Frequent Genomic Loss Research Articles

Abstract 2557: Slit2 inhibits small cell lung cancer by targeting tumor microenvironment

Abstract Small cell lung cancer (SCLC) is the deadliest subtype of lung cancer with a 5-year survival rate of <5% in extensive-state disease and is designated by US Congress as a “recalcitrant cancer”. Recently approved by FDA, immune checkpoint blockade (ICB) immunotherapy showed marginally improved survival and only in a few patients, suggesting an inert immune tumor microenvironment (TME) and the existence of SCLC subtypes. Therefore, there is a significant need to develop more effective therapies for SCLC. In addition to the nearly universal biallelic inactivation of Rb and Tp53 genes in human SCLC tumors, comprehensive genome-wide screening studies have identified major co-occurring mutations in PTEN, CREBBP, EP300, SLIT2, MLL, COBL, and EPHA7 genes. Among them, SLIT2 showed the highest level of genomic abnormalities defined by a pronounced clustering of mutations and frequent genomic loss. However, the role of Slit2 in SCLC has not been defined yet. By using genetically engineered human SCLC cells overexpressing Slit2 or recombinant Slit2 (rSlit2) protein, we show that Slit2 inhibits SCLC cells proliferation, migration, and invasion properties and tumor sphere formation ability of drug-resistant SCLC cells in vitro. The mechanistic studies have identified inhibition of AKT and β-catenin mediated signaling pathways by Slit2 overexpression. Next, we implanted human SCLC cell lines into the immunocompromised NSG mice subcutaneously followed by treatment with rSlit2 or sterile saline. Analysis of tumor volume over the 4 weeks showed that rSlit2 treatment significantly reduced the growth of SCLC tumors. Further analysis of tumors showed that rSlit2 treated tumors harbor less M2-like CD206+ macrophages. Overall, these results suggest that Slit2 possesses tumor suppressive properties and could be developed as a novel therapeutic agent against SCLC. Citation Format: Dinesh K. Ahirwar, Manish Charan, Swati Misri, Konstantin Shilo, Ramesh Ganju. Slit2 inhibits small cell lung cancer by targeting tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2557.

