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1.  LRIG1 as a Potential Novel Marker for Neoplastic Transformation in Ocular Surface Squamous Neoplasia 
PLoS ONE  2014;9(4):e93164.
The leucine rich repeats and immunoglobulin-like protein 1 (LRIG1) is a newly discovered negative regulator of epidermal growth factor receptor (EGFR) and a proposed tumor suppressor. It is not universally downregulated in human cancers, and its role in neoplastic transformation and tumorigenesis is not well-documented. In this study, we show the expression of LRIG1 as a novel potential marker for neoplastic transformation in ocular-surface squamous neoplasia (OSSN). The following two groups were included in this study: 1) benign group (3 cases; 1 with papilloma and 2 with dysplasia) and 2) malignant group (3 cases with squamous cell carcinoma (SCC)). In both groups, immunofluorescence analysis was firstly performed for keratins 4, 12, 13, and 15 to characterize the state of differentiation, and for Ki67 to evaluate the proliferation activity. Subsequently, LRIG1 and EGFR expression was analyzed. Either keratin 4 and/or 13, both non-keratinized epithelial cell markers, were generally expressed in both groups, except for 1 severe SCC case. Keratin 15, an undifferentiated basal cell marker, was more strongly expressed in the malignant cases than in the benign cases. The Ki67 index was significantly higher (P<0.002) in the malignant group (33.2%) than in the benign group (10.9%). LRIG1 expression was limited to basal epithelial cells in normal corneal epithelial tissue. Interestingly, LRIG1 was expressed throughout the epithelium in all the benign cases. In contrast, its expression was limited or totally disappeared in the malignant cases. Inversely, EGFR staining was faintly expressed in the benign cases, yet strongly expressed in the malignant cases. Malignant tissue with proliferative potential presented EGFR overexpression and inverse downregulation of LRIG1, consistent with LRIG1 being a suppressor of neoplastic transformation by counteracting the tumor growth property of EGFR. Our findings indicate that downregulation of LRIG1 is possibly a novel potential marker of transformation and tumorigenesis in OSSN cases.
PMCID: PMC3977825  PMID: 24709893
2.  Depletion of Cutaneous Macrophages and Dendritic Cells Promotes Growth of Basal Cell Carcinoma in Mice 
PLoS ONE  2014;9(4):e93555.
Basal cell carcinoma (BCC) belongs to the group of non-melanoma skin tumors and is the most common tumor in the western world. BCC arises due to mutations in the tumor suppressor gene Patched1 (Ptch). Analysis of the conditional Ptch knockout mouse model for BCC reveals that macrophages and dendritic cells (DC) of the skin play an important role in BCC growth restraining processes. This is based on the observation that a clodronate-liposome mediated depletion of these cells in the tumor-bearing skin results in significant BCC enlargement. The depletion of these cells does not modulate Ki67 or K10 expression, but is accompanied by a decrease in collagen-producing cells in the tumor stroma. Together, the data suggest that cutaneous macrophages and DC in the tumor microenvironment exert an antitumor effect on BCC.
PMCID: PMC3972151  PMID: 24691432
3.  Large Genomic Rearrangements of BRCA1 and BRCA2 among Patients Referred for Genetic Analysis in Galicia (NW Spain): Delimitation and Mechanism of Three Novel BRCA1 Rearrangements 
PLoS ONE  2014;9(3):e93306.
In the Iberian Peninsula, which includes mainly Spain and Portugal, large genomic rearrangements (LGRs) of BRCA1 and BRCA2 have respectively been found in up to 2.33% and 8.4% of families with hereditary breast and/or ovarian cancer (HBOC) that lack point mutations and small indels. In Galicia (Northwest Spain), the spectrum and frequency of BRCA1/BRCA2 point mutations differs from the rest of the Iberian populations. However, to date there are no Galician frequency reports of BRCA1/BRCA2 LGRs. Here we used multiplex ligation-dependent probe amplification (MLPA) to screen 651 Galician index cases (out of the 830 individuals referred for genetic analysis) without point mutations or small indels. We identified three different BRCA1 LGRs in four families. Two of them have been previously classified as pathogenic LGRs: the complete deletion of BRCA1 (identified in two unrelated families) and the deletion of exons 1 to 13. We also identified the duplication of exons 1 and 2 that is a LGR with unknown pathogenicity. Determination of the breakpoints of the BRCA1 LGRs using CNV/SNP arrays and sequencing identified them as NG_005905.2:g.70536_180359del, NG_005905.2:g.90012_97270dup, and NC_000017.10:g.41230935_41399840delinsAluSx1, respectively; previous observations of BRCA1 exon1-24del, exon1-2dup, and exon1-13del LGRs have not characterized them in such detail. All the BRCA1 LGRs arose from unequal homologous recombination events involving Alu elements. We also detected, by sequencing, one BRCA2 LGR, the Portuguese founder mutation c.156_157insAluYa5. The low frequency of BRCA1 LGRs within BRCA1 mutation carriers in Galicia (2.34%, 95% CI: 0.61–7.22) seems to differ from the Spanish population (9.93%, 95% CI: 6.76–14.27, P-value = 0.013) and from the rest of the Iberian population (9.76%, 95% CI: 6.69–13.94, P-value = 0.014).
