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1.  Toward precision medicine in glioblastoma: the promise and the challenges 
Neuro-Oncology  2015;17(8):1051-1063.
Integrated sequencing strategies have provided a broader understanding of the genomic landscape and molecular classifications of multiple cancer types and have identified various therapeutic opportunities across cancer subsets. Despite pivotal advances in the characterization of genomic alterations in glioblastoma, targeted agents have shown minimal efficacy in clinical trials to date, and patient survival remains poor. In this review, we highlight potential reasons why targeting single alterations has yielded limited clinical efficacy in glioblastoma, focusing on issues of tumor heterogeneity and pharmacokinetic failure. We outline strategies to address these challenges in applying precision medicine to glioblastoma and the rationale for applying targeted combination therapy approaches that match genomic alterations with compounds accessible to the central nervous system.
doi:10.1093/neuonc/nov031
PMCID: PMC4490873  PMID: 25934816
clinical trial; genomics; glioblastoma; precision medicine; targeted therapy
2.  Dual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific for small cell carcinoma of the ovary, hypercalcaemic type 
The Journal of Pathology  2015;238(3):389-400.
Abstract
Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a lethal and sometimes familial ovarian tumour of young women and children. We and others recently discovered that over 90% of SCCOHTs harbour inactivating mutations in the chromatin remodelling gene SMARCA4 with concomitant loss of its encoded protein SMARCA4 (BRG1), one of two mutually exclusive ATPases of the SWI/SNF chromatin remodelling complex. To determine the specificity of SMARCA4 loss for SCCOHT, we examined the expression of SMARCA4 by immunohistochemistry in more than 3000 primary gynaecological tumours. Among ovarian tumours, it was only absent in clear cell carcinoma (15 of 360, 4%). In the uterus, it was absent in endometrial stromal sarcomas (4 of 52, 8%) and high‐grade endometrioid carcinomas (2 of 338, 1%). Recent studies have shown that SMARCA2 (BRM), the other mutually exclusive ATPase of the SWI/SNF complex, is necessary for survival of tumour cells lacking SMARCA4. Therefore, we examined SMARCA2 expression and discovered that all SMARCA4‐negative SCCOHTs also lacked SMARCA2 protein by IHC, including the SCCOHT cell lines BIN67 and SCCOHT1. Among ovarian tumours, the SMARCA4/SMARCA2 dual loss phenotype appears completely specific for SCCOHT. SMARCA2 loss was not due to mutation but rather from an absence of mRNA expression, which was restored by treatment with the histone deacetylase inhibitor trichostatin A. Re‐expression of SMARCA4 or SMARCA2 inhibited the growth of BIN67 and SCCOHT1 cell lines. Our results indicate that SMARCA4 loss, either alone or with SMARCA2, is highly sensitive and specific for SCCOHT and that restoration of either SWI/SNF ATPase can inhibit the growth of SCCOHT cell lines. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
doi:10.1002/path.4633
PMCID: PMC4832362  PMID: 26356327
small cell carcinoma; hypercalcaemic type; rhabdoid tumour; SMARCA4/BRG1; SMARCA2/BRM; SMARCB1/INI1; SWI/SNF; HDAC inhibitor; trichostatin A; epigenetic silencing
3.  Assessing Combinational Drug Efficacy in Cancer Cells by Using Image-based Dynamic Response Analysis 
Cancer Informatics  2016;14(Suppl 5):33-43.
The landscape of translational research has been shifting toward drug combination therapies. Pairing of drugs allows for more types of drug interaction with cells. In order to accurately and comprehensively assess combinational drug efficacy, analytical methods capable of recognizing these alternative reactions will be required to prioritize those drug candidates having better chances of delivering appreciable therapeutic benefits. Traditional efficacy measures are primarily based on the “extent” of drug inhibition, which is the percentage of cells being killed after drug exposure. Here, we introduce a second dimension of evaluation criterion, speed of killing, based on a live cell imaging assay. This dynamic response trajectory approach takes advantage of both “extent” and “speed” information and uncovers synergisms that would otherwise be missed, while also generating hypotheses regarding important mechanistic modes of drug action.
doi:10.4137/CIN.S30799
PMCID: PMC4786100  PMID: 26997864
combinational drug; synergism; drug response; dynamics; cell imaging
4.  Nonsense Mutations in the Shelterin Complex Genes ACD and TERF2IP in Familial Melanoma 
Background:
The shelterin complex protects chromosomal ends by regulating how the telomerase complex interacts with telomeres. Following the recent finding in familial melanoma of inactivating germline mutations in POT1, encoding a member of the shelterin complex, we searched for mutations in the other five components of the shelterin complex in melanoma families.
