Cancer Genetics

FANCM, RAD1, CHEK1 and TP53I3 act as BRCA-like tumor suppressors and are mutated in hereditary ovarian cancer Publication date: Available online 9 May 2019 Source: Cancer Genetics Author(s): Jaime L. Lopes, Sophia Chaudhry, Guilherme S. Lopes, Nancy K. Levin, Michael A. Tainsky Abstract Although 25% of ovarian cancer cases are due to inherited factors, most of the genetic risk remains unexplained. We previously identified candidate genes through germline whole exome sequencing of BRCA1/BRCA2negative ovarian cancer patients with familial risk. Here, we performed functional assessment to determine whether they act as BRCA-like tumor suppressors. Seven candidate risk genes were targeted by siRNA for mRNA depletion followed by functional assays for clonogenic survival, cytotoxicity to DNA damaging agents, and involvement in homologous recombination repair. BRCA1 and BRCA1 were targeted as standards for loss of function outcome. Knockdown of various candidate genes led to tumor suppressor phenotypes also observed in BRCA1/BRCA2 deficient cells. Deficiency of CHEK1, FANCM and TP53I3 led to reduced homologous recombination repair efficiency. Knockdown of RAD1, CHEK1 or FANCM led to a decrease in cellular viability and cells deficient in CHEK1, RAD1 or TP53I3 displayed increased sensitivity to cisplatin. Functional studies of candidate genes identified by whole exome sequencing complements bioinformatics techniques and aid the implication of novel risk loci. The results of this study suggest that genes found mutated in hereditary ovarian cancer, FANCM, RAD1, CHEK1 and TP53I3, act as BRCA-like tumor suppressors.

Somatic mutation panels: Time to clear their names Publication date: Available online 26 April 2019 Source: Cancer Genetics Author(s): Amy M. Trottier, Marcela Cavalcante de Andrade Silva, Zejuan Li, Lucy A. Godley Abstract With improvements in DNA sequencing technologies and the consequent reduction in costs, next generation sequencing is being utilized increasingly in panel-based testing to perform molecular profiling of tumors. Such tumor-based panels are often referred to as 'somatic' panels, but this term is misleading and should not be used, since not all DNA variants within a tumor are somatic in nature. Every cell in a person's body contains that person's germline DNA, including tumor cells. Moreover, tumor samples are invariably contaminated with blood, a tissue that can contain somatic mutations itself in a process now called clonal hematopoiesis. Differentiating between germline variants or tumor-associated somatic mutations versus clonal hematopoiesis can be challenging. In this review, we address how to interpret the results of somatic mutation panels, how to differentiate between germline and truly somatic events, and discuss the importance of this distinction.

Outcomes of disease-specific next-generation sequencing gene panel testing in adolescents and young adults with colorectal cancer Publication date: Available online 26 April 2019 Source: Cancer Genetics Author(s): Maureen E. Mork, Andrea Rodriguez, Sarah A. Bannon, Patrick M. Lynch, Miguel A. Rodriguez-Bigas, Selvi Thirumurthi, Y. Nancy You, Eduardo Vilar Abstract Purpose Adolescents and young adults with colorectal cancer (CRC) have attracted recent attention, with a hereditary syndrome identified in one-third of patients diagnosed ≤ 35. We aimed to study this population to determine if a CRC-specific gene panel increased the yield of testing. Methods Patients with CRC ≤ 35 evaluated from 05/2014–11/2017 were identified from the genetic counseling database. Records were reviewed for personal/family history and genetic counseling outcomes. Results One hundred forty-three patients with CRC ≤ 35 were included. One hundred four (72.7%) underwent CRC panel testing. Thirty-nine (27.2%) had syndrome-directed testing, declined, or were lost to follow-up. Forty-two patients had a genetic syndrome (29.4%). Twenty-four of the 42 hereditary patients (57.1%) were identified via syndrome-directed testing. Mutations identified via panel testing were consistent with patient personal/family history. Thirty-three patients had at least one variant of uncertain significance. Conclusion Hereditary syndromes were identified in 29.4% of patients. Panel testing in patients without a phenotype did not increase diagnostic yield, but identified variants in one-third. Disease-specific panel testing is of low yield in young patients without a suggestive personal/family history. Testing broader panels may increase the yield of mutation pick-up in this population, although at the expense of identifying variants.

