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Background: Black patients with triple negative breast cancer (TNBC) have worse survival outcomes, even after adjusting for stage at diagnosis, income, insurance status and other socioeconomic factors. Little is known regarding anti-tumor immune responses in Black patients and how these differences affect responses to treatment in TNBC. Limited data exists regarding the stromal tumor infiltrating lymphocytes (sTILs, which are strongly prognostic in TNBC) distribution based on race and ethnicity. Here we evaluate the prevalence, distribution, and prognostic impact of sTILs in TNBC by race/ethnicity from 2 prospective clinical trials of adjuvant anthracycline/taxane-based chemotherapy (E2197 and E1199). Methods: Full-face hematoxylin and eosin-stained sections of 481 tumors from ECOG-ACRIN trials E2197 and E1199 were previously evaluated for density of sTILs and shown to be associated with disease-free survival (DFS), distant recurrence-free interval (DRFI), and overall survival (OS) (Adams, et al JCO 2014). Further analyses were undertaken to evaluate the impact of race/ethnicity. Results: The majority of the 481 TNBC were from White patients (82.3%, n=403); with 12.3% (n=59) Black patients, 1.6% (n=14) other (9 Hispanic, 3 Asian, 2 Other), and 0.5% (n=5) unknown race. Age distribution (mean 49.2 for White and 49.2 for Black) and node negative disease (White 68/403 (42%), Black 24/59 (41%)) were similar. However, tumor size ≤2cm was seen more commonly in White patients (34%, 137/403) compared with Black patients (20%, 12/59). Black patients had a higher proportion of high sTILs (≥30%) with 23.7% (14/59) compared to White patients (11.4%, 46/403). The association of continuous stromal TILs with DFS (hazard ratio for a 10-point difference) was 0.84 (95% CI 0.72, 0.98) for White patients and 0.94 (95% CI 0.73, 1.20) for Black patients [159 DFS events for Whites, 26 DFS events for Blacks]. Conclusions: This is the first dataset from prospective clinical trials evaluating sTILs in TNBC in Black patients. Prevalence of high sTILs was greater in Black patients compared to White patients. The association between increasing sTILs and improved invasive disease-free survival across racial/ethnic groups must be investigated in larger datasets. Table 1.Race/EthnicityTotal (n=481)White (n=403)Black (n=59)Other (n=19)Mean age49.049.249.245.6T1 (tumor 2 and Citation Format: Natalie Klar, Robert J Gray, Sylvia Adams, Joseph A Sparano, Lori J Goldstein, Angela M DeMichele, Antonio C Wolff, Nancy E Davidson, George W Sledge, Sunil S Badve. Stromal tumor infiltrating lymphocytes analysis by race and ethnicity in triple negative breast cancers from 2 phase III randomized adjuvant breast cancer trials: ECOG-ACRIN E2197 and E1199 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-35.

Supplemental Methods. Supplemental Table 1: ââ,¬â€¹Genomic Data Characterization by Center. Supplemental Table 2: ââ,¬â€¹Gene Panels Submitted by Each Center. Figure S1: Number of putative germline SNPs per sample, before and after uniform germline filtering. Figure S2ââ,¬â€¹. Distribution of total somatic mutation burden per sample stratified by sequencing panel. Figure S3: ââ,¬â€¹Log-scale comparison of mutation frequencies at hotspot sites between GENIE (data aggregated from all sequencing panels) and cancerhotspots.org (CHS) using a binomial test. Figure S4:ââ,¬â€¹ Comparison of mutation frequencies at hotspot sites in each GENIE sequencing panel with cancerhotspots.org (CHS) using a binomial test.

Supplemental Fig S5. Imprinted genes display approximately 50% DNA methylation. Gene regions known to be imprinted were chosen to confirm the validity of the Methyl-Seq assay. For each gene a β value was calculated for each window. The frequency of each particular β value is graphically represented for each gene.

Table S4

Supplemental Figure S2. All samples from both time points were used in a hierarchal clustering analysis. 10 samples at the early time point and 11 samples at the late time point were included.(E=early, L=late, P=parous, NP=nulliparous)

Supplemental Fig S8. Hypermethylation of the Igf1r aligns with sites of histone methylation. (A) The UCSC Genome Browser was used to align the Igf1r with other epigenetic modifications. The red bar indicates the region of differential DNA methylation identified in the SureSelectXT Methyl-Seq Assay. Black bars represent sites of histone methylation, the intensity corresponds to the degree of methylation. (B) Igf1r bisulfite sequencing data are depicted. Each animal is numbered and each horizontal line represents DNA from a separate clone from that animal. The percent methylation of each animal is listed under each CpG site. Open circles = unmethylated, and closed circles = methylated. The lower panel represents a combined result from all clones in all animals.