Methods:
Next-generation sequencing techniques were used to screen 510 melanoma families (with unknown genetic etiology) and control cohorts for mutations in shelterin complex encoding genes: ACD, TERF2IP, TERF1, TERF2, and TINF 2. Maximum likelihood and LOD [logarithm (base 10) of odds] analyses were used. Mutation clustering was assessed with χ2 and Fisher’s exact tests. P values under .05 were considered statistically significant (one-tailed with Yates’ correction).
Results:
Six families had mutations in ACD and four families carried TERF2IP variants, which included nonsense mutations in both genes (p.Q320X and p.R364X, respectively) and point mutations that cosegregated with melanoma. Of five distinct mutations in ACD, four clustered in the POT1 binding domain, including p.Q320X. This clustering of novel mutations in the POT1 binding domain of ACD was statistically higher (P = .005) in melanoma probands compared with population control individuals (n = 6785), as were all novel and rare variants in both ACD (P = .040) and TERF2IP (P = .022). Families carrying ACD and TERF2IP mutations were also enriched with other cancer types, suggesting that these variants also predispose to a broader spectrum of cancers than just melanoma. Novel mutations were also observed in TERF1, TERF2, and TINF2, but these were not convincingly associated with melanoma.
Conclusions:
Our findings add to the growing support for telomere dysregulation as a key process associated with melanoma susceptibility.
doi:10.1093/jnci/dju408
PMCID: PMC4334787  PMID: 25505254
5.  Loss of the tumor suppressor SMARCA4 in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) 
Rare Diseases  2014;2(1):e967148.
Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a rare and understudied cancer with a dismal prognosis. SCCOHT's infrequency has hindered empirical study of its biology and clinical management. However, we and others have recently identified inactivating mutations in the SWI/SNF chromatin remodeling gene SMARCA4 with concomitant loss of SMARCA4 protein in the majority of SCCOHT tumors.1–4 Here we summarize these findings and report SMARCA4 status by targeted sequencing and/or immunohistochemistry (IHC) in an additional 12 SCCOHT tumors, 3 matched germlines, and the cell line SCCOHT-1. We also report the identification of a homozygous inactivating mutation in the gene SMARCB1 in one SCCOHT tumor with wild-type SMARCA4, suggesting that SMARCB1 inactivation may also play a role in the pathogenesis of SCCOHT. To date, SMARCA4 mutations and protein loss have been reported in the majority of 69 SCCOHT cases (including 2 cell lines). These data firmly establish SMARCA4 as a tumor suppressor whose loss promotes the development of SCCOHT, setting the stage for rapid advancement in the biological understanding, diagnosis, and treatment of this rare tumor type.
doi:10.4161/2167549X.2014.967148
PMCID: PMC4755243  PMID: 26942101
BRG1; chromatin remodeling; small cell carcinoma of the ovary hypercalcemic type (SCCOHT); SMARCA4; SMARCB1; SNF5; ovarian cancer; SWI/SNF; tumorigenesis
6.  8q24 Risk Alleles and Prostate Cancer in African-Barbadian Men 
The Prostate  2014;74(16):1579-1588.
BACKGROUND
African American men (AA) exhibit a disproportionate share of prostate cancer (PRCA) incidence, morbidity and mortality. Several genetic association studies have implicated select 8q24 loci in PRCA risk in AA. The objective of this investigation is to evaluate the association between previously reported 8q24 risk alleles and PRCA in African-Barbadian (AB) men known to have high rates of PRCA.