PMCID: PMC3970959  PMID: 24686251
4.  Aurora-A V57I (rs1047972) Polymorphism and Cancer Susceptibility: A Meta-Analysis Involving 27,269 Subjects 
PLoS ONE  2014;9(3):e90328.
The association between Aurora-A V57I (rs1047972, G>A) polymorphism and cancer susceptibility has been widely studied. However, the results are inconsistent.
Methodology/Principal Findings
To obtain a more precise evaluation of the relationship, we performed a meta-analysis of 14 case-control studies involving a total of 11,245 cancer cases and 16,024 controls. Our results demonstrated that there was a borderline evidence of an association between the Aurora-A V57I polymorphism and the decreased risk of overall cancer in two genetic models: AA vs. GA+GG and AA vs. GG. In a stratified analysis by cancer type, significant association between Aurora-A V57I polymorphism and the decreased risk of breast cancer was identified in one genetic model: AA vs. GG. In a stratified analysis by ethnicity, in three genetic models, significant decreased cancer risk was observed among Caucasians (AA vs. GA+GG; AA vs. GG and A vs. G) instead of Asians. Furthermore, a stratified analysis by ethnicity in breast cancer subgroup, five genetic models (AA+GA vs. GG; AA vs. GA+GG; AA vs. GG; AA vs. GA and A vs. G), significant decreased cancer risk was observed among Caucasians, but not among Asians. A slight publication bias was observed in our meta-analysis, thus nonparametric “trim-and-fill” method was utilized to detect the stability of our results. The adjusted odds ratios and confidence intervals showed that Aurora-A V57I polymorphism might be a protective factor for cancer risk, suggesting the reliability of our findings.
In summary, this meta-analysis suggests that Aurora-A V57I polymorphism may be a protective factor for cancer risk.
PMCID: PMC3943872  PMID: 24598702
5.  Loss of Grainy Head-Like 1 Is Associated with Disruption of the Epidermal Barrier and Squamous Cell Carcinoma of the Skin 
PLoS ONE  2014;9(2):e89247.
The Grainyhead-like 1 (GRHL1) transcription factor regulates the expression of desmosomal cadherin desmoglein 1 (Dsg1) in suprabasal layers of the epidermis. As a consequence, the epidermis of Grhl1-null mice displays fewer desmosomes that are abnormal in structure. These mice also exhibit mild chronic skin barrier defects as evidenced by altered keratinocyte terminal differentiation, increased expression of inflammatory markers and infiltration of the skin by immune cells. Exposure of Grhl1−/− mice to a standard chemical skin carcinogenesis protocol results in development of fewer papillomas than in wild type control animals, but with a rate of conversion to squamous cell carcinoma (SCC) that is strikingly higher than in normal littermates. The underlying molecular mechanism differs from mice with conditional ablation of a closely related Grhl family member, Grhl3, in the skin, which develop SCC due to the loss of expression of phosphatase and tensin homolog (PTEN) and activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) signaling pathway.
PMCID: PMC3930704  PMID: 24586629
6.  A Gene Expression and Pre-mRNA Splicing Signature That Marks the Adenoma-Adenocarcinoma Progression in Colorectal Cancer 
PLoS ONE  2014;9(2):e87761.
It is widely accepted that most colorectal cancers (CRCs) arise from colorectal adenomas (CRAs), but transcriptomic data characterizing the progression from colorectal normal mucosa to adenoma, and then to adenocarcinoma are scarce. These transition steps were investigated using microarrays, both at the level of gene expression and alternative pre-mRNA splicing. Many genes and exons were abnormally expressed in CRAs, even more than in CRCs, as compared to normal mucosae. Known biological pathways involved in CRC were altered in CRA, but several new enriched pathways were also recognized, such as the complement and coagulation cascades. We also identified four intersectional transcriptional signatures that could distinguish CRAs from normal mucosae or CRCs, including a signature of 40 genes differentially deregulated in both CRA and CRC samples. A majority of these genes had been described in different cancers, including FBLN1 or INHBA, but only a few in CRC. Several of these changes were also observed at the protein level. In addition, 20% of these genes (i.e. CFH, CRYAB, DPT, FBLN1, ITIH5, NR3C2, SLIT3 and TIMP1) showed altered pre-mRNA splicing in CRAs. As a global variation occurring since the CRA stage, and maintained in CRC, the expression and splicing changes of this 40-gene set may mark the risk of cancer occurrence from analysis of CRA biopsies.
