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Background: Diesel exhaust is a complex mixture, including polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs (nitro-PAH), many of which are potent mutagens and possible bladder carcinogens. To explore the association between diesel exposure and bladder carcinogenesis, we examined the relationship between exposure and somatic mutations and mutational signatures in bladder tumors. Methods: Targeted sequencing was conducted in bladder tumors from the New England Bladder Cancer Study. Using data on 797 cases and 1,418 controls, two-stage polytomous logistic regression was used to evaluate etiologic heterogeneity between bladder cancer subtypes and quantitative, lifetime estimates of respirable elemental carbon (REC), a surrogate for diesel exposure. Poisson regression was used to evaluate associations between REC and mutational signatures. Results: We observed significant heterogeneity in the diesel-bladder cancer risk relationship, with a strong positive association among cases with high-grade, nonmuscle invasive TP53-mutated tumors compared with controls [ORTop Tertile vs.Unexposed, 4.8; 95% confidence interval (CI), 2.2–10.5; Ptrend Conclusions: The relationship between diesel exhaust and bladder cancer was heterogeneous based on the presence of TP53 mutations in tumors, further supporting the link between PAH exposure and TP53 mutations in carcinogenesis. Future studies that can identify nitro-PAH signatures in exposed tumors are warranted to add human data supporting the link between diesel and bladder cancer. Impact: This study provides additional insight into the etiology and possible mechanisms related to diesel exhaust-induced bladder cancer.

Supplemental tables

Supplementary figures

HPV16 is the most oncogenic type of human papillomaviruses (HPV). Integration of HPV into the human genome is an important mechanism of carcinogenesis but is absent in at least 30% of HPV16+ tumors. We applied long-read whole-genome sequencing (WGS) to cervical cancer cell lines and tumors to characterize HPV16 carcinogenesis in the absence of integration. Large tandem arrays of full-length and unique truncated viral genomes integrated into multiple chromosomes were identified in two HPV16+ cell lines. The dispersion of characteristic viral variants to multiple integration sites indicates that viral deletions formed as extrachromosomal DNA (a phenomenon we term HPV superspreading). In addition, we identified an HPV16+ cell line with unintegrated (episomal) DNA that has tandem arrays of full-length, truncated, and rearranged HPV16 genomes (multimer episomes). Cytogenetic analysis of this cell line shows intense extrachromosomal HPV staining, including structures resembling double-minute chromosomes. WGS of HPV16+ cervical tumor samples from Latin America revealed that 11 of 20 tumors with only episomal HPV (EP) had intact monomer episomes. The remaining nine EP tumors had multimer and rearranged HPV genomes. The majority (80%) of HPV rearrangements and deletions disrupted the E1 and E2 genes, and EP tumors overexpressed the E6 and E7 viral oncogenes, a similar profile to tumors with HPV integration. Tumors with putative multimer HPV integrations display HPV multimers and concatemers of human and viral sequences. Our data uncovered a novel mechanism for HPV16 to cause cancer without integration through aberrant episomal replication, forming rearranged, mutated, and multimer episomes.Significance:Multimers of the HPV genome are generated in cervical tumors replicating as extrachromosomal episomes, which is associated with deletion and rearrangement of the HPV genome and provides a mechanism for oncogenesis without integration.

Cosine similarities for all signatures

Case-case associations between REC and TP53 mutation, NEBCS

specific TP53 mutations and ever diesel

Background:Diesel exhaust is a complex mixture, including polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs (nitro-PAH), many of which are potent mutagens and possible bladder carcinogens. To explore the association between diesel exposure and bladder carcinogenesis, we examined the relationship between exposure and somatic mutations and mutational signatures in bladder tumors.Methods:Targeted sequencing was conducted in bladder tumors from the New England Bladder Cancer Study. Using data on 797 cases and 1,418 controls, two-stage polytomous logistic regression was used to evaluate etiologic heterogeneity between bladder cancer subtypes and quantitative, lifetime estimates of respirable elemental carbon (REC), a surrogate for diesel exposure. Poisson regression was used to evaluate associations between REC and mutational signatures.Results:We observed significant heterogeneity in the diesel-bladder cancer risk relationship, with a strong positive association among cases with high-grade, nonmuscle invasive TP53-mutated tumors compared with controls [ORTop Tertile vs.Unexposed, 4.8; 95% confidence interval (CI), 2.2–10.5; Ptrend Pheterogeneity = 0.002]. In muscle-invasive tumors, we observed a positive association between diesel exposure and the nitro-PAH signatures of 1,6-dintropyrene (RR, 1.93; 95% CI, 1.28–2.92) and 3-nitrobenzoic acid (RR, 1.97; 95% CI, 1.33–2.92).Conclusions:The relationship between diesel exhaust and bladder cancer was heterogeneous based on the presence of TP53 mutations in tumors, further supporting the link between PAH exposure and TP53 mutations in carcinogenesis. Future studies that can identify nitro-PAH signatures in exposed tumors are warranted to add human data supporting the link between diesel and bladder cancer.Impact:This study provides additional insight into the etiology and possible mechanisms related to diesel exhaust-induced bladder cancer.

Ever diesel and nitro-PAH signatures adjusted for smoking

study characteristics

Supplementary Table Legend from The 6q22.33 Locus and Breast Cancer Susceptibility

Supplementary tables 1-7

The ubiquitin C (UBC) network in bladder cancer.

Supplementary Tables 1-6. Supplemental Table 1. Primers used in Ion Torrent sequencing of HPV. Supplemental Table 2. Predicted somatic mutations in PIK3CA discovered by targeted sequencing of cervical tumors from Mexico, Venezuela, and Guatemala. Supplementary Table 3. Predicted somatic mutations in 5 candidate genes discovered by targeted sequencing of cervical tumors. Supplementary Table 4. Association of HPV type and PIK3CA mutation. Supplementary Table 5. PIK3CA mutations in histologic subtype in this and other published studies Supplementary Table 6. Fig.3A_B_C raw data

Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence.Significance:This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.

Recently, we identified a novel breast cancer susceptibility locus at 6q22.33 following a genome-wide association study in the Ashkenazi Jewish genetic isolate. To replicate these findings, we did a case-control association analysis on 6q22.33 (rs2180341) in an additional 487 Ashkenazi Jewish breast cancer cases and in an independent non-Jewish, predominantly European American, population of 1,466 breast cancer cases and 1,467 controls. We confirmed the 6q22.33 association with breast cancer risk in the replication cohorts [per-allele odds ratio (OR), 1.18; 95% confidence interval (95% CI), 1.04-1.33; P = 0.0083], with the strongest effect in the aggregate meta-analysis of 3,039 breast cancer cases and 2,616 Ashkenazi Jewish and non-Jewish controls (per-allele OR, 1.24; 95% CI, 1.13-1.36; P = 3.85 × 10-7). We also showed that the association was slightly stronger with estrogen receptor–positive tumors (per-allele OR, 1.35; 95% CI, 1.20-1.51; P = 2.2 × 10-5) compared with estrogen receptor–negative tumors (per-allele OR, 1.19; 95% CI, 0.97-1.47; P = 0.1). Furthermore, this study provides a novel insight into the functional significance of 6q22.33 in breast cancer susceptibility. Due to the stronger association of 6q22.33 with estrogen receptor–positive breast cancer, we examined the effect of candidate genes on estrogen receptor response elements. Upon transfection of overexpressed RNF146 in the MCF-7 breast cancer cell line, we observed diminished expression of an estrogen receptor response element reporter construct. This study confirms the association of 6q22.33 with breast cancer, with slightly stronger effect in estrogen receptor–positive tumors. Further functional studies of candidate genes are in progress, and a large replication analysis is being completed as part of an international consortium. (Cancer Epidemiol Biomarkers Prev 2009;18(9):2468–75)

Purpose: Genetic analysis of bladder cancer has revealed a number of frequently altered genes, including frequent alterations of the telomerase (TERT) gene promoter, although few altered genes have been functionally evaluated. Our objective is to characterize alterations observed by exome sequencing and sequencing of the TERT promoter, and to examine the functional relevance of histone lysine (K)–specific demethylase 6A (KDM6A/UTX), a frequently mutated histone demethylase, in bladder cancer.Experimental Design: We analyzed bladder cancer samples from 54 U.S. patients by exome and targeted sequencing and confirmed somatic variants using normal tissue from the same patient. We examined the biologic function of KDM6A using in vivo and in vitro assays.Results: We observed frequent somatic alterations in BRCA1 associated protein-1 (BAP1) in 15% of tumors, including deleterious alterations to the deubiquitinase active site and the nuclear localization signal. BAP1 mutations contribute to a high frequency of tumors with breast cancer (BRCA) DNA repair pathway alterations and were significantly associated with papillary histologic features in tumors. BAP1 and KDM6A mutations significantly co-occurred in tumors. Somatic variants altering the TERT promoter were found in 69% of tumors but were not correlated with alterations in other bladder cancer genes. We examined the function of KDM6A, altered in 24% of tumors, and show depletion in human bladder cancer cells, enhanced in vitro proliferation, in vivo tumor growth, and cell migration.Conclusions: This study is the first to identify frequent BAP1 and BRCA pathway alterations in bladder cancer, show TERT promoter alterations are independent of other bladder cancer gene alterations, and show KDM6A loss is a driver of the bladder cancer phenotype. Clin Cancer Res; 20(18); 4935–48. ©2014 AACR.