Uptake of genetic testing for germline BRCA1/2 pathogenic variants in a predominantly Hispanic population Publication date: Available online 24 April 2019 Source: Cancer Genetics Author(s): Julia E. McGuinness, Meghna S. Trivedi, Thomas Silverman, Awilda Marte, Jennie Mata, Rita Kukafka, Katherine D. Crew Abstract Genetic counseling is under-utilized in women who meet family history criteria for BRCA1 and BRCA2 (BRCA1/2) testing, particularly among racial/ethnic minorities. We evaluated the uptake of BRCA1/2 genetic testing among women presenting for screening mammography in a predominantly Hispanic, low-income population of Washington Heights in New York City. We administered the Six-Point Scale (SPS) to women presenting for screening mammography at Columbia University Irving Medical Center (CUIMC) in the Washington Heights neighborhood of New York, NY. The SPS is a family history screener to determine eligibility for BRCA1/2 genetic testing based upon U.S. Preventive Services Task Force (USPSTF) guidelines that has been validated in low-income, multiethnic populations. Among women who underwent screening mammography at CUIMC between November 2014 and June 2016, 3,055 completed the SPS family history screener. Participants were predominantly Hispanic (76.7%), and 12% met family history criteria for BRCA1/2 testing, of whom <5% had previously undergone testing. In a multiethnic population, a significant proportion met family history criteria for BRCA1/2 testing, but uptake of genetic testing was low. Such underutilization of BRCA1/2 genetic testing among minorities further underscores the need to develop programs to engage high-risk women from underrepresented populations in genetic testing services.

Retrotransposon elements among initial sites of hepatitis B virus integration into human genome in the HepG2-NTCP cell infection model Publication date: Available online 24 April 2019 Source: Cancer Genetics Author(s): Ranjit Chauhan, Yoshimi Shimizu, Koichi Watashi, Takaji Wakita, Masayoshi Fukasawa, Tomasz I Michalak Abstract Integration of hepatitis B virus (HBV) DNA into host's genome is evident in all stages and models of HBV infection. Investigations of the initial virus-host junctions have been just recently initiated since their nature may promote liver oncogenesis immediately following infection. We examined the time-frame and host sites at which HBV integrates in HepG2 cells overexpressing sodium taurocholate co-transporting polypeptide (NTCP) receptor mediating HBV entry. HepG2-NTCP cells were analyzed from 15 min to 13 days post-infection (p.i.). The results showed that except for 15 min p.i., HBV-host integrations were detected at all time points thereafter. At 30 min p.i., virus junctions with retrotransposon SINE and with neuroblastoma breakpoint family member 1 gene were detected. At one-hour p.i., HBV integration with retrotransposon THE-1B-LTR was identified, while virus insertions into proline-rich protein and protein kinase cGMP-dependent type 1 encoding genes were found at 3 h p.i. Fusion with runt-related transcription factor 1 was detected at 24 h p.i. and merges with 9 different genes at 13 day p.i. The data showed that retrotransposon elements are frequent among first-hit sites of HBV insertion. This may suggest a mechanism by which HBV DNA may spread across host's genome from earliest stages of infection.