METHODS
Ten previously reported candidate tag SNPs were genotyped and/or imputed in the 8q24 region in 532 AB men with PRCA and 513 AB controls from the Prostate Cancer in a Black Population (PCBP) study.
RESULTS
Rs2124036 was significant in AB men, (OR = 2.7, 95% CI (1.3–5.3), P=0.005, Empirical (max(T), corrected for multiple testing) P = 0.03) for the homozygous C/C genotype. Only a single SNP from this region remained statistically significant in our analysis of our AB population. These results may indicate the presence of a founder effect or due to the chosen SNPs not tagging an ancestral haplotype bearing the 8q24 risk allele(s) in this population or could reflect inadequate power to detect an association. We conducted a meta-analysis including our AB population along with two additional African Caribbean populations from Tobago and Jamaica for SNPs rs16901979 and rs1447295. Meta-analysis results were most significant for rs16901979 A allele (Z score 2.73; p=0.006) with a summary OR= 1.31 (95% CI: 1.09–1.58).
CONCLUSIONS
Additional studies are needed to provide deeper genotype coverage to further interrogate the 8q24 region to understand its contribution to PRCA in this population.
doi:10.1002/pros.22871
PMCID: PMC4322001  PMID: 25252079
7.  Development of potent autophagy inhibitors that sensitize oncogenic BRAF V600E mutant melanoma tumor cells to vemurafenib 
Autophagy  2014;10(6):1120-1136.
Autophagy is a dynamic cell survival mechanism by which a double-membrane vesicle, or autophagosome, sequesters portions of the cytosol for delivery to the lysosome for recycling. This process can be inhibited using the antimalarial agent chloroquine (CQ), which impairs lysosomal function and prevents autophagosome turnover. Despite its activity, CQ is a relatively inadequate inhibitor that requires high concentrations to disrupt autophagy, highlighting the need for improved small molecules. To address this, we screened a panel of antimalarial agents for autophagy inhibition and chemically synthesized a novel series of acridine and tetrahydroacridine derivatives. Structure-activity relationship studies of the acridine ring led to the discovery of VATG-027 as a potent autophagy inhibitor with a high cytotoxicity profile. In contrast, the tetrahydroacridine VATG-032 showed remarkably little cytotoxicity while still maintaining autophagy inhibition activity, suggesting that both compounds act as autophagy inhibitors with differential effects on cell viability. Further, knockdown of autophagy-related genes showed no effect on cell viability, demonstrating that the ability to inhibit autophagy is separate from the compound cytotoxicity profiles. Next, we determined that both inhibitors function through lysosomal deacidification mechanisms and ultimately disrupt autophagosome turnover. To evaluate the genetic context in which these lysosomotropic inhibitors may be effective, they were tested in patient-derived melanoma cell lines driven by oncogenic BRAF (v-raf murine sarcoma viral oncogene homolog B). We discovered that both inhibitors sensitized melanoma cells to the BRAF V600E inhibitor vemurafenib. Overall, these autophagy inhibitors provide a means to effectively block autophagy and have the potential to sensitize mutant BRAF melanomas to first-line therapies.
doi:10.4161/auto.28594
PMCID: PMC4091172  PMID: 24879157
autophagy; melanoma; vemurafenib; PLX-4032; lysosome; antimalarial; chloroquine; quinacrine
8.  Genome-Wide Association Study for Serum Complement C3 and C4 Levels in Healthy Chinese Subjects 
PLoS Genetics  2012;8(9):e1002916.