PMCID: PMC3916340  PMID: 24516561
7.  Longer Telomeres Are Associated with Cancer Risk in MMR-Proficient Hereditary Non-Polyposis Colorectal Cancer 
PLoS ONE  2014;9(2):e86063.
Aberrant telomere length measured in blood has been associated with increased risk of several cancer types. In the field of hereditary non-polyposis colorectal cancer (CRC), and more particularly in Lynch syndrome, caused by germline mutations in the mismatch repair (MMR) genes, we recently found that cancer-affected MMR gene mutation carriers had shorter telomeres and more pronounced shortening of telomere length with age than controls and unaffected MMR gene mutation carriers. Here we evaluate blood telomere length in MMR-proficient hereditary non-polyposis CRC, i.e. familial CRC type X (fCRC-X). A total of 57 cancer-affected and 57 cancer-free individuals from 34 Amsterdam-positive fCRC-X families were analyzed and compared to the data previously published on 144 cancer-affected and 100 cancer-free MMR gene mutation carriers, and 234 controls. Relative telomere length was measured using a monochrome multiplex quantitative PCR method, following strict measures to avoid sources of bias and adjusting by age. Despite the retrospective nature of our study, the results show that longer telomeres associate with cancer risk in fCRC-X, thus identifying different patterns of telomere length according to the status of the MMR system.
PMCID: PMC3911901  PMID: 24498269
8.  Genetic Diversity of the KIR/HLA System and Outcome of Patients with Metastatic Colorectal Cancer Treated with Chemotherapy 
PLoS ONE  2014;9(1):e84940.
To explore genes of the killer-cell immunoglobulin-like receptor (KIR) and of the HLA ligand and their relationship with the outcome of metastatic colorectal cancer (mCRC) patients treated with first-line 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI).
A total of 224 mCRC patients were screened for KIR/HLA typing. The determination of the KIR/HLA combinations was based upon the gene content and variants. Genetic associations with complete response (CR), time to progression (TTP) and overall survival (OS) were evaluated by calculating odds and hazard ratios. Multivariate modeling with prognostic covariates was also performed.
For CR, the presence of KIR2DL5A, 2DS5, 2DS1, 3DS1, and KIR3DS1/HLA-Bw4-I80 was associated with increased CR rates, with median ORs ranging from 2.1 to 4.3, while the absence of KIR2DS4 and 3DL1 was associated with increased CR rates (OR 3.1). After univariate analysis, patients that underwent resective surgery of tumor, absence of KIR2DS5, and presence of KIR3DL1/HLA-Bw4-I80 showed a significant better OS (HR 1.5 to 2.8). Multivariate analysis identified as parameters independently related to OS the type of treatment (surgery; HR 2.0) and KIR3DL1/HLA-Bw4-I80 genotype (HR for T-I80 2.7 and for no functional KIR/HLA interaction 1.8). For TTP, no association with KIR/HLA genes was observed.
This study, for the first time, evidences that the genotyping for KIR-HLA pairs are found predictive markers associated with complete response and improves overall survival prediction of FOLFIRI treatment response in metastatic colorectal cancer. These results suggest a role of the KIR/HLA system in patient outcome, and guide new research on the immunogenetics of mCRC through mechanistic studies and clinical validation.
PMCID: PMC3908861  PMID: 24497922
9.  Analysis of BRCA1 Variants in Double-Strand Break Repair by Homologous Recombination and Single-Strand Annealing 
Human mutation  2012;34(3):439-445.
Missense substitutions of uncertain clinical significance in the BRCA1 gene are a vexing problem in genetic counseling for women who have a family history of breast cancer. In this study, we evaluated the functions of 29 missense substitutions of BRCA1 in two DNA repair pathways. Repair of double-strand breaks by homology-directed recombination (HDR) had been previously analyzed for 16 of these BRCA1 variants, and 13 more variants were analyzed in this study. All 29 variants were also analyzed for function in double-strand break repair by the single-strand annealing (SSA) pathway. We found that among the pathogenic mutations in BRCA1, all were defective for DNA repair by either pathway. The HDR assay was accurate because all pathogenic mutants were defective for HDR, and all nonpathogenic variants were fully functional for HDR. Repair by SSA accurately identified pathogenic mutants, but several nonpathogenic variants were scored as defective or partially defective. These results indicated that specific amino acid residues of the BRCA1 protein have different effects in the two related DNA repair pathways, and these results validate the HDR assay as highly correlative with BRCA1-associated breast cancer.
