Association of SULT1A1 Arg²¹³His polymorphism with male breast cancer risk: results from a multicenter study in Italy.

Male breast cancer (MBC) is rare and poorly understood. Like female breast cancer (FBC), MBCs are highly sensitive to hormonal changes, and hyperestrogenism, specifically, represents a major risk factor for MBC. MBC is considered similar to late-onset, post-menopausal estrogen/progesteron receptors positive FBC (ER+/PR+). Sulfotransferase 1A1 (SULT1A1) is an enzyme involved in the metabolism of estrogens. Recently, SULT1A1 common functional polymorphism Arg(213)His (638G>A) variant has been found to be associated with increased breast cancer (BC) risk, particularly in post-menopausal women. For this reason, we decided to explore whether SULT1A1 Arg(213)His could exert an effect on MBC development. The primary aim of this study was to evaluate the influence of the SULT1A1 Arg(213)His polymorphism on MBC risk. The secondary aim was to investigate possible associations with relevant clinical-pathologic features of MBC. A total of 394 MBC cases and 786 healthy male controls were genotyped for SULT1A1 Arg(213)His polymorphism by PCR-RFLP and high-resolution melting analysis. All MBC cases were characterized for relevant clinical-pathologic features. A significant difference in the distribution of SULT1A1 Arg(213)His genotypes was found between MBC cases and controls (P < 0.0001). The analysis of genotype-specific risk showed a significant increased MBC risk in individuals with G/A (OR 1.97, 95% CI 1.50-2.59; P < 0.0001) and A/A (OR 3.09, 95% CI 1.83-5.23; P < 0.0001) genotypes in comparison to wild-type genotype, under co-dominant model. A significant association between SULT1A1 risk genotypes and HER2 status emerged. Results indicate that SULT1A1 Arg(213)His may act as a low-penetrance risk allele for developing MBC and could be associated with a specific tumor subtype associated with HER2 overexpression.

Risk of desmoid tumours after open and laparoscopic colectomy in patients with familial adenomatous polyposis.

BACKGROUND: Desmoid tumour (DT) is a main cause of death after prophylactic colectomy in patients with familial adenomatous polyposis (FAP). The purpose of this study was to evaluate the impact of prophylactic laparoscopic colectomy on the risk of developing DT in patients with FAP. METHODS: The database of a single institution was reviewed. Patients with classical FAP with defined genotype who underwent either open or laparoscopic colectomy between 1947 and 2011 were included in the study. The impact of various demographic and clinical features on the risk of developing DT was assessed. RESULTS: A total of 672 patients underwent prophylactic colectomy: 602 by an open and 70 by a laparoscopic approach. With a median (range) follow-up of 132 (0-516) months in the open group and 60 (12-108) months in the laparoscopic group, 98 patients (16·3 per cent) developed DT after an open procedure compared with three (4 per cent) following laparoscopic surgery. The estimated cumulative risk of developing DT at 5 years after surgery was 13·0 per cent in the open group and 4 per cent in the laparoscopic group (P = 0·042). In multivariable analysis, female sex (hazard ratio (HR) 2·18, 95 per cent confidence interval 1·40 to 3·39), adenomatous polyposis coli mutation distal to codon 1400 (HR 3·85, 1·90 to 7·80), proctocolectomy (HR 1·67, 1·06 to 2·61), open colectomy (HR 6·84, 1·96 to 23·98) and year of surgery (HR 1·04, 1·01 to 1·07) were independent risk factors for the diagnosis of DT after prophylactic surgery. CONCLUSION: Laparoscopic surgery decreased the risk of DT after prophylactic colectomy in patients with FAP.

Association of low-penetrance alleles with male breast cancer risk and clinicopathological characteristics: results from a multicenter study in Italy.

It is well-known that male breast cancer (MBC) susceptibility is mainly due to high-penetrance BRCA1/2 mutations. Here, we investigated whether common low-penetrance breast cancer (BC) susceptibility alleles may influence MBC risk in Italian population and whether variant alleles may be associated with specific clinicopathological features of MBCs. In the frame of the Italian Multicenter Study on MBC, we genotyped 413 MBCs and 745 age-matched male controls at 9 SNPs annotating known BC susceptibility loci. By multivariate logistic regression models, we found a significant increased MBC risk for 3 SNPs, in particular, with codominant models, for rs2046210/ESR1 (OR = 1.71; 95 % CI: 1.43-2.05; p = 0.0001), rs3803662/TOX3 (OR = 1.59; 95 % CI: 1.32-1.92; p = 0.0001), and rs2981582/FGFR2 (OR = 1.26; 95 % CI: 1.05-1.50; p = 0.013). Furthermore, we showed that the prevalence of the risk genotypes of ESR1 tended to be higher in ER- tumors (p = 0.062). In a case-case multivariate analysis, a statistically significant association between ESR1 and ER- tumors was found (OR = 1.88; 95 % CI: 1.03-3.49; p = 0.039). Overall, our data, based on a large and well-characterized MBC series, support the hypothesis that common low-penetrance BC susceptibility alleles play a role in MBC susceptibility and, interestingly, indicate that ESR1 is associated with a distinct tumor subtype defined by ER-negative status.

Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome linked to inherited defects in human mismatch repair (MMR) genes. Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref. 3), making functional characterization of mutations in hMLH1 critical to the development of genetic testing for HNPCC. Here, we describe a new method for detecting mutations in hMLH1 using a dominant mutator effect of hMLH1 cDNA expressed in Saccharomyces cerevisiae. The majority of hMLH1 missense mutations identified in HNPCC patients abolish the dominant mutator effect. Furthermore, PCR amplification of hMLH1 cDNA from mRNA from a HNPCC patient, followed by in vivo recombination into a gap expression vector, allowed detection of a heterozygous loss-of-function missense mutation in hMLH1 using this method. This functional assay offers a simple method for detecting and evaluating pathogenic mutations in hMLH1.

Unclassified variants in BRCA genes: guidelines for interpretation.

In the last few years, several studies have focused on the interpretation of unclassified variants (UVs) of BRCA1 and BRCA2 genes. Analysis of UVs through a unique approach is not sufficient to understand their role in the development of tumors. Thus, it is clear that assembling results from different sources (genetic and epidemiological data, histopathological features, and in vitro and in silico analyses) represents a powerful way to classify such variants. Building reliable integrated models for UV classification requires the joining of many working groups to collaborative consortia, allowing data exchange and improvements of methods. This will lead to improvement in the predictivity of gene testing in BRCA1 and BRCA2 and, consequently, to an increase in the number of families that can be correctly classified as linked or unlinked to these genes, allowing more accurate genetic counseling and clinical management.

COGENT (COlorectal cancer GENeTics): an international consortium to study the role of polymorphic variation on the risk of colorectal cancer.

It is now recognised that a part of the inherited risk of colorectal cancer (CRC) can be explained by the co-inheritance of low-penetrance genetic variants. The accumulated experience to date in identifying these variants has served to highlight difficulties in conducting statistically and methodologically rigorous studies and follow-up analyses. The COGENT (COlorectal cancer GENeTics) consortium includes 20 research groups in Europe, Australia, the Americas, China and Japan. The overarching goal of COGENT is to identify and characterise low-penetrance susceptibility variants for CRC through association-based analyses. In this study, we review the rationale for identifying low-penetrance variants for CRC and our proposed strategy for establishing COGENT.

The TP53 Arg72Pro and MDM2 309G>T polymorphisms are not associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers.

BACKGROUND: The TP53 pathway, in which TP53 and its negative regulator MDM2 are the central elements, has an important role in carcinogenesis, particularly in BRCA1- and BRCA2-mediated carcinogenesis. A single nucleotide polymorphism (SNP) in the promoter region of MDM2 (309T>G, rs2279744) and a coding SNP of TP53 (Arg72Pro, rs1042522) have been shown to be of functional significance. METHODS: To investigate whether these SNPs modify breast cancer risk for BRCA1 and BRCA2 mutation carriers, we pooled genotype data on the TP53 Arg72Pro SNP in 7011 mutation carriers and on the MDM2 309T>G SNP in 2222 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analysed using a Cox proportional hazards model within a retrospective likelihood framework. RESULTS: No association was found between these SNPs and breast cancer risk for BRCA1 (TP53: per-allele hazard ratio (HR)=1.01, 95% confidence interval (CI): 0.93-1.10, P(trend)=0.77; MDM2: HR=0.96, 95%CI: 0.84-1.09, P(trend)=0.54) or for BRCA2 mutation carriers (TP53: HR=0.99, 95%CI: 0.87-1.12, P(trend)=0.83; MDM2: HR=0.98, 95%CI: 0.80-1.21, P(trend)=0.88). We also evaluated the potential combined effects of both SNPs on breast cancer risk, however, none of their combined genotypes showed any evidence of association. CONCLUSION: There was no evidence that TP53 Arg72Pro or MDM2 309T>G, either singly or in combination, influence breast cancer risk in BRCA1 or BRCA2 mutation carriers.