Recurrent Copy Number Variants Are Highly Prevalent in Acute Myeloid Leukemia

Introduction: Large chromosomal copy number variants (CNVs) detected by conventional karyotyping are common in AML; however, small segmental CNVs or copy-neutral loss of heterozygosity (CN-LOH) are not detected by conventional techniques and require sequence-based approaches. Specifically, acquired CN-LOH occurs through duplication of genetic material and subsequent loss of the reciprocal chromosome resulting in a hemizygous state. CN-LOH has been demonstrated in AML, mostly occurring in regions of known recurrent mutations (ex: FLT3 , JAK2 , TET2 ), but its overall prevalence and involvement in AML pathogenesis has yet to be determined. Initial studies regarding the significance of CNVs suggest possible prognostic impact. We aimed to explore the prevalence of segmental CNVs and CN-LOH in a cohort of pediatric and adult AML specimens that underwent clinical comprehensive genomic profiling (CGP) (FoundationOne Heme Sequencing).Methods: CGP for targeted DNA and RNA sequencing was performed on 932 patients with AML (n=179 pediatric age ≤ 18 years, n=753 adult age ≥ 19 years). Specimen sites included bone marrow, peripheral blood, and biopsy of extramedullary disease. Following strict quality control (QC) measures to exclude any samples with transplant history, contamination, low purity, and lack of aneuploidy, all level CNV and LOH events were reported using a proprietary algorithm. A total of 323 samples met QC standards for LOH events, with all level copy number analyses available for 236 (73%) patients. We analyzed the loci of 91 genes to determine the type and prevalence of CNV events for amplifications (CNA; CN>2), deletions (CND; CN<2), CN-LOH, copy-loss LOH (CL-LOH) events at the genomic or chromosome level excluding sex chromosomes.Results: Among the 323 samples (n=59 pediatric, n=264 adult), the prevalence of LOH events was 91% (294). Identified LOH events included single gene LOH or chromosomal LOH, including arm or whole chromosome. Chromosomal events occurred in 172 (53%) of patients, and most frequently involved the following arms: 7q (n=59), 7p (n=57), 13q (n=25), 18q (n=23), 18p (n=22), 5q (n=19), and 17p (n=19). Among patients with chromosomal LOH events, 131 (76%) had events in the 7 chromosomal arms with the highest frequency (Fig 1A). Genomic LOH events were detected in 287 (89%) patients, and were detected in all 91 of the genes analyzed, with a frequency of 1-58. Further, CN-LOH events were detected in 162 (50%) patients occurring in 89 (98%) of genes analyzed (Fig 1B), with a frequency of 1-17. Among the 20 genes with highest frequency of LOH, 11 of them occurred at the 7q and 13q regions, which is consistent with previous findings that these are areas of frequent genomic losses. LOH, including CN-LOH, events overall commonly occurred in genes located in the 5q, 7p, 13q, 16q, and 17p regions. Recurrent LOH events in 3 of the most commonly affect genes occurred in approximately 31% of patients (EZH2 , n=103, BRAF , n=102, and KMT2C , n=99), all located on 7q. CN-LOH events were detected in oncogenes such as CDK6 , MET , FLT3 , JAK2 , ABL1 .Among the 236 samples evaluable for CNA assessment, 217 (92%) had at least one CNA or CND detected. CNAs were detected in 163 patients (69%) and occurred in all 91 of the genes analyzed (Fig 1C). Among samples with CNAs detected, the median number of somatically acquired CNAs was 5 (range 1-91). Recurring CNAs were detected across all chromosomes, but were most common in genes located on chromosome 1q, 8, 11q, 13q, 17q, 19, and 21q. CNDs were detected in 165 patients (70%) and occurred in 83 of genes (91%) analyzed. Among samples with CNDs detected, the median number of CNDs was 6 (range 1-58).Conclusion: CNVs and LOH events were commonly detected in this sample of 323 AML specimens. We show that LOH is highly prevalent, with genomic events detected in 89% of cases analyzed and CN-LOH events specifically detected in 50%. We found highly recurrent genes located at a few distinct chromosomal loci, suggesting some regions may be more prone to chromosomal instability. Future research in AML is needed to understand the mechanisms behind genomic and large-scale chromosomal disruptions, including CN-LOH. Identification of segmental CNVs and CN-LOH may identify cryptic variants that might aid in more appropriate diagnosis, management, disease monitoring, as well aid in the interpretation of mutation data as these events might alter the variant allele fraction of sequence variants. [Display omitted] DisclosuresHe:Foundation Medicine: Employment, Other: stock. Sun:Foundation Medicine: Employment, Other: Shareholder. Severson:Foundation Medicine: Employment, Other: Stock. Bailey:Foundation Medicine: Employment, Other: stock. Morley:Foundation Medicine: Employment, Other: Stock. Balasubramanian:Foundation Medicine: Employment, Other: stock. Erlich:Foundation Medicine: Employment, Other: stock. Lipson:Foundation Medicine: Employment, Other: stock. Otto:Foundation Medicine: Employment, Other: stock. Vergillo:Foundation Medicine: Employment, Other: stock. Ross:Foundation Medicine Inc: Employment, Other: stockholder. Mughal:Foundation Medicine Inc: Other: stockholder. Stephens:Foundation Medicine: Employment, Other: stock. Miller:Foundation Medicine Inc: Employment, Other: stockholder. He:Foundation Medicine Inc: Employment, Other: stockholder.

Exome sequencing identifies frequent genomic loss of TET1 in IDH-wild-type glioblastoma

Glioblastoma (GBM) is the most common and malignant brain tumor in adults. Genomic and epigenomic alterations of multiple cancer-driving genes are frequent in GBM. To identify molecular alterations associated with epigenetic aberrations, we performed whole exome sequencing-based analysis of DNA copy number variations in 55 adult patients with IDH-wild-type GBM. Beside mutations in common GBM driver genes such as TERTp (76%), TP53 (22%) and PTEN (20%), 67% of patients were affected by amplifications of genes associated with RTK/Rb/p53 cell signaling, including EGFR (45%), CDK4 (13%), and MDM2/4 (both 7%). The minimal deleted region at chromosome 10 was detected at the DNA demethylase TET1 (93%), mainly due to a loss-of-heterozygosity of complete chromosome 10 (53%) or by a mono-allelic microdeletion at 10q21.3 (7%). In addition, bi-allelic TET1 deletions, detected in 18 patients (33%), frequently co-occurred with EGFR amplification and were associated with low levels of TET1 mRNA expression, pointing at loss of TET1 activity. Bi-allelic TET1 loss was not associated with global concentrations of 5-hydroxymethylcytosine, indicating a site-specific effect of TET1 for DNA (de)methylation. Focal amplification of EGFR positively correlated with overall mutational burden, tumor size, and poor long-term survival. Bi-allelic TET1 loss was not an independent prognostic factor, but significantly associated with poor survival in patients with concomitant EGFR amplification. Rates of genomic TET1 deletion were significantly lower in a cohort of IDH1-mutated patients. Despite the relevance of TET1 for DNA demethylation and as potential therapeutic target, a frequent genomic loss of TET1 has not previously been reported in GBM.