PMCID: PMC3906639  PMID: 23161852
BRCA1; homologous recombination; single-strand annealing; centrosome; VUS
10.  On the Reproducibility of TCGA Ovarian Cancer MicroRNA Profiles 
PLoS ONE  2014;9(1):e87782.
Dysregulated microRNA (miRNA) expression is a well-established feature of human cancer. However, the role of specific miRNAs in determining cancer outcomes remains unclear. Using Level 3 expression data from the Cancer Genome Atlas (TCGA), we identified 61 miRNAs that are associated with overall survival in 469 ovarian cancers profiled by microarray (p<0.01). We also identified 12 miRNAs that are associated with survival when miRNAs were profiled in the same specimens using Next Generation Sequencing (miRNA-Seq) (p<0.01). Surprisingly, only 1 miRNA transcript is associated with ovarian cancer survival in both datasets. Our analyses indicate that this discrepancy is due to the fact that miRNA levels reported by the two platforms correlate poorly, even after correcting for potential issues inherent to signal detection algorithms. Corrections for false discovery and microRNA abundance had minimal impact on this discrepancy. Further investigation is warranted.
PMCID: PMC3906208  PMID: 24489963
11.  Identification of a Recurrent STRN/ALK Fusion in Thyroid Carcinomas 
PLoS ONE  2014;9(1):e87170.
Thyroid carcinoma is the most common endocrine malignant tumor and accounts for 1% of all new malignant diseases. Among all types and subtypes of thyroid cancers that have been described so far, papillary thyroid carcinoma is the most frequent. The standard management treatment of these tumors consists of surgery, followed by radioiodine treatment in case of high risk of relapse. The most aggressive forms are commonly treated by chemotherapy, radiotherapy or experimental drug testing. We recently reported the case of a patient presenting an anaplastic thyroid carcinoma with lung metastases. Fluorescence in situ hybridization analysis allowed us to detect a rearrangement of the anaplastic lymphoma kinase (ALK) gene in both tumors. The patient was treated with crizotinib and presented an excellent drug response. We present here the subsequent investigations carried out to further characterize this genetic alteration and to assess the prevalence of ALK rearrangements in thyroid lesions. High resolution array-comparative genomic hybridization data complemented by RT-PCR and sequencing analyses, allowed us to demonstrate the presence of a STRN/ALK fusion. The STRN/ALK transcript consisted of the fusion between exon 3 of STRN and exon 20 of ALK. Subsequent screening of 75 various thyroid tumors by RT-PCR revealed that 2 out of 29 papillary thyroid carcinomas exhibited the same fusion transcript. None was detected in other types of malignant or benign thyroid lesions analyzed. These findings could pave the way for the development of new targeted therapeutic strategies in the treatment of papillary thyroid carcinomas and point to ALK inhibitors as promising agents that merit rapid evaluation.
PMCID: PMC3903624  PMID: 24475247
12.  Assessment of Whole Genome Amplification for Sequence Capture and Massively Parallel Sequencing 
PLoS ONE  2014;9(1):e84785.
Exome sequence capture and massively parallel sequencing can be combined to achieve inexpensive and rapid global analyses of the functional sections of the genome. The difficulties of working with relatively small quantities of genetic material, as may be necessary when sharing tumor biopsies between collaborators for instance, can be overcome using whole genome amplification. However, the potential drawbacks of using a whole genome amplification technology based on random primers in combination with sequence capture followed by massively parallel sequencing have not yet been examined in detail, especially in the context of mutation discovery in tumor material. In this work, we compare mutations detected in sequence data for unamplified DNA, whole genome amplified DNA, and RNA originating from the same tumor tissue samples from 16 patients diagnosed with non-small cell lung cancer. The results obtained provide a comprehensive overview of the merits of these techniques for mutation analysis. We evaluated the identified genetic variants, and found that most (74%) of them were observed in both the amplified and the unamplified sequence data. Eighty-nine percent of the variations found by WGA were shared with unamplified DNA. We demonstrate a strategy for avoiding allelic bias by including RNA-sequencing information.