Circulating cell-free DNA integrity as a diagnostic and prognostic marker for breast and prostate cancers Publication date: Available online 23 April 2019 Source: Cancer Genetics Author(s): Benjamin Arko-Boham, Nii Ayite Aryee, Richard Michael Blay, Ewurama Dedea Ampadu Owusu, Emmanuel Ayitey Tagoe, Eshirow-Sam Doris Shackie, Ama Boatemaa Debrah, Nii Armah Adu-Aryee Abstract Background Cancer incidence and its related mortality is rising and is currently the second leading cause of death globally. In Africa, breast and prostate cancer in females and males, respectively, are the worst globally. However, biomarkers for their early detection and prognosis are not well developed. This study sought to investigate circulating cell-free DNA (ccfDNA) integrity and its potential utility as diagnostic and/or prognostic biomarker. Circulating cell-free DNA (ccfDNA) is degraded DNA fragments released into the blood plasma. In healthy individuals, the source of ccfDNA is solely apoptosis, producing evenly sized shorter DNA fragments. In cancer patients, however, necrosis produces uneven longer cell-free DNA fragments in addition to the shorter fragments originating from apoptosis. DNA integrity, expressed as the ratio of longer fragments to total DNA, may be clinically useful for the detection of breast and prostate cancer progression. Methods Sixty-four (64) females, consisting of 32 breast cancer patients and 32 controls, and 61 males (31 prostate cancer patients and 30 controls) were included in the study. Each participant donated 5 ml peripheral blood from which sera were separated. Real-time qPCR was performed on the sera to quantify ALU 115 and 247 levels, and DNA integrity (ALU247/ALU115) determined. Results & Conclusion ALU species 115 and 247 levels in serum were elevated in breast and prostate cancer patients compared to their counterpart healthy controls. DNA integrity was higher in prostate cancer patients than in the control, but in breast cancer patients was lower compared to their controls. In prostate but not in breast cancers, DNA integrity increased with disease severity and higher staging.

Prevalence and characteristics of likely-somatic variants in cancer susceptibility genes among individuals who had hereditary pan-cancer panel testing Publication date: Available online 13 April 2019 Source: Cancer Genetics Author(s): Thomas P. Slavin, Bradford Coffee, Ryan Bernhisel, Jennifer Logan, Hannah C. Cox, Guido Marcucci, Jeffrey Weitzel, Susan L. Neuhausen, Debora Mancini-DiNardo Abstract Next-generation sequencing (NGS) hereditary pan-cancer panel testing can identify somatic variants, which exhibit lower allele frequencies than do germline variants and may confound hereditary cancer predisposition testing. This analysis examined the prevalence and characteristics of likely-somatic variants among 348,543 individuals tested using a clinical NGS hereditary pan-cancer panel. Variants showing allele frequencies between 10% and 30% were interpreted as likely somatic and identified in 753 (0.22%) individuals. They were most frequent in TP53, CHEK2 and ATM, commonly as C-to-T transitions. Among individuals who carried a likely-somatic variant and reported no personal cancer history, 54.2% (78/144) carried a variant in TP53, CHEK2 or ATM. With a reported cancer history, this percentage increased to 81.1% (494/609), predominantly in CHEK2and TP53. Their presence was associated with age (OR=3.1, 95% CI 2.5, 3.7; p<0.001) and personal history of cancer (OR=3.3, 95% CI 2.7, 4.0; p<0.001), particularly ovarian cancer. Germline ATM pathogenic variant carriers showed significant enrichment of likely-somatic variants (OR=2.8, 95% CI 1.6, 4.9; p = 0.005), regardless of cancer status. The appearance of likely-somatic variants is consistent with clonal hematopoiesis, possibly influenced by cancer treatment. These findings highlight the precision required of diagnostic laboratories to deliver accurate germline testing results.

A comparison of survival analysis methods for cancer gene expression RNA-Sequencing data Publication date: Available online 12 April 2019 Source: Cancer Genetics Author(s): Pichai Raman, Samuel Zimmerman, Komal S. Rathi, Laurence de Torrenté, Mahdi Sarmady, Chao Wu, Jeremy Leipzig, Deanne M. Taylor, Aydin Tozeren, Jessica C. Mar Abstract Identifying genetic biomarkers of patient survival remains a major goal of large-scale cancer profiling studies. Using gene expression data to predict the outcome of a patient's tumor makes biomarker discovery a compelling tool for improving patient care. As genomic technologies expand, multiple data types may serve as informative biomarkers, and bioinformatic strategies have evolved around these different applications. For categorical variables such as a gene's mutation status, biomarker identification to predict survival time is straightforward. However, for continuous variables like gene expression, the available methods generate highly-variable results, and studies on best practices are lacking. We investigated the performance of eight methods that deal specifically with continuous data. K-means, Cox regression, concordance index, D-index, 25th-75th percentile split, median-split, distribution-based splitting, and KaplanScan were applied to four RNA-sequencing (RNA-seq) datasets from the Cancer Genome Atlas. The reliability of the eight methods was assessed by splitting each dataset into two groups and comparing the overlap of results. Gene sets that had been identified from the literature for a specific tumor type served as positive controls to assess the accuracy of each biomarker using receiver operating characteristic (ROC) curves. Artificial RNA-Seq data were generated to test the robustness of these methods under fixed levels of gene expression noise. Our results show that methods based on dichotomizing tend to have consistently poor performance while C-index, D-index and k-means perform well in most settings. Overall, the Cox regression method had the strongest performance based on tests of accuracy, reliability, and robustness.

