESIGN: Tumor tissue positive for HER2(mut) 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 HER2(mut)). RESULTS: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2(mut) (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2(mut) cases were identified locally. Twenty-one of these 22 HER2(mut) 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 HER2(mut) 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 HER2(mut) variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression. CONCLUSIONS: Neratinib is active in HER2(mut), nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2(mut) cancers for clinical trial participation.

Background Overexpression of fibroblast growth factor receptor 1 (FGFR1), found in ≤8% of hormone receptor–positive (HR+), human epidermal growth factor receptor 2–negative (HER2−) breast cancer cases, is correlated with decreased overall survival and resistance to endocrine therapy (ET). Dovitinib, a potent FGFR inhibitor, has demonstrated antitumor activity in heavily pretreated patients with FGFR pathway–amplified breast cancer. Methods In this randomized, placebo-controlled phase II trial, we evaluated whether the addition of dovitinib to fulvestrant would improve outcomes in postmenopausal patients with HR+, HER2− advanced breast cancer that had progressed during or after prior ET. Patients were stratified by FGF pathway amplification and presence of visceral disease, and they were randomized 1:1 to receive fulvestrant plus dovitinib or placebo. The primary endpoint was progression-free survival (PFS). Results From 15 May 2012 to 26 November 2014, 97 patients from 36 centers were enrolled. The frequency of FGF pathway amplification was lower than anticipated, and the study was terminated early owing to slow accrual of patients with FGF pathway amplification. The median PFS (95% CI) was 5.5 (3.8–14.0) months vs 5.5 (3.5–10.7) months in the dovitinib vs placebo arms, respectively (HR, 0.68; did not meet predefined efficacy criteria). For the FGF pathway–amplified subgroup (n = 31), the median PFS (95% CI) was 10.9 (3.5–16.5) months vs 5.5 (3.5–16.4) months in the dovitinib vs placebo arms, respectively (HR, 0.64; met the predefined superiority criteria). Frequently reported adverse events in the dovitinib (diarrhea, nausea, vomiting, asthenia, and headache) and placebo (diarrhea, fatigue, nausea, and asthenia) arms were mostly low grade. Conclusions The safety profile of dovitinib plus fulvestrant was consistent with the known safety profile of single-agent dovitinib. Dovitinib in combination with fulvestrant showed promising clinical activity in the FGF pathway–amplified subgroup. However, the data reported herein should be interpreted with caution, given that fewer PFS events occurred in the FGF pathway–amplified patients than was expected and that an effect of dovitinib regardless of FGR pathway amplification status cannot be excluded, because the population was smaller than expected. Trial registration ClinicalTrials.gov identifier: NCT01528345. Registered 31 January 2012. Electronic supplementary material The online version of this article (doi:10.1186/s13058-017-0807-8) contains supplementary material, which is available to authorized users.

Premenopausal women treated for early stage breast cancer (ESBC) are at risk for chemotherapy-related amenorrhea (CRA). Prospectively-validated, predictive markers of CRA are needed.Premenopausal women with ESBC and planned chemotherapy (/= 25% risk of amenorrhea) were evaluated. Follicle stimulating hormone (FSH), estradiol, Inhibin A and B, anti-Müllerian hormone (AMH), and quality of life (QOL) were prospectively evaluated pre-, post-, 6 months and 1 year post-chemotherapy and correlated with age and menstrual status. CRA was defined as absence of menses 1 year post-chemotherapy.Forty-four women were evaluated at the time of analysis. Median age at diagnosis and FSH 1 year post-chemotherapy were higher among women with CRA (44 yrs [33-51] vs. 40 yrs [31-43]; p = 0.03; 39.8 vs. 5.0 mLU/mL, p = 0.0058, respectively). Median estradiol 1 year post-chemotherapy was higher among women who resumed menses (108.3 vs. 41.3 pg/mL, p = 0.01). Pre-chemotherapy median Inhibin B and AMH were lower among women with CRA (33.2 vs. 108.8 pg/mL; p = 0.03; 0.16 vs. 1.09 ng/mL, p = 0.02, respectively). The risk of CRA was increased among women with lower pre-chemotherapy Inhibin B (RR = 1.67, p = 0.15) and AMH (RR = 1.83, p = 0.05). Amongst women whose pre-chemotherapy Inhibin B and AMH values were below the median, the incidence of CRA was 87.5%.RESULTS indicate that pre-chemotherapy Inhibin B and AMH are lower among women experiencing CRA and may be predictive of CRA among premenopausal women facing chemotherapy for ESBC.

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.

Background Patients with HER2-overexpressing metastatic breast cancer, despite initially benefiting from the monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib, will eventually have progressive disease. HER2-based vaccines induce polyclonal antibody responses against HER2 that demonstrate enhanced anti-tumor activity when combined with lapatinib in murine models. We wished to test the clinical safety, immunogenicity, and activity of a HER2-based cancer vaccine, when combined with lapatinib. Methods We immunized women (n = 12) with metastatic, trastuzumab-refractory, HER2-overexpressing breast cancer with dHER2, a recombinant protein consisting of extracellular domain (ECD) and a portion of the intracellular domain (ICD) of HER2 combined with the adjuvant AS15, containing MPL, QS21, CpG and liposome. Lapatinib (1250 mg/day) was administered concurrently. Peripheral blood antibody and T cell responses were measured. Results This regimen was well tolerated, with no cardiotoxicity. Anti-HER2-specific antibody was induced in all patients whereas HER2-specific T cells were detected in one patient. Preliminary analyses of patient serum demonstrated downstream signaling inhibition in HER2 expressing tumor cells. The median time to progression was 55 days, with the majority of patients progressing prior to induction of peak anti-HER2 immune responses; however, 300-day overall survival was 92% (95% CI: 77-100%). Conclusions dHER2 combined with lapatinib was safe and immunogenic with promising long term survival in those with HER2-overexpressing breast cancers refractory to trastuzumab. Further studies to define the anticancer activity of the antibodies induced by HER2 vaccines along with lapatinib are underway. Trial registry ClinicalTrials.gov NCT00952692