PMCID: PMC3883664  PMID: 24409309
13.  Allele-specific imbalance mapping identifies HDAC9 as a candidate gene for cutaneous squamous cell carcinoma 
More than 3.5 million non-melanoma skin cancers were treated in 2006; of these 700,000 were cutaneous squamous cell carcinomas (cSCC). Despite clear environmental causes for cSCC, studies also suggest genetic risk factors. A cSCC susceptibility locus, Skts5, was identified on mouse chromosome 12 by linkage analysis. The orthologous locus to Skts5 in humans maps to 7p21 and 7q31. These loci show copy number increases in approximately 10% of cSCC tumors. Here we show that an additional 15-22% of tumors exhibit copy-neutral loss of heterozygosity. Furthermore, our previous data identified microsatellite markers on 7p21 and 7q31 that demonstrate preferential allelic imbalance (PAI) in cSCC tumors. Based on these results, we hypothesized that the human orthologous locus to Skts5 would house a gene important in human cSCC development and that tumors would demonstrate allele-specific somatic alterations. To test this hypothesis, we performed quantitative genotyping of 108 single nucleotide polymorphisms (SNPs) mapping to candidate genes at human SKTS5 in paired normal and tumor DNAs. Nine SNPs in HDAC9 (rs801540, rs1178108, rs1178112, rs1726610, rs10243618, rs11764116, rs1178355, rs10269422, and rs12540872) showed PAI in tumors. These data suggest that HDAC9 variants may be selected for during cSCC tumorigenesis.
PMCID: PMC3831612  PMID: 23784969
HDAC9; cutaneous squamous cell carcinoma; allelic-specific imbalance; Skts5
14.  Joint Effect of Multiple Common SNPs Predicts Melanoma Susceptibility  
PLoS ONE  2013;8(12):e85642.
Single genetic variants discovered so far have been only weakly associated with melanoma. This study aims to use multiple single nucleotide polymorphisms (SNPs) jointly to obtain a larger genetic effect and to improve the predictive value of a conventional phenotypic model. We analyzed 11 SNPs that were associated with melanoma risk in previous studies and were genotyped in MD Anderson Cancer Center (MDACC) and Harvard Medical School investigations. Participants with ≥15 risk alleles were 5-fold more likely to have melanoma compared to those carrying ≤6. Compared to a model using the most significant single variant rs12913832, the increase in predictive value for the model using a polygenic risk score (PRS) comprised of 11 SNPs was 0.07(95% CI, 0.05-0.07). The overall predictive value of the PRS together with conventional phenotypic factors in the MDACC population was 0.69 (95% CI, 0.64-0.69). PRS significantly improved the risk prediction and reclassification in melanoma as compared with the conventional model. Our study suggests that a polygenic profile can improve the predictive value of an individual gene polymorphism and may be able to significantly improve the predictive value beyond conventional phenotypic melanoma risk factors.
PMCID: PMC3877376  PMID: 24392023
15.  Identification of Candidate Driver Genes in Common Focal Chromosomal Aberrations of Microsatellite Stable Colorectal Cancer 
PLoS ONE  2013;8(12):e83859.
Colorectal cancer (CRC) is a leading cause of cancer deaths worldwide. Chromosomal instability (CIN) is a major driving force of microsatellite stable (MSS) sporadic CRC. CIN tumours are characterised by a large number of somatic chromosomal copy number aberrations (SCNA) that frequently affect oncogenes and tumour suppressor genes. The main aim of this work was to identify novel candidate CRC driver genes affected by recurrent and focal SCNA. High resolution genome-wide comparative genome hybridisation (CGH) arrays were used to compare tumour and normal DNA for 53 sporadic CRC cases. Context corrected common aberration (COCA) analysis and custom algorithms identified 64 deletions and 32 gains of focal minimal common regions (FMCR) at high frequency (>10%). Comparison of these FMCR with published genomic profiles from CRC revealed common overlap (42.2% of deletions and 34.4% of copy gains). Pathway analysis showed that apoptosis and p53 signalling pathways were commonly affected by deleted FMCR, and MAPK and potassium channel pathways by gains of FMCR. Candidate tumour suppressor genes in deleted FMCR included RASSF3, IFNAR1, IFNAR2 and NFKBIA and candidate oncogenes in gained FMCR included PRDM16, TNS1, RPA3 and KCNMA1. In conclusion, this study confirms some previously identified aberrations in MSS CRC and provides in silico evidence for some novel candidate driver genes.
PMCID: PMC3867468  PMID: 24367615
16.  SEPP1 Influences Breast Cancer Risk among Women with Greater Native American Ancestry: The Breast Cancer Health Disparities Study 
PLoS ONE  2013;8(11):e80554.