Krüppel-Like Factor 6 Is Required for Oxidative and Oncogene-Induced Cellular Senescence.

Krüppel-like factor 6 (KLF6) is a transcription factor involved in the regulation of several cellular processes. Regarding its role in tumorigenesis, KLF6 is considered a tumor suppressor. Numerous reports demonstrate its frequent genomic loss or down-regulation, implying a functional inactivation in a broad range of human cancers. Previous work from our laboratory showed that the down-regulation of KLF6 expression in normal fibroblasts leads to cellular transformation, while its ectopic expression interferes with the oncogenic transformation triggered by activated Ras through a cell cycle arrest. We hypothesize that the growth suppressor activity of KLF6 may involve the induction of cellular senescence thereby helping to prevent the proliferation of cells at risk of neoplastic transformation. Here, we explored the association of KLF6 up-regulation in two different cellular senescence scenarios. We found that KLF6 silencing bypasses both oxidative and oncogene-induced senescence. In this context, KLF6 expression per se was capable to trigger cellular senescence in both normal and tumoral contexts. As such, the findings presented in this report provide insights into a potential mechanism by which KLF6 may play a suppressing role of uncontrolled or damaged cell proliferation.

Abstract LB-165: Multiple mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma

Abstract The let-7 microRNA family are known tumor suppressors often deregulated in cancer, yet the underlying mechanisms of let-7 disruption remain poorly understood. Neuroblastoma, a neural crest derived tumor, is defined in part by poor prognosis associated with genetic amplification of MYCN, itself a let-7 target. The let-7 biogenesis inhibitor LIN28B has recently been implicated as a critical regulator of MYCN, but through CRISPR-mediated gene disruption we show that LIN28B is dispensable for both MYCN protein expression and growth of MYCN-amplified neuroblastoma cell lines despite robust de-repression of let-7, prompting us to explore additional mechanisms for let-7 disruption. Consequently, we have found a novel non-coding role for amplified MYCN mRNA as a potent let-7 sponge that through exceptionally high expression defines a sub-class of self-sponging amplified-competing-endogenous-RNA (aceRNA) and reconciling the dispensability of LIN28B in neuroblastoma cell lines. Furthermore, by analyzing a large cohort of tumor samples from patients, we observe frequent genomic loss of let-7 that inversely associates with MYCN-amplification, providing a functional explanation for the known MYCN-amplification-independent pattern of chromosome 3p and 11q loss, which harbor let-7g and let-7a2, respectively. We thus propose a model whereby let-7 disruption by genetic loss, LIN28B expression, or aceRNA sponging is a unifying mechanism of neuroblastoma pathogenesis. Indeed, our data show that the majority of neuroblastomas have at least one let-7 disruption event and that genetic loss in non-MYCN-amplified tumors marks decreased survival, further underscoring its importance. The inverse selective relationship between allelic loss and sponging of let-7 from highly expressed or amplified oncogenes may have broad implications for oncogenesis. Citation Format: John T. Powers, Kaloyan Tsanov, Frederik Roels, Catherine Spina, Richard Ebright, Marc Seligson, Yvanka de Soysa, Patrick Cahan, Daniel Pearson, Jessica Theißen, Ho-Chou Tu, Grace LaPier, Jihan Osborne, Samantha Ross, James Collins, Frank Berthold, George Daley. Multiple mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-165.

