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Supplemental Table 3 Race and Survival Predictor Regulon Gene Enrichment Ontology Processes

Supplemental Figure 5 Overall breast cancer survival based on median income of county of diagnosis in European American (EA) (A) compared to African American (AA) patients (B). CI, confidence interval.

Supplemental Table 1 Survival and Race Predictor Regulon Gene Descriptions & Regulons

Supplemental Table 2 Race and 3-year Survival Regulon RFS

Supplemental Figure 4 Quantitative correlation of biomarker expression stratified by race.

Supplemental Figure 3 Racial difference in survival in (A) Luminal A compared to (B) TNBC breast cancer. CI, confidence interval.

Supplemental Figure 1 Comparison of subtype distribution by ancestry in the current cohort study

Purpose:Compared with their European American (EA) counterparts, African American (AA) women are more likely to die from breast cancer in the United States. This disparity is greatest in hormone receptor–positive subtypes. Here we uncover biological factors underlying this disparity by comparing functional expression and prognostic significance of master transcriptional regulators of luminal differentiation.Experimental Design:Data and biospecimens from 262 AA and 293 EA patients diagnosed with breast cancer from 2001 to 2010 at a major medical center were analyzed by IHC for functional biomarkers of luminal differentiation, including estrogen receptor (ESR1) and its pioneer factors, FOXA1 and GATA3. Integrated comparison of protein levels with network-level gene expression analysis uncovered predictive correlations with race and survival.Results:Univariate or multivariate HRs for overall survival, estimated from digital IHC scoring of nuclear antigen, show distinct differences in the magnitude and significance of these biomarkers to predict survival based on race: ESR1 [EA HR = 0.47; 95% confidence interval (CI), 0.31–0.72 and AA HR = 0.77; 95% CI, 0.48–1.18]; FOXA1 (EA HR = 0.38; 95% CI, 0.23–0.63 and AA HR = 0.53; 95% CI, 0.31–0.88), and GATA3 (EA HR = 0.36; 95% CI, 0.23–0.56; AA HR = 0.57; CI, 0.56–1.4). In addition, we identify genes in the downstream regulons of these biomarkers highly correlated with race and survival.Conclusions:Even within clinically homogeneous tumor groups, regulatory networks that drive mammary luminal differentiation reveal race-specific differences in their association with clinical outcome. Understanding these biomarkers and their downstream regulons will elucidate the intrinsic mechanisms that drive racial disparities in breast cancer survival.

Supplemental data

Background: KRAS activating mutations are reported in 42-54% and inactivation of CDKN2A/p16 in 14-30% of human small intestinal adenocarcinomas, suggesting a role in small intestine adenocarcinogenesis. The phenotypic effects of oncogenic KRAS and p16 loss have not been well characterized in small intestine (SI) adenocarcinogenesis. We targeted LGR5-expressing progenitor cells residing in intestinal crypts for conditional activation of Kras and/or Cdkn2a/p16 deletion to generate a murine model to study the single and combined effects of KRAS and p16 alterations driving gastrointestinal tumorigenesis. We hypothesized that oncogenic Kras and loss of Cdkn2a/p16 drives adenoma-adenocarcinoma sequence in murine SI. Methods: We generated mice with conditional knockout of Cdkn2a/p16 (p16del: IL1bwt/tg;Lgr5-Cre+/−;p16fl/fl;Kraswt/wt), conditional expression of oncogenic KrasG12D (IL1bwt/tg;Lgr5-Cre+/−;KrasG12D/wt), or both alterations (p16del+KrasG12D: IL1bwt/tg;Lgr5-Cre+/−;p16fl/fl;KrasG12D/wt), in mice carrying the human interleukin 1 beta (IL1B) and expressing modified Cre under the Lgr5 promoter. Our control murine model expresses wild type p16 and KRAS (p16wt+Kraswt: IL1bwt/tg;Lgr5-Cre+/−;p16wt/wt;Kraswt/wt). These mice were initially generated to study esophageal adenocarcinogenesis and express the IL1b transgene in esophagus but not in SI. All mice were given bile acid in drinking water daily and were treated with tamoxifen to activate Cre in Lgr5-positive gastrointestinal epithelial progenitor cells. Mice were euthanized and gross and histologic alterations in the SI were examined from 4 to 21 months of age. 3D organoids were isolated from the SI of each mouse model. Results: We examined 89 mice from 4 to 12 months of age. Mice with KRAS activation (KrasG12D) alone infrequently developed SI duodenal adenomas (6.25%: 1/16) compared to 50% (9/18) of mice carrying the combined P16del+KrasG12D genotypes (P=0.005). No adenomas were seen in 27 control and 28 p16del mice. Adenomas with high-grade dysplasia, intramucosal adenocarcinoma, and invasive adenocarcinoma were seen only in older p16del+KrasG12D mice (13-21 months). Single or multiple adenomas and adenocarcinomas were observed only in the duodenal segment of SI and not in the colon. 3D organoids isolated from each mouse model reproduce the intestinal histologic phenotype. Conclusions: Loss of Cdkn2a/p16 and KRASG12D expression synergistically advances adenoma-adenocarcinoma tumorigenesis in the SI duodenum, providing a murine model that recapitulates SI adenocarcinogenesis in vivo. 3D SI organoids provide useful in vitro models for mechanistic studies of SI adenocarcinogenesis. These in vivo and in vitro models will enable identification of key molecular pathways that may be targeted with novel therapies for this rare cancer. Citation Format: Tyler Seckar, Jing Sun, Elena V. Komissarova, Jorge L. Sepulveda, Timothy L. Wang, Antonia R. Sepulveda. Oncogenic KrasG12D and Cdkn2a/p16 knockout in LGR5 expressing progenitor cells synergize to advance adenoma and adenocarcinoma phenotypes in murine small intestine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 24.

