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Purpose HER2 overexpression has a central role in breast cancer carcinogenesis and is associated with poor prognosis if untreated. Lately, identification of HER2-low breast cancer has been proposed to select patients for novel HER2-directed chemotherapy and includes cancers with immunohistochemistry 1 + or 2 + with negative FISH, encompassing approximately 55–60% of all breast carcinomas. In early-stage breast cancer, the prognostic significance of HER2 low-disease is less well understood, with a particular paucity of data evaluating the prevalence and implications of HER2-low status in invasive lobular carcinoma (ILC). Methods We evaluated 666 stage I-III ILC tumors from a prospectively maintained institutional database, comparing clinicopathologic features and disease-free survival (DFS) using a multivariable Cox proportional hazards model. Results HER2-low status was common in this cohort of patients with ILC, but most clinicopathologic features did not differ between HER2-low and HER2-negative cases. However, when adjusting for tumor size, number of positive nodes, ER/PR status, and local therapy received, patients with HER2-low status had worse disease-free survival (DFS) than those with HER2-negative tumors (hazard ratio 2.0, 95% confidence interval 1.0–4.1, p = 0.05). Conclusion This difference in DFS supports the notion that HER2-low and HER2-negative early stage ILC may differ clinically, despite similar clinicopathologic features. Further investigation into the potential benefit of HER2 targeted therapy in HER2-low early-stage breast cancer, and specifically lobular cancer, is warranted to ensure optimal outcomes in this distinct tumor subtype.

PURPOSE Chemotherapy has not demonstrated benefit over adjuvant endocrine therapy alone for postmenopausal patients with node-positive breast cancer with a 21-gene breast recurrence score (RS) of 25 or below (RS ≤ 25). We tested whether combined results from RS and the sensitivity to endocrine therapy (SET2,3) index of endocrine-related transcription (SETER/PR) adjusted for baseline prognostic index (BPI) improve prognostic assessment, and whether SET2,3 predicted benefit from anthracycline-based chemotherapy. METHODS A blinded retrospective clinical validation of SET2,3 in two randomized treatment arms from the SWOG S8814 trial comparing adjuvant anthracycline-based chemotherapy followed by tamoxifen endocrine therapy for 5 years, versus tamoxifen alone. SET2,3 assay was calibrated and measured using whole-transcriptome RNA sequence of tumor samples already tested for RS. The primary end point was disease-free survival (DFS). RESULTS There were 106 events in 283 patients over a median follow-up of 8.99 years. Proportional hazards assumptions were met during the first 5 years only. SET2,3 index and RS were not correlated (r = –0.04) and were independently prognostic (SET2,3: hazard ratio [HR], 0.48 per unit; 95% CI, 0.34 to 0.68; P < .001; RS: HR, 1.28 per 10 units; 95% CI, 1.14 to 1.44; P < .001). SET2,3 index did not predict chemotherapy benefit (interaction P = .77). SET2,3 was high in 93/175 (53%) patients with RS ≤ 25 (concordant low-risk), with 5-year DFS 97%. SET2,3 was low in 55/108 (51%) patients with RS > 25 (concordant high-risk), with 5-year DFS 53%. Both components of SET2,3 index were prognostic after adjustment for RS: SETER/PR (HR, 0.65; 95% CI, 0.46 to 0.92) and BPI (HR, 0.45; 95% CI, 0.31 to 0.64). CONCLUSION SET2,3 index was not correlated with RS, demonstrated additive prognostic performance, and was not chemopredictive in this subset of patients from S8814. The SETER/PR and BPI components of SET2,3 each added prognostic information to RS.

PURPOSE Triple-negative breast cancer (TNBC) is defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) expression. In this study, we compared response to neoadjuvant chemotherapy and survival between patients with TNBC and non-TNBC. PATIENTS AND METHODS Analysis of a prospectively collected clinical database was performed. We included 1,118 patients who received neoadjuvant chemotherapy at M.D. Anderson Cancer Center for stage I-III breast cancer from 1985 to 2004 and for whom complete receptor information were available. Clinical and pathologic parameters, pathologic complete response rates (pCR), survival measurements, and organ-specific relapse rates were compared between patients with TNBC and non-TNBC. RESULTS Two hundred fifty-five patients (23%) had TNBC. Patients with TNBC compared with non-TNBC had significantly higher pCR rates (22% v 11%; P = .034), but decreased 3-year progression-free survival rates ( P < .0001) and 3-year overall survival (OS) rates ( P < .0001). TNBC was associated with increased risk for visceral metastases ( P = .0005), lower risk for bone recurrence ( P = .027), and shorter postrecurrence survival ( P < .0001). Recurrence and death rates were higher for TNBC only in the first 3 years. If pCR was achieved, patients with TNBC and non-TNBC had similar survival ( P = .24). In contrast, patients with residual disease (RD) had worse OS if they had TNBC compared with non-TNBC ( P < .0001). CONCLUSION Patients with TNBC have increased pCR rates compared with non-TNBC, and those with pCR have excellent survival. However, patients with RD after neoadjuvant chemotherapy have significantly worse survival if they have TNBC compared with non-TNBC, particularly in the first 3 years.