Abstract LB-290: Multiple distinct mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma

Abstract The let-7 microRNA family are known tumor suppressors often deregulated in cancer, yet the underlying mechanisms of let-7 disruption remain poorly understood. Neuroblastoma, a neural crest derived tumor, is defined in part by poor prognosis associated with genetic amplification of MYCN, itself a let-7 target. The let-7 biogenesis inhibitor LIN28B has recently been implicated as a critical regulator of MYCN, but we have employed siRNA and CRISPR-mediated gene disruption to show that LIN28B is dispensable for both MYCN protein expression and growth of MYCN-amplified neuroblastoma cell lines despite robust de-repression of let-7, which prompted us to explore additional mechanisms for let-7 disruption. Consequently, we have found that amplified MYCN mRNA is a potent let-7 sponge that through exceptionally high expression defines a sub-class of self-sponging amplified-competing-endogenous-RNA (aceRNA), which reconciles the dispensability of LIN28B in NB cell lines. In addition, by analyzing a large cohort of tumor samples from patients, we observe frequent genomic loss of let-7 that inversely associates with MYCN-amplification, providing a functional explanation for the known MYCN-amplification-independent pattern of chromosome 3p and 11q loss, which harbor let-7g and let-7a2, respectively. We thus propose a model whereby let-7 disruption by genetic loss, LIN28B expression, or aceRNA sponging is a unifying mechanism of neuroblastoma pathogenesis. Indeed, our data show that the majority of neuroblastomas have at least one let-7 disruption event and that genetic loss in non-MYCN-amplified tumors marks decreased survival, further underscoring its importance. The inverse selective relationship between allelic loss and sponging of let-7 from highly expressed or amplified oncogenes may have broad implications for oncogenesis. Note: This abstract was not presented at the meeting. Citation Format: John T. Powers, Kaloyan M. Tsanov, Frederik Roles, Richard Ebright, Marc Seligson, Yvanka de Soysa, Patrick Cahan, Jessica Theissen, Grace S. LaPier, Dan S. Pearson, Frank Berthold, George Q. Daley. Multiple distinct mechanisms disrupt let-7 miRNA biogenesis and function in neuroblastoma. [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 LB-290. doi:10.1158/1538-7445.AM2015-LB-290

Genetic Polymorphism of PRKCDBP is Associated with an Increased Risk of Endometrial Cancer

PRKCDBP is a putative tumor suppressor located at 11p15.4, where frequent genomic loss has been observed in human cancers. We explored the possible association between an intra-exonic single nucleotide polymorphism (SNP), rs1051992, that results in a Leu to Pro substitution, and risk for endometrial carcinogenesis. We assessed the genotype of rs1051992 in endometrial cancer tissues from 147 patients and normal endometrial tissue from 191 healthy individuals by restriction endonuclease PvuII-based genotyping. Allele frequencies in the cancer specimens were compared with those in the healthy controls. We also evaluated the association between polymorphisms at this locus and histopathological features of endometrial cancer.

A Global View of the Oncogenic Landscape in Nasopharyngeal Carcinoma: An Integrated Analysis at the Genetic and Expression Levels

Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Therefore a genome wide analysis of chromosome copy number and gene expression was performed in tumour cells micro-dissected from the same NPC biopsies. Cellular tumour suppressor and tumour-promoting genes (TSG, TPG) and Epstein-Barr Virus (EBV)-encoded oncogenes were examined. The EBV-encoded genome maintenance protein EBNA1, along with the putative oncogenes LMP1, LMP2 and BARF1 were expressed in the majority of NPCs that were analysed. Significant downregulation of expression in an average of 76 cellular TSGs per tumour was found, whilst a per-tumour average of 88 significantly upregulated, TPGs occurred. The expression of around 60% of putative TPGs and TSGs was both up-and down-regulated in different types of cancer, suggesting that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of context-dependent onco-suppressors may be more extensive than previously recognised. No significant enrichment of TPGs within regions of frequent genomic gain was seen but TSGs were significantly enriched within regions of frequent genomic loss. It is suggested that loss of the FHIT gene may be a driver of NPC tumourigenesis. Notwithstanding the association of TSGs with regions of genomic loss, on a gene by gene basis and excepting homozygous deletions and high-level amplification, there is very little correlation between chromosomal copy number aberrations and expression levels of TSGs and TPGs in NPC.