Selenoproteins are a class of proteins containing a selenocysteine residue, many of which have been shown to have redox functions, acting as antioxidants to decrease oxidative stress. Selenoproteins have previously been associated with risk of various cancers and redox-related diseases. In this study we evaluated possible associations between breast cancer risk and survival and single nucleotide polymorphisms (SNPs) in the selenoprotein genes GPX1, GPX2, GPX3, GPX4, SELS, SEP15, SEPN1, SEPP1, SEPW1, TXNRD1, and TXNRD2 among Hispanic/Native American (2111 cases, 2597 controls) and non-Hispanic white (NHW) (1481 cases, 1586 controls) women in the Breast Cancer Health Disparities Study. Adaptive Rank Truncated Product (ARTP) analysis was used to determine both gene and pathway significance with these genes. The overall selenoprotein pathway PARTP was not significantly associated with breast cancer risk (PARTP = 0.69), and only one gene, GPX3, was of borderline significance for the overall population (PARTP =0.09) and marginally significant among women with 0-28% Native American (NA) ancestry (PARTP=0.06). The SEPP1 gene was statistically significantly associated with breast cancer risk among women with higher NA ancestry (PARTP=0.002) and contributed to a significant pathway among those women (PARTP=0.04). GPX1, GPX3, and SELS were associated with Estrogen Receptor-/Progesterone Receptor+ status (PARTP = 0.002, 0.05, and 0.01, respectively). Four SNPs (GPX3 rs2070593, rsGPX4 rs2074451, SELS rs9874, and TXNRD1 rs17202060) significantly interacted with dietary oxidative balance score after adjustment for multiple comparisons to alter breast cancer risk. GPX4 was significantly associated with breast cancer survival among those with the highest NA ancestry (PARTP = 0.05) only. Our data suggest that SEPP1 alters breast cancer risk among women with higher levels of NA ancestry.
PMCID: PMC3835321  PMID: 24278290
17.  Integrative Analysis of Hereditary Nonpolyposis Colorectal Cancer: the Contribution of Allele-Specific Expression and Other Assays to Diagnostic Algorithms 
PLoS ONE  2013;8(11):e81194.
The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC) is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS). Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE), nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR) genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI) and immunohistochemical (IHC) MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5%) probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.
PMCID: PMC3835792  PMID: 24278394
18.  TERT Promoter Mutations Are Frequent in Cutaneous Basal Cell Carcinoma and Squamous Cell Carcinoma 
PLoS ONE  2013;8(11):e80354.
Activating mutations in the TERT promoter were recently identified in up to 71% of cutaneous melanoma. Subsequent studies found TERT promoter mutations in a wide array of other major human cancers. TERT promoter mutations lead to increased expression of telomerase, which maintains telomere length and genomic stability, thereby allowing cancer cells to continuously divide, avoiding senescence or apoptosis. TERT promoter mutations in cutaneous melanoma often show UV-signatures. Non-melanoma skin cancer, including basal cell carcinoma and squamous cell carcinoma, are very frequent malignancies in individuals of European descent. We investigated the presence of TERT promoter mutations in 32 basal cell carcinomas and 34 cutaneous squamous cell carcinomas using conventional Sanger sequencing. TERT promoter mutations were identified in 18 (56%) basal cell carcinomas and in 17 (50%) cutaneous squamous cell carcinomas. The recurrent mutations identified in our cohort were identical to those previously described in cutaneous melanoma, and showed a UV-signature (C>T or CC>TT) in line with a causative role for UV exposure in these common cutaneous malignancies. Our study shows that TERT promoter mutations with UV-signatures are frequent in non-melanoma skin cancer, being present in around 50% of basal and squamous cell carcinomas and suggests that increased expression of telomerase plays an important role in the pathogenesis of these tumors.
PMCID: PMC3832433  PMID: 24260374
19.  Deciphering Genomic Alterations in Colorectal Cancer through Transcriptional Subtype-Based Network Analysis 
PLoS ONE  2013;8(11):e79282.
Both transcriptional subtype and signaling network analyses have proved useful in cancer genomics research. However, these two approaches are usually applied in isolation in existing studies. We reason that deciphering genomic alterations based on cancer transcriptional subtypes may help reveal subtype-specific driver networks and provide insights for the development of personalized therapeutic strategies. In this study, we defined transcriptional subtypes for colorectal cancer (CRC) and identified driver networks/pathways for each subtype. Applying consensus clustering to a patient cohort with 1173 samples identified three transcriptional subtypes, which were validated in an independent cohort with 485 samples. The three subtypes were characterized by different transcriptional programs related to normal adult colon, early colon embryonic development, and epithelial mesenchymal transition, respectively. They also showed statistically different clinical outcomes. For each subtype, we mapped somatic mutation and copy number variation data onto an integrated signaling network and identified subtype-specific driver networks using a random walk-based strategy. We found that genomic alterations in the Wnt signaling pathway were common among all three subtypes; however, unique combinations of pathway alterations including Wnt, VEGF and Notch drove distinct molecular and clinical phenotypes in different CRC subtypes. Our results provide a coherent and integrated picture of human CRC that links genomic alterations to molecular and clinical consequences, and which provides insights for the development of personalized therapeutic strategies for different CRC subtypes.