Introduction Breast cancer is uncommon in men. Almost all male breast cancers are hormone receptor-positive, HER2-negative, although the pathogenesis is not always attributable to an endocrine condition. A few studies have compared biological characteristics or molecular signatures with breast cancers in women. We sought to evaluate whether hormone receptor-related gene expression is different in cancers from men compared to equivalent cancers from women. SET2,3 index measures non-proliferative hormone receptor-related transcriptional activity in the cancer (SET-ER/PR index) and adjusts this for a Baseline Prognosis Index (BPI) that combines the measurements of tumor and nodal stage with a 4-gene molecular subtype (ESR1, PGR, ERBB2, and AURKA). Methods We received aliquots of total RNA from male patients with breast cancer included in the retrospective cohort study of the EORTC 10085/BCG/TBCRC/BIG/NCTN International Male Breast Cancer Program (NCT01101425). SET2,3 assay was performed using the QuantiGene assay (Thermo Fisher) using bead-based hybridization and laser spectroscopy (Luminex). The statistical analyses were performed by the EORTC statistician. The primary objective of the study was the assessment of the prognostic value of the SET2,3 index score in patients with early-stage hormone receptor-positive, HER2-negative male breast cancer, treated with endocrine therapy. Clinical outcomes (recurrence-free survival – RFS; overall survival – OS) were estimated by Kaplan-Meier curves and secondarily compared using multivariable Cox models adjusted for continuous SET2,3 index, tumor size, nodal status, age, and chemotherapy and radiotherapy use. An exploratory analysis to compare the SET2,3 index scores distribution in female and male breast cancer patients was also performed using results from the same assay performed on cancers from women selected on the same inclusion criteria. Due to the low numbers of male patients treated with neoadjuvant treatment (N=6), this analysis was restricted to patients treated with adjuvant treatment (n=315 male and 660 female). Results Of the 321 male patients with breast cancer analyzed, treated between 1990 and 2010, 211 (65.7%) were categorized as high SET2,3 index score, reflecting a high endocrine activity in the cancer and low risk of recurrence, and 110 patients (34.3%) categorized as being low score, reflecting low endocrine activity and high risk of recurrence. At 5 years, the RFS was 75.0% (95% CI, 67.4-81.1) in the high SET2,3 group versus 60.7% (95% CI, 49.1-70.5) in the low SET2,3 group (HR univariate, 0.49; 95% CI, 0.34-0.70; P< 0.0001). The 5-year OS rate among patients with a high SET2,3 index was 84.3% (95% CI, 45.5-73.8), in contrast of 67.8% (95% CI, 56.6-76.7) in the low SET2,3 group (HR univariate, 0.44; 95% CI, 0.30-0.65; P< 0.0001). SET2,3 was independently prognostic for OS, but not RFS in multivariable Cox models. In patients classified as low SET2,3, the addition of neo/adjuvant chemotherapy to adjuvant endocrine therapy was associated with 5-year OS of 76.0% (95% CI, 59.5-86.4) and in patients who received endocrine therapy alone the 5-year OS was 61.3% (95% CI, 45.5-73.8), an absolute difference of 14.7 percentage points. Overall, we did not observe a difference in the distributions (median, interquartile range) of SET2,3 index between men (2.4, 1.9–2.6) and women (2.3, 2.0–2.7). Conclusion SET2,3 index measurements of endocrine-related transcriptional activity in male patients with breast cancer were not different from measurements in female patients with breast cancer. SET2,3 was prognostic in male breast cancer and our exploratory analysis suggests that chemotherapy might improve the poor prognosis for men with breast cancer that has low SET2,3 index. This study was funded by the Breast Cancer Research Foundation (BCRF). Citation Format: Danielle B. Zakon, Coralie Poncet, Fatima Cardoso, Neven Anouk, Vicente Valero, Stefan Aebi, Kim Benstead, Oliver Bogler, Lissandra Dal Lago, Judith Fraser, Carmela Caballero, Ingrid A. Hedenfalk, Larissa A. Korde, Barbro Linderholm, John WM Martens, Lavinia P. Middleton, Melissa Murray, Catherine M. Kelly, Cecilia Nilsson, Monika Nowaczyk, Stephanie Peeters, Melanie Beauvois, Peggy Porter, Carolien P. Schroder, Isabel T. Rubio, Kathryn Ruddy, Christi van Asperen, Danielle Van Den Weyngaert, Carolien HM van Deurzen, Elise van Leeuwen-Stok, Joanna M. Vermeij, John MS Bartlett, Antonio C. Wolff, Sharon H. Giordano, W. Fraser Symmans. PD6-11 Evaluation of the Sensitivity to Endocrine Therapy Index (SET2,3) in Early Male Breast Cancer: Results from an analysis in the EORTC 10085/TBCRC/BIG/NCTN International Male Breast Cancer Program [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD6-11.