505 MIR-218 ON THE GENOMIC LOSS REGION OF CHROMOSOME 4P15.31 FUNCTIONS AS A TUMOR SUPPRESSOR IN BLADDER CANCER

INTRODUCTION AND OBJECTIVES: MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs and display aberrant expression patterns and functional abnormalities in human diseases including cancers. While some miRNAs function as tumor suppressors and are down-regulated in cancer cells, other miRNAs act as oncogenes. However, genetic or epigenetic regulations of these miRNAs are not fully elucidated as of now. We hypothesize that chromosomal loss region harbors down-regulated miRNAs that have a tumor suppressive function in bladder cancer (BC). METHODS: On the basis of our previous comparative genomic hybridization (CGH) array data in BC cell lines, we focused on chromosome 4 because it was the frequent genomic loss region, and 23 miRNAs were coded there. The expression levels of 19 miRNAs, whose PCR primers were commercially available, were evaluated by a real-time PCR in four BC cell lines and 22 clinical samples. The miRNA s transfected BC cell lines (BOY and T24) were subjected to cell viability assays by cell growth, wound healing, and matrigel invasion assay; and apoptosis assay by flow cytometory. To find which genes were differentially regulated by the miRNAs, the gene expression profile of the miRNA s transfectants was performed. RESULTS: Among the 19 miRNAs on chromosome 4, the expression levels of miR-95, miR-218 and miR-578 were markedly repressed in the four BC cell lines. In clinical samples, miR-218 located on chromosome 4p15.31 was actually down-regulated in 16 clinical BC specimens in comparison with six normal bladder epitheliums (NBEs) (p 0.0004), whereas no significant difference was observed in miR-95 and miR-578 expressions among the groups. Significant cell viability inhibitions of cell growth, invasion and migration were observed in miR-218 transfectants (each, p 0.005). The flow cytometory demonstrated that the apoptotic fraction was significantly greater in the miR-218 transfectants than in the controls (each, p 0.005). Oligomicroarray analyses identified that a total of 337 down-regulated genes and 162 up-regulated genes were differently expressed in the transfectants. Interestingly, anti-apoptotic TMX1 gene and signaling pathway regulator RGS20 gene, which have miR-218 conserved binding sites in their mRNA, were listed in the top 20 down-regulated genes. CONCLUSIONS: Our data suggest that miR-218 may be a tumor suppressive miRNA and that its expression was generally down-regulated in BC due to the chromosomal loss region where the miRNA harbors.

Frequent genomic loss at chr16p13.2 is associated with poor prognosis in colorectal cancer

Genomic alterations play important roles in colorectal cancer (CRC) carcinogenesis. Here, we aimed to identify and characterize recurrent copy-number alterations (CNAs) associated with clinical outcome of CRC by the use of single nucleotide polymorphism arrays, genomic quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH). Colorectal neoplasia specimens and paired germline samples from 144 patients (40 adenomas and 104 carcinomas) as well as 40 CRC cell lines were investigated. This large dataset revealed frequent loss, including homozygous loss, at chr16p13.2 (from 5.9 to 7.42Mb). The loss was observed in 30% of adenomas and even more frequently in carcinomas, 56%, indicating that the loss define a subset of adenomas with a propensity for invasion. Consistent with this, the loss occurred twice as frequent in villous (40%) as in tubular adenomas (20%). The loss occurred independently of microsatellite stability and could be validated by qPCR in an independent sample cohort (n = 71). In Stage II/III, microsatellite stable (MSS) CRC it was associated with poor recurrence free survival (hazard ratio 2.4; p = 0.02; Multivariate Cox regression analysis). No transcriptional consequences of the losses were observed, and the only gene, A2BP1, located in the region showed no mutations. Correlation with other CNAs was established for chr3p22 in carcinomas and chr20p (inverse) in adenomas. FISH documented the chr16p13.2 region to be involved in complex structural rearrangements that included translocation to chr3p22 in some cases. The findings indicate that structural rearrangements involving chr16p13.2 are very frequent in colorectal neoplasia, often lead to homozygous deletion, and are associated with poor clinical outcome.