Purpose:Exome- and whole-genome sequencing of muscle-invasive bladder cancer has revealed important insights into the molecular landscape; however, there are few studies of non–muscle-invasive bladder cancer with detailed risk factor information.Experimental Design:We examined the relationship between smoking and other bladder cancer risk factors and somatic mutations and mutational signatures in bladder tumors. Targeted sequencing of frequently mutated genes in bladder cancer was conducted in 322 formalin-fixed paraffin-embedded bladder tumors from a population-based case–control study. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI), evaluating mutations and risk factors. We used SignatureEstimation to extract four known single base substitution mutational signatures and Poisson regression to calculate risk ratios (RR) and 95% CIs, evaluating signatures and risk factors.Results:Non-silent KDM6A mutations were more common in females than males (OR = 1.83; 95% CI, 1.05–3.19). There was striking heterogeneity in the relationship between smoking status and established single base substitution signatures: current smoking status was associated with greater ERCC2-Signature mutations compared with former (P = 0.024) and never smoking (RR = 1.40; 95% CI, 1.09–1.80; P = 0.008), former smoking was associated with greater APOBEC-Signature13 mutations (P = 0.05), and never smoking was associated with greater APOBEC-Signature2 mutations (RR = 1.54; 95% CI, 1.17–2.01; P = 0.002). There was evidence that smoking duration (the component most strongly associated with bladder cancer risk) was associated with ERCC2-Signature mutations and APOBEC-Signature13 mutations among current (Ptrend = 0.005) and former smokers (P = 0.0004), respectively.Conclusions:These data quantify the contribution of bladder cancer risk factors to mutational burden and suggest different signature enrichments among never, former, and current smokers.

Bladder cancer network containing debiquitinating enzymes (DUBs), chromosome remodeling genes and genes associated with VEGF signaling.

Supplementary Figures and legends

Introduction: Recent pan-cancer whole-genome sequencing studies revealed the rich landscape of structural variants (SV), from simpler indels to complex events involving multiple breakpoints and sequence gain/loss. SVs may contribute to tumorigenesis through direct modification of coding sequence or deregulation from copy number alterations, enhancer hijacking, or topological domain modification. A substantial part of the variation in the human genome is not accessible to short reads due to mapping ambiguities. Recent benchmarking studies reported that the best short-read methods only have 30-70% SV sensitivity. Long-read sequencing (such as PacBio or Oxford Nanopore) can overcome the limitations of short reads, however the current methods were not designed for the analysis of rearranged cancer genomes with complex copy number profiles. Methods: We developed BGA (Breakpoint Graph Assembler), a method that combines the ideas from long-read assembly and breakpoint graph frameworks. BGA detects abnormally mapped reads and builds a breakpoint graph that characterizes the structure of derived cancer karyotypes. Complex events with multiple breakpoints form connectivity clusters and are classified based on the subgraph properties. BGA also takes advantage of phased haplotypes and can incorporate multiple related datasets (such as in tumor-normal comparison or multi-site tumor sampling). BGA is freely available at: https://github.com/KolmogorovLab/BGA. Results: We first analyzed three cancer cell lines and corresponding matching normal DNAs (HCC1954, H2009, and COLO829). In each cell line, we identified 8-56 somatic rearrangement clusters involving more than two breakpoints and at least 1kb of sequence. In H2009, we identified a chromoplexy event involving chr13 and chr1, consistent with previous FISH experiments. COLO829 showed the lowest number of somatic rearrangement clusters (n=8), including translocation and inversion events between chr3, chr10, and chr12 within the RARB, BICC1, and TRHDE genes. Homologous recombination deficient HCC1954 has the highest number of complex events including the chr17q arm which hosts ERBB2, chromoplexy between chr8, chr5, and chromothripsis in chr21. In addition, we analyzed three other HPV-infected cell lines (CaSki, SCC152, SNU1000). In each of them, we observed complex clusters of HPV-HPV and HPV-human breakpoints that formed cycles, suggesting extrachromosomal amplification. The HPV fragments had many interactions with chromosomal DNA in CaSki and SC152, but not in mostly episomal SNU1000 cells. We also observed karyotype-scale changes that did not involve HPV sequences, such as the simultaneous exchange of six chromosome arms of chr2, chr7, and chr17 in CaSki. Citation Format: Ayse Keskus, Tanveer Ahmad, Ataberk Donmez, Yi Xie, Isabel Rodriguez, Rose Milano, Nicole Rossi, Hong Lou, Laksh Malik, Kimberley Billingsley, Cornelis Blauwendraat, Michael Dean, Mikhail Kolmogorov. Long-read, assembly-based characterization of rearranged cancer karyotypes. [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 4289.

Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence. Significance: This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.

Background: Second to cigarette smoking, occupational exposures are a leading risk factor for the development of bladder cancer. Occupational exposure to diesel exhaust in particular has been linked to increased bladder cancer risk in several epidemiologic studies. To provide insight into possible mechanisms that influence this association, we examined the relationship between diesel exhaust and somatic mutations in bladder tumors. Methods: Targeted sequencing of frequently mutated genes in bladder cancer was conducted in formalin-fixed paraffin-embedded bladder tumors from a population-based case-control study in New England. Lifetime occupational histories combined with exposure-oriented questions were used to estimate cumulative exposure to respirable elemental carbon (REC), a primary index for diesel exhaust. REC exposure was modeled both continuously (per 100 μg/m3-yrs) and categorically (by tertiles). Two-stage polytomous logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) to evaluate etiologic heterogeneity by tumor subtypes using data on 797 cases and 1,418 controls. Results: We observed significant heterogeneity in the relationship between diesel exhaust and risk of bladder cancer by TP53 mutation status (p-heterogeneity=0.013). When comparing patients with TP53+ tumors to controls, there was a strong association with increasing cumulative REC exposure (ORTertile 1 vs. Unexposed=1.4, 95% CI=0.7, 2.8; ORTertile 2 vs. Unexposed=1.3, 95% CI=0.6, 2.7; ORTertile 3 vs. Unexposed=2.8, 95% CI=1.4, 5.7; p-trend=0.003), while there was no apparent association between cumulative REC exposure and bladder cancer among patients with wild-type TP53 tumors (p-trend=0.603). Subtype analyses that incorporate clinical features (stage: non-muscle invasive/muscle-invasive and grade: low-grade/high-grade) with TP53 mutation, also indicate etiologic heterogeneity in the relationship between diesel exhaust and bladder cancer (p-het=0.002), with the strongest association observed between increasing cumulative REC and risk of bladder cancer among cases with non-muscle invasive, high-grade tumors (including carcinoma in situ) with TP53 mutations (OR per 100 μg/m3-yrs =1.14, 95% CI=1.00, 1.29). Conclusions: Our results suggest that diesel engine exhaust may influence bladder cancer risk by impacting the cell cycle, especially among patients with high-risk non-muscle invasive tumors that are more likely to progress to invasive disease. More work will be needed to understand the impact of diesel-generated oxidative stress on tumor suppressor genes like TP53 and cell cycle regulation in the development of bladder cancer. Citation Format: Nicole Gonzalez, Nina Rao, Michael Dean, Ludmila Prokunina-Olsson, Dalsu Baris, Molly Schwenn, Alison Johnson, Nathaniel Rothman, Debra Silverman, Stella Koutros. Diesel engine exhaust is associated with TP53 mutations and high-risk non-muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 861.

Purpose: Cervical cancer is a significant cause of mortality in Guatemala due to inefficient screening and late diagnosis. We are developing new technologies to improve the analysis of HPV-positive cervical tumors in Guatemala. The goal is to enhance understanding of HPV infection, tumor progression, and therapeutics development. Methods: We established a cohort of 700 Guatemalan cervical cancer patients with blood, fresh-frozen tumors, and clinical data. To optimize techniques, we extracted DNA and RNA from 20 cervical cancer cell lines. Long-read whole genome and transcriptome sequencing, and long-range PCR, were performed on cervical cancer DNA. HPV and HLA gene sequences were aligned to reference genomes. Results: Complete HPV sequences were compiled, HPV subtypes determined, and integration sites identified in cell lines with single and multiple integration events. HLA types of the class I genes were determined from the complete gene sequence, mutations detected, and HLA homozygosity. RNA was used for long-read whole transcriptome analysis of HPV and cellular genes. These results establish the use of long-read sequencing technology to characterize both HLA and host genomes. Paired Guatemalan tumor and blood DNA was successfully used for full HLA gene sequencing and mutation analysis. In addition, the complete HPV genome sequence was resolved. Binding of HPV peptides was predicted using the HPV and HLA sequences. These peptides identify the specific HPV epitopes potentially presented on the cell surface. Conclusion: Long-read RNA and DNA sequencing technology allow the determination of the complete HPV sequence, integration, and HLA class I typing and mutation analysis. HLA typing provides essential information on an individual's immune response. These techniques and analyses are being applied to tumor samples from women in Guatemala to aid in the development of targeted treatments. These methods are portable and could be employed in low- and middle-income countries. Citation Format: Nicole Rossi, Michael Dean. Characterization of Human Papillomavirus (HPV) Sequence and Integration and Analysis of Immune Response Genes for Application in Guatemalan Cervical Cancer [abstract]. In: Proceedings of the 9th Annual Symposium on Global Cancer Research; Global Cancer Research and Control: Looking Back and Charting a Path Forward; 2021 Mar 10-11. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2021;30(7 Suppl):Abstract nr 48.