RNA sequencing (RNAseq) analysis is emerging as a clinical research or diagnostic approach for cytologic samples, but there is need for formal comparison of different sample preparation methods in the cytology laboratory to identify which pre-analytic methods could provide alternatives to formalin-fixed paraffin-embedded (FFPE) sections.We prepared 13 malignant effusions (metastatic estrogen receptor-positive breast cancer) in the cytology laboratory using 6 routine cytologic methods: FFPE cell block, Carnoy's solution, 95% ethanol (EtOH), air-dried and Diff-Quik, ThinPrep, and SurePath preparations. Measurements of RNA quality, expression of 2 multigene expression signatures, molecular subtype, and 4 common activating mutation sites in each preparation were compared with fresh frozen (FF) cell pellet in RNA preservative using distribution of fragment length and concordance correlation coefficient (CCC).The fraction of RNA fragments measuring 200 bases or more (DV200) were 24% higher from cytospins fixed in Carnoy's solution or 95% EtOH than DV200 from FFPE cell blocks. SurePath samples failed RNAseq quality control. There was high concordance of gene expression measurements with FF samples using cytospins fixed in Carnoy's solution, 95% EtOH, Diff-Quik (CCC = 0.829, 0.812, 0.760, respectively), or ThinPrep (CCC = 0.736), but lower using FFPE cell block (CCC = 0.564). The proportion of mutant transcripts was concordant between FF and any cytologic preparation methods.Cytospin preparations fixed with Carnoy's or 95% ETOH then Papanicolaou stained produced RNAseq results that were equivalent to FF samples and superior to FFPE cell block sections.

Purpose: To evaluate prediction of response and event-free survival (EFS) following neoadjuvant endocrine therapy by SET2,3 index of nonproliferation gene expression related to estrogen and progesterone receptors adjusted for baseline prognosis. Experimental Design: A correlative study was conducted of SET2,3 measured from gene expression profiles of diagnostic tumor (Agilent microarrays) in 379 women with cStage II–III breast cancer from the American College of Surgeons Oncology Group Z1031 neoadjuvant aromatase inhibitor trial SET2,3 was dichotomized using the previously published cutoff. Fisher exact test was used to assess the association between SET2,3 and low proliferation at week 2–4 [Ki67 ≤ 10% or complete cell-cycle arrest (CCCA; Ki67 ≤ 2.7%)] and PEPI-0 rate in cohort B, and the association between SET2,3 and ypStage 0/I in all patients. Cox models were used to assess EFS with respect to SET2,3 excluding cohort B patients who switched to chemotherapy. Results: Patients with high SET2,3 had higher rate of pharmacodynamic response than patients with low SET2,3 (Ki67 ≤ 10% in 88.2% vs. 56.9%, P < 0.0001; CCCA in 50.0% vs. 26.2%, P = 0.0054), but rate of ypStage 0/I (24.0% vs. 20.4%, P = 0.4580) or PEPI = 0 (28.4% vs. 20.6%, P = 0.3419) was not different. Patients with high SET2,3 had longer EFS than patients with low SET2,3 (HR, 0.52, 95% confidence interval: 0.34–0.80; P = 0.0026). Conclusions: This exploratory analysis of Z1031 data demonstrated a higher rate of pharmacodynamic suppression of proliferation and longer EFS in high SET2,3 disease relative to low SET2,3 disease. The ypStage 0/I rate and PEPI = 0 rate were similar with respect to SET2,3.

Context.— Neoadjuvant systemic therapy refers to the use of systemic agent(s) for malignancy prior to surgical treatment and has recently emerged as an option for most breast cancer patients eligible for adjuvant systemic therapy. Consequently, treated breast carcinomas have become routine specimens in pathology practices. A standard protocol has not yet been universally adopted for the evaluation and reporting of these specimens. The American Joint Committee on Cancer staging system recognizes the challenges in staging breast carcinomas after neoadjuvant treatment and provides important data points but does not currently provide detailed guidance in estimating the residual tumor burden in the breast and lymph nodes. The Residual Cancer Burden system is the only Web-based system that quantifies treatment response as a continuous variable using residual tumor burden in the breast and the lymph nodes. Objective.— To provide clarifications and guidance for evaluation and reporting of postneoadjuvant breast specimens, discuss issues with the current staging and reporting systems, and provide specific suggestions for future modifications to the American Joint Committee on Cancer system and the Residual Cancer Burden calculator. Data Sources.— English-language literature on the subject and the data from the I-SPY 2, a multicenter, adaptive randomization phase 2 neoadjuvant platform trial for early-stage, high-risk breast cancer patients. Conclusions.— This article highlights challenges in the pathologic evaluation and reporting of treated breast carcinomas and provides recommendations and clarifications for pathologists and clinicians. It also provides specific recommendations for staging and discusses future directions.