PMCID: PMC3829853  PMID: 24260186
20.  A Common Variant in the SIAH2 Locus Is Associated with Estrogen Receptor-Positive Breast Cancer in the Chinese Han Population 
PLoS ONE  2013;8(11):e79365.
Genome-wide association studies (GWAS) have identified many loci associated with breast cancer risk. These studies have primarily been conducted in populations of European descent.
To determine whether previously reported susceptibility loci in other ethnic groups are also risk factors for breast cancer in a Chinese population.
We genotyped 21 previously reported single nucleotide polymorphisms (SNPs) within a female Chinese cohort of 1203 breast cancer cases and 2525 healthy controls using the Sequenom iPlex platform. Fourteen SNPs passed the quality control test. These SNPs were subjected to statistical analysis for the entire cohort and were further analyzed for estrogen receptor (ER) status. The associations of the SNPs with disease susceptibility were assessed using logistic regression, adjusting for age. The Bonferroni correction was used to conservatively account for multiple testing, and the threshold for statistical significance was P<3.57×10−3 (0.05/14).
Although none of the SNPs showed an overall association with breast cancer, an analysis of the ER status of the breast cancer patients revealed that the SIAH2 locus (rs6788895; P = 5.73×10−4, odds ratio [OR] = 0.81) is associated with ER-positive breast cancer.
A common variant in the SIAH2 locus is associated with ER-positive breast cancer in the Chinese Han population. The replication of published GWAS results in other ethnic groups provides important information regarding the genetic etiology of breast cancer.
PMCID: PMC3823686  PMID: 24244489
21.  Characteristics and Prognostic Implications of High-Risk HPV-Associated Hypopharyngeal Cancers 
PLoS ONE  2013;8(11):e78718.
High-risk human papillomavirus (HPV) is an oncogenic virus that causes oropharyngeal cancers, and it has a favorable outcome after the treatment. Unlike in oropharyngeal cancer, the prevalence and role of high-risk HPV in the etiology of hypopharyngeal squamous cell carcinoma (HPSCC) is uncertain.
The aim of the present study was to evaluate the effect and prognostic significance of high-risk HPV in patients with HPSCC.
The study included 64 subjects with HPSCC who underwent radical surgery with or without radiation-based adjuvant therapy. Primary tumor sites were the pyriform sinus in 42 patients, posterior pharyngeal wall in 19 patients, and postcricoid area in 3 patients. High-risk HPV in situ hybridization was performed to detect HPV infection.
The positive rate of high-risk HPV in situ hybridization was 10.9% (7/64). There was a significant difference in the fraction of positive high-risk HPV among pyriform sinus cancer (16.7%), posterior pharyngeal wall cancer (0%), and postcricoid area cancer (0%) (p = 0.042). The laryngoscopic examination revealed a granulomatous and exophytic appearance in 85.7% (6/7) of patients with high-risk HPV-positive pyriform sinus cancer, but in only 31.4% (11/35) of patients with high-risk HPV-negative pyriform sinus cancer (p = 0.012). Significant correlations were found between positive high-risk HPV and younger age (p = 0.050) and non-smoking status (p = 0.017). HPV-positive patients had a significantly better disease-free survival (p = 0.026) and disease-specific survival (p = 0.047) than HPV-negative patients.
High-risk HPV infection is significantly related to pyriform sinus cancer in patients with HPSCC.
PMCID: PMC3823884  PMID: 24244346
22.  Arg72Pro Polymorphism of TP53 Gene and the Risk of Skin Cancer: a Meta-Analysis 
PLoS ONE  2013;8(11):e79983.
TP53 gene is one of the most important tumor suppressor genes. We undertook this meta-analysis to explore the association between TP53 Arg72Pro polymorphism and the risk of skin cancer mainly in Caucasians.
We searched PubMed for case-control studies published up to March 2013. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association.
A total of 5276 skin cancer cases and 5315 controls from 20 studies were included. Overall, no significant association between TP53 Arg72Pro polymorphism and skin cancer was observed in all genetic contrast models (Pro/Pro versus Arg/Arg, Pro/Arg versus Arg/Arg, Pro/Pro + Pro/Arg versus Arg/Arg, Pro/Pro versus Arg/Arg + Pro/Arg, Pro allele versus Arg allele). Similar results were obtained in the stratified analysis by ethnicity and histological types of skin cancer, such as melanoma, squamous cell carcinoma and basal cell carcinoma. Power calculations indicated that some studies were underpowered. No publication bias was found by using the funnel plot and Egger's test.