The immune system has several mechanisms to recognize tumor cells as foreign and eliminate them. Somatically altered genes can create peptides, or neoantigens, that class I HLA (HLA-I) molecules can present to cytotoxic T-cells. As a result, people with suppressed immune systems commonly have a higher rate of cancer. PanCancer data of 9423 tumor exomes from the Cancer Genome Atlas project identified 299 frequently mutated cancer genes, 4 of which (HLA-A, HLA-B, beta-2-microglobulin (B2M), and caspase-8 (CASP8) are associated with the neoantigen presentation system. By mutating genes essential to immune recognition and cytotoxicity, tumors can escape the immune system. Tumors with a high mutation burden (MB) and thus more neoantigens experience even greater pressure to mutate HLA-I genes to escape detection. Therefore, the immune system shapes tumor evolution. Tumors with a higher mutation burden respond more effectively to immune checkpoint inhibitors (ICI) such as anti-PD-L1 and anti-CTLA-4 drugs. However, there are many PD-L1 positive patients who fail to respond. These patients may have immune escape (IE) mutations inhibiting neoantigen presentation to T-cells, and a database of immune escape mutations would help inform cancer treatment and improve response to immunotherapy. From the TCGA Pan-Cancer dataset we identified samples with mutations in HLA-A, HLA-B, HLA-C and mutations or loss of B2M or CASP8. We also identified samples with microsatellite instability (MSI) and mutations in DNA Polymerase E (POLE), both of which are associated with high mutation burden. We predicted individual HLA genotypes using multiple programs and aligned tumor and normal reads to these alleles, and called mutations. We used the Integrative Genome Viewer, to validate the mutations and used multiple programs to identify potential functional effects of missense mutations. Finally, we used chi-squared tests to find associations between genes and phenotypes. There were substantially more mutations in HLA-A and HLA-B genes than in HLA-C. Loss-of-function (LoF) mutations accounted for over 50% of the mutations in HLA-A and 54% in HLA-B, but are significantly suppressed (25%) in HLA-C (X2=21; P=3 × 10-5). Since all HLA-C alleles are ligands for natural killer (NK) cell receptors (KIR) this indicates that NK cell recognition of tumor cells is important. Over 90% of HLA-I missense variants were predicted to be functional (likely to impact the stability, membrane localization, B2M binding, peptide binding of the protein, the signal peptide or T cell recognition). Cervical, head and neck, stomach, colon and lung cancer have the highest level of IE mutations (9-20%). Immune escape mutations were significantly higher in MSI (45%) and POLE mutated (72%) tumors as compared to MSI- and POLE wild type tumors (3.9%, X2=470 P< Citation Format: Megan Ren, Mathias Viard, Arman Bashirova, Meredith Yeager, Lisa Mirabello, Mary Carrington, Michael Dean. Immune escape mutations in HLA Class I, B2M and CASP8 genes shape tumor evolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2160.

Purpose: Genetic analysis of bladder cancer has revealed a number of frequently altered genes, including frequent alterations of the telomerase (TERT) gene promoter, although few altered genes have been functionally evaluated. Our objective is to characterize alterations observed by exome sequencing and sequencing of the TERT promoter, and to examine the functional relevance of histone lysine (K)–specific demethylase 6A (KDM6A/UTX), a frequently mutated histone demethylase, in bladder cancer. Experimental Design: We analyzed bladder cancer samples from 54 U.S. patients by exome and targeted sequencing and confirmed somatic variants using normal tissue from the same patient. We examined the biologic function of KDM6A using in vivo and in vitro assays. Results: We observed frequent somatic alterations in BRCA1 associated protein-1 (BAP1) in 15% of tumors, including deleterious alterations to the deubiquitinase active site and the nuclear localization signal. BAP1 mutations contribute to a high frequency of tumors with breast cancer (BRCA) DNA repair pathway alterations and were significantly associated with papillary histologic features in tumors. BAP1 and KDM6A mutations significantly co-occurred in tumors. Somatic variants altering the TERT promoter were found in 69% of tumors but were not correlated with alterations in other bladder cancer genes. We examined the function of KDM6A, altered in 24% of tumors, and show depletion in human bladder cancer cells, enhanced in vitro proliferation, in vivo tumor growth, and cell migration. Conclusions: This study is the first to identify frequent BAP1 and BRCA pathway alterations in bladder cancer, show TERT promoter alterations are independent of other bladder cancer gene alterations, and show KDM6A loss is a driver of the bladder cancer phenotype. Clin Cancer Res; 20(18); 4935–48. ©2014 AACR.

The tumor microenvironment (TME) is composed of tumor cells, as well as immune and stromal cells. Cancer cells can interact with the tumor microenvironment to suppress anticancer immunity and host inflammatory cells can modulate that immune response to the lesion. To better understand the TME across cancer types we used single-cell RNA-seq analysis of 224,330 cells from 77 patients with breast, gastric, nasopharyngeal carcinoma (NPC), diffuse large B-cell lymphoma (DLBCL), pancreatic, colorectal and lung cancer. The tumor cells and immune cells from individual patients were analyzed simultaneously at the single-cell level to identify distinct cell clusters and subtypes. Comparison of the profile of T cells, macrophages, dendritic cells (DC), B cells, mast cells, granulocytes and cancer-associated fibroblasts (CAFs) identifies unique profiles by tumor type. Breast tumors have high numbers of macrophages, colorectal tumors are elevated for CD4+-T cells, DLBCLs are low in CAFs, gastric cancers have high levels of mast cells, lung tumors high levels of CD4+-T cells and natural killer (NK) T cells, NPC have elevated levels of B cells and DCs and pancreatic cancers high levels of CAFs and granulocytes and low levels of DC and CD8+-T cells. However, within a tumor type, there is also considerable heterogeneity. Understanding how the TME evolves and changes with therapy or predicts response to treatment and survival may provide insight into tumorigenesis and cancer progression. Note: This abstract was not presented at the meeting. Citation Format: Michael Dean, Guibo Li, Jianhua Jin, Yong Hou, Kui Wu, Shida Zhu, Hanlin Zhou, Ruqian Lv, Feng Lin, Si Liu, Shichen Dong, Lei Wang, Cuijuan Zhang, Yi Zhao. Analysis of the tumor microenvironment in seven cancer types by single-cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 148.

The tumor microenvironment is composed of numerous cell types, including tumor, immune and stromal cells. Cancer cells interact with the tumor microenvironment to suppress anticancer immunity. In this study, we molecularly dissected the tumor microenvironment of breast cancer by single-cell RNA-seq. The breast tumor microenvironment was profiled by analyzing the single-cell transcriptomes of 52,163 cells from the tumor tissues of 15 breast cancer patients. The tumor cells and immune cells from individual patients were analyzed simultaneously at the single-cell level. Among more than 40,000 tumor cells, we identified distinct cell clusters with different molecular subtypes and gene signatures in the same tumor, which revealed intra-tumor heterogeneity in breast cancer. We comprehensively profiled the functional state of T cells, macrophages, B cells, CAFs in breast cancer tumor microenvironment, which highlight the immune cell and CAF diversity in the breast cancer TME. Moreover, we analyzed intercellular communication in the breast cancer TME using a dataset of human ligand receptor pairs, which showed distinct cell interaction models across tumors. The evolution of the tumor microenvironment during clinical treatment was dissected by comparing tumor microenvironment of breast cancer pre- and post-chemotherapy. This study investigated the diversity of the cell types in the tumor microenvironment and provided information on the mechanisms of escape from clearance by immune cells in breast cancer, which could be a beneficial resource for immunotherapy development. Citation Format: Jianhua Yin, Hanlin Zhou, Chen Yan, Ruqian lv, Ting Wang, Chenyang He, Chen Huang, Yong Hou, Shida Zhu, Ling Wang, Michael Dean, Kui Wu, Hong Hu, Guibo Li. Comprehensive analysis of immune evasion in breast cancer by single-cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 140.