Purpose: Women of African ancestry in the United States are more likely to die from breast cancer than their European counterparts. While prior studies suggest differences in the frequency of hormone receptor-negative disease as an underlying cause, recent studies report higher race-based mortality rates in patients with hormone receptor-positive, luminal breast cancer. Here we explore biologic factors that may underly this disparity by comparing racial differences in the level, functional activity, and prognostic significance of 3 master transcriptional regulators of mammary luminal differentiation. Patients and Methods: Medical records and tissues from 555 patients (293 European and 262 African ancestry) diagnosed with Stage 0 to IV breast cancer, from 2001 to 2010 at a major medical center in East North Carolina, were analyzed for the expression of functional biomarkers of luminal differentiation including estrogen receptor (ESR1), and pioneer transcription factors FOXA1 and GATA3. Differential comparison of protein expression was integrated with network-level gene expression analysis (22% of cohort) to define predictive correlations with race and survival. Results: Univariate and multivariate odds ratios combined with area under the curve receiver operator characteristics show significant differences in predictive activity of these functional biomarkers based on race and survival—ESR1 (EA OR= 0.47, p = 5e-04; AA OR= 0.77, p = 0.22), FOXA1 (EA OR= 0.38, p= 1.4e-04; AA OR = 0.53, p = 1.3e-02), and GATA3 (EA OR= 0.36, p= 3.7e-06, AA OR= 0.57, p= 0.51)—and uncover genes in the downstream regulons of these biomarkers that strongly correlate either with genetic ancestry or overall survival. Conclusion: Transcriptional regulatory networks linked to mammary luminal differentiation reveal race-specific differences in master regulatory activity that may underlie tumor characteristics contributing to racial disparities in outcome. These biomarkers and their downstream regulons represent important targets to explore intrinsic mechanisms that drive breast cancer survival disparities. Citation Format: Jung S. Byun, Sandeep K. Singhal, Sam Park, Dae Ik Yi, Tingfen Yan, Ambar Caban, Alana Jones, Partha Mukhopadhyay, Sara Gil Hernandez, Stephen Hewitt, Lisa A. Newman, Melissa Davis, Jorge Sepulveda, Adrianna De Siervi, Anna Napoles, Nasreen Vohra, Kevin Gardner. Racial differences in the associations between luminal master regulator transcription factor expression and breast cancer survival [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C033.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common cancers in the United States. The five-year survival rate for patients with PDAC remains dismal. Identification of biomarkers for early diagnosis of PDAC and pre-cancer pancreatic intraepithelial neoplasia (PanIN) lesions with risk of progression to PDAC is critically needed. We hypothesized that differentially expressed genes and regulatory pathways in PanIN and PDAC compared to normal duct epithelium (ND) may represent biomarkers of development of malignancy. We used Affymetrix Human Transcriptome Arrays 2.0 to establish gene expressionprofiles in ND, low-grade PanIN, and PDAC epithelium. Total RNA was isolated after laser capture microdissection (LCM) of frozen tissue sections and then used for producing hybridization-ready DNA. Hybridization quality control was performed with Expression Console 1.4 software and background corrected/normalized data were analyzed with Transcriptome Analysis Console (TAC) 3.1 and the ASSIGN algorithm. We tested RNA from 22 LCM samples (9 PDACs, 5 PanINs, and 8 ND), including 4 matched trios of ND, PanIN and PDAC from the same patients. Differential expression analysis with one-way between subject ANOVA revealed over 2000 genes differentially expressed in PDAC and PanIN vs. ND group (filter criteria up/down >2; ANOVA p1.5; ANOVA p Citation Format: Elena V. Komissarova, Jorge Sepulveda, Sarawut Kongkarnka, Maryam Shirazi, Brynn Levy, Claudia Cujar, Antonia R. Sepulveda. Human transcriptome alterations in pre-cancer and cancer epithelium identify candidate biomarkers of progression to pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 714. doi:10.1158/1538-7445.AM2017-714