This meta-analysis indicated that TP53 Arg72Pro polymorphism probably had little association with skin cancer susceptibility mainly in Caucasians. However, larger sample-size studies are required to verify the conclusion as low statistical powers.
PMCID: PMC3832645  PMID: 24260330
23.  The Homeobox Gene MEIS1 Is Methylated in BRAFp.V600E Mutated Colon Tumors 
PLoS ONE  2013;8(11):e79898.
Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAFp.V600E mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAFp.V600E mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAFp.V600E with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10-9). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAFp.V600E and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1D27, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAFp.V600E tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAFp.V600E stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAFp.V600E colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression.
PMCID: PMC3820613  PMID: 24244575
24.  Identification and Characterization of CDH1 Germline Variants in Sporadic Gastric Cancer Patients and in Individuals at Risk of Gastric Cancer 
PLoS ONE  2013;8(10):e77035.
To screen and characterize germline variants for E-cadherin (CDH1) in non-hereditary gastric cancer (GC) patients and in subjects at risk of GC.
59 GCs, 59 first degree relatives (FDRs) of GC, 20 autoimmune metaplastic atrophic gastritis (AMAGs) and 52 blood donors (BDs) were analyzed for CDH1 by direct sequencing, structural modelling and bioinformatics. Functional impact on splicing was assessed for intronic mutations. E-cadherin/β-catenin immunohistochemical staining and E-cadherin mRNA quantification using RT-PCR were performed.
In GCs, 4 missense variants (p.G274S; p.A298T; p.T470I; p.A592T), 1 mutation in the 5′UTR (−71C>G) and 1 mutation in the intronic IVS12 (c.1937-13T>C) region were found. First pathogenic effect of p.A298T mutation was predicted by protein 3D modelling. The novel p.G274S mutation showed a no clear functional significance. Moreover, first, intronic IVS12 (c.1937-13T>C) mutation was demonstrated to lead to an aberrant CDH1 transcript with exon 11 deletion. This mutation was found in 2 GCs and in 1 BD. In FDRs, we identified 4 variants: the polymorphic (p.A592T) and 3 mutations in untranslated regions with unidentified functional role except for the 5′UTR (−54G>C) that had been found to decrease CDH1 transcription. In AMAGs, we detected 2 alterations: 1 missense (p.A592T) and 1 novel variant (IVS1 (c.48+7C>T)) without effect on CDH1 splicing. Several silent and polymorphic substitutions were found in all the groups studied.
Overall our study improves upon the current characterization of CDH1 mutations and their functional role in GC and in individuals at risk of GC. Mutations found in untranslated regions and data on splicing effects deserve a particular attention like associated with a reduced E-cadherin amount. The utility of CDH1 screening, in addition to the identification of other risk factors, could be useful for the early detection of GC in subjects at risk (i.e. FDRs and AMAGs), and warrants further study.
PMCID: PMC3812172  PMID: 24204729
25.  BRCA1 R1699Q variant displaying ambiguous functional abrogation confers intermediate breast and ovarian cancer risk 
Journal of medical genetics  2012;49(8):10.1136/jmedgenet-2012-101037.
Clinical classification of rare sequence changes identified in the breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. We previously showed that variant BRCA1 c.5096G>A p. Arg1699Gln in the BRCA1 transcriptional transactivation domain demonstrated equivocal results from a series of functional assays, and proposed that this variant may confer low to moderate risk of cancer.
Measures of genetic risk (report of family history, segregation) were assessed for 68 BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) families recruited through family cancer clinics, comparing results with 34 families carrying the previously classified pathogenic BRCA1 c.5095C>T p.Arg1699Trp (R1699W) mutation at the same residue, and to 243 breast cancer families with no BRCA1 pathogenic mutation (BRCA-X).
Comparison of BRCA1 carrier prediction scores of probands using the BOADICEA risk prediction tool revealed that BRCA1 c.5096G>A p.Arg1699Gln variant carriers had family histories that were less ‘BRCA1-like’ than BRCA1 c.5095C>T p.Arg1699Trp mutation carriers (p<0.00001), but more ‘BRCA1-like’ than BRCA-X families (p=0.0004). Further, modified segregation analysis of the subset of 30 families with additional genotyping showed that BRCA1 c.5096G >A p. Arg1699Gln had reduced penetrance compared with the average truncating BRCA1 mutation penetrance (p=0.0002), with estimated cumulative risks to age 70 of breast or ovarian cancer of 24%.
Our results provide substantial evidence that the BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) variant, demonstrating ambiguous functional deficiency across multiple assays, is associated with intermediate risk of breast and ovarian cancer, highlighting challenges for risk modelling and clinical management of patients of this and other potential moderate-risk variants.
PMCID: PMC3810416  PMID: 22889855

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