Characterizing the evolution of clonal cell populations in tumor progression is a challenging task given pervasive intratumor heterogeneity (ITH). Bulk DNA sequencing remains the dominant technology for large cohorts, but requires computationally deconvolving clonal substructure from bulk data, an error-prone process with limited resolution. Single-cell sequencing (SCS) is a promising alternative but is not yet practical at the scales needed for large cohorts. To address limitations of both bulk and SCS approaches, we developed strategies for deconvolving ITH by combining bulk sequencing with limited SCS data with specific focus on copy number alterations (CNAs), which are amenable to low-depth SCS and particularly challenging for deconvolution. We introduce two methods based on non-negative matrix factorization (NMF) of bulk data. One method extends the NMF optimization objective with a penalty for deviation of inferred clones from small numbers of observed SCS samples. The other combines clonal deconvolution with tumor phylogeny inference, balancing deconvolution quality against a minimum evolution cost for incorporating inferred and observed single cells into a reconstruction of the history of clonal evolution. We validated the methods on a set of semi-synthetic data derived from true low-depth SCS data consisting of 393 single cells derived from three regions each of two human glioblastoma cases. These data were used to call mean copy numbers at 9934 genomic loci. We artificially mixed the data to generate synthetic bulk samples of known clonal composition and applied each method to correctly reconstruct unobserved single cells and their clonal structures from three, six, or nine semi-synthetic bulk samples plus six single cells each per trial. For three/six/nine bulk samples, we achieved mean RMSD of copy number inference of 1.507/1.483/1.406 for pure NMF without SCS, 0.657/0.641/0.588 for the phylogeny-free SCS method, and 0.479/0.497/0.462 for the phylogeny-based SCS method. RMSD of mixture fractions describing the clonal composition were 0.245/0.242/0.243 for pure NMF without SCS, 0.215/0.198/0.206 for the phylogeny-free SCS method, and 0.215/0.215/0.224 for the phylogeny-based SCS method. The results show that we can substantially improve on bulk CNA deconvolution using limited SCS data, providing a way to balance advantages of pure bulk and pure SCS. The work also supports the value of a principled evolutionary model in inferring accurate clonal structure. Citation Format: Haoyun Lei, Bochuan Lyu, E. Michael Gertz, Alejandro A. Schaeffer, Xulian Shi, Kui Wu, Guibo Li, Liqin Xu, Yong Hou, Michael Dean, Russell Schwartz. Deconvolution of copy number alterations combining bulk and single-cell genomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1650.

Exome and whole-genome sequencing of muscle-invasive (MI) bladder cancer has revealed important insights into the molecular landscape; however, there are few studies of non-muscle invasive (NMI) bladder cancer with detailed information on bladder cancer risk factors. We used deep, targeted amplicon sequencing of the coding regions of 44 genes frequently mutated in bladder cancer (average 630x coverage for tumor and 350x for germline DNA) to characterize somatic mutations and the mutational spectrum in 307 formalin fixed paraffin embedded bladder tumors (n=260 NMI and n=47 MI) from a population-based case-control study with detailed information on smoking and other risk factors. Variants identified by mutation calling pipelines were validated by manual review of aligned sequences. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) to evaluate somatic mutations and risk factors. We used SignatureEstimation to extract the four known single base substitution mutational signatures (COSMIC: Sig1, Sig2, Sig13, ERCC2-Signature) and Poisson regression to calculate risk ratios (RRs) and 95%CIs to evaluate signatures and risk factors. The most frequently mutated gene in NMI tumors was FGFR3 (52% mutated) and in MI tumors was TP53 (51% mutated). FGFR3 (89%), STAG2 (81%), and PIK3CA (78%) mutations were significantly higher in Ta tumors compared to T1 or MI tumors. Mutations in histone/chromatin regulating genes (KDM6A, KMT2D, KMT2C, CREBBP, ARID1A, and EP300) were present at a high frequency in low grade Ta tumors. Non-silent KDM6Amutations were significantly more common in females compared to males (OR=1.79,95%CI:1.03-3.11); in females these mutations largely occurred in Ta tumors (86%). There was striking heterogeneity in the relationship between smoking status (current, former, never) and the established signatures: only current smoking was associated with greater ERCC2-Signature variants compared to both former smoking (p-value=0.015) and never smoking (p-value=0.023); only former smoking was associated with APOBEC-Sig13 variants (RR=1.83,95%CI: 1.10-3.04,p-value=0.019); and only never smoking was associated with the APOBEC-Sig2 variants (RR=1.52,95%CI: 1.15-2.01,p-value=0.003). Further, there was significant evidence that smoking duration, the component of smoking most strongly associated with bladder cancer risk, was associated with ERCC2-Signature variants and APOBEC-Sig13 variants among current (p-trend=0.013) and former smokers (p=0.002), respectively. Exploration of insertion/deletions revealed that both status and duration of smoking was significantly associated with presence of any 1bp deletions (p-trend=0.007); these were enriched for deletions in KDM6A (p=0.0003). In conclusion, these data quantify the contribution of bladder cancer risk factors to mutational burden in a population-based series of tumors and reveal distinct signature contributions among never, former and current smokers. Citation Format: Stella Koutros, Nina Rao, Lee E. Moore, Michael L. Nickerson, Donghyuk Lee, Bin Zhu, Larissa Pardo, Dalsu Baris, Molly Schwenn, Alison Johnson, Kristine Jones, Montserrat Garcia-Closas, Ludmila Prokunina-Olsson, Debra T. Silverman, Nathaniel Rothman, Michael Dean. Integrative, targeted deep sequencing of bladder tumors reveals novel associations between cancer gene mutations and mutational signatures with major risk factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-051.

Hereditary breast and ovarian cancer syndrome (HBOC) is an autosomal dominant disease that represents approximately 5-10% of all patients with breast cancer. This syndrome is mainly associated to high-risk pathogenic alleles in BRCA1 and BRCA2 genes, but only for 25% of HBOC cases. This work aimed to find new pathogenic alleles in a panel of 143 cancer-predisposing genes in 300 Mexican cancer patients with suspicion of HBOC and 31 non-cancer patients with a severe family history of cancer, using massive parallel sequencing. We found 15% (45/300) patients with pathogenic variants in the group of cancer patients; 12% (35/300) harbored variants with unknown clinical significance (VUS) and 73% (220/300) were negative. The non-cancer group had a 32% (10/31) of patients with pathogenic variants, 3% (1/31) had VUS and 65% (20/31) were negative. Moreover, the most recurrent mutation was the Mexican founder deletion of exons 9-12 in BRCA1, found in 5 of 16 cancer patients with alterations in this gene. Private or rare VUS variants with potential impact at protein level were found in 22 genes, being CHEK2 the one with most VUS (6/39). Noteworthy, our results show for the first time in the Mexican population an equal contribution of pathogenic alleles in other susceptibility cancer genes (50%) as in BRCA1/2 (50%). This highlights the high locus heterogeneity of HBOC and the necessity of expanding genetic tests for this disease to broader gene panels. Further studies need to be conducted to define the clinical impact of the pathogenic alleles and VUS identified. Citation Format: Felipe Vaca-Paniagua, Rosalía Quezada-Urban, Clara E. Díaz-Velásquez, Rina Gitler, María P. Rojo-Castillo, Max Sirota-Toporek, Andrea Figueroa-Morales, Oscar Moreno-García, Lizbeth García Esquivel, Gabriela Torres-Mejía, Michael Dean, Ivan Delgado-Enciso, Héctor Ochoa-Díaz-López, Fernando Rodriguez-León, Virginia Jan, Victor H. Hugo Garzón-Barrientos, Pablo Ruiz-Flores, Perla K. Espino-Silva, Jorge Haro-Santa Cruz, Héctor Martínez-Gregorio, Ernesto Rojas-Jiménez, Rosa M. Álvarez-Gómez, Luis A. Herrera, Isabelle Romieu, Luis I. Terrazas, Yolanda I. Chirino, Cecilia Frecha, Javier Oliver, Sandra Perdomo. Comprehensive analysis of germline variants in Mexican patients with hereditary breast and ovarian cancer susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1240.

In order to identify novel inhibitors of the ATP-binding cassette transporter, ABCG2, a high-throughput assay measuring the accumulation of the ABCG2 substrate pheophorbide a in ABCG2-overexpressing NCI-H460 MX20 cells was used to screen libraries of compounds. Out of a library of 7,325 natural products and synthetic compounds from the National Cancer Institute/Developmental Therapeutics Program collection, 18 were found to inhibit ABCG2 at 10 μmol/L. After eliminating flavonoids and compounds of limited availability from the 18 original compounds, 10 of the 11 remaining compounds reversed mitoxantrone resistance in NCI-H460/MX20 cells and prevented ABCG2-mediated BODIPY-prazosin transport in ABCG2-transfected HEK293 cells, confirming an interaction with ABCG2. Based on the activity profiles and the availability of materials, five inhibitors were examined for their ability to compete with [125I]iodoarylazidoprazosin labeling of ABCG2, increase binding of the anti-ABCG2 antibody 5D3, and prevent P-glycoprotein or multidrug resistance protein 1–mediated transport. At a concentration of 20 μmol/L, all of the compounds reduced iodoarylazidoprazosin labeling by 50% to 80% compared with controls. All five compounds also increased 5D3 labeling of ABCG2, indicating that these compounds are inhibitors but not substrates of ABCG2. None of the compounds affected P-glycoprotein–mediated rhodamine 123 transport, whereas three affected multidrug resistance protein-1–mediated calcein transport at 25 μmol/L, suggesting that the compounds are relatively specific for ABCG2. These five novel inhibitors of ABCG2 activity may provide a basis for further investigation of ABCG2 function and its relevance in multidrug resistance. [Mol Cancer Ther 2007;6(12):3271–8]

Nearly one in eight US women will develop breast cancer in their lifetime. Most breast cancer is not associated with a hereditary syndrome, occurs in postmenopausal women, and is estrogen and progesterone receptor-positive. Estrogen exposure is an epidemiologic risk factor for breast cancer and estrogen is a potent mammary mitogen. We studied single nucleotide polymorphisms (SNPs) in estrogen receptors in 615 healthy subjects and 1011 individuals with histologically confirmed breast cancer, all from New York City. We analyzed 13 SNPs in the progesterone receptor gene (PGR), 17 SNPs in estrogen receptor 1 gene (ESR1), and 8 SNPs in the estrogen receptor 2 gene (ESR2). We observed three common haplotypes in ESR1 that were associated with a decreased risk for breast cancer [odds ratio (OR), ∼ O.4; 95% confidence interval (CI), 0.2–0.8; P < 0.01]. Another haplotype was associated with an increased risk of breast cancer (OR, 2.1; 95% CI, 1.2–3.8; P < 0.05). A unique risk haplotype was present in ∼7% of older Ashkenazi Jewish study subjects (OR, 1.7; 95% CI, 1.2–2.4; P < 0.003). We narrowed the ESR1 risk haplotypes to the promoter region and first exon. We define several other haplotypes in Ashkenazi Jews in both ESR1 and ESR2 that may elevate susceptibility to breast cancer. In contrast, we found no association between any PGR variant or haplotype and breast cancer. Genetic epidemiology study replication and functional assays of the haplotypes should permit a better understanding of the role of steroid receptor genetic variants and breast cancer risk.