The BOADICEA model of genetic susceptibility to breast and ovarian cancers: updates and extensions.

Multiple genetic loci confer susceptibility to breast and ovarian cancers. We have previously developed a model (BOADICEA) under which susceptibility to breast cancer is explained by mutations in BRCA1 and BRCA2, as well as by the joint multiplicative effects of many genes (polygenic component). We have now updated BOADICEA using additional family data from two UK population-based studies of breast cancer and family data from BRCA1 and BRCA2 carriers identified by 22 population-based studies of breast or ovarian cancer. The combined data set includes 2785 families (301 BRCA1 positive and 236 BRCA2 positive). Incidences were smoothed using locally weighted regression techniques to avoid large variations between adjacent intervals. A birth cohort effect on the cancer risks was implemented, whereby each individual was assumed to develop cancer according to calendar period-specific incidences. The fitted model predicts that the average breast cancer risks in carriers increase in more recent birth cohorts. For example, the average cumulative breast cancer risk to age 70 years among BRCA1 carriers is 50% for women born in 1920-1929 and 58% among women born after 1950. The model was further extended to take into account the risks of male breast, prostate and pancreatic cancer, and to allow for the risk of multiple cancers. BOADICEA can be used to predict carrier probabilities and cancer risks to individuals with any family history, and has been implemented in a user-friendly Web-based program (http://www.srl.cam.ac.uk/genepi/boadicea/boadicea_home.html).

Classification of BRCA1 missense variants of unknown clinical significance.

BACKGROUND: BRCA1 is a tumour suppressor with pleiotropic actions. Germline mutations in BRCA1 are responsible for a large proportion of breast-ovarian cancer families. Several missense variants have been identified throughout the gene but because of lack of information about their impact on the function of BRCA1, predictive testing is not always informative. Classification of missense variants into deleterious/high risk or neutral/low clinical significance is essential to identify individuals at risk. OBJECTIVE: To investigate a panel of missense variants. METHODS AND RESULTS: The panel was investigated in a comprehensive framework that included (1) a functional assay based on transcription activation; (2) segregation analysis and a method of using incomplete pedigree data to calculate the odds of causality; (3) a method based on interspecific sequence variation. It was shown that the transcriptional activation assay could be used as a test to characterise mutations in the carboxy-terminus region of BRCA1 encompassing residues 1396-1863. Thirteen missense variants (H1402Y, L1407P, H1421Y, S1512I, M1628T, M1628V, T1685I, G1706A, T1720A, A1752P, G1788V, V1809F, and W1837R) were specifically investigated. CONCLUSIONS: While individual classification schemes for BRCA1 alleles still present limitations, a combination of several methods provides a more powerful way of identifying variants that are causally linked to a high risk of breast and ovarian cancer. The framework presented here brings these variants nearer to clinical applicability.

Breast and ovarian cancer risks to carriers of the BRCA1 5382insC and 185delAG and BRCA2 6174delT mutations: a combined analysis of 22 population based studies.

A recent report estimated the breast cancer risks in carriers of the three Ashkenazi founder mutations to be higher than previously published estimates derived from population based studies. In an attempt to confirm this, the breast and ovarian cancer risks associated with the three Ashkenazi founder mutations were estimated using families included in a previous meta-analysis of populatrion based studies. The estimated breast cancer risks for each of the founder BRCA1 and BRCA2 mutations were similar to the corresponding estimates based on all BRCA1 or BRCA2 mutations in the meta-analysis. These estimates appear to be consistent with the observed prevalence of the mutations in the Ashkenazi Jewish population.

Cell surface antigens of chemically induced fibrosarcomas: detection by a monoclonal antibody of a tumor-restricted Mr 12,000 protein gag antigen encoded by a dual-tropic murine leukemia virus.