Complement C3 and C4 play key roles in the main physiological activities of complement system, and their deficiencies or over-expression are associated with many clinical infectious or immunity diseases. A two-stage genome-wide association study (GWAS) was performed for serum levels of C3 and C4. The first stage was conducted in 1,999 healthy Chinese men, and the second stage was performed in an additional 1,496 subjects. We identified two SNPs, rs3753394 in CFH gene and rs3745567 in C3 gene, that are significantly associated with serum C3 levels at a genome-wide significance level (P = 7.33×10−11 and P = 1.83×10−9, respectively). For C4, one large genomic region on chromosome 6p21.3 is significantly associated with serum C4 levels. Two SNPs (rs1052693 and rs11575839) were located in the MHC class I area that include HLA-A, HLA-C, and HLA-B genes. Two SNPs (rs2075799 and rs2857009) were located 5′ and 3′ of C4 gene. The other four SNPs, rs2071278, rs3763317, rs9276606, and rs241428, were located in the MHC class II region that includes HLA-DRA, HLA-DRB, and HLA-DQB genes. The combined P-values for those eight SNPs ranged from 3.19×10−22 to 5.62×10−97. HBsAg-positive subjects have significantly lower C3 and C4 protein concentrations compared with HBsAg-negative subjects (P<0.05). Our study is the first GWAS report which shows genetic components influence the levels of complement C3 and C4. Our significant findings provide novel insights of their related autoimmune, infectious diseases, and molecular mechanisms.
Author Summary
The complement system plays important roles in the innate and adaptive immune functions. C3 and C4 participate in almost all physiological activities and activated pathways as key complement members and host defense proteins. Identifying the genes that influence serum levels of C3 and C4 may help to elucidate the factors and mechanisms underlying the complement system. The genome-wide association studies (GWAS) have shown great success in revealing robust associations in both quantitative and qualitative traits. In this study, we performed a two-stage GWAS in a large cohort from the Chinese male population to examine the roles of common genetic variants on serum C3 and C4 levels. Our research identified genetic determinants associated with the quantitative levels of C3 and C4. Overall, our study highlights an intricate regulation of complement levels and potentially reveals novel mechanisms that may be followed up with additional functional studies.
doi:10.1371/journal.pgen.1002916
PMCID: PMC3441730  PMID: 23028341
9.  Small cell carcinoma of the ovary, hypercalcemic type, displays frequent inactivating germline and somatic mutations in SMARCA4 
Nature genetics  2014;46(5):427-429.
Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is an extremely rare, aggressive cancer affecting children and young women. We identified germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 69% (9/13) of SCCOHT cases in addition to SMARCA4 protein loss in 82% (14/17) of SCCOHT tumors but in only 0.4% (2/485) of other primary ovarian tumors. These data implicate SMARCA4 in SCCOHT oncogenesis.
doi:10.1038/ng.2928
PMCID: PMC4332808  PMID: 24658001
10.  Characterization of X Chromosome Inactivation Using Integrated Analysis of Whole-Exome and mRNA Sequencing 
PLoS ONE  2014;9(12):e113036.
In females, X chromosome inactivation (XCI) is an epigenetic, gene dosage compensatory mechanism by inactivation of one copy of X in cells. Random XCI of one of the parental chromosomes results in an approximately equal proportion of cells expressing alleles from either the maternally or paternally inherited active X, and is defined by the XCI ratio. Skewed XCI ratio is suggestive of non-random inactivation, which can play an important role in X-linked genetic conditions. Current methods rely on indirect, semi-quantitative DNA methylation-based assay to estimate XCI ratio. Here we report a direct approach to estimate XCI ratio by integrated, family-trio based whole-exome and mRNA sequencing using phase-by-transmission of alleles coupled with allele-specific expression analysis. We applied this method to in silico data and to a clinical patient with mild cognitive impairment but no clear diagnosis or understanding molecular mechanism underlying the phenotype. Simulation showed that phased and unphased heterozygous allele expression can be used to estimate XCI ratio. Segregation analysis of the patient's exome uncovered a de novo, interstitial, 1.7 Mb deletion on Xp22.31 that originated on the paternally inherited X and previously been associated with heterogeneous, neurological phenotype. Phased, allelic expression data suggested an 83∶20 moderately skewed XCI that favored the expression of the maternally inherited, cytogenetically normal X and suggested that the deleterious affect of the de novo event on the paternal copy may be offset by skewed XCI that favors expression of the wild-type X. This study shows the utility of integrated sequencing approach in XCI ratio estimation.
doi:10.1371/journal.pone.0113036
PMCID: PMC4264736  PMID: 25503791
11.  Loss-of-Function Fibroblast Growth Factor Receptor-2 Mutations in Melanoma 
We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer.
doi:10.1158/1541-7786.MCR-08-0021
PMCID: PMC4233648  PMID: 19147536
12.  Assessment of PALB2 as a Candidate Melanoma Susceptibility Gene 
PLoS ONE  2014;9(6):e100683.