Background: Acquired ESR1 mutations are a dominant driver of distant metastasis in metastatic breast cancer, inducing a basal-like phenotype that is relatively resistant to ER antagonists with decreased progression free survival. The majority of ER+ patients also express the androgen receptor (AR), and although AR expression is associated with better outcomes, high AR expression has also been associated with resistance to endocrine therapy (ET). Thus, there is currently a conundrum on how best to target AR, and to define when it is participating as a proliferative/metastatic driver, or when it may be a potential tumor suppressor associated with good prognosis. Methods: We generated ESR1 Y537S or D538G homozygous mutations in MCF-7 cells using CRISPR Cas-9 technology. ChIP-Seq, transcriptome analyses, and quantitative analysis of ER ChIP-Seq profiles from patient biopsies were integrated. Nuclear fractionation and immunoblot analyses were performed. Results: Correlation and gene set enrichment analyses demonstrated that the androgen response pathway was significantly reduced in ESR1 mutant, compared to wild-type (WT) ER cells. A dramatic redistribution in AR binding sites to heterochromatin was observed in ESR1 mutant cells. AR and ER co-occupied DNA binding sites were redistributed only to ERE motifs present in a restricted set of gene enhancer regions. Quantitative analysis of ER DNA binding profiles from ER+ patients showed that the highest recruitment of ER to AR/ER binding sites were in patients with metastatic breast cancer. Integration of AR/ER co-bound sites with the differentially-expressed mutant transcriptome identified a gene signature that predicted poor disease-free and overall survival in ER+ primary breast cancer patients from the METABRIC database. Elevated gene expression of these AR/ER co-regulated genes, including NCOA3, BMP7, N4BP3, and FOXA1, were validated in a cohort of metastatic tumors (N=97), compared to primary tumors (N=276). AR protein levels were elevated in ESR1 mutant tumors. Mechanistic studies demonstrated that elevated AR phosphorylation at specific sites (pS515, pS650) and decreased phosphorylation (pS308) occurs in mutant cells. These sites are known to affect AR transcriptional activity, protein stability, and nuclear export. Decreased K48-AR ubiquitination was observed, suggesting that elevated AR protein levels result from altered post-transcriptional modification of AR in mutant cells. Conclusions: We propose a two-prong genomic activation mechanism in ESR1 mutant tumors with AR redistribution to heterochromatin, and genomic co-activation of AR and ER. We hypothesize that the loss of AR tumor suppressor function may help drive metastasis in ESR1 mutant tumors via AR’s collaboration with ESR1 mutant oncogenic activity. Efficacious targeting of AR may require disruption of this driver AR/ER-regulated transcriptional program. Citation Format: Sandra L. Grimm, Guowei Gu, Sarah K. Herzog, Thomas L. Gonzalez, Hangqing Lin, Amanda R. Beyer, Yassine Rechoum, Tasneem Bawa-Khalfe, Ashfia F. Khan, Lili Du, W. Fraser Symmans, Ralf Kittler, Cristian Coarfa, Suzanne A.W. Fuqua. Mutant ESR1 receptors antagonize the tumor suppressor function of androgen receptors [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 PD1-07.

This supplemental file is the data base of all 2492 patients with their clinical and pathological factors..

Purpose: The clinical relevancy of the 7-subtype classification of triple-negative breast cancer (TNBC) reported by Lehmann and colleagues is unknown. We investigated the clinical relevancy of TNBC heterogeneity by determining pathologic complete response (pCR) rates after neoadjuvant chemotherapy, based on TNBC subtypes.Experimental Design: We revalidated the Lehmann and colleagues experiments using Affymetrix CEL files from public datasets. We applied these methods to 146 patients with TNBC with gene expression microarrays obtained from June 2000 to March 2010 at our institution. Of those, 130 had received standard neoadjuvant chemotherapy and had evaluable pathologic response data. We classified the TNBC samples by subtype and then correlated subtype and pCR status using Fisher exact test and a logistic regression model. We also assessed survival and compared the subtypes with PAM50 intrinsic subtypes and residual cancer burden (RCB) index.Results: TNBC subtype and pCR status were significantly associated (P = 0.04379). The basal-like 1 (BL1) subtype had the highest pCR rate (52%); basal-like 2 (BL2) and luminal androgen receptor had the lowest (0% and 10%, respectively). TNBC subtype was an independent predictor of pCR status (P = 0.022) by a likelihood ratio test. The subtypes better predicted pCR status than did the PAM50 intrinsic subtypes (basal-like vs. non basal-like).Conclusions: Classifying TNBC by 7 subtypes predicts high versus low pCR rate. We confirm the clinical relevancy of the 7 subtypes of TNBC. We need to prospectively validate whether the pCR rate differences translate into long-term outcome differences. The 7-subtype classification may spur innovative personalized medicine strategies for patients with TNBC. Clin Cancer Res; 19(19); 5533–40. ©2013 AACR.