AURORA aims to study the processes of relapse in metastatic breast cancer (MBC) by performing multi-omics profiling on paired primary tumors and early-course metastases. Among 381 patients (primary tumor and metastasis pairs: 252 targeted gene sequencing, 152 RNA sequencing, 67 single nucleotide polymorphism arrays), we found a driver role for GATA1 and MEN1 somatic mutations. Metastases were enriched in ESR1, PTEN, CDH1, PIK3CA, and RB1 mutations; MDM4 and MYC amplifications; and ARID1A deletions. An increase in clonality was observed in driver genes such as ERBB2 and RB1. Intrinsic subtype switching occurred in 36% of cases. Luminal A/B to HER2-enriched switching was associated with TP53 and/or PIK3CA mutations. Metastases had lower immune score and increased immune-permissive cells. High tumor mutational burden correlated to shorter time to relapse in HR+/HER2− cancers. ESCAT tier I/II alterations were detected in 51% of patients and matched therapy was used in 7%. Integration of multi-omics analyses in clinical practice could affect treatment strategies in MBC.Significance:The AURORA program, through the genomic and transcriptomic analyses of matched primary and metastatic samples from 381 patients with breast cancer, coupled with prospectively collected clinical data, identified genomic alterations enriched in metastases and prognostic biomarkers. ESCAT tier I/II alterations were detected in more than half of the patients.This article is highlighted in the In This Issue feature, p. 2659

File with the supplementary material & methods, supplementary tables and supplementary figure.

Primers used for quantitative PCR and bisulfite sequencing.

Supplemental Fig S7. Igf1r is central to a top signaling network in Ingenuity pathway analysis. Ingenuity pathway analysis (IPA) identified this signaling network to be significantly altered by DNA methylation. The hyper and hypomethylated genes identified to be persistently differentially methylated by the meta-analysis were input into IPA (Supplemental Data 1). Red symbols represent hypermethylation, green symbols represent hypomethylation and intensity depicts methylation difference between nulliparous and parous.

AACR GENIE Data Guide

Supplemental Fig S9. Differentially methylated regions of IGF pathway genes align with histone methylation sites. The UCSC Genome Browser was used to align the Irs1, Igf1, and Igfbp4 with other epigenetic modifications. The red bars indicate the regions of differential DNA methylation identified in the SureSelectXT Methyl-Seq Assay. Black bars represent sites of histone methylation, the intensity corresponds to the degree of methylation.

The most effective natural prevention against breast cancer is an early first full-term pregnancy. Understanding how the protective effect is elicited will inform the development of new prevention strategies. To better understand the role of epigenetics in long-term protection, we investigated parity-induced DNA methylation in the mammary gland. FVB mice were bred or remained nulliparous and mammary glands harvested immediately after involution (early) or 6.5 months following involution (late), allowing identification of both transient and persistent changes. Targeted DNA methylation (109 Mb of Ensemble regulatory features) analysis was performed using the SureSelectXT Mouse Methyl-seq assay and massively parallel sequencing. Two hundred sixty-nine genes were hypermethylated and 128 hypomethylated persistently at both the early and late time points. Pathway analysis of the persistently differentially methylated genes revealed Igf1r to be central to one of the top identified signaling networks, and Igf1r itself was one of the most significantly hypermethylated genes. Hypermethylation of Igf1r in the parous mammary gland was associated with a reduction of Igf1r mRNA expression. These data suggest that the IGF pathway is regulated at multiple levels during pregnancy and that its modification might be critical in the protective role of pregnancy. This supports the approach of lowering IGF action for prevention of breast cancer, a concept that is currently being tested clinically. Cancer Prev Res; 8(10); 1000–9. ©2015 AACR.

Supplemental Figure S3. Mapping efficiency was above 50 for all samples in both time points. One sample was an outlier in the early time point (P12) and one at the late time point (NP4) and were removed from further analysis. Both Pearson Correlation plots and Principal Component Analysis are displayed.

Supplementary file with the listing of the liver genes excluded from transcriptomic analyses.

All Supplementary Figure legends are included in this document.

Supplemental Figure S6. Supervised hierarchical clustering and cell type content analysis. (A) Heatmaps displaying supervised hierarchical clustering of hypermethylated and hypomethylated windows including dendrograms (Red=Methylated, Green=Unmethylated).