Partner and localizer of BRCA2 (PALB2) interacts with BRCA2 to enable double strand break repair through homologous recombination. Similar to BRCA2, germline mutations in PALB2 have been shown to predispose to Fanconi anaemia as well as pancreatic and breast cancer. The PALB2/BRCA2 protein interaction, as well as the increased melanoma risk observed in families harbouring BRCA2 mutations, makes PALB2 a candidate for melanoma susceptibility. In order to assess PALB2 as a melanoma predisposition gene, we sequenced the entire protein-coding sequence of PALB2 in probands from 182 melanoma families lacking pathogenic mutations in known high penetrance melanoma susceptibility genes: CDKN2A, CDK4, and BAP1. In addition, we interrogated whole-genome and exome data from another 19 kindreds with a strong family history of melanoma for deleterious mutations in PALB2. Here we report a rare known deleterious PALB2 mutation (rs118203998) causing a premature truncation of the protein (p.Y1183X) in an individual who had developed four different cancer types, including melanoma. Three other family members affected with melanoma did not carry the variant. Overall our data do not support a case for PALB2 being associated with melanoma predisposition.
doi:10.1371/journal.pone.0100683
PMCID: PMC4065098  PMID: 24949998
13.  A BAP1 Mutation in a Danish Family Predisposes to Uveal Melanoma and Other Cancers 
PLoS ONE  2013;8(8):e72144.
Truncating germline mutations in the tumor suppressor gene BRCA-1 associated protein-1 (BAP1) have been reported in families predisposed to developing a wide range of different cancer types including uveal melanoma and cutaneous melanoma. There has also been an association between amelanotic tumor development and germline BAP1 mutation suggesting a possible phenotypic characteristic of BAP1 mutation carriers. Though there have been many types of cancer associated with germline BAP1 mutation, the full spectrum of disease association is yet to be ascertained. Here we describe a Danish family with predominantly uveal melanoma but also a range of other tumor types including lung, neuroendocrine, stomach, and breast cancer; as well as pigmented skin lesions. Whole-exome sequencing identified a BAP1 splice mutation located at c.581-2A>G, which leads to a premature truncation of BAP1 in an individual with uveal melanoma. This mutation was carried by several other family members with melanoma or various cancers. The finding expands on the growing profile of BAP1 as an important uveal and cutaneous melanoma tumor suppressor gene and implicates its involvement in the development of lung, and stomach cancer.
doi:10.1371/journal.pone.0072144
PMCID: PMC3747051  PMID: 23977234
14.  Association of prostate cancer risk with SNPs in regions containing androgen receptor binding sites captured by ChIP-on-chip analyses 
The Prostate  2011;72(4):376-385.
Background
Genome-wide association studies (GWAS) have identified approximately three dozen single nucleotide polymorphisms (SNPs) consistently associated with prostate cancer (PCa) risk. Despite the reproducibility of these associations, the molecular mechanism for most of these SNPs has not been well elaborated as most lie within non-coding regions of the genome. Androgens play a key role in prostate carcinogenesis. Recently, using ChIP-on-chip technology, 22,447 androgen receptor (AR) binding sites have been mapped throughout the genome, greatly expanding the genomic regions potentially involved in androgen-mediated activity.
Methodology/Principal findings
To test the hypothesis that sequence variants in AR binding sites are associated with PCa risk, we performed a systematic evaluation among two existing PCa GWAS cohorts; the Johns Hopkins Hospital and the Cancer Genetic Markers of Susceptibility (CGEMS) study population. We demonstrate that regions containing AR binding sites are significantly enriched for PCa risk-associated SNPs, i.e. more than expected by chance alone. In addition, compared with the entire genome, these newly observed risk-associated SNPs in these regions are significantly more likely to overlap with established PCa risk-associated SNPs from previous GWAS. These results are consistent with our previous finding from a bioinformatics analysis that one-third of the 33 known PCa risk-associated SNPs discovered by GWAS are located in regions of the genome containing AR binding sites.