Glutathione S-transferase (GST), a member of the phase II group of xenobiotic metabolizing enzymes, has been intensively studied at the levels of phenotype and genotype. The GST μ 1 (GSTM1) and GST θ 1 (GSTT1) genes have a null-allele variant in which the entire gene is absent. The null genotype for both enzymes has been associated with many different types of tumors. The aim of this study was to determine the possible differences in increased oxidative stress susceptibility to smoking within the GSTM1 and GSTT1 genotypes and the impact of high tea drinking on this. We designed a Phase II randomized, controlled, three-arm tea intervention trial to study the effect of high consumption (4 cups/day) of decaffeinated green or black tea, or water on oxidative DNA damage, as measured by urinary 8-hydroxydeoxyguanosine (8-OHdG), among heavy smokers over a 4-month period and to evaluate the roles of GSTM1 and GSTT1 genotypes as effect modifiers. A total of 133 heavy smokers (100 females and 33 males) completed the intervention. GSTM1 and GSTT1 genotype statuses were determined with a PCR-based approach. Multiple linear regression models were used to estimate the main effects and interaction effect of green and black tea consumption on creatinine-adjusted urinary 8-OHdG, with or without adjustment for potential confounders. Finally, we studied whether the effect of treatment varied by GSTM1 and GSTT1 status of the individual. Although there were no differences in urinary 8-OHdG between the groups at baseline, the between-group 8-OHdG levels at month 4 were statistically significant for GSTM1-positive smokers (P = 0.05) and GSTT1-positive smokers (P = 0.02). GSTM1-positive and GSTT1-positive smokers consuming green tea showed a decrease in urinary 8-OHdG levels after 4 months. Assessment of urinary 8-OHdG after adjustment for baseline measurements and other potential confounders revealed significant effect for green tea consumption (P = 0.001). The change from baseline was significant in both GSTM1-positive (t = −2.99; P = 0.006) and GSTT1-positive (P = 0.004) green tea groups, but not in the GSTM1-negative (P = 0.07) or GSTT1-negative (P = 0.909) green tea groups. Decaffeinated black tea consumption had no effect on urinary 8-OHdG levels among heavy smokers. Our data show that consumption of 4 cups of tea/day is a feasible and safe approach and is associated with a significant decrease in urinary 8-OHdG among green tea consumers after 4 months of consumption. This finding also suggests that green tea intervention may be effective in the subgroup of smokers who are GSTM1 and/or GSTT1 positive.

Germline mutations in the BRCA1 and BRCA2 genes result in predisposition to breast and ovarian cancer. Detection of BRCA mutation carriers can lead to improved prevention and therapeutic interventions such as the targeted therapy using poly ADP ribose polymerase (PARP) inhibitors, which are particularly effective in BRCA mutation carriers. The spectrum of BRCA mutations varies depending on geographic origin, population, and ethnic group; however, the prevalence of BRCA mutations in non-Caucasian populations has been poorly characterized, particularly in a population-based setting. The goal of this study was to characterize the spectrum of germline mutations in BRCA1/2 and PALB2 in unselected breast cancer cases who were seen in Sarawak General Hospital, Malaysia, where 93% of the breast cancer patients in Sarawak are treated. We performed targeted sequencing using a validated AmpliSeq panel on 467 cases with available risk factor questionnaire and clinical follow-up data. Breast cancer subtypes were defined by the joint expression of ER, PR, and HER2. Common variants with frequency >1% in any public database (1000 Genome, Exome Sequencing Project, The Exome Aggregation Consortium) were excluded. Pathogenic variants included known pathogenic variants in ClinVar, loss of function variants (frameshift and stop-gain), and variants that alter the first or second base of the splice site. Variants of unknown significance (VUS) were also defined using the ClinVar classification. We found 10 BRCA1 pathogenic variants in 12 patients, 10 BRCA2 pathogenic variants in 15 patients, and 4 PALB2 pathogenic variants in 4 patients, which gave a BRCA mutation prevalence of 5.78% among the unselected breast cancer cases in this population. All these variants were extremely rare in the general population ( Citation Format: Xiaohong (Rose) Yang, Beena Devi, Hyuna Sung, Jennifer Guida, Yanzi Xiao, Lisa Garland, Nan Hu, Maria Rodriguez-Herrera, Chaoyu Wang, Kristine Jones, Wen Luo, Belynda Hicks, Tieng Swee Tang, Karobi Moitra, Mike Dean. Prevalence and spectrum of germline rare variants in BRCA1/2 and PALB2 among breast cancer cases in Sarawak, Malaysia [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 1294. doi:10.1158/1538-7445.AM2017-1294

In urothelial bladder cancer accurate identification of grade and stage is critical for optimal treatment to achieve robust disease control and long-term survival. However, among the initially superficial tumors, which are non-invasive and highly treatable, 20 to 25 % recur, progress to invasive tumors, and metastasize during the patients’ lifetime. Thus, the challenge is to provide risk stratification during the initial diagnosis in order to identify those patients who are unlikely to progress while offering radical therapy to those who are at risk. To address this issue, the field has heavily focused on the discovery of few mutations in potential driver genes. However, recent findings indicate that deregulation of enhancers can play a major role in cancer progression. In this study, we employed DNase-Seq to detect enhancer activities genome-wide in 16 bladder cancer cell lines (BLCs) that included three lineages (T24, UMUC3, 253J lineages) with different tumorigenic and metastatic potentials, and thus represent models of cancer progression and metastasis development. We analyzed the gain and loss of enhancer activity in each BLC lineages as well as in metastatic cell lines relative to non-metastatic cell lines. Our analysis in the T24 lineage revealed a striking feature, a dramatic loss of intronic and distal DHSs (DNase I Hypersensitive sites), during the initial transition to tumorigenic type. During metastatic progression, new enhancer classes emerged and there was an enrichment for association with target organ-specific genes. The analysis of differential DHSs between metastatic and non-metastatic BLCs identified an enhancer signature enriched with lost DHSs in metastatic BLCs where nearby genes were associated with cellular movement/invasion functions. Lost DHSs were also present near key factors such as PPARG, RXRA, FOXA1, TP63, and GATA3, which play a role in urothelial development and differentiation, and the transcription activity of those genes were correlated with the change of enhancer activity. This study identified a set of DHSs that are associated with cancer progression in bladder cancer, and provides a potential clinical application for developing prognostic markers to predict the risk of developing aggressive disease. Citation Format: Sohyoung Kim, Lyuba Varticovski, Qizong Lao, Songjoon Baek, Michael L. Nickerson, Myong-Hee Sung, Lars Grontved, Thompson Bethtrice, Dan Theodorescu, Piyush K. Agarwal, Michael Dean, Gordon Hager. Genome-wide enhancer identify signature predictive of metastatic phenotypes in bladder cancers [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 3352. doi:10.1158/1538-7445.AM2017-3352

