Shiv Srivastava

In prostate cancer, emerging data highlight the role of DNA damage repair genes (DDRGs) in aggressive forms of the disease. However, DDRG mutations in African American men are not yet fully defined. Here, we profile germline mutations in all known DDRGs (N = 276) using whole genome sequences from blood DNA of a matched cohort of patients with primary prostate cancer comprising of 300 African American and 300 European Ancestry prostate cancer patients, to determine whether the mutation status can enhance patient stratification for specific targeted therapies. Here, we show that only 13 of the 46 DDRGs identified with pathogenic/likely pathogenic mutations are present in both African American and European ancestry patients. Importantly,RADfamily genes (RAD51, RAD54L, RAD54B), which are potentially targetable, as well asPMS2andBRCA1, are among the most frequently mutated DDRGs in African American, but not in European Ancestry patients.

Multiparametric MRI provides prognostic information, and pairing information from MRI with molecular characterization from biopsy samples may assist prostate cancer risk stratification. Background Although prostate MRI is routinely used for the detection and staging of localized prostate cancer, imaging-based assessment and targeted molecular sampling for risk stratification are a...

Background: Despite a growing number of studies evaluating cancer of prostate (CaP) specific gene alterations, oncogenic activation of the ETS Related Gene (ERG) by gene fusions remains the most validated cancer gene alteration in CaP. Prevalent gene fusions have been described between the ERG gene and promoter upstream sequences of androgen-inducible genes, predominantly TMPRSS2 (transmembrane protease serine 2). Despite the extensive evaluations of ERG genomic rearrangements, fusion transcripts and the ERG oncoprotein, the prognostic value of ERG remains to be better understood. Using gene expression dataset from matched prostate tumor and normal epithelial cells from an 80 GeneChip experiment examining 40 tumors and their matching normal pairs in 40 patients with known ERG status, we conducted a cancer signaling-focused functional analysis of prostatic carcinoma representing moderate and aggressive cancers stratified by ERG expression.

We hypothesized that specific ERG splice forms in TMPRSS2-ERG fusion transcripts are selectively expressed in CaP cells and are functionally relevant in CaP. The specific aims of this study were to characterize full length sequences of TMPRSS2-ERG transcripts; to quantitatively evaluate selected TMPRSS2-ERG variants in CaP specimens and their prognostic features; and to defined the functional significance of specific splice variants of the rearranged ERG locus in CaP. We have identified two types of TMPRSS2-ERG,Type I, which encodes full-length ERG protein consisting SAM and ETS domains (ERG1, ERG2, ERG3), and Type II, encoding ERG proteins lacking the ETS domain (ERG8 and a new variant, TEPC). Increased ratio of Type I over Type II variants showed a correlation with poorly differentiated pathology /high Gleason score and outcome. We found that ERG3 a product of Type I splice variant, transcriptionally activates gene expression through ETS-regulated enhancers and ERG8 encoded by the Type II splice variant abrogated the transcriptional activator function of ERG3. We have generated an anti-ERG antibodies to detect the protein products of both Type I and Type II splice variants and demonstrated the correlation between detecting ERG genomic rearrangement and ERG oncoprotein human prostate tumors. We showed that Type I/Type II ERG ratios may play a role in defining the levels of C-MYC oncogene in a TMPRSS2-ERG fusion harboring prostate cancer cell culture model and revealed that Type I/Type II ratio correlated with C-MYC levels, higher Gleason sum and poor overall prognosis of prostate cancer patients.

The long arm of chromosome 6 is frequently deleted in diverse human neoplasms. Our previous study showed a minimum deletion region between markers D6S1056 and D6S300 on chromosome 6q in primary prostate cancer (CaP). In this study, we further refined a 200-kb minimal region of deletion (6qTSG1) centered around D6S1013 marker. The 6qTSG1 transcripts contained complex multiple splicing variants with low or absent expression in CaP cells. None of the transcripts identified contained open reading frames that code for a protein in the NCBI database. The expression of 6qTSG transcripts revealed interesting hormonal regulation relevant to CaP biology. Expression of 6q TSG transcript was induced in LNCaP cells that were cultured in charcoal-stripped serum medium suggesting an upregulation of 6qTSG transcript by androgen ablation and cell growth inhibition/apoptosis. Induction of 6qTSG1 expression in response to androgen ablation was abrogated in androgen-independent derivatives of LNCaP cells. In summary, we have defined a candidate CaP suppressor locus on chromosome 6q16.1, and deletions of this locus are frequently associated with prostate tumorigenesis. In the light of emerging role of noncoding RNAs in cancer biology including CaP, future investigations of 6qTSG11 locus is warranted.

P16 (MTS 1) and p15 (MTS 2) are negative regulators of cell cycle progression at the G1 cell cycle checkpoint and function as tumor suppressor genes (TSG). Both p15 and p16 are located on chromosome 9p21 and alterations have been demonstrated in a variety of human malignancies and human cancer cell lines. In testicular germ cell tumors (TGCT) loss of heterozygosity of 9p21 to 23 has been observed in 41% to 72% of informative cases. The aim of our study was to examine TGCT and testicular cancer cell lines for deletions and mutations of the p15 and p16 genes. 24 testicular germ cell tumors, 4 testicular cancer cell lines (TERA I, TERA II, HTE, HTH), 8 benign testicular tumors and 9 samples of normal testicular parenchyma were examined. Examinations for loss of heterozygosity (LOH) on 9p21 were performed for IFNA, D9S171, D9S126, D9S161, D9S1748 and PKY9 locus using Southern Blot analysis. Southern Blot analysis of the p16 gene to examine gross alterations was done using random primer labeled p16 cDNA. In addition all samples were examined for mutations of exons 1 to 3 of p16 and exons 1 to 2 of p15 by polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) analysis on MDE-gels. All observed band shifts on SSCP were further evaluated by direct DNA sequencing techniques. No major deletions were detected for p16 and p15.42% of all TGCT demonstrated LOH of the IFNA locus, 16% of the D9S161 locus and 5.5% of the D9S171 locus. No LOH was seen for the D9S126 locus. 5/24 TGCT (21%) and 1 testicular cancer cell line (TERA II) demonstrated band shifts for exon 2, 8/24 TGCT (33%) and 1 testicular cancer cell line (TERA I) showed band shifts for exon 3; 4 TGCT had an allelic loss for exon 3 on SSCP. DNA sequencing analysis for exon 2 demonstrated a single base substitution (G->A) in codon 140 resulting in an amino acid exchange (ala->thre) in 4 cases and a single base deletion with frame shift mutation in codon 142 in 2 cases. DNA sequencing analysis for exon 3 demonstrated a polymorphism in intron 2 in all cases. For p15 no band shifts were observed for exons 1 to 2 in TGCT or testicular cancer cell lines; none of benign testicular tumors or normal control tissues demonstrated any band shifts for p15 or p16. We demonstrated a high frequency of mutations for exon 2 in specimens of primary testicular germ cell tumors, but not in benign tumors or normal controls. We therefore believe that p16 might be involved in the pathogenesis of human TGCT. The polymorphisms described for exon 3 might be utilized for further LOH studies. The absence of mutations in p15 gene in TGCT specimens suggests that p15 might not play an important role in the pathogenesis of testicular germ cell tumors.