Supplementary Figures S5-S6 from Microtubule-Associated Protein-tau is a Bifunctional Predictor of Endocrine Sensitivity and Chemotherapy Resistance in Estrogen Receptor–Positive Breast Cancer

Supplementary Data from Development of Candidate Genomic Markers to Select Breast Cancer Patients for Dasatinib Therapy

Background: Differences in gene or protein expression patterns between breast cancers according to race/ethnicity and cancer subtype.Methods: Transcriptional profiling was performed using Affymetrix HG-U133A platform in 376 patients and reverse phase protein array analysis (RPPA) was done for 177 proteins in 255 patients from a separate cohort. Unsupervised clustering was conducted, as well as supervised comparison by race and tumor subtype. Standard statistical methods, BRB-Array tools, and Ingenuity Pathways software packages were used to analyze the data.Results: Median age was 50 years in both the cohorts. In the RPPA cohort, 54.5% of the tumors were hormone receptor–positive (HR-positive), 20.7% HER2-positive, and 24.71% triple-negative (TNBC). One hundred and forty-seven (57.6%), 47 (18.43%), and 46 (18.1%) of the patients were White, Hispanic, and Black, respectively. Unsupervised hierarchical clustering of the protein expression data showed no distinct clusters by race (P values were 0.492, 0.489, and 0.494 for the HR-positive, HER2-positive, and TNBC tumors respectively). In the gene expression cohort, 54.2% of the tumors were HR-positive, 16.5% HER2-positive, and 29.3% TNBC. Two hundred and sixteen (57.5%), 111 (29.52%), and 32 (8.52%) patients were White, Hispanic, and Black, respectively. No probe set with a false discovery rate (FDR) of Conclusions: We did not detect a significant variation in RNA or protein expression comparing different race/ethnicity groups of women with breast cancer.Impact: More research on the complex network of factors that result in outcomes differences among race/ethnicities is needed. Cancer Epidemiol Biomarkers Prev; 23(2); 316–23. ©2013 AACR.

Purpose: Estrogen receptor-positive (ER+) and -negative (ER) breast cancers are molecularly distinct diseases. We hypothesized that p53 mutations may lead to different transcriptional changes and carry different prognostic value in these two different types of cancers.Experimental Design: We developed a 39-gene p53 signature derived from 213 ER+ and a separate 30-gene signature from 38 ER− cancers with known mutation status and tested their prognostic and chemotherapy response predictive values in ER+ and ER− cancers, respectively.Results: External validation to predict p53 status (n = 103) showed sensitivity and specificity of 89% and 54% for the 39-gene signature, and 82% and 61% for the 30-gene signature. The 39-gene signature was predictive of worse distant metastasis free survival in ER+ cancers in two separate prognostic data sets (n = 255, HR: 2.3, P = 0.005 and n = 198, HR: 2.17, P = 0.09). It also predicted for poor prognosis even with adjuvant tamoxifen therapy (n = 277, HR = 2.43, P < 0.0001) but it was not prognostic in ER− cancers. It was also associated with higher chemotherapy sensitivity in ER+ but not in ER− cancers. The prognostic and predictive values remained significant in multivariate analysis. The 30-gene, ER−, p53 signature showed no prognostic or predictive values in ER+ cancers but it was associated with better prognosis in ER− cancers. It also had no chemotherapy response predictive value in ER− or ER+ cancers.Conclusions: P53 dysfunction is prognostically most relevant in ER+ cancers and supports the hypothesis that different predictive or prognostic markers will be needed for different molecular subsets of breast cancer. Clin Cancer Res; 17(8); 2591–601. ©2011 AACR.