Conclusions/Significance
The results to date provide novel statistical evidence suggesting an androgen-mediated mechanism by which some PCa associated SNPs act to influence PCa risk. However, these results are hypothesis generating and ultimately warrant testing through in-depth molecular analyses.
doi:10.1002/pros.21439
PMCID: PMC3366362  PMID: 21671247
AR; prostate cancer; GWAS; pathway association study
15.  The BioIntelligence Framework: a new computational platform for biomedical knowledge computing 
Breakthroughs in molecular profiling technologies are enabling a new data-intensive approach to biomedical research, with the potential to revolutionize how we study, manage, and treat complex diseases. The next great challenge for clinical applications of these innovations will be to create scalable computational solutions for intelligently linking complex biomedical patient data to clinically actionable knowledge. Traditional database management systems (DBMS) are not well suited to representing complex syntactic and semantic relationships in unstructured biomedical information, introducing barriers to realizing such solutions. We propose a scalable computational framework for addressing this need, which leverages a hypergraph-based data model and query language that may be better suited for representing complex multi-lateral, multi-scalar, and multi-dimensional relationships. We also discuss how this framework can be used to create rapid learning knowledge base systems to intelligently capture and relate complex patient data to biomedical knowledge in order to automate the recovery of clinically actionable information.
doi:10.1136/amiajnl-2011-000646
PMCID: PMC3555311  PMID: 22859646
Database management systems (L01.224.900.280); access to information (L01.143.024); knowledge (L01.535); data; information; knowledge; bioinformatics; databases; research; computer; science; biomedical; informatics; trent1952; genomics; knowledge; hypergraph; cancer; database
16.  Contribution of HPC1 (RNASEL) and HPCX variants to prostate cancer in a founder population 
The Prostate  2010;70(15):1716-1727.
Background
Prostate cancer is a genetically complex disease with locus and disease heterogeneity. The RNASEL gene and HPCX locus have been implicated in hereditary prostate cancer; however, their contributions to sporadic forms of this malignancy remain uncertain.
Methods
Associations of prostate cancer with two variants in the RNASEL gene (a founder mutation, 471delAAAG, and a non-synonymous SNP, rs486907), and with five microsatellite markers in the HPCX locus, were examined in 979 cases and 1,251 controls of Ashkenazi Jewish descent. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression models.
Results
There was an inverse association between RNASEL rs486907 and prostate cancer in younger men (<65 years) and those with a first-degree relative with prostate cancer; men with AA genotype had ORs of 0.64 and 0.47 (both p<0.05), respectively, in comparison to men with GG genotype. Within the HPCX region, there were positive associations for allele 135 of bG82i1.1 marker (OR=1.77, p=0.01) and allele 188 of DXS1205 (OR=1.65, p=0.02). In addition, allele 248 of marker D33 was inversely associated (OR=0.65, p=0.05) with Gleason score ≥7 tumors.
Conclusions
Results suggest that variants in RNASEL contribute to susceptibility to early onset and familial forms of prostate cancer, whereas HPCX variants are associated with prostate cancer risk and tumor aggressiveness. The observation that a mutation predicted to completely inactivate RNASEL protein was not associated with prostate cancer, but that a missense variant was associated, suggests that the effect is due to either partial inactivation of the protein, and/or acquisition of a new protein activity.
doi:10.1002/pros.21207
PMCID: PMC3404133  PMID: 20564318
17.  Cancer of the ampulla of Vater: analysis of the whole genome sequence exposes a potential therapeutic vulnerability 
Genome Medicine  2012;4(7):56.
Background
Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scale clinical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers.
Methods
We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNA from a 63 year-old man who underwent a pancreaticoduodenectomy by whole genome sequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations.
Results
We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition.
Conclusions
Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers.
doi:10.1186/gm357
PMCID: PMC3580412  PMID: 22762308
18.  High-throughput RNAi screening identifies a role for TNK1 in growth and survival of pancreatic cancer cells 
Molecular cancer research : MCR  2011;9(6):724-732.