Abstract 922: Association of the hSRBC gene polymorphism with endometrial cancer

Abstract Background: hSRBC is a putative tumor suppressor located at 11p15.4, at which frequent genomic loss has been observed in several human malignancies. We previously reported that hSRBC expression is frequently downregulated in many types of human cancers due to aberrant promoter CpG sites hypermethylation, and hSRBC activation induces cell cycle arrest and apoptosis by enhancing the protein stability of p53. Interestingly, our recent studies suggest that hSRBC might be a novel transcription target of TNFα, which plays a crucial role in inflammation and tumorigenesis. We explored the possible association of an intreaexonic SNP (T509C), which results in a Leu to Pro substitution, with risk for endometrial cancer development. Methods: TNFα regulation of hSRBC expression was examined using semi-quantitative RT-PCR and immunoblot assays. Endometrial tissues from 147 cancer patients and 191 healthy individuals were included for test for SNP T509C by restriction endonuclease PvuII-based genotyping. Allele frequencies in cancer specimens were compared with those in healthy controls. Results: Allelic frequencies of T and C at the T509C position was 30.27% and 69.73% in healthy control and 37.17% and 62.83% in cancer specimens, respectively. Allelic frequency at T509C in cancer specimens showed no significant difference compared to controls. However, genotype frequencies of TT, CT, and CC in healthy controls were 16.75%, 40.84%, and 42.412%, respectively, while cancer specimens displayed 14.97%, 30.61%, and 54.42%. The CC genotype frequency in cancer specimens was significantly higher (p &amp;lt; 0.05) compared with that in healthy controls. Conclusion: Although the number of specimens used in this study is not enough, this study raises the possibility that the T509C SNP of hSRBC, a putative novel transcription target of TNFα and proapoptotic tumor suppressor, might be associated with endometrial cancer development. Further genotype screening of several other SNPs within hSRBC in cancer patients would provide valuable diagnostic and prognostic information for human cancers. 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 922.

Abstract 320: Fine structure genomic mapping of chromosome 10q23 interstitial deletions in prostate cancer reveals the PTEN gene locus as the minimum region deleted

Abstract Interstitial genomic deletions of the PTEN tumor suppressor gene and neighboring loci at 10q23 are detected in approximately 40% prostate cancer (CaP). Previously we have shown that the presence of PTEN genomic losses at surgery can be predictive of a shorter time to biochemical recurrence and that homozygous PTEN deletions portend early recurrence and metastatic disease. Although PTEN inactivation is strongly associated with CaP onset and progression, the genetic and molecular events leading to PTEN deletion are poorly understood. The reported breakpoints of PTEN genomic deletions in CaP vary significantly but at the present time there has been no systematic mapping of genomic losses at 10q23 and neighboring regions in tumors with interstitial deletions. This study examined the breakpoint regions associated with PTEN deletions and analyzed involvement of flanking genomic regions using fluorescence in situ hybridization (FISH) analysis of CaP tissue microarrays (n = 132 patients) and available online SNP databases (n = 27 metastases; from 9 patients) to determine the genomic locations of the most frequent tumor-specific 10q23 alterations. Four-color FISH was performed on the tissue microarrays using 6 bacterial artificial chromosome (BAC) clones spanning both flanking PTEN genomic regions (88.2 - 90.8 Mb) and the gene locus (89.61 - 89.72 Mb), with a centromeric DNA probe (CEP10) for region 10p11.1-q11.1. The configuration of the FISH probes allowed us to determine the genomic size of deletion events at cytoband 10q23 in CaP. FISH analyses showed that the most frequent genomic losses (including both hemi- and homozygous deletions) at 10q23 are interstitial and restricted to the PTEN gene locus. The second most frequent class of deletion is larger, and includes the PTEN gene and both up- and downstream flanking genomic regions. Similarly, in silico copy number analysis of the 10q23 region in the metastatic dataset identified PTEN and both flanking genomic regions as the most prominent deletion. Furthermore, the smallest overlapping region of deletion is a partial deletion of the PTEN gene and maps specifically to the BAC that is located at 89.66 - 89.84 Mb. One of the downstream consequences of the deletion acquisition and concomitant loss of PTEN functional activity is the activation of the AKT pathway. Elevated levels of activated AKT leads to the initiation of tumor promoting pathways, including proliferation, survival, and genomic instability, contributing to additional genomic aberrations such as formation of TMPRSS2:ETS fusion genes and loss of the second PTEN allele. These findings draw attention to the impact characterization of PTEN genomic loss, and complementary or independent candidate flanking genes involved will have on CaP. 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 320.

A Gynecologic Oncology Group study of frequent copy number aberrations in DNA repair genes and other genomic regions in stage I serous ovarian cancers