Supplemental Data 1. This spreadsheet contains the results of the differential methylation analysis, the meta-analysis and the pathway analysis. In the meta-analysis sheets, the first 3 columns represent the chromosomal locations, followed by the significance level (stat, p-value, and FDR). Weight.1 and weight.2 represent the weight of early and late data respectively. For the meta-analysis we weighted both as 1. The effect size represents the difference in DNA methylation between NP and P at the early and late time points (effect size.1=early, effect size.2=late), and the p-value for each is next depicted. We then filtered the data by trimmed means as in the Materials and Methods section and the new values are listed in columns N and O. We then annotated the gene name, which genomic feature the loci resides in, and the distance to the nearest promoter (distance to feature). We then annotated the mean coverage and the number of CpG sites in the differentially methylated region. The differential methylation sheets include similar data without the columns pertaining to the meta-analysis. The 3Ingenuity Pathway Analysis (IPA) spreadsheets contain the pathway analysis result. The pathway name, followed by the statistical significance, and molecules represented in our data for each pathway are detailed on each sheet. The analysis was first conducted on the hyper and hypomethylated genes separately, then the combined hyper and hypermethylated genes were analyzed together.

Supplementary Figure S1. Experimental design and quality control analysis for Methyl-Seq data. (A) FVB mice were bred to become pregnant (parous, P) and underwent a complete pregnancy (Pr), lactation (L), and Involution (I). Animals were then euthanized immediately after involution (Early, E), or 6.5 months post-involution (Late, Lt). Nulliparous (NP) age matched controls were included at each time point. Mammary glands were harvested and genomic DNA was isolated and processed for input into the SureSelectXT Mouse Methy-Seq Enrichment Platform for DNA methylation analysis. (B) Quantitative RT-PCR of cell type specific transcripts. Each data point represents an animal, and bars represent mean ? SEM.

Cellular tumor purity for biopsies is greater than 70%. Tumor purity from biopsies was analyzed with the Estimate Package from Bioconductor using R language. The majority (85%) of biopsies have an estimated cellular purity of over 70%, with small differences observed between pre and post-tx biopsies. Patient 17 had both 8-week and 6-months post-tx biopsies. Tx: therapy.

PDF file - 20K, Triple negative lower expression.

Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal carcinoma (IDC) and characterized by the loss of E-cadherin–mediated adherens junctions. Despite displaying unique histologic and clinical features, ILC still remains a chronically understudied disease, with limited knowledge gleaned from available laboratory research models. Here we report a comprehensive 2D and 3D phenotypic characterization of four estrogen receptor–positive human ILC cell lines: MDA-MB-134, SUM44, MDA-MB-330, and BCK4. Compared with the IDC cell lines MCF7, T47D, and MDA-MB-231, ultra-low attachment culture conditions revealed remarkable anchorage independence unique to ILC cells, a feature not evident in soft-agar gels. Three-dimensional Collagen I and Matrigel culture indicated a generally loose morphology for ILC cell lines, which exhibited differing preferences for adhesion to extracellular matrix proteins in 2D. Furthermore, ILC cells were limited in their ability to migrate and invade in wound-scratch and transwell assays, with the exception of haptotaxis to Collagen I. Transcriptional comparison of these cell lines confirmed the decreased cell proliferation and E-cadherin–mediated intercellular junctions in ILC while uncovering the induction of novel pathways related to cyclic nucleotide phosphodiesterase activity, ion channels, drug metabolism, and alternative cell adhesion molecules such as N-cadherin, some of which were differentially regulated in ILC versus IDC tumors. Altogether, these studies provide an invaluable resource for the breast cancer research community and facilitate further functional discoveries toward understanding ILC, identifying novel drug targets, and ultimately improving the outcome of patients with ILC.Significance: These findings provide the breast cancer research community with a comprehensive assessment of human invasive lobular carcinoma (ILC) cell line signaling and behavior in various culture conditions, aiding future endeavors to develop therapies and to ultimately improve survival in patients with ILC. Cancer Res; 78(21); 6209–22. ©2018 AACR.