Fusion products of spleen cells of W/FuDp rats immunized with a methylcholanthrene-induced BALB/c sarcoma, CA-2, and mouse myeloma cells were screened in an attempt to identify a monoclonal antibody defining the individually distinct tumor-specific transplantation antigen of CA-2. A hybridoma, MP/69/04, was isolated which produces an IgG2a monoclonal antibody that recognized a tumor-restricted antigen of CA-2. In direct binding assay, MP/69/04 reacted only with 2 of 15 methylcholanthrene induced BALB/c sarcomas tested. Thymus, spleen, lymph nodes, bone marrow, brain, adult lung fibroblasts, newborn muscle fibroblasts and 3T3 cells were negative. Absorption tests revealed, however, expression of the MP/69/04 determinant on 8 of the 12 murine leukemia virus (MuLV) producer BALB/c sarcoma tested. The antigen was not detected on any of the three non-producer sarcomas tested nor on a wide range of normal tissues and cell lines. An N-dualtropic MuLV was isolated from CA-2, and cell lines susceptible to infection by this virus were shown to express the MP/69/04 epitope. By Western blotting, the MP/69/04 epitope was identified as being expressed on the MuLV structural protein with a molecular weight of 12,000, present in CA-2 cells and in the purified CA-2 MuLV. These results indicate the MP/69/04 antigen is not a unique tumor-specific transplantation antigen but is a gag product of a recombinant retrovirus which is expressed on the cell surface of many MuLV + methylcholanthrene-induced BALB/c fibrosarcomas.

Genetic evidence for an independent origin of multiple preneoplastic and neoplastic lung lesions.

Patients with a primary cancer in the lung or in the upper aerodigestive tract have an increased risk of developing synchronous or metachronous second primary lung tumors. This phenomenon has been related to the chronic exposure of the bronchial tree to carcinogens through a so-called "field cancerization" process. This study was designed to investigate at the somatic level the genetic basis of the field cancerization effect in patients having multiple simultaneous neoplastic and preneoplastic lesions of the lung. The pattern of specific genetic changes occurring with high frequency and in early stages of lung carcinogenesis including p53 mutations, deletions of chromosome 3p, and K-ras mutations, was investigated by immunocytochemical, cytogenetic, and molecular approaches in 11 synchronous lesions of five patients with multiple lung cancers. Different genetic lesions were observed in all of the pathological specimens analyzed from each patient. The pattern of these changes was different both in topographically distant or adjacent lesions and in tumors with the same histopathological diagnosis supporting their independent origin. The present data provide further evidence of the clinical relevance of the field cancerization process, and support the use of genetic markers in the differential diagnosis of recurrence or metastasis versus second primaries of the lung.

Human TRK proto-oncogene maps to chromosome 1q32-q41.

The chromosomal localization of TRK, a gene coding for a putative receptor molecule with an associated tyrosine kinase activity that we have found activated in 25% of patients with papillary thyroid carcinoma, was determined by Southern blot analysis of a panel of human-rodent somatic cells using a cDNA clone containing the entire human TRK proto-oncogene (Martin-Zanca et al., 1986). The TRK gene was assigned to chromosome 1. One hybrid that had retained only the short arm of the human chromosome 1 was negative. Subsequently, in situ hybridization of the same probe to human metaphase chromosomes localized the TRK gene to 1q32-q41.

HRAS1 proto-oncogene polymorphisms in human malignant melanoma: TaqI defined alleles significantly associated with the disease.

We have analysed the DNA of peripheral blood leukocytes (PBL) from 55 melanoma patients and 53 healthy individuals and failed to find any significant association between melanoma and rare HRAS1 alleles defined by MspI/HpaII digestion. However, the analysis of the same DNAs for a different polymorphism based on the presence of additional TaqI sites in the variable tandem repeat region of HRAS1 showed that the total frequency of a group of allelic variants, named Tp, was significantly higher in melanoma patients than in normal donors.

The human tropomyosin gene involved in the generation of the TRK oncogene maps to chromosome 1q31.

The chromosomal localization of hTMnm, a gene coding for a cytoskeletal tropomyosin non-muscle isoform involved in the activation of the TRK proto-oncogene in various human tumors, was determined by Southern blot analysis of a panel of human-rodent somatic cell hybrids. Using as a probe an Alu-free intronic fragment related to the tropomyosin sequence fused to the TRK tyrosine kinase domain, the hTMnm gene was assigned to the long arm of chromosome 1. Subsequently, in situ hybridization of the same probe to human metaphase chromosomes localized the hTMnm gene to 1q31. Since we have recently assigned the TRK locus to chromosome 1q32-q41, the generation of the hybrid transforming sequence tropomyosin-TRK may be due to an intrachromosomal rearrangement of the long arm of chromosome 1.

Refined localization to contiguous regions on chromosome 10q of the two genes (H4 and RET) that form the oncogenic sequence PTC.