The detection of circulating tumor cells (CTCs) in peripheral blood may have important prognostic and predictive implications in breast cancer treatment. A limitation in this field has been the lack of a validated method of accurately measuring CTCs. While sensitivity has improved using RT-PCR, specificity remains a major challenge. The goal of this paper is to present a sensitive and specific methodology of detecting CTCs in women with HER-2-positive metastatic breast cancer, and to examine its role as a marker that tracks disease response during treatment with trastuzumab-containing regimens. The study included patients with HER-2-positive metastatic breast cancer enrolled on two different clinical protocols using a trastuzumab-containing regimen. Serial CTCs were measured at planned time points and clinical correlations were made. Immunomagnetic selection of circulating epithelial cells was used to address the specificity of tumor cell detection using cytokeratin 19 (CK19). In addition, the extracellular domain of the HER-2 protein (HER-2/ECD) was measured to determine if CTCs detected by CK19 accurately reflect tumor burden. The presence of CTCs at first restaging was associated with disease progression. We observed an association between CK19 and HER-2/ECD. The association of HER-2/ECD with clinical response followed a similar pattern to that seen with CK19. Finally, the absence of HER-2/ECD at best overall response and a change of HER-2/ECD from positive at baseline to negative at best overall response was associated with favorable treatment response. Our study supports the prognostic and predictive role of the detection of CTCs in treatment of HER-2-positive metastatic breast cancer patients. The association between CK19 and markers of disease burden is in line with the concept that CTCs may be a reliable measure of tumor cells in the peripheral blood of patients with metastatic breast cancer. The association of CTCs at first restaging with treatment failure indicates that CTCs may have a role as surrogate markers to monitor treatment response.

Fibrin formation is required for tumor angiogenesis, metastasis, and invasion. D-dimer, a fibrin degradation product, is produced when crosslinked fibrin is degraded by plasmin. The current study prospectively examined D-dimer levels in patients with metastatic colorectal carcinoma treated in a Phase II randomized trial comparing bevacizumab (Avastin, Genentech, South San Francisco, CA) plus 5-fluorouracil/leucovorin (5-FU/LV) with 5-FU/LV alone.At least one circulating D-dimer level was evaluable in 98 of the 104 previously untreated patients with metastatic colorectal carcinoma in the current trial. Plasma D-dimer levels were determined using a quantitative immunoassay kit at enrollment, before each treatment, and at the time of trial completion or disease progression.At trial enrollment, 86 of 104 patients (88%) had elevated D-dimer levels (20 ng/mL), and 86 of 102 patients (84%) had elevated carcinoembryonic antigen (CEA) levels (3 ng/mL). Baseline D-dimer levels were correlated with the following baseline characteristics: CEA (Pearson coefficient, 0.31; P = 0.002), albumin levels (Pearson coefficient, -0.32; P = 0.002), tumor burden (Pearson coefficient, 0.30; P = 0.003), and number of metastatic sites (Pearson coefficient, 0.21; P = 0.04). At the time of progression, plasma D-dimer levels reached a maximum postbaseline value in 51 of 61 patients (84%), whereas the CEA level was at its maximum postbaseline value in 39 of 55 patients (71%). Baseline D-dimer levels were a strong predictor of overall survival on univariate analysis (P = 0.008) and multivariate analysis (P = 0.03). Overall, treatment with bevacizumab (5 mg/kg) and baseline D-dimer levels were the only predictors of overall survival (P0.05).The current study indicates that fibrin remodeling is an important prognostic feature in metastatic colorectal carcinoma. D-dimer levels should be incorporated into prognostic models, and D-dimer may represent a useful biomarker for patients treated with antiangiogenic agents.

Introduction Neoadjuvant chemotherapy has become the standard of care for the diverse population of women diagnosed with locally advanced breast cancer. Serum biomarker levels are increasingly being investigated for their ability to predict therapy response and aid in the development of individualized treatment regimens. Multianalyte profiles may offer greater predictive power for neoadjuvant treatment response than the individual biomarkers currently in use. Methods Serum samples were collected from 44 patients enrolled in a phase I–II, open-label study of liposomal doxorubicin and paclitaxel in combination with whole breast hyperthermia for the neoadjuvant treatment of locally advanced breast cancer (stage IIB or stage III). Samples were collected prior to each of four rounds of treatment and prior to definitive surgery. Samples were assayed by Luminex assay for 55 serum biomarkers, including cancer antigens, growth/angiogenic factors, apoptosis-related molecules, metastasis-related molecules, adhesion molecules, adipokines, cytokines, chemokines, hormones, and other proteins. Results Biomarker levels were compared retrospectively with clinical and pathologic treatment responses. Univariate analysis of the data identified several groups of biomarkers that differed significantly among treatment outcome groups early in the course of neoadjuvant chemotherapy. Multivariate statistical analysis revealed multibiomarker panels that could differentiate between treatment response groups with high sensitivity and specificity. Conclusion We demonstrate here that serum biomarker profiles may offer predictive power concerning treatment response and outcome in the neoadjuvant setting. The continued development of these findings will be of considerable clinical utility in the design of treatment regimens for individual breast cancer patients. Trial registration #NCT00346229.