Purpose: To conduct an exploratory analysis of the relationship between gene expression and recurrence in patients with operable triple-negative breast cancer (TNBC) treated with adjuvant doxorubicin-containing chemotherapy.Experimental Design: RNA was extracted from archived tumor samples derived from 246 patients with stage I-III TNBC treated with adjuvant doxorubicin-containing chemotherapy, and was analyzed by quantitative reverse transcriptase PCR for a panel of 374 genes. The relationship between gene expression and recurrence was evaluated using weighted Cox proportional hazards model score tests.Results: Growth factor receptor bound protein 7 (GRB7) was the only gene for which higher expression was significantly associated with increased recurrence in TNBC (Korn's adjusted P value = 0.04). In a Cox proportional hazards model adjusted for clinicopathologic features, higher GRB7 expression was associated with an increased recurrence risk (HR = 2.31; P = 0.04 using the median as the split). The 5-year recurrence rates were 10.5% [95% confidence intervals (CI), 7.8–14.1] in the low and 20.4% (95% CI, 16.5–25.0) in the high GRB7 groups. External validation in other datasets indicated that GRB7 expression was not prognostic in two adjuvant trials including variable systemic therapy, but in two other trials showed that high GBR7 expression was associated with resistance to neoadjuvant doxorubicin and taxane therapy.Conclusions:GRB7 was associated with an increased risk of recurrence in TNBC, suggesting that GRB7 or GRB7-dependent pathways may serve as potential biomarkers for therapeutic targets. Therapeutic targeting of one or more factors identified which function as interaction nodes or effectors should also be considered. Clin Cancer Res; 17(22); 7194–203. ©2011 AACR.

Purpose: To perform an exploratory analysis of the relationship between gene expression and recurrence in operable hormone receptor (HR)–positive, human epidermal growth factor receptor 2 (HER2)–normal breast cancer patients treated with adjuvant doxorubicin-containing chemotherapy.Experimental Design: RNA was extracted from archived tumor samples derived from 378 patients with stage I to III HR-positive, HER2-normal breast cancer and analyzed by reverse transcription-PCR for a panel of 374 genes, including the 21-gene recurrence score (RS). Patients were randomized to receive adjuvant doxorubicin plus cyclophosphamide or docetaxel in trial E2197, with no difference in recurrence seen in the treatment arms. All available recurrent cases were selected plus a nonrecurrent cohort. Cox proportional hazard models were used to identify relationships between gene expression and recurrence.Results: TOP2A expression exhibited the strongest association with increased recurrence risk (P = 0.01), and was significantly associated with recurrence (P = 0.008) in a multivariate analysis adjusted for clinicopathologic features. Elevated TOP2A expression above the median was associated with a 2.6-fold increase (95% confidence interval, 1.3-5.2; P = 0.008) in risk of recurrence if the RS was P = 0.003) if there was an intermediate RS of 18 to 30.Conclusions: In patients with HR-positive, HER2-normal breast cancer, a population known to have a low incidence of TOP2A gene alterations thought to be predictive of anthracycline benefit, there is a range of TOP2A RNA expression that is strongly associated with recurrence after adjuvant anthracyclines, which provides information complementary to RS, indicating that it merits further evaluation as a prognostic and predictive marker. (Clin Cancer Res 2009;15(24):7693–700)

Lists of upregulated and downregulated pathways in ILC versus IDC cell lines.

Table S1. Sequence of targeted amplification primers. Table S2. Sequence of ddPCR primer and probes. Table S3. Cellularity and location of bone metastases.

Supplementary Data from Heterogeneity of Breast Cancer Metastases: Comparison of Therapeutic Target Expression and Promoter Methylation Between Primary Tumors and Their Multifocal Metastases

Supplementary Table 4 - Summary of SNPs with p

Supplementary Table 3 - Summary of SNPs with p

PDF file, 67K

Global and promoter CpG island demethylation in available paired pre- and post-treatment (8 weeks) hormone-resistant and triple-negative tumor biopsies.

Purpose: A comprehensive comparison of biomarker expression between patients' primary breast carcinoma (PBC) and their metastatic breast carcinomas (MBC) has not been done.Experimental Design: We did rapid autopsies (postmortem intervals, 1-4 hours) on 10 consenting patients who died of MBC. We constructed single-patient tissue microarrays from the patients' archived PBC and multiple different MBCs harvested at autopsy, which were immunohistochemically labeled for multiple biomarkers. Methylation of multiple gene promoters was assessed quantitatively on dissected PBC and MBC samples.Results: Extensive heterogeneity was observed between PBC and their paired MBC, as well as among multiple MBC from the same patient. Estrogen and progesterone receptors tended to be uniformly down-regulated in metastases. E-cadherin was down-regulated in a subset of the MBC of one case. Variable overexpression in MBC compared with the PBC was observed for cyclooxygenase-2 (five cases), epidermal growth factor receptor (EGFR; four cases), MET (four cases), and mesothelin (four cases). No case strongly overexpressed HER-2/neu by immunohistochemistry, but eight cases showed variable protein expression ranging from negative to equivocal (2+) in different MBC. In one case, variable low-level HER-2/neu gene amplification was found. EGFR and MET overexpression were restricted to the four basal-type cancers. EGFR protein overexpression did not correlate with EGFR gene amplification. Multigene promoter hypermethylation of RASSF1a, HIN1, cyclin D2, Twist, estrogen receptor α, APC1, and RARβ was overall very similar in the PBC and all MBCs in all cases.Conclusions: Therapeutic targets identified in the PBC or even some MBC may not reflect targets present in all metastatic sites.