e16504 Background: A proof of principle array-based comparative genomic hybridization (aCGH) analysis was performed in archival formalin-fixed and paraffin-embedded (FFPE) stage I ovarian cancers (EOC) to determine if frequent (&gt;40%) copy number aberrations (CNAs) can be detected in DNA repair genes including the Fanconi anemia complementation group (FANC) and RAD51 families compared with the rest of the genome. Methods: Tumor DNA was isolated from 22 serous cancers from the GOG-175 virtual tissue bank. RPCI 19K BAC arrays were hybridized (GeneTAC HybStation) and scanned (Gene Pix 4200AL Laser Scanner). Spot fluorescence values were quantified using ImaGene, vetted for quality and loess corrected with adjustments for chip-specific spatial effects. The genome was segmented to identify regions with common copy number means (DNAcopy software). Posterior aberration probabilities for the regions were obtained using CGHcall and data was visualized and annotated using iGenomicViewer in R. Results: Several genes associated with the Fanconi DNA-damage response pathway were frequently altered in stage I serous ovarian carcinomas. A RAD51 homology DMC1 was amplified in 55% of the specimens. Genomic losses were observed in FANC-D1 (BRCA2), and RAD51L3 in 41%, and 27% of specimens, respectively. In contrast, frequent genomic losses or gains involved 13q33.1; 13 q21.31; 17p12; 17q22; 18q12.3; 9p11.2; 9p22.2–22.3; 9q33.1; 8p23.2; 21q21.3; and 5q14.2; or 8q24.3; 3q29; 12p11.1; 17q25.1; 17q25.3; 20q13.33; 20q11.21; 20q11.23; 19p13.13; and 19p13.2, respectively. Conclusions: The GOG-0175 virtual tissue bank yielded high quality DNA for detecting and mapping CNAs in archival FFPE specimens with high resolution. Frequent genomic losses and gains were observed in DNA repair genes and other genomic regions in stage I serous ovarian cancer which may promote genomic instability, resistance, metastasis and aggressiveness of this disease. No significant financial relationships to disclose.

Frequent epigenetic inactivation of hSRBC in gastric cancer and its implication in attenuated p53 response to stresses

hSRBC is a putative tumor suppressor located at 11p15.4, at which frequent genomic loss has been observed in several human malignancies. To explore the candidacy of hSRBC as a suppressor of gastric tumorigenesis, we analyzed the expression and mutation status of hSRBC in gastric tissues and cell lines. hSRBC transcript was expressed in all normal and benign tumor tissues examined, but undetectable or very low in 73% (11/15) cancer cell lines and 41% (46/111) primary tumors. Loss or reduction of hSRBC expression was tumor-specific and correlated with stage and grade of tumors. While allelic loss or somatic mutations of the gene were infrequent, its expression was restored in tumor cells by 5-aza-2'-deoxycytidine treatment and aberrant hypermethylation of 23 CpG sites in the promoter region showed a tight association with altered expression. Transient or stable expression of hSRBC led to a G(1) cell cycle arrest and apoptosis of tumor cells, and strongly suppresses colony forming ability and xenograft tumor growth. In addition, hSRBC elevated apoptotic sensitivity of tumor cells to genotoxic agents, such as 5-FU, etoposide and ultraviolet. Interestingly, hSRBC increased the protein stability of p53 and expression of p53 target genes, such as p21(Waf1), PUMA and NOXA, while hSRBC-mediated cell cycle arrest and apoptosis were abolished by blockade of p53 function. Our findings suggest that hSRBC is a novel tumor suppressor whose epigenetic inactivation contributes to the malignant progression of gastric tumors, in part, through attenuated p53 response to stresses.

Genome-wide profiling of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common malignancy, the incidence of which is particularly high in some Asian countries due to the geographically linked areca quid (AQ) chewing habit. In this study, array-based comparative genomic hybridization was used to screen microdissected OSCCs for genome-wide alterations. The highest frequencies of gene gain were detected for TP63, Serpine1, FGF4/FGF3, c-Myc and DMD. The highest frequencies of deletion were detected for Caspase8 and MTAP. Gained genes, classified by hierarchical clustering, were mainly on 17q21-tel; 20q; 11q13; 3q27-29 and the X chromosome. Among these, gains of EGFR at 7p, FGF4/FGF3, CCND1 and EMS1 at 11q13, and AIB1 at 20q were significantly associated with lymph node metastasis. The genomic profiles of FHIT and EXT1 in AQ-associated and non-AQ-associated OSCCs exhibited the most prominent differences. RT-PCR confirmed the significant increase of TP63 and Serpine1 mRNA expression in OSCC relative to non-malignant matched tissue. A significant increase in Serpine1 immunoreactivity was observed from non-malignant matched tissue to OSCC. However, there was no correlation between the frequent genomic loss of Caspase 8 and a significant decrease in Caspase8 expression. These data demonstrate that genomic profiling can be useful in analysing pathogenetic events involved in the genesis or progression of OSCC.