Supplementary Table S1 from Breast Cancer Molecular Subtypes Respond Differently to Preoperative Chemotherapy

Purpose: Pathologic complete response (pCR) to neoadjuvant chemotherapy reflects the cytotoxic efficacy of a drug, but patient survival is influenced by many other factors. The purpose of this study was to assess the relationship between increased pCR rate and trial-level survival benefit in triple-negative breast cancer (TNBC).Experimental Design: We used bootstrap resampling from a neoadjuvant trial to simulate trials with different pCR rates. We used estimates from Adjuvant!Online to simulate trial populations with different baseline prognosis and estimated survival improvements associated with changes in pCR rate.Results: Assuming that survival is similar for patients with pCR regardless of treatment arm, a linear relationship exists between increasing pCR rate and increasing recurrence-free survival (RFS). The slope is equal to the difference in survival between those with pCR and residual disease, which in turn is influenced by (i) the baseline prognosis of the trial population, (ii) interactions between prognostic variables and pCR, and (iii) the efficacy of the postneoadjuvant therapies. For example, if the pCR rates are 30% and 60% (OR = 3.5) and the 10-year RFS of the control arm is 0.74, the trial would require 3,550 patients per arm, whereas if the RFS is 0.54, the trial would require only 425 patients per arm to detect significant survival benefit.Conclusions: We provide a framework for understanding the relationship between pCR and overall survival benefit that can help inform the design of neoadjuvant trials aiming to demonstrate improved survival from a regimen that results in higher pCR rate. Clin Cancer Res; 22(1); 26–33. ©2015 AACR.See related commentary by Berry, p. 3

Supplementary Figure 1: Examples of staining and coring system for cyclin E; Supplementary Figure 2: Examples of LMW-E cyclin E staining; Supplementary Figure 3: REMARK diagram of biospecimen availability for the MDA Lab00-222, NCI TMA, MDA TMA, and UK TMA cohorts.

Supplementary Figure A1 PDF file 73K, Subgroup analysis of the extended validation cohort

R File codes pertaining to NCI cohort

PDF - 160KB, Supplementary Figure 1 -Histograms of BUM model to the P-values from the ANOVA test of race/ethnicity by breast cancer subtype in 177 proteins. Supplementary Figure 2 - Histograms of BUM model to the P-values from the ANOVA test of race/ethnicity by breast cancer subtype in 16,438 probe sets. Supplementary Figure 3 - Unsupervised clustering of the significant 384 probe sets with FDR

Purpose: We examined in a prospective, randomized, international clinical trial the performance of a previously defined 30-gene predictor (DLDA-30) of pathologic complete response (pCR) to preoperative weekly paclitaxel and fluorouracil, doxorubicin, and cyclophosphamide (T/FAC) chemotherapy, and assessed if DLDA-30 also predicts increased sensitivity to FAC-only chemotherapy. We compared the pCR rates after T/FAC versus FACx6 preoperative chemotherapy. We also did an exploratory analysis to identify novel candidate genes that differentially predict response in the two treatment arms.Experimental Design: Two hundred and seventy-three patients were randomly assigned to receive either weekly paclitaxel × 12 followed by FAC × 4 (T/FAC, n = 138), or FAC × 6 (n = 135) neoadjuvant chemotherapy. All patients underwent a pretreatment fine-needle aspiration biopsy of the tumor for gene expression profiling and treatment response prediction.Results: The pCR rates were 19% and 9% in the T/FAC and FAC arms, respectively (P < 0.05). In the T/FAC arm, the positive predictive value (PPV) of the genomic predictor was 38% [95% confidence interval (95% CI), 21-56%], the negative predictive value was 88% (95% CI, 77-95%), and the area under the receiver operating characteristic curve (AUC) was 0.711. In the FAC arm, the PPV was 9% (95% CI, 1-29%) and the AUC was 0.584. This suggests that the genomic predictor may have regimen specificity. Its performance was similar to a clinical variable–based predictor nomogram.Conclusions: Gene expression profiling for prospective response prediction was feasible in this international trial. The 30-gene predictor can identify patients with greater than average sensitivity to T/FAC chemotherapy. However, it captured molecular equivalents of clinical phenotype. Next-generation predictive markers will need to be developed separately for different molecular subsets of breast cancers. Clin Cancer Res; 16(21); 5351–61. ©2010 AACR.

Purpose: To examine gene expression differences between pre- and post-neoadjuvant systemic therapy (NST) specimens of breast cancers and identify biologic changers that may lead to new therapeutic insights.Methods: Gene expression data from prechemotherapy fine needle aspiration specimens were compared with resected residual cancers in 21 patients after 4 to 6 months of NST. We removed stroma-associated genes to minimize confounding effects. PAM50 was used to assign molecular class. Paired t test and gene set analysis were used to identify differentially expressed genes and pathways.Results: The ER and HER2 status based on mRNA expression remained stable in all but two cases, and there were no changes in proliferation metrics (Ki67 and proliferating cell nuclear antigen expression). Molecular class changed in 8 cases (33.3%), usually to normal-like class, which was associated with low residual cancer cell cellularity. The expression of 200 to 600 probe sets changed between baseline and post-NST samples. In basal-like cancers, pathways driven by increased expression of phosphoinositide 3-kinase, small G proteins, and calmodulin-dependent protein kinase II and energy metabolism were enriched, whereas immune cell–derived and the sonic hedgehog pathways were depleted in residual cancer. In non–basal-like breast cancers, notch signaling and energy metabolism (e.g., fatty acid synthesis) were enriched and sonic hedgehog signaling and immune-related pathways were depleted in residual cancer. There was no increase in epithelial–mesenchymal transition or cancer stem cell signatures.Conclusions: Our data indicate that energy metabolism related processes are upregulated and immune-related signals are depleted in residual cancers. Targeting these biologic processes may represent promising adjuvant treatment strategies for patients with residual cancer. Clin Cancer Res; 18(4); 1109–19. ©2012 AACR.