PDF file, 85K - Supplementary Figure 1 Pre and Post Vorinostat Values of Other Gene Expression using the Oncotype DX 21-gene Assay.

Supplementary Figure 1. Principal component analysis for patients in ECOG-5103 Supplementary Figure 2. Regional association (locuszoom) plots of rs3125923.

Purpose: Agents that target the epigenome show activity in breast cancer models. In preclinical studies, the histone deacetylase inhibitor vorinostat induces cell-cycle arrest, apoptosis, and differentiation. We evaluated biomarker modulation in breast cancer tissues obtained from women with newly diagnosed invasive disease who received vorinostat and those who did not.Experimental Design: Tumor specimens were collected from 25 women who received up to 6 doses of oral vorinostat 300 mg twice daily and from 25 untreated controls in a nonrandomized study. Candidate gene expression was analyzed by reverse transcription PCR (RT-PCR) using the Oncotype DX 21-gene assay, and by immunohistochemistry for Ki-67 and cleaved caspase-3. Matched samples from treated women were analyzed for gene methylation by quantitative multiplex methylation-specific PCR (QM-MSP). Wilcoxon nonparametric tests were used to compare changes in quantitative gene expression levels pre- and post-vorinostat with changes in expression in untreated controls, and changes in gene methylation between pre- and post-vorinostat samples.Results: Vorinostat was well tolerated and there were no study-related delays in treatment. Compared with untreated controls, there were statistically significant decreases in the expression of proliferation-associated genes Ki-67 (P = 0.003), STK15 (P = 0.005), and Cyclin B1 (P = 0.03) following vorinostat, but not in other genes by the Oncotype DX assay, or in expression of Ki-67 or cleaved caspase-3 by immunohistochemistry. Changes in methylation were not observed.Conclusions: Short-term vorinostat administration is associated with a significant decrease in expression of proliferation-associated genes in untreated breast cancers. This demonstration of biologic activity supports investigation of vorinostat in combination with other agents for the management of breast cancer. Clin Cancer Res; 19(14); 4008–16. ©2013 AACR.

Supplementary Figure 2 - PDF file 9846K, Patient Flow Diagram for N9831 MYC IHC Analysis

Supplementary Data from Relationship between Topoisomerase 2A RNA Expression and Recurrence after Adjuvant Chemotherapy for Breast Cancer

Purpose: Taxane-induced peripheral neuropathy (TIPN) is an important survivorship issue for many cancer patients. Currently, there are no clinically implemented biomarkers to predict which patients might be at increased risk for TIPN. We present a comprehensive approach to identification of genetic variants to predict TIPN.Experimental Design: We performed a genome-wide association study (GWAS) in 3,431 patients from the phase III adjuvant breast cancer trial, ECOG-5103 to compare genotypes with TIPN. We performed candidate validation of top SNPs for TIPN in another phase III adjuvant breast cancer trial, ECOG-1199.Results: When evaluating for grade 3–4 TIPN, 120 SNPs had a P value of −4 from patients of European descent (EA) in ECOG-5103. Thirty candidate SNPs were subsequently tested in ECOG-1199 and SNP rs3125923 was found to be significantly associated with grade 3–4 TIPN (P = 1.7 × 10−3; OR, 1.8). Race was also a major predictor of TIPN, with patients of African descent (AA) experiencing increased risk of grade 2–4 TIPN (HR, 2.1; P = 5.6 × 10−16) and grade 3–4 TIPN (HR, 2.6; P = 1.1 × 10−11) compared with others. An SNP in FCAMR, rs1856746, had a trend toward an association with grade 2–4 TIPN in AA patients from the GWAS in ECOG-5103 (OR, 5.5; P = 1.6 × 10−7).Conclusions: rs3125923 represents a validated SNP to predict grade 3-4 TIPN. Genetically determined AA race represents the most significant predictor of TIPN. Clin Cancer Res; 21(22); 5082–91. ©2015 AACR.

Genome wide genotyping of E5103 samples. Quality controls for GWAS.

XLS file - 31K, Patient characteristics by GRB7 expression.

Supplementary Table and Figure Legends