Prognostic significance of CDC25C in lung adenocarcinoma: An analysis of TCGA data Publication date: April 2019 Source: Cancer Genetics, Volumes 233–234 Author(s): Zengfei Xia, Wen Ou-yang, Ting Hu, Ketao Du Abstract Objective Cell division cycle 25C (CDC25C) is involved in the regulation of the G2/M phase transition and is associated with various cancers, including non-small cell lung cancer. We evaluated its prognostic value in lung adenocarcinoma (LUAD) based on data from The Cancer Genome Atlas (TCGA). Methods Kruskal–Wallis test, Wilcoxon signed-rank test, and logistic regression were used to evaluate relationships between clinical-pathologic features and CDC25C expression. Cox regression analyses and the Kaplan–Meier method were used to evaluate factors contributing to prognosis. Gene set enrichment analysis (GSEA) was performed. Results High CDC25C expression in LUAD was associated with a high tumor extent (odds ratio (OR) = 2.23 (1.52–3.29), P < 0.001), regional lymph node invasion (OR = 2.18 (1.48–3.22), P < 0.001), OR = advanced stage (OR = 2.47 (1.72–3.59), P < 0.001), and poor status (OR = 1.87 (1.19–2.96), P = 0.007). A univariate analysis showed that high CDC25C expression is associated with a short overall survival (OS) (HR: 1.873; 95% CI: 1.385–2.535; P < 0.001) and poor progression-free survival (HR: 1.503; 95% CI: 1.173–1.926; P = 0.0012). In a multivariate analysis, high CDC25C expression was associated with poor OS (HR = 2.193; CI: 1.394–3.452, P = 0.001). GSEA showed the enrichment of cell cycle, apoptosis, p53-dependent G1 DNA damage response, S-phase, mitotic M-M G1 phases, and FA-mediated cell death in the CDC25C high-expression phenotype. Conclusions CDC25C predicts poor prognosis in LUAD and may function in cell cycle regulation and FAS-mediated apoptosis.

Aberrant methylation status of SPG20 promoter in hepatocellular carcinoma: A potential tumor metastasis biomarker Publication date: April 2019 Source: Cancer Genetics, Volumes 233–234 Author(s): Lifeng He, Xiaoxiao Fan, Yirun Li, Bin Cui, Zhaoqi Shi, Daizhan Zhou, Hui Lin Abstract Purpose The aim of this study is to analyze the methylation levels of SPG20 promotor region and explore the association between the methylation levels and clinical features in hepatocellular carcinoma (HCC). Materials and methods We collected paired of HCC and adjacent non-cancerous tissues (ANT) from 160 HCC patients and analyze the methylation levels through MassARRAY Analyzer 4. The statistical calculations were performed using SPSS version 22.0. Real-time-quantification PCR was performed to assess expression levels of SPG20 in HCC cell lines. Wound healing assay and transwell assay was used to measure cell migration capacity. Result We found that mean methylation level of SPG20 in tumor tissues was significantly higher than that in ANT (7.3% vs. 16.2%, P<0.0013). There was a significantly negative correlation between expression level and methylation level of SPG20 (P<0.01). In addition, the methylation levels in HCC were correlated with age and HBV infection. Meanwhile, micro-satellite tumors (P = 0.016) and tumor number (P = 0.018) was found significantly associated with increased methylation levels of several CpG sites and the mean levels of SPG20 promotor in ANT. In addtion, the capacity of cell migration was significantly enhanced in SPG20 knock-down HCC cells. Conclusion The hypermethylation status of SPG20 gene promoter is significantly associated with intra-hepatic metastasis and contribute to HCC metastasis.

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