Citations

Abstract Introduction: Diffuse Large B cell Lymphoma (DLBCL) is up to 80-fold more likely to occur and follows a more aggressive clinical course in HIV positive (+) individuals compared to HIV negative (-) individuals. Moreover, the vast majority frequently present at advanced stages with high tumor burdens with poor prognosis. The molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood; however, it is suspected to depend on decreased host immune surveillance and alternative mechanisms of tumorogenesis. In this study, we sought to identify the differences in gene expression between HIV(+) and HIV(-) DLBCL cohorts that may contribute to the unique features of HIV(+) DLBCL. Methods: We performed a retrospective gene expression profiling (GEP) study using a total of 70 DLBCL cases, 31 HIV(+) obtained from AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 39 HIV(-) institutional cases. The H&Es for all cases were reviewed by a hematopathologist and tumor content determined. Cases that did not meet the minimum tumor content threshold of 70% were macro-dissected. RNA was extracted from 4x 5µm formalin fixed, paraffin embedded (FFPE) sections using the Qiagen AllPrep DNA/RNA FFPE Kit and quantified using a NanoDrop. The Lymph2Cx assay, which accurately subtypes DLBCL in FFPE tissues (Scott et al 2014), was used to determine the cell of origin (COO) subtypes: Germinal Center B cell (GCB), Activated B cell (ABC) and Unclassifiable (UNC). Digital GEP was performed using the NanoString PanCancer Pathways and Immunology panels, targeting a total of over 1300 genes with known roles in cancer or immune signaling. Results: Of the 31 HIV(+) cases, Lymph2Cx subtyping showed 74% (23/31) were GCB, 13% (4/31) were ABC and 13% (4/31) were UNC. In a similar distribution to the HIV(+), and comparable to prior literature, 54% (21/39) of HIV(-) cases were GCB, 31% (12/39) were ABC and 15% (6/39) were UNC. Differential gene expression was observed between HIV(+) and HIV(-) cases, but no association with COO subtype was found. When the data was re-analyzed within the GCB subtype alone, stratification by HIV status was observed. Particularly, reduced expression of genes associated with immune regulation was observed in HIV(+) GCB cases compared to HIV(-) GCB cases. In contrast, expression of genes associated with cell cycle regulation and homologous recombination (the predominate double stranded DNA repair pathway employed during the cell cycle), were higher in HIV(+) cases compared to HIV(-) GCB cases. Conclusions: The results show, that within the GCB subtype, marked differences in gene expression are associated with HIV status. Our results suggest that the aggressive nature of DLBCL in HIV(+) patients may, in part, be mediated by enhanced cell proliferation potentiated by aberrant DNA repair. Disclosures Fogel: Natural Selection Inc: Employment. Liu: Natural Selection Inc: Employment. Lamers: Bioinfoexperts LLC: Employment.

Abstract Background: The BRCA1 tumour suppressor gene is mutated in the germline of women who are predisposed to developing breast cancer. Gene expression profiling has identified at least five different breast cancer subtypes with BRCA- mutated breast tumours clustering with triple negative breast cancers. The majority of BRCA1-mutated breast tumours are characterised as being negative for the estrogen receptor-α (ER-α), however, the underlying molecular biology of these tumours has not yet been fully determined. The aim of this study is to identify and characterise novel proliferation-associated BRCA1 target genes that are activated in BRCA1 mutated estrogen receptor negative (ER-ve) breast tumours. Methods: Gene expression profiling and data analysis was performed on a cohort of 46 FFPE (Formalin Fixed Paraffin Embedded) derived BRCA1 mutated (ER-ve) breast tumours and matched sporadic controls using the Almac Diagnostics Breast DSA research tool. Profiling was also performed on a panel of 15 breast cancer cell lines. Bioinformatics analysis was performed using Oncomine, DAVID and Metacore. High throughput siRNA screens using HiPerFect were performed on the Qiagen Flexiplate siRNA. Validation of gene targets was performed by qRT-PCR and ChIP assay. Clonogenic assays were performed to independently validate the effect of selected target genes on cell survival. Results: A list of differentially expressed transcripts was derived from the comparison of 23 (ER-ve) BRCA1 mutated breast tumours and 23 matched sporadic controls. A genome-wide ChIP-Chip promoter analysis was also performed in MCF7 breast cancer cells. By overlapping these two datasets, a list of tumour derived BRCA1 promoter bound target genes was identified. Functional analysis of this gene list has identified the main pathways and processes that are deregulated in BRCA1 mutated (ER-ve) breast cancer including: (1) immune response (2) induction of the epithelial to mesenchymal transition (EMT) (3) cell cycle regulation and (4) apoptosis and survival. Hierarchical clustering of these 46 breast tumours and 15 breast cancer cell lines was performed and the BRCA1 mutated (ER-ve) breast cancer cell lines (MDA436 and SUM149) were identified as those that best reflect the biology BRCA1 mutated (ER-ve) tumours. High throughput siRNA screening in these cell lines has identified a set of transcripts that when inhibited have a negative impact on cellular proliferation. Independent validation by qRT-PCR, ChIP assay, western blotting and clonogenic assays have confirmed HE4 (WFDC2) as a novel BRCA1 target gene that provides a growth advantage in BRCA1 mutated (ER-ve) breast cancer cells. Conclusions: Gene expression profiling of an extensive cohort of BRCA1 mutated (ER-ve) breast tumours and matched sporadic controls has identified a profile of BRCA1 mutated (ER-ve) breast cancer. This list has been further refined to generate a list of BRCA1 promoter bound transcriptional target genes. High throughout siRNA screening has revealed a panel of genes that are implicated in the proliferation of BRCA1 mutated (ER-ve) breast cancer. HE4 has been identified as a novel BRCA1 transcriptional target gene that promotes the survival of BRCA1 mutated (ER-ve) breast tumours. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD10-05.