In order to identify novel targets in pancreatic cancer cells, we utilized high-throughput RNAi (HT-RNAi) to identify genes that, when silenced, would decrease viability of pancreatic cancer cells. The HT-RNAi screen involved reverse transfecting the pancreatic cancer cell line BxPC3 with a siRNA library targeting 572 kinases. From replicate screens, approximately thirty-two kinases were designated as hits, of which twenty-two kinase targets were selected for confirmation and validation. One kinase identified as a hit from this screen was tyrosine kinase non-receptor 1 (TNK1), a kinase previously identified as having tumor suppressor-like properties in embryonic stem cells. Silencing of TNK1 with siRNA showed reduced proliferation in a panel of pancreatic cancer cell lines. Furthermore, we demonstrated that silencing of TNK1 led to increased apoptosis through a caspase-dependent pathway and that targeting TNK1 with siRNA can synergize with gemcitabine treatment. Despite previous reports that TNK1 affects Ras and NFκB signaling, we did not find similar correlations with these pathways in pancreatic cancer cells. Our results suggest that TNK1 in pancreatic cancer cells does not possess the same tumor suppressor properties seen in embryonic cells, but appears to be involved in growth and survival. The application of functional genomics using HT-RNAi screens has allowed us to identify TNK1 as a growth-associated kinase in pancreatic cancer cells.
doi:10.1158/1541-7786.MCR-10-0436
PMCID: PMC3137903  PMID: 21536687
HT-RNAi; TNK1; gemcitabine; pancreatic cancer
20.  A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma 
Nature  2011;480(7375):99-103.
So far, two familial melanoma genes have been identified, accounting for a minority of genetic risk in families. Mutations in CDKN2A account for approximately 40% of familial cases1, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds2. To identify other familial melanoma genes, here we conducted whole-genome sequencing of probands from several melanoma families, identifying one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF). Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log odds ratio (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant. Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case–control sample. Likewise, it was similarly associated in an independent case–control sample from the United Kingdom. In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both. The variant allele was also associated with increased naevus count and non-blue eye colour. Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets. These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.
doi:10.1038/nature10630
PMCID: PMC3266855  PMID: 22080950
21.  Evidence for two independent prostate cancer risk associated loci in the HNF1B gene at 17q12 
Nature genetics  2008;40(10):1153-1155.
A fine mapping study in the HNF1B gene at 17q12 among two study populations revealed a second prostate cancer locus, ~26 kb centromeric to the first known locus (rs4430796); these are separated by a recombination hotspot. A SNP in the second locus (rs11649743) was confirmed in five additional populations, and P=1.7×10−9 for an allelic test in the seven combined studies. The association at each SNP remains significant after adjusting for the other SNP.
doi:10.1038/ng.214
PMCID: PMC3188432  PMID: 18758462
22.  Loss of Inositol Polyphosphate-5-Phosphatase is an Early Event in Development of Cutaneous Squamous Cell Carcinoma 
Cutaneous squamous cell carcinoma (SCC) occurs commonly and can metastasize. Identification of specific molecular aberrations and mechanisms underlying the development and progression of cutaneous SCC may lead to better prognostic and therapeutic approaches and more effective chemoprevention strategies. To identify genetic changes associated with early stages of cutaneous SCC development, we analyzed a series of 40 archived skin tissues ranging from normal skin to invasive SCC. Using high-resolution array-based comparative genomic hybridization (aCGH) we identified deletions of a region on chromosome 10q harboring INPP5A gene in 24% of examined SCC tumors. Subsequent validation by immunohistochemistry (IHC) on an independent sample set of 71 SCC tissues demonstrated reduced INPP5A protein levels in 72% of primary SCC tumors. Decrease in INPP5A protein levels appears to be an early event in SCC development as it also is observed in 9 of 26 (35%) examined actinic keratoses (AK), the earliest stage in SCC development. Importantly, further reduction of INPP5A levels is seen in a subset of SCC patients as the tumor progresses from primary to metastatic stage. The observed frequency and pattern of loss indicate that INPP5A, a negative regulator of inositol signaling, may play a role in development and progression of cutaneous SCC tumors.