Background: Aflatoxin, which is a risk factor for developing hepatocellular carcinoma, may also cause gallbladder cancer (GBC). We hypothesized that GBC patients have higher aflatoxin exposure than gallstone patients. Methods: We used samples and data from the Shanghai Biliary Tract Cancer Case-control Study conducted from 1997-2001. Of the 267 GBC and 730 gallstone participants with plasma available, 250 GBC patients and 250 controls with gallstones were randomly selected for measurement of aflatoxin B1 (AFB1)-lysine plasma adducts using isotope dilution mass spectrometry. In 54 GBC patients, tumor tissue was examined for the aflatoxin-related R249S TP53 mutation through targeted sequencing. Results: We calculated age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals (CIs), as well as the population attributable fraction (PAF). AFB1-lysine adduct detection was nearly 3-fold higher in GBC patients (OR, 2.96; 95% CI, 1.90 to 4.61). Among the 85 GBC patients (34%) with detectable AFB1-lysine adduct levels, the median was 4.6 pg/mg, compared to 1.2 pg/mg among the 37 gallstone-patient controls (15%) with detectable levels, and the OR was 9.97(95% CI, 2.73 to 36.44) for quartile 4 versus 1 in GBC compared to gallstone patients. The PAF for cancer related to aflatoxin was 22% (95% CI: 17%-27%). In 54 GBC patients, tumor tissue was examined for the aflatoxin-related R249S TP53 mutation through targeted sequencing, however, all patients were negative for the mutation. Conclusions: Aflatoxin is significantly associated with GBC, although it does not appear to be correlated with the R249S TP53 mutation in the gallbladder. If aflatoxin is shown to cause GBC, it may have accounted for up to 22% of the gallbladder cancers in Shanghai during the study period and may account for an even higher proportion in high-risk areas. If verified, reducing aflatoxin exposure may reduce GBC incidence. Citation Format: Jill E. Koshiol, Yu-Tang Gao, Michael Dean, Patricia Egner, Chirag Nepal, Kristine Jones, Bingsheng Wang , Asif Rashid, Wen Lou, Alison Van Dyke, Catterina Ferreccio, Michael Malasky, Ming-Chang Shen, Bin zhu, Jesper Andersen, Allan Hildesheim, Ann W. Hsing, John Groopman. Association of aflatoxin and gallbladder cancer in Shanghai [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 LB-157. doi:10.1158/1538-7445.AM2017-LB-157

Immunotherapy has emerged as a novel and promising strategy for cancer treatment, while available markers for immunotherapy have poor effectiveness and safety. The advance of next-generation sequencing (NGS) technology enables us to identify tumor-specific neoantigen which could be more effective and safer for personalized immunotherapy. In this study, we demonstrated a novel workflow for the development of liver cancer personalized vaccine. Xenograft models for liver cancer were built in mice using Hepa1-6 cell lines. Tumor-specific candidate neoantigen were determined by performed whole exome sequencing (WES) and used a comprehensive bioinformatics pipeline integrating multiple factors such as MHCI-binding affinity fold change (FC), variant allele frequencies (VAFs) of somatic mutation, mRNA expression and hydrophobicity of peptides. The results showed that peptide vaccine of Gpr55 neoantigen not only triggered striking neo-antigen specific CD8+ T cell response, but also significantly inhibited the growth of tumors in both prophylactic and therapeutic models. Moreover, our results demonstrated that VAFs and FC were the effective and safety property for immunogenicity. The identified Gpr55 neoantigen was an ideal vaccine for Hepa1-6 induced xenograft models considering the immunogenicity, anti-tumor effect and safety evaluation. It was also possible to identify and develop highly effective personalized cancer vaccine by our workflow within 13 weeks. The results illustrated huge potential of tumor-specific neoantigen as a potent vaccine for clinical tumor therapy and the workflow built in this study could be used to develop more specific, safer, more effective treatments to all types of cancer patients. Note: This abstract was not presented at the meeting. Citation Format: Cheng-chi Chao, Fengjiao Yuan, Geng Liu, Guanglei Li, Bo Li, Lian Lu, Yaomei Tian, Dongli Li, Bailing Zhou, Ting An, Yingzi Fan, Chongming Jiang, Yang Shu, Honggang Lin, Heng Xu, Hanshuo Yang, Rui zhang, Xueyan Zhang, Zhen Cheng, Rong Huang, Michael Dean, Ke Men, Xiuqing Zhang, Chaoheng Yu, Jian Wang, Shuang Cheng, Huanming Yang, Yantai Wang, Yong Hou, Yuquan Wei, Li Yang. A novel neoepitope screening platform for generating effective and safe neoantigen-based vaccines and immunotherapeutics in a hepatocellular carcinoma model [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 LB-202. doi:10.1158/1538-7445.AM2017-LB-202

The Androgen Receptor (AR), a DNA-binding transcription factor plays a key role in the development and maintenance of prostate epithelia by modulating expression of growth-promoting genes. AR has been proposed to regulate gene expression by enhancing the efficiency of RNA polymerase II (RNAPII) dependent transcription elongation. Dysregulation of this AR-dependent transcriptional activity has been implicated not only in prostate cancer but also androgen-independent prostate cancer. Recently, we showed that protein expression of Lemur Tyrosine Kianse-2 (LMTK2), a serine-threonine kinase is downregulated in prostate cancer. Importantly, our study ascribed a novel role for LMTK2, as a negative regulator of AR-dependent transcriptional activity in prostate epithelial cells. However, the mechanism through which LMTK2 is able to regulate AR remains to be determined. Here, we show that LMTK2 is not only present in cytoplasmic fraction but also, within the nucleus of mammalian cells where it co-localizes with AR, newly transcribed mRNA and with RNA polymerase II (RNAPII). Interestingly, our data reveals that LMTK2 colocalizes with elongating RNAPII, phosphorylated on serine 2 of the carboxyl-terminal domain. This presents an interesting possibility that LMTK2 might be able to regulate transcription of AR-dependent genes by interacting with the elongating RNAPII and transcription & epigenetic factors. As such, our study potentially identifies LMTK2 as an important player regulating the AR-RNAPII mediated transcription machinery in mammalian cells. Citation Format: Kalpit Shah, Justin Foley, Michael L. Nickerson, Michael Dean, Neil Bradbury. Nuclear lemur tyrosine kinase-2 regulates RNA polymerase II dependent transcription in prostate 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 2379. doi:10.1158/1538-7445.AM2017-2379

Molecular markers for metastatic prostate cancer (PCa) can be identified by sequencing metastatic tumor genomes. We recently identified cancer gene mutations in a patient with metastatic disease using next generation sequencing (NGS) of DNA from 5 metastatic tumors and blood. We characterized a somatic non-conservative substitution in the methylcytosine dioxygenase ten-eleven translocation 2 (TET2) in all metastatic tumors but not the blood or primary adenocarcinoma. Analysis of metastatic tumors from additional patients revealed frequent somatic loss and sequence alteration of TET2, which was previously observed to be altered in 5-15% of myeloid, kidney and colon cancer. Genome-wide association studies previously identified non-coding risk variants associated with PCa and melanoma, and rare germline missense variants are observed in African-Americans with PCa. We sought to further investigate the role of TET2 in PCa. We performed fine-mapping of PCa risk across the TET2 locus using genotypes from the PEGASUS case-control cohort and identify six new risk variants in introns 1 and 2. Electrophoretic mobility shift assays show two risk SNPs are bound by the transcription factor octamer-binding protein 1 (Oct1). Full length TET2 (2002 aa) is expressed in normal prostate and cancer tissue and is significantly reduced in a subset of the Cancer Genome Atlas (TCGA) PCa tumors that are associated with metastatic disease and reduced disease-free survival. TET2-loss drives a cancer phenotype as siRNA-mediated knockdown (KD) significantly increases LNCaP and DU145 cell proliferation, and LNCaP transwell migration and wound healing. Affinity chromatography, mass spectrometry and co-immunoprecipitation confirm that endogenous TET2 binds the androgen receptor (AR) in LNCaP cell extracts. TET2 KD alters the expression of a subset of androgen-responsive genes including increased prostate-specific antigen (KLK3/PSA) expression, and published data indicate TET-catalyzed hydroxymethylcytosine (hmC) and TET2 binding sites proximal to KLK3. Analysis of TCGA PCa tumor RNA-seq reveals TET2 expression is co-regulated with the expression of genes encoding functions metabolizing 2-oxoglutarate and succinate including the lysine demethylase KDM6A, BRCA1-associated BAP1, and citric acid cycle enzymes IDH1-2, SDHA-B and FH. Co-expression is conserved across all 31 TCGA cancers examined. Examination of genomic locations associated with TET2-binding and hmC, and gene expression changes during androgen signaling indicate a putative role for TET2 as an energy sensor that modifies androgen-AR signaling. Decreased TET2 mRNA expression in TCGA PCa tumors that are associated with reduced patient survival indicates TET2 expression may be an informative biomarker of PCa disease progression. Citation Format: Michael L. Nickerson, Sudipto Das, Kate Im, Sevilay Turan, Sonja Berndt, Hongchuan Li, Hong Lou, Seth Brodie, Kalpit Shah, Jean-Noel Billaud, Tongwu Zhang, Joseph Boland, Stephen Anderson, Meredith Yeager, Michael Dean, Thorkell Andresson. TET2-loss modifies androgen signaling in prostate 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 355. doi:10.1158/1538-7445.AM2017-355