Abstract Background: Approximately 5-10% of all breast cancers are hereditary and the majority of these arise due to germline mutations in the BRCA1 and BRCA2 tumour suppressor genes. BRCA1 is involved in multiple cellular pathways including DNA damage signalling, DNA repair, cell cycle regulation, protein ubiquitination, chromatin remodelling, transcriptional regulation and apoptosis. Several distinct pathological features can be used to estimate the likelihood of the presence of a BRCA1 mutation, however, it is not yet possible to fully categorise a BRCA1 mutated tumour. BRCA1-associated breast cancers are generally defined as being ER (Estrogen Receptor) negative and indeed triple negative for ER, PR and HER2. However, approximately 10-36% of BRCA1 mutated breast cancers are, in fact, ER positive. These tumours less frequently demonstrate the characteristics more commonly associated with BRCA 1 -associated breast cancers. Initial molecular evidence also suggests that there is heterogeneity within BRCA1-associated breast tumours and this is dependent on the presence or absence of the estrogen receptor. The aims of this study are to investigate the underlying biology of BRCA1-mutated (ER positive) and BRCA1-mutated (ER negative) breast tumours. Methods: Extensive gene expression profiling and data analysis was performed on a cohort of 70 FFPE (Formalin Fixed Paraffin Embedded) derived BRCA1 mutated breast tumours and matched sporadic controls using the ALMAC Breast Cancer DSA™ research tool. Within this dataset we have generated molecular profiles of: (1) BRCA 1 -mutated ER positive and (2) BRCA1-mutated ER negative breast cancer. Functional analysis was performed using DAVID and METACORE. Validation of gene targets was performed by qRT-PCR and Western blotting. Results: A list of differentially expressed transcripts was derived from the comparison of 35 BRCA1 mutant breast tumours and 35 matched sporadic controls. Further analysis based on the presence and absence of ER identified a set of transcripts defining BRCA1-mutated (ER positive) and BRCA1-mutated (ER negative) breast cancer. Functional analysis of these two datasets has identified the main pathways and processes that are deregulated. Specifically, BRCA1-deficiency in the absence of ER was associated with deregulation of pathways implicated in immune response whereas BRCA 1 deficiency in the presence of ER was associated with pathways implicated in cell adhesion and cytoskeletal remodelling. Validation of the key genes underlying these two BRCA1-deficient breast cancer subtypes has been performed. Discussion: This approach has revealed significant heterogeneity within BRCA1 mutated breast cancer based on the presence or absence of ER. Significant differences in the transcripts and molecular processes underlying BRCA1-mutated (ER positive) and BRCA1-mutated (ER negative) breast tumours have been identified. The ability to identify BRCA 1 -deficiency by gene expression profiling from FFPE derived breast tissue may also have significant clinical application. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-14.

Abstract Background The BRCA 1 tumour suppressor gene is mutated in a significant proportion of hereditary breast cancer cases. Additionally, downregulation of BRCA1 mRNA and protein expression is reported in approximately one third of sporadic breast cancers. BRCA1 is strongly implicated in the maintenance of genomic stability by its involvement in multiple cellular pathways including: DNA damage signalling, DNA repair, cell cycle regulation, protein ubiquitination, chromatin remodelling, transcriptional regulation and apoptosis. To date, gene expression profiling has identified: (1) at least five breast cancer subtypes and (2) that BRCA1 mutant tumours segregate with basal-like breast cancers. These studies also provide evidence that breast cancers with germline mutations in BRCA1 are different from non BRCA1-related tumours. The main aim of this study is to investigate the underlying biology of BRCA1-mutated breast cancer. Methods Extensive gene expression profiling and data analysis were performed on a cohort of 70 FFPE (Formalin Fixed Paraffin Embedded) derived BRCA 1 mutated breast tumours and matched sporadic controls using the Almac Breast Cancer DSATM research tool. Functional analysis was performed with DAVID and METACORE. Validation of gene targets was performed by qRT-PCR and Western blotting. Results A list of differentially expressed transcripts has been derived from the comparison of these BRCA1 mutant breast tumours and matched sporadic controls. Functional analysis of this gene list has identified the key genes and molecular pathways that are deregulated in these tumours. BRCA1 deficiency was associated with deregulation of pathways involved in: (1) immune response, (2) metastasis and invasion, (3) cytoskeletal remodelling, (4) spindle assembly and chromosome separation, (5) apoptosis and survival. Validation of the key genes underlying this BRCA1-deficient breast cancer profile has been performed. Conclusions This approach has revealed a set of transcripts that could potentially be used to identify both hereditary and sporadic breast cancer patients with BRCA1- deficiency. The ability to perform gene expression profiling from FFPE derived breast tissue could also have significant clinical application. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-02.