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R File codes pertaining to UK cohort

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Purpose: The clinical outcome for patients with breast cancer is influenced by the metastatic competence of the cancer and its sensitivity to endocrine therapy and chemotherapy. A molecular marker may be prognostic of outcome or predictive of response to therapy, or a combination of both.Experimental Design: We examined separately the prognostic and predictive values of tau mRNA expression in estrogen receptor (ER)–positive primary breast cancers in three patient cohorts. We used gene expression data from 209 untreated patients to assess the pure prognostic value of tau, data from 267 patients treated with adjuvant tamoxifen to assess predictive value for endocrine therapy, and data from 82 patients treated with preoperative paclitaxel followed by 5-fluorouracil, doxorubicin, and cyclophosphamide (paclitaxel/FAC) to assess predictive value for chemotherapy response. Wilcoxon rank sum test was used to compare tau expression between different outcome groups.Results: Higher tau mRNA expression showed borderline nonsignificant association with better prognosis in the absence of systemic adjuvant therapy. Higher tau mRNA expression was significantly associated with no recurrence (at 5 and 10 years, P = 0.005 and P = 0.05, respectively) in patients treated with tamoxifen, indicating a predictive value for endocrine therapy. Tau expression was significantly lower in patients who achieved pathologic complete response to paclitaxel/FAC chemotherapy (P < 0.001).Conclusion: This study suggests that high tau mRNA expression in ER-positive breast cancer indicates an endocrine-sensitive but chemotherapy-resistant disease. In contrast, low tau expression identifies a subset of ER-positive cancers that have poor prognosis with tamoxifen alone and may benefit from taxane-containing chemotherapy.

R File codes pertaining to the MDACC-TMA cohort

Purpose: Several different multivariate prediction models using routine clinical variables or multigene signatures have been proposed to predict pathologic complete response to combination chemotherapy in breast cancer. Our goal was to compare the performance of four conceptually different predictors in an independent cohort of patients.Experimental Design: Gene expression profiling was done on fine-needle aspirations of 100 stage I to III breast cancers before preoperative paclitaxel, 5-fluorouracil, doxorubicin, and cyclophosphamide combination chemotherapy. Pathologic response was correlated with prediction results from a clinical nomogram, a human cancer–derived genomic predictor (DLDA30), a cell line–based genomic predictor [in vitro coexpression extrapolation (COXEN)], and an optimized cell line–derived (in vivo COXEN) predictor. None of the 100 test cases were used in the development of these predictors.Results: The in vitro COXEN using a combination of four individual drug sensitivity predictions derived from cell lines was not predictive [area under the receiver operator characteristic curve (AUC), 0.5; 95% confidence interval, (95% CI), 0.41-0.59]. The clinical nomogram (AUC, 0.73; 95% CI, 0.65-0.80) and the DLDA30 (AUC, 0.73; 95% CI, 0.66-0.80) genomic predictor had similar performances. The in vivo COXEN that used informative genes from cell lines but was trained on a separate human data set also showed significant predictive value (AUC, 0.67; 95% CI, 0.60-0.74). These three different prediction scores correlated with each other and were significant in univariate but not in multivariate analysis.Conclusions: Three conceptually different predictors performed similarly in this validation study and tended to identify the same patients as responders. A genomic predictor that relied solely on a composite of individual drug sensitivity predictions from cell lines did not show any predictive value. Clin Cancer Res; 16(2); 711–8

Information of Microarray Data - PDF file 31K, Gene expression data have been deposited into the GEO database

Figure legends pertaining to supplemental figures 1-3

XLS file - 15K, DLDA30 probe set interaction with treatment using logistic regression.