Next-generation sequencing (NGS) of urologic tumor genomes has identified frequently altered cancer genes. We recently examined the exomes of bladder tumors (BCa) from 54 U.S. patients and 99 Chinese patients to identify novel, altered cancer genes associated with clinical characteristics of disease. Exome capture of DNA from tumor and normal urothelial tissue using probes for ~180,000 protein-coding exons and microRNA loci; DNA fragment libraries were sequenced on a HiSeq 2000 platform (Illumina); and base calls on paired-end, 100 bp reads were generated using the Genome Analyzer Pipeline, v. 1.3 and standard parameters. Gene expression was analyzed using quantitative PCR, telomere length by a standard assay, and KDM6A function using cell-based assays in T24T BCa cells. Copy number variation (CNV), aneuploidy, and CpG methylation were analyzed as recently described. Telomerase (TERT) was the most frequently altered by somatic sequence changes in 37/54 (69%) of U.S. patient tumors. The TERT promoter was altered by frequent germline nucleotide changes in 30/54 (56%) tumors. Most of the TERT germline variants were novel (19/20) and three variants were confirmed in distinct tumors as either somatic or germline, including the most frequent variant, c. –245 T>C (rs2853669). The somatic TERT promoter alterations did not correlate with other somatic BCa gene alterations, but were associated with increased TERT expression and with significantly shorter telomeres in tumors as compared to matched normal tissue. The stromal antigen 2 (STAG2) gene was the most frequently altered novel BCa gene in Chinese patients, by somatic, deleterious sequence changes in 11% of tumors; CNV deletions (5%); and promoter CpG hypermethylation leading to presumed expression silencing (7/30, 23%). STAG2 encodes a component of the cohesion complex involved in sister chromatid cohesion and segregation. STAG2 mutations were associated with an increased number of chromosomal arm copy number changes corresponding to higher tumor cell aneuploidy, and mutations were associated with a significantly reduced patient survival by Kaplan-Meier survival analysis. In total, 32/99 (32%) of tumors displayed SCCS pathway gene alterations. The most frequently altered epigenetic gene was the histone lysine-specific demethylase 6A (KDM6A/UTX) in 24% of U.S. and 30% of Chinese patient tumors. Experimentally, we show KDM6A loss in human BCa T24T cells enhanced in vitro proliferation and cell migration, and in vivo tumor growth in mice. Re-expression reversed these effects, confirming KDM6A loss drives the BCa phenotype. Somatic KDM6A alterations were associated with somatic BRCA1-associated protein-1 (BAP1) alterations observed in 15% of U.S. tumors. Somatic, deleterious sequence changes occurred preferentially in U.S. Caucasian as compared to Chinese BCa patient tumors; are associated with papillary histologic features in a subset of tumors; and contribute to a significant number of tumors (10/14, 71%) that displayed alteration of genes encoding BRCA DNA repair pathway proteins. Genes encoding BRCA pathway proteins, including BAP1, BRCA1, BRCA2, PALB2, and ATM were altered by a combination of somatic and rare germline variants. We identified altered genes associated with clinical characteristics of BCa, including ‘immortalization' (TERT), aneuploidy (STAG2), proliferation (KDM6A), and papillary features in tumors (BAP1). STAG2 alterations are associated with a reduced patient survival, indicating a lethal subtype of disease; and altered X chromosome genes (STAG2 and KDM6A) are single copy in males and likely contribute to the cancer gender bias. Finally, we identified BRCA pathway alterations in BCa that are similar to those in breast, ovarian, prostate and kidney tumors, that have a common DNA repair deficiency that can be exploited by therapy targeting poly (ADP ribose) polymerase. Citation Format: Mike Nickerson, Kate M. Im, Guangwu Guo, Yaoting Gui, Sevilay Turan, James C. Costello, Quan Zhou, Zhiming Cai, Song Wu, M. Scott Lucia, Lee E. Moore, Michael Dean, Dan Theodorescu. Gene alterations associated with clinical characteristics of bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A52.

Bladder cancer (BCa) is the 5th most prevalent cancer in the US, and the metastatic disease has very poor prognosis with 150,000 deaths per year worldwide. The use of molecular markers to predict disease progression has been largely unsuccessful, with only few therapeutically approachable mutations. Over 2,300 alterations in the coding sequences have been described in high-grade bladder cancer, encompassing frequent alterations in genes involving chromatin modifiers. Thus, chromatin organization may play a critical role in this tumor type. The development of tumor progression models from transitional carcinoma samples that are poorly tumorigenic and non-metastatic to highly metastatic to the lung or liver by serial in vivo passaging allows analysis of the mechanisms associated with tumor progression. We examined whole exome sequencing to identify mutations, gene expression, and DNase I hypersensitivity followed by deep sequencing to identify sites of chromatin modification during tumor progression. Bioinformatic analysis of was performed using algorithms developed at LRBGE. Comparative analysis combining multiple data sets was performed using Ingenuity Pathways. Mutations in several previously identified genes associated with bladder cancer: TP53, RAS and TERT, and mutations/deletions in several chromatin modifiers KDM6A and MLL2/3 were found in parental cells. Only few additional mutations, none identifiable as cancer driver mutations marked the progression to metastatic phenotype. Changes in gene expression were already evident in progression to tumorigenic state, with high score in cells in cell-to-cell interaction pathway marked by a decrease in cell adhesion, increase in genes associated with EMT and genes associated with cancer. Changes in global chromatin landscape occurred at many known genes correlated with pathways detected by expression analysis, but many were in novel sites distant to promoters, suggestive of novel distant enhancer and confirm massive reprogramming of regulatory networks. Many of these sites were unique for each stage of tumor progression and have potential clinical application. Specific chromatin modifications were found in NFkB and inflammatory pathways. Thus, in the tumorigenic stage, these cells acquired not only the capacity for tumor formation, but also some of the characteristics associated with metastasis. Combination of exome sequencing with the gene expression and unbiased analysis of global chromatin landscape provided valuable new information on bladder cancer biology and tumor progression. Citation Format: Lyuba Varticovski, Sohyoung Kim, Michael L. Nickerson, Bethtrice Thompson, Qizong Lao, Lars Grøntved, Songjoon Baek, Myong-Hee Sung, Dan Theodorescu, Michael Dean, Gordon L. Hager. Novel molecular markers of bladder cancer progression identified by global chromatin profiling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4783. doi:10.1158/1538-7445.AM2015-4783

Next generation sequencing (NGS) has identified frequently altered cancer genes in urologic cancers including bladder cancer (BCa). We examined BCa tumors from 54 U.S. patients using exome NGS and targeted sequencing, and compared these to 99 extensively characterized Chinese patient tumors (Guo et al., 2013). We are first to show that telomerase (TERT) is the gene most frequently altered by somatic sequence changes in 37/54 (69%) of tumors and that TERT is also altered by rare and common germline variants in 30/54 (56%) tumors. Most TERT germline variants were novel (19/20) and three variants were confirmed as both somatic and germline in distinct tumors, including c. - 245 T>C (rs2853669), the most recurrent variant. A subset of variants created or altered transcription factor binding sites and promoter CpGs. Telomere length was significantly shorter in tumor versus adjacent normal tissue from the same patient (p = 0.004) indicating TERT minimally lengthens telomeres in rapidly proliferating BCa cells. Somatic TERT alterations were not associated with stage, grade, or alterations in other BCa genes identified by exome sequencing. STAG2 is altered by somatic mutations in 20/153 (13%) tumors and by X chromosome deletions and promoter hypermethylation. STAG2 mutant tumors had higher levels of aneuploidy (p = 0.01) and Kaplan-Meier analysis indicated a poor prognosis due to significantly shorter survival times (p < 0.001). Somatic BAP1 alterations were associated with papillary histologic features in 15% of Caucasian patient tumors but were infrequently observed in Asian patients (p = 0.003), which remained marginally significant after accounting for differences in tumor grade and stage (p = 0.037). BAP1 mutations co-occurred with histone 3 lysine 27-specific demethylase (KDM6A) mutations (p = 0.017), indicating the cancer genes might provide complementary survival advantages to cancer cells. We find that cancer gene alterations are highly predictive of canonical features of the BCa phenotype and ∼4-5 tumor suppressors are altered for each oncogene. X chromosome cancer genes such as STAG2 and KDM6A have a single copy in men and their lethality (as shown for STAG2) likely contributes to the gender bias in cancer. We speculate that TERT alterations are early events in BCa and identification of somatic alterations in urine may provide a biomarker for early BCa diagnosis. BAP1 mutations contribute to frequent BRCA pathway alterations in BCa and indicate treatment with poly(ADP ribose) polymerase inhibitors would be effective. Citation Format: Michael L. Nickerson, Kate M. Im, Sevilay Turan, Lee E. Moore, Michael Dean, Dan Theodorescu. Clinical associations between altered bladder cancer genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1105. doi:10.1158/1538-7445.AM2015-1105

Metastatic prostate cancer (mPCa) is a highly lethal disease and molecular markers identifying disease subtypes and/or therapeutic targets are needed. We sequenced the exomes of five metastatic tumors and healthy kidney tissue from an index patient with castration resistant mPCa to identify lesions associated with disease progression and metastasis. A somatic missense TET2 alteration predicted to be deleterious to the protein was observed at the metastatic stage of disease. We sequenced TET2 in additional mPCa tumors and PCa cell lines and detected somatic missense alterations, frequent LOH, and a frameshift truncation in PCa cell line, DU-145, derived from a mPCa brain tumor. We also observed complex combinations of frequent and rare germline missense SNPs, which may contribute to a previously described association of TET2 with PCa risk by GWAS. In this study, we confirm transcription factor binding to TET2 alleles associated with disease by GWAS. We examine TET2 binding partners in prostate cells by affinity purification, mass spectrometry, and forward and reverse immunoprecipitation. We confirmed a protein complex containing TET2, OGT, and two transcriptional co-activators/splicing factors implicated in receptor-mediated gene expression. Functional analysis shows TET2 loss alters expression of a unique set of genes, increases cell proliferation, and therefore is a driver of PCa. TET2 loss may facilitate progression to castration resistant mPCa and appears to define a subset of cases with metastatic disease. Citation Format: Michael L. Nickerson, Sudipto Das, Hong Lou, Hongchuan Li, Sevilay Turan, Kate Im, Stephen Anderson, Thorkell Andresson, Michael Dean. TET2 alterations facilitate progression of metastatic prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 454. doi:10.1158/1538-7445.AM2014-454

