The Marker State Space (MSS) Method for Classifying Clinical Samples

The development of accurate clinical biomarkers has been challenging in part due to the diversity between patients and diseases. One approach to account for the diversity is to use multiple markers to classify patients, based on the concept that each individual marker contributes information from its respective subclass of patients. Here we present a new strategy for developing biomarker panels that accounts for completely distinct patient subclasses. Marker State Space (MSS) defines "marker states" based on all possible patterns of high and low values among a panel of markers. Each marker state is defined as either a case state or a control state, and a sample is classified as case or control based on the state it occupies. MSS was used to define multi-marker panels that were robust in cross validation and training-set/test-set analyses and that yielded similar classification accuracy to several other classification algorithms. A three-marker panel for discriminating pancreatic cancer patients from control subjects revealed subclasses of patients based on distinct marker states. MSS provides a straightforward approach for modeling highly divergent subclasses of patients, which may be adaptable for diverse applications. © 2013 Fallon et al

Germline Mutations in HOXB13 and Prostate-Cancer Risk

Family history is a significant risk factor for prostate cancer, although the molecular basis for this association is poorly understood. Linkage studies have implicated chromosome 17q21-22 as a possible location of a prostate-cancer susceptibility gene

Paired Tumor and Normal Whole Genome Sequencing of Metastatic Olfactory Neuroblastoma

Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression.Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects.This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations

Abstract A74: Differential Wnt signaling in African American and Caucasian women with triple-negative breast cancer

Abstract In the U.S., breast cancer (BC) incidences among African American (AA) and Caucasian (CA) women are similar, however, AA have a significantly higher mortality rate (20%). In addition, AA women often present with tumors at a younger age, with a higher grade/later stage, and are more likely to be diagnosed with the highly aggressive triple-negative breast cancer (TNBC) subtype. Although multiple factors may contribute to the observed health disparities in TNBC, it is essential that we identify the molecular characteristics and underlying biological differences between CA and AA TNBC. We initially conducted a gene expression study using archived formalin-fixed, paraffin-embedded (FFPE) samples obtained from CA and AA women diagnosed with Node 0 TNBC. Briefly; total RNA was isolated from 10 μm scrolls from each FFPE block, cDNA synthesis, and each sample was hybridized to a breast-enriched gene expression array (Affymetrix, BC DSA Research Tool). Expression analysis was conducted using GeneSpring 12.1 analytical software. PCA analysis revealed that the samples clustered well with respect to ethnicity and unsupervised hierarchical clustering analysis resulted in distinct subgroups based on ethnicity. Differentially expressed genes (DEG) from each cohort were selected using ANOVA analysis (fold change > 2.0, p value <.05) followed by the Benjamin/Hochberg for multiple-testing correction. Finally, the DEG was imported into GeneGo MetaCore, which revealed that the majority of functionally enriched pathways were associated with the Wnt/β-catenin signaling pathway in the AA-TNBC cohort. Additionally, TNC, CAV1, TCF4 and FOX03A, genes associated with this pathway, were significantly upregulated in the AA-TNBC cohort. We have thus far validated these findings for CAV1 and FOX03A by qRTPCR, using an independent cohort of Node 0 TNBC FFPE samples from AA and CA women. These observations are being investigated in-vitro, using a multi-ethnic TNBC cell line panel, and siRNA-mediated knockdown of the Wnt-associated genes. The effects will be evaluated through a series of functional assays including proliferation, migration, invasion, apoptosis, and epithelial-mesenchymal transition (EMT). We believe that our observations suggest that the Wnt/β-catenin pathway may contribute to the more aggressive phenotype observed in AA women diagnosed with TNBC. These studies have important implications for further understanding of TNBC and the associated health disparities that exist in AA women diagnosed with this disease; ultimately leading to the development of targeted therapies for treating this aggressive subtype of BC. Citation Format: Julie E. Getz, Davyd B. Teoh, Sara Nasser, Tembe Waibhav, Legendre R. Christophe, Venkata Yellapantula, Mary Ellen Ahearn, Carmen R. Gomez, Merce Jorda, Mark D. Pegram, John D. Carpten, Lisa L. Baumbach. Differential Wnt signaling in African American and Caucasian women with triple-negative breast cancer. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A74

Abstract C54: High caveolin-1 expression in a cohort of African American women with triple-negative breast cancer

Abstract In the U.S., the incidence rates of breast cancer (BC) among Caucasian (CA) women are lower than those of African American (AA), however, AA women have a significantly higher mortality rate. AA women often present at a younger age, with later stage and higher grade tumors. In fact, AA women are 3x more likely to be diagnosed with the more aggressive molecular subtype, triple-negative breast cancer (TNBC), than CA women. Socioeconomic and lifestyle factors may be contributory; however, it is imperative that we investigate the underlying molecular biology that may be the cause of health disparities between AA and CA with TNBC. In this study, gene expression profiling, using the Almac BC DSA Research Tool, was performed on archived FFPE samples, obtained from CA and AA women diagnosed with early stage (Node 0) TNBC. Unsupervised hierarchical clustering revealed a pattern of differential gene expression in the AA cohort compared to CA. Using the TNBC type: A Subtyping Tool for TNBC, we found a distinct distribution pattern of TNBC molecular subtypes in the AA cohort, which was very different than the CA cohort; Basal-like (14%), Immunomodulatory (43%) and Mesenchymal (43%). Gene expression analyses, comparing the AA and CA cohort (fold change > 2.0, p-value <.05), resulted in 190 differentially expressed genes (DEG). Pathway enrichment analysis conducted in MetaCore GeneGo, revealed that the DEGs were over-represented in cytoskeletal remodeling, cell adhesion, tight junctions, and immune response in the AA TNBC cohort. Furthermore, several genes in the Wnt/β-catenin pathways were over-expressed in the top 10-enrichment pathways. We validated our results using RT-qPCR and identified Caveolin-1 (CAV1) as being significantly expressed in the AA-TNBC cohort (p-value 1.22 x 10-05). An independent cohort of FFPE samples, from AA and CA women with early stage TNBC, was used to create a tissue microarray (TMA). Immunohistochemistry results showed no difference in localization of Cav1 between the AA and CA cohorts, however, the AA cohort had significantly higher levels of Cav1 staining (p-value 0.04). Additionally, using RT-qPCR, we demonstrated that CAV1 mRNA was significantly higher in the AA TNBC Cohort (p-value 0.48). Furthermore, endogenous Cav1 was shown to be highly expressed in a cell line panel of TNBC, in particular, those of the mesenchymal and basal-like molecular subtype. Finally, using siRNA, we demonstrated that CAV1 silencing resulted in a significant decrease in cell proliferation, for each of the TNBC cell lines while it showed no effect on the luminal ER+ cell lines. Our combined study results suggest that CAV1 over-expression may be a biological contributor to the observed health disparity between AA women and CA diagnosed with early stage TNBC. Citation Format: Julie E. Getz, Davyd B. Teoh, Sara Nasser, Christophe R. Legendre, Waibhav Tembe, Venkata Yellapantula, Mary Ellen Ahearn, Carmen R. Gomez, Merce Jorda, Shuk Mei Wong, Mark D. Pegram, John D. Carpten, Lisa L. Baumbach-Reardon. High caveolin-1 expression in a cohort of African American women with triple-negative breast cancer. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C54