In this report we confirm the localization of the human RET proto-oncogene to chromosome 10q11.2, both by Southern blot analysis of a panel of human-rodent somatic cell hybrids and by in situ hybridization on human metaphase chromosomes. Previously, we had assigned to the same chromosome region the gene termed H4. In about 25% of papillary thyroid carcinomas, this gene was shown to rearrange with RET to give rise to the transforming sequence PTC. The analysis of different cell hybrids containing subfragments of chromosome 10, in conjunction with pulse field gel electrophoresis, established that H4 is mapped distally to RET at a distance not less than 280 kb. These findings suggest that intrachromosomal rearrangements are responsible for PTC activation in papillary thyroid carcinomas.

Screening for mutations in exon 11 of the BRCA1 gene in 70 Italian breast and ovarian cancer patients by protein truncation test.

The most common mutations in the familial breast and ovarian cancer susceptibility gene BRCA1 are frameshift and nonsense mutations, which lead to the synthesis of truncated proteins. On this ground, we have analysed BRCA1 exon 11, which includes about 61% of coding region, in germline DNA from 70 Italian breast and/or ovarian cancer patients, using the protein truncation test (PTT). BRCA1 mutations were identified in nine of 29 (approximately 31%) patients with a family history of cancer and in three of 41 (approximately 7%) women with early-onset breast carcinomas, and were subsequently characterized by sequence analysis. In addition, BRCA1 mutations were also detected in six affected relatives of two positive index cases. The observed frequencies of mutations were not significantly different from those expected on the basis of the phenotypic characteristics of patients and their families, indicating that PTT is a rapid and sensitive method that can be used for a first BRCA1 mutational screening. The histological findings in BRCA1 mutated cases showed that eight of nine (approximately 89%) breast carcinomas were of grade III and nine of 9 (100%) ovarian carcinomas were of the endometrioid type (eight of grade III and one of grade II). This suggests that specific histological characteristics may represent additional criteria for selection of cases eligible to BRCA1 mutational analysis.

A novel zinc finger gene is fused to EWS in small round cell tumor.

Ewing sarcoma family of tumors share recurrent translocations that fuse EWS from 22q12 to five different members of transcription factors namely FLI-1, ERG, ETV1, E1AF and FEV. Different classes of DNA binding proteins, ATF1, WT1 and CHOP are fused to EWS generating distinct tumor phenotypes: clear cell sarcoma, desmoplastic small round cell tumor, and myxoid liposarcoma, respectively. We have cloned a novel gene located at 22q12 fused to EWS by a submicroscopic inversion of 22q in a small round cell sarcoma showing a translocation (t(1;22)(p36.1;q12). The gene, designated ZSG (Zinc finger Sarcoma Gene), is a putative Cys2-His2 zinc finger protein which contains a POZ transcriptional repressor-like domain at the N-terminus. The rearrangement involves intron 8 of EWS and exon 1 of ZSG creating a chimeric sequence containing the transactivation domain of EWS fused to zinc finger domain of ZSG. This product lacks the transcriptional repressor domain at the N-terminus of ZSG. A rearrangement of the second ZSG allele was also found in tumor cells. This is the first example of an intra-chromosomal rearrangement of chromosome 22, undetectable by cytogenetics, activating EWS in soft tissue sarcoma.

Multiple approach to the exploration of genotype-phenotype correlations in familial adenomatous polyposis.

PURPOSE: Familial adenomatous polyposis (FAP), caused by a mutation in the APC gene, is a colorectal cancer predisposition syndrome associated with several other clinical conditions. The severity of the FAP is related to the position of the inherited mutation in the APC gene. We analyzed a large series of FAP patients to identify associations among major clinical manifestations and to correlate the mutation site with specific disease manifestations. MATERIALS AND METHODS: APC mutations were identified in 953 FAP patients from 187 families. We used unconditional logistic regression models and a method involving generalized estimating equations to investigate the association between genotype and phenotype. We used multiple correspondence analysis to represent the interrelationships of a multiway contingency table of the considered variables. RESULTS: APC germline mutations were located between codons 156 and 2011 of the APC gene. Mutations spanning the region between codons 543 and 1309 were variable, but strongly associated with congenital hypertrophy of retinal pigment epithelium. Mutations between codons 1310 and 2011 were associated with a six-fold risk of desmoid tumors relative to the low-risk reference region (159 to 495). Mutations at codon 1309 were associated with early development of colorectal cancer. Mutations between codons 976 and 1067 were associated with a three- to four-fold increased risk of duodenal adenomas. The cumulative frequency of extracolonic manifestations was highest for mutations between codons 976 and 1067, followed by mutations between 1310 and 2011. CONCLUSION: Analysis of the relation between APC mutation site and phenotype identifies subgroups of FAP patients at high risk for major extracolonic disease, which is useful for surveillance and prevention.