Bladder cancer is the 4th most common cancer in men in the US and metastatic disease has a poor prognosis. To date, only few mutations in cancer driving genes have been identified in this tumor. Altered gene expression involving an estimate of >2,300 coding sequences is reported in high-grade bladder cancer. In addition to previously reported mutations in KRAS, FGFR3 and TPp53, recent data showed frequent deletions and mutations in genes involving several chromatin modifiers, such as UTX (KDM6A). These data indicate that chromatin organization may play an important role in bladder cancer development and progression. The process of in vivo passaging of a poorly tumorigenic bladder cells (T24) with subsequent injection into mice and selection for tumorigenic and metastatic phenotype specifically to the lung and liver, provides a unique opportunity for analysis of mechanisms associated with tumor development and progression. We compared whole exome sequencing and mRNA expression with analysis of DNase I hypersensitivity combined with deep sequencing (DHS-seq) on multiple cell lines developed by in vivo selection. Bioinformatics of DHS-seq was performed using algorithms developed at LRBGE. Analysis of microarray expression was performed using Ingenuity Pathways (IPA). Mutations in several genes, including TP53, RAS, TERT and chromatin remodeler UTX (KDM6A), were detected in parent cell line, T24. Only few additional mutations marked the progression to the metastatic phenotype. Changes in gene expression were already evident in progression from parental cells that were weakly tumorigenic (T24) to cells that were capable to form tumors in mice, but rarely formed metastasis (T24T). The highest score was assigned to genes involved in Cell-to-Cell interaction. Prominent changes included genes involved in cell adhesion and genes associated with EMT, followed by changes in genes associated with cancer. Thus, during the progression from weakly tumorigenic to fully tumorigenic but weakly metastatic cell type, bladder cancer cells acquired gene expression and chromatin landscape changes associated with tumor formation and metastasis. Gene expression profile for most genes correlated with nearby chromatin remodeling of regulatory regions. In addition, DHS analysis identified many novel areas of changes, termed “hot spots” which were located in upstream or downstream enhancer regions. Some of these changes were unique for each stage of tumor progression. Genome-wide analysis of DHS showed large-scale changes in chromatin landscape during tumorigenesis indicating massive reprogramming of regulatory networks during cancer development and progression. Thus, combination of exome sequencing with microarray gene expression and analysis of global unbiased chromatin landscape provides valuable new information on bladder cancer biology and tumor progression. Citation Format: Sohyoung Kim, Lyuba Varticovski, Lars Grontved, Songjoon Baek, Bethrice Thompson, Michael Dean, Daniel Theodoresco, Michael L. Nickerson, Gordon L. Hager. Changes in global chromatin landscape identify bladder cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 474. doi:10.1158/1538-7445.AM2014-474

The retinoblastoma tumor suppressor gene (RB1) is a key regulator of cell cycle control, most notably through E2F transcription factor binding. Deregulation of RB1 is important in many cancers. Survivors of hereditary retinoblastoma, caused by a germline mutation in RB1, experience substantially elevated risks for subsequent neoplasms, particularly bone and soft tissue sarcomas and melanomas. Oncogenic RB1 mutations arise from a range of DNA alterations and occur throughout the gene. No previous study has evaluated how the specific location and functional consequences of these RB1 mutations may differentially confer risk for specific subsequent neoplasms. As part of a biomarker study nested within a long-term follow-up study of the risk of subsequent cancers in retinoblastoma survivors, we identified germline RB1 mutations in 76/80 (95%) survivors of hereditary retinoblastoma who developed at least one subsequent neoplasm and had sufficient DNA for mutation testing (median follow-up time=47 years, range 7-71 years). Survivors were classified into four groups according to whether they ever developed a soft tissue sarcoma (STS, N=47), melanoma (without STS, N=10), bone tumor (without STS, N=5), or another type of subsequent malignant neoplasm (N=14). The identified mutations were distributed throughout RB1, from the promoter through exon 25. Nonsense, frameshift, or splice mutations that resulted in premature termination of the RB1 residue sequence were identified in 51 (67%) survivors, with the remaining classified as having loss of multiple exons (N=6), loss or rearrangement of a single exon (N=5), a splice mutation (N=7), a missense mutation (N=4), or an intronic substitution with unknown consequence (N=3). Preliminary analyses indicated that RB1 mutations resulting in loss of >2 exons were more common in survivors with a subsequent STS (40/47, 85%) than those with a subsequent melanoma (6/10, 60%) or bone tumor (2/5, 40%; Fisher's exact P Citation Format: Lindsay M. Morton, Jeannette R. Wong, Brenda L. Gallie, David H. Abramson, Johanna M. Seddon, Michael Dean, Bert Gold, Alisa M. Goldstein, Ruth A. Kleinerman, Margaret A. Tucker. Specific RB1 mutations and risk of subsequent neoplasms among survivors of hereditary retinoblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1333. doi:10.1158/1538-7445.AM2013-1333

Background: Prostate cancer (PCa) is a common malignancy and a leading cause of cancer death among African American men. The hereditary prostate cancer 1 gene (HPC1-1q24-25) was initially implicated as a PCa susceptibility locus in 1996, but was not replicated in the African American Hereditary Prostate Cancer (AAHPC) study, which recruited African American families with four or more affected family members (n=95 families). The objective of our study is to identify and confirm PCa genetic susceptibility loci in African Americans. Methods: Short tandem repeat polymorphism (STR) scans implicated several loci, but no specific family had a logarithm of odds score higher than 1.8. We sought to refine implicated loci through single nucleotide polymorphism microarray. Therefore, we devised a prioritization scoring system where families with multiple affected and unaffected brothers within a likely onset age bracket scored the highest. After Illumina 660K microarray run, Bayesian prior likelihoods will be coupled with family based statistics to analyze the data in 6 families (n=32). Results: Scoring criteria was effective at identifying families (n=31, 14 cases, 17 controls) most informative at identifying genes and SNPs associated PCa. A total of 51 SNPs in 13 genes were found to be associated with PCa Risk at the p Conclusions: This study is a first step in identifying PCa genes that warrant further follow-up in the rest of the AAHPC families. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A69.

Exome libraries were prepared from five metastatic tumors and healthy tissue from an Ashkenazi Jewish man who died from metastatic prostate cancer. A total of 6.77 × 109 bases were sequenced using a 454 FLX. Germline alteration and somatic loss of heterozygosity of the BRCA1 tumor suppressor gene were observed. Novel, somatic variants in the catalytic domain of two kinases and alteration of two oncogenes were also potential drivers of this cancer. We identified an insertion in the TMPRSS2 gene in addition to fusion with ERG. Alteration of several signaling pathways was predicted as a result of a surprising combination of germline and somatic alterations. Clonal as well as subclonal variants were identified that allow a ‘natural history’ of this individual's cancer to be described. Several alterations, including BRCA1 loss, suggest therapeutic options that may have been effective in this patient. This talk is also presented as Poster B17.

Insight into the cancer genome has recently increased in an unprecedented way due to high throughput (HT) sequencing technology to examine the genome, exome, and transcriptome. We sought new genetic biomarkers for cancer by using HT sequencing to examine highly characterized primary sporadic tumors and tumor-derived cell lines. Several tumors/cell lines represented the most common subtype of kidney cancer, clear cell renal cell carcinoma, as well as NCI-60 panel cell lines from breast (MCF7) and prostate (PC3). Incorporation of NCI-60 panel cell lines was advantageous due to extensive prior characterization of these reagents, including gene expression, comparative genome hybridization, SNPs, and drug cytotoxicity. The study was performed using three major platforms (454, Illumina, ABI) to compare sequencing quality, cost, quantity, and computational amenability. Different methodologies were examined including exome, transcriptome, and whole genome sequencing. Alignment of sequence to a consensus genome (hg 18) allowed identification of novel and previously documented genomic variants. Various filters were implemented to sort the large number of variants detected, including dbSNP, personal genome variants, and 1000 genomes data. An average of 65 million 35-50 base pair sequences were generated from transcriptomes of two primary clear cell RCC tumors. Over 2 million 375 base pair sequences were generated from each of four cell line exomes. Differences were observed in extent and depth of coverage of a given target region by various experimental approaches. SNP array genotyping showed 1% discordance with sequencing. Previously characterized mutations were confirmed in HT sequences. We identified multiple regions of homozygosity at an unprecedented resolution. For example, in the clear cell renal cancer cell line, 786-0, a known mutation in VHL (311delG) was identified, and LOH at SNP positions across the entire length of chromosome 3 was confirmed. Alignment to a reference genome (hg 18) permitted identification of 2583-5307 genomic sequence variants, including 92-297 novel non-synonymous variants in the exomes of each cell line. Variants include frameshift and nonsense mutations. Novel non-synonymous variants were identified in cancer-related genes and these are being examined in additional tumors. Potential functions of newly identified mutated genetic loci suggest perturbation of signaling pathways and are candidates for targeted therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2198.