doi:10.1158/1940-6207.CAPR-10-0058
PMCID: PMC2955780  PMID: 20876729
Inositol polyphosphate-5-phosphatase (INPP5A); Squamous Cell Carcinoma (SCC); Actinic Keratosis (AK); array based Comparative Genomic Hybridization (aCGH); Immunohistochemistry (IHC)
23.  Common sequence variants on 20q11.22 confer melanoma susceptibility 
Nature genetics  2008;40(7):838-840.
We conducted a genome-wide association pooling study for cutaneous melanoma and performed validation in samples totalling 2019 cases and 2105 controls. Using pooling we identified a novel melanoma risk locus on chromosome 20 (rs910873, rs1885120), with replication in two further samples (combined P <1 × 10-15). The odds ratio is 1.75 (1.53, 2.01), with evidence for stronger association in early onset cases.
doi:10.1038/ng.163
PMCID: PMC2755512  PMID: 18488026
24.  Optimization of oligonucleotide microarray fabricated by spotting 65-mer 
Analytical biochemistry  2007;368(1):61-69.
DNA microarrays currently provide measurements of sufficiently high quality to allow a wide variety of sound inferences about gene regulation and the coordination of cellular processes to be drawn. Nonetheless, a desire for greater precision in the measurements continues to drive the microarray research community to seek higher measurement quality through improvements in array fabrication and sample labeling and hybridization. We prepared oligonucleotide microarrays by printing 65-mer on aldehyde functional group derivatized slides as described in the previous study xxx. We could improve the reliability of data by removing enzymatic bias during probe labeling and hybridizing under a more stringent condition. This optimized method was used to profile gene expression patterns for nine different mouse tissues and organs, and MDS analysis of data showed both strong similarity between like samples and a clear, highly reproducible separation between different tissue samples. Three other microarrays were fabricated on commercial substrates and hybridized following the manufacturer’s instructions. The data was then compared with in-house microarray data and RT-PCR data. The microarray printed on the custom aldehyde slide was superior to microarrays printed on commercially available substrate slides in terms of signal intensities, background and hybridization characteristics. The data from the custom substrate microarray generally showed good agreement in quantitative changes up to 100-fold changes of transcript abundance with RT-PCR data. However, more accurate comparisons will be come as more genomic sequence information is gathered in public data domain.
doi:10.1016/j.ab.2007.06.005
PMCID: PMC2697255  PMID: 17618862
25.  PERFORMANCE CHARACTERISTICS OF 65-MER OLIGONUCLEOTIDE MICROARRAYS 
Analytical biochemistry  2007;368(1):70-78.
Microarray fabrication using pre-synthesized long oligonucleotide is becoming increasingly important, but a study of large-scale array productions is not published yet. We addressed the issue of fabricating oligonucleotide microarrays by spotting commercial, pre-synthesized 65-mers with 5′ amines representing 7500 murine genes. Amine-modified oligonucleotides were immobilized on glass slides having aldehyde groups via transient Schiff base formation followed by reduction to produce a covalent conjugate. When RNA derived from the same source was used for Cy3 and Cy5 labeling and hybridized to the same array, signal intensities spanning three orders of magnitude were observed, and the coefficient of variation between the two channels for all spots was 8–10%. To ascertain the reproducibility of ratio determination of these arrays, two triplicate hybridizations (with fluorochrome reversal) comparing RNAs from a fibroblast (NIH3T3) and a breast cancer (JC) cell line were carried out. The 95% confidence interval for all spots in the six hybridizations was 0.60 – 1.66. This level of reproducibility allows use of the full range of pattern finding and discriminant analysis typically applied to cDNA microarrays. Further comparative testing was carried out with oligonucleotide microarrays, cDNA microarrays and RT-PCR assays to examine the comparability of results across these different methodologies.
doi:10.1016/j.ab.2007.05.010
PMCID: PMC2697254  PMID: 17617369

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