Gastric adenocarcinoma (GC) is the 5th most common cancer worldwide but is the 3rd leading cause of cancer death. FLI1 (Friend leukemia integration-1) is an ETS family transcription factor that regulates genes involved in proliferation and differentiation. FLI1 is implicated in tumorigenesis, such as in Ewing’s sarcoma where a translocation creates an EWS-FLI1 oncogenic fusion protein. However, few studies have examined the role of FLI1 in carcinomas. In human breast cancer, overexpression of FLI1 led to inhibition of apoptosis, thereby promoting survival and malignant potential. In functional studies in a murine breast cancer model, however, downregulation of FLI1 increased malignant potential. In human GCs, we recently reported that FLI1 expression is inversely correlated with its promoter CpG methylation of the FLI1. To determine if decreased expression occurs in GC epithelial cells or background non-epithelial cells, we analyzed 91 human GC tumors by immunohistochemistry (IHC) for FLI1 and compared them to normal gastric mucosa and intestinal metaplasia (IM) using an IHC composite scoring system accounting for intensity and percentage of epithelial cells expressing FLI1. We found that FLI1 is strongly expressed in normal gastric glandular epithelium and in IM, and that decreased expression was seen in most human GCs (P < 1x10-17 vs normal, P < 1x10-26 vs IM). These findings suggest that FLI1 acts as a tumor suppressor gene in GC. To test this hypothesis, we used an invasion assay and the human GC cell lines NUGC3 and SNU638, which have little to no FLI1 expression, respectively. Cultured GC cells were transduced to overexpress FLI1 or control, along with an eGFP reporter from an IRES (Lv203, Genecopoeia). After selection by puromycin, these GC cells were plated in serum-free media in the upper chamber on a Matrigel coated 8µm pore opaque membrane. Complete media with 10% fetal calf serum was plated in the lower chamber. Images were obtained of the lower membrane with an inverted fluorescent microscope and cellSens imaging software. Overexpression of FLI1 significantly decreased invasion by NUGC3 cells at 24 hours (P = 0.013) but not at 48 hours (P = 0.268) as compared to control. Overexpression of FLI1 significantly decreased invasion by SNU638 cells at both 24 and 48 hours (P = 0.027 and 0.012, respectively) as compared to control. Since NUGC3 cells have low FLI1 expression, we knocked down FLI1 by using a FLI1 shRNA lentiviral system with mCherry from an IRES as a reporter (LvRU6MP, Genecopoeia). Using the same invasion assay, knockdown of FLI1 trended towards a significant increase in invasion as compared to control at 48 hrs (P = 0.14), but not at 24 hrs (P = 0.73). In summary, the combined observations in human GC tissue samples and the functional analyses in GC cells support a tumor suppressor role for FLI1 in human GC and also suggest that FLI1 and/or its target genes may be involved in regulatory mechanisms driving invasive properties of GC. Citation Format: Armando Del Portillo, Elena V. Komissarova, Anne Koehne de Gonzalez, Aqiba Bokhari, Helen Remotti, Jorge Sepulveda, Antonia Sepulveda. Functional role of Friend Leukemia Integration-1 (FLI1) in gastric carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5532. doi:10.1158/1538-7445.AM2017-5532