Purpose: Patients with triple-negative breast cancer (TNBC) and residual disease after neoadjuvant chemotherapy generally have worse outcome; however, some patients with residual tumor after neoadjuvant chemotherapy do not relapse. We hypothesize that there are subgroups of patients with chemoresistant TNBC with different prognosis.Experimental Design: Forty-nine chemoresistant cases from 111 patients with TNBC treated with neoadjuvant chemotherapy (M.D. Anderson Cancer Center, Houston, TX) constituted the discovery cohort, and 25 chemoresistant samples from 47 neoadjuvant chemotherapy-treated TNBC (The Methodist Hospital, Houston, TX) were chosen for validation. Extended validation was carried out in 269 operable TNBC predicted to be chemoresistant by expression pattern from published datasets.Results: We established a seven-gene prognostic signature using dChip and gene set enrichment analyses. In the independent validation cohort, the classifier predicted correctly with positive predictive value of 75.0% and negative predictive value (i.e., relapse-free survival; RFS) of 76.9% at 3 years. Those predicted to relapse had a HR of 4.67 [95% confidence interval (CI): 1.27–17.15] for relapse in 3 years. In extended validation, patients predicted not to relapse exhibited 3-year RFS of 78.9%, whereas the 3-year RFS was 48.5% for patients predicted to relapse, with HR of 2.61 (95% CI: 1.52–4.49). The TNBC subgroup that predicted to have relatively favorable prognosis was characterized by high expression of “luminal-like” genes [androgen-receptor (AR) and GATA3], whereas the subgroup with worse prognosis was characterized by expression of cancer stem-cell markers.Conclusion: We developed a clinically relevant signature for patients with chemoresistant TNBC. For these women, new therapeutic strategies like targeting AR activation or cancer stem cells may need to be developed. Clin Cancer Res; 19(10); 2723–33. ©2013 AACR.

Supplementary Table from Evaluation of Sensitivity to Endocrine Therapy Index (SET2,3) for Response to Neoadjuvant Endocrine Therapy and Longer-Term Breast Cancer Patient Outcomes (Alliance Z1031)

Patient Characteristics

Purpose: We used high-resolution oligonucleotide comparative genomic hybridization (CGH) arrays and matching gene expression array data to identify dysregulated genes and to classify breast cancers according to gene copy number anomalies.Experimental Design: DNA was extracted from 106 pretreatment fine needle aspirations of stage II-III breast cancers that received preoperative chemotherapy. CGH was done using Agilent Human 4 × 44K arrays. Gene expression data generated with Affymetrix U133A gene chips was also available on 103 patients. All P values were adjusted for multiple comparisons.Results: The average number of copy number abnormalities in individual tumors was 76 (range 1-318). Eleven and 37 distinct minimal common regions were gained or lost in >20% of samples, respectively. Several potential therapeutic targets were identified, including FGFR1 that showed high-level amplification in 10% of cases. Close correlation between DNA copy number and mRNA expression levels was detected. Nonnegative matrix factorization (NMF) clustering of DNA copy number aberrations revealed three distinct molecular classes in this data set. NMF class I was characterized by a high rate of triple-negative cancers (64%) and gains of 6p21. VEGFA, E2F3, and NOTCH4 were also gained in 29% to 34% of triple-negative tumors. A gain of ERBB2 gene was observed in 52% of NMF class II and class III was characterized by a high rate of estrogen receptor–positive tumors (73%) and a low rate of pathologic complete response to preoperative chemotherapy (3%).Conclusion: The present study identified dysregulated genes that could classify breast cancer and may represent novel therapeutic targets for molecular subsets of cancers.

Supplementary Table 1 Legend from Reproducibility of Gene Expression Signature–Based Predictions in Replicate Experiments

XLS file - 57K, Top 206 candidate probesets on the U133A array with logistic regression interaction p-value

Supplementary Data from Molecular Characterization of Breast Cancer with High-Resolution Oligonucleotide Comparative Genomic Hybridization Array

Power calculations for extreme scenario

Patient selection is important for targeted therapies, yet phase I/II trials are often underpowered for developing predictors of drug response. The goal of this research was to define genomic predictors for dasatinib that could be prospectively tested in early-phase clinical trials.Gene expression profiles of dasatinib-sensitive and dasatinib-resistant cell lines (n = 23) were compared to develop a dasatinib-sensitivity index (modified DS index). A Src pathway activity index (revised Src index) was defined using genes induced by the Src transfection of mammary epithelial cells and was optimized to be reproducible across cell lines and human specimens. A dasatinib target index was devised using the weighted sum of 19 kinases that bind to dasatinib with variable affinity. The performance of these prediction models was assessed in independent cell lines with known dasatinib sensitivity. The feasibility of applying these genomic tests to human samples was evaluated on 133 biopsies of primary breast cancers.The modified DS index showed 90% accuracy in independent breast cancer cell lines (n = 12) and the target index, but not the revised Src index signature, also distinguished dasatinib-sensitive and dasatinib-resistant cells (P = 0.0024). The genomic predictors showed acceptable reproducibility in replicate cell line and human gene expression data. When all three predictors were applied to the same 133 patient samples, the predictors identified different patient subsets as potentially sensitive.We defined three conceptually different potential predictors of dasatinib response that were reproducible across cell lines and human data. These candidate markers are being tested in a clinical trial to determine their utility. Mol Cancer Ther; 9(5); 1120–7. ©2010 AACR.

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