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2.
Cancer Genet ; 254-255: 1-10, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33516942

RESUMO

A MSH6 3'UTR variant (c.*23_26dup) was found in 13 unrelated families consulted for Lynch/Muir-Torre Syndrome. This variant, which is very rare in the genomic databases, was absent in healthy controls and strongly segregated with the disease in the studied pedigrees. All tumors were defective for MSH2/MSH6/MSH3 proteins expression, but only MSH2 somatic pathogenic mutations were found in 5 of the 12 sequenced tumors. Moreover, we had no evidence of MSH6 transcript decrease in carriers, whereas MSH2 transcript was downregulated. Additional evaluations performed in representative carriers, including karyotype, arrayCGH and Linked-Reads whole genome sequencing, failed to evidence any MSH2 germline pathogenic variant. Posterior probability of pathogenicity for MSH6 c.*23_26dup was obtained from a multifactorial analysis incorporating segregation and phenotypic data and resulted >0.999, allowing to classify the variant as pathogenic (InSiGHT Class 5). Carriers shared a common haplotype involving MSH2/MSH6 loci, then a cryptic disease-associated variant, linked with MSH6 c.*23_26dup, cannot be completely excluded. Even if it is not clear whether the MSH6 variant is pathogenic per se or simply a marker of a disease-associated MSH2/MSH6 haplotype, all data collected on patients and pedigrees prompted us to manage the variant as pathogenic and to offer predictive testing within these families.

3.
Int J Mol Sci ; 21(24)2020 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-33352687

RESUMO

Homologous Recombination Deficiency (HRD) is a frequent feature of high-grade epithelial ovarian carcinoma (EOC), associated with sensitivity to PARP-inhibitors (PARPi). The best characterized causes of HRD in EOCs are germline or somatic mutations in BRCA1 and BRCA2 genes. Although promoter methylation is a well-known mechanism of gene transcriptional repression, few data have been published about BRCA gene methylation in EOCs. In this retrospective study, we quantitatively analyzed by pyrosequencing a selected series of 90 formalin-fixed (FFPE) primary EOCs without BRCA germline mutations. We identified 20/88 (22.7%) EOCs showing BRCA promoter methylation, including 17/88 (19.3%) in BRCA1 and 4/86 (4.6%) in BRCA2 promoters, one of which showing concomitant BRCA1 methylation. Mean methylation levels were 49.6% and 45.8% for BRCA1 and BRCA2, respectively, with methylation levels ≥50% in 10/20 methylated EOCs. Constitutive BRCA methylation was excluded by testing blood-derived DNA. In conclusion, pyrosequencing methylation analysis of BRCA genes is a robust, quantitative and sensitive assay applicable to FFPE samples. Remarkably, a considerable subset of germline BRCA-negative EOCs showed somatic methylation and, likely, HRD. A subpopulation of women with BRCA methylation, even without BRCA mutations, could potentially benefit from PARP-inhibitors; further clinical studies are needed to clarify the predictive role of somatic BRCA methylation of PARP-therapy response.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Biomarcadores Tumorais/genética , Metilação de DNA , Mutação , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Adenocarcinoma de Células Claras/tratamento farmacológico , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patologia , Adulto , Idoso , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Prognóstico , Regiões Promotoras Genéticas , Estudos Retrospectivos , Taxa de Sobrevida
4.
Tumori ; 106(6): NP67-NP72, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32635821

RESUMO

INTRODUCTION: The relationship between endocervical cancer and cancer susceptibility syndromes is not yet fully understood. We present 2 cases of endocervical cancer: 1 arising in a patient carrier with a pathogenic BRCA1 variant and the second detected in a Lynch syndrome family carrying the MSH2 germline pathogenic variant. CASE DESCRIPTION: Somatic analyses including loss of heterozygosity and fluorescent in situ hybridization demonstrated that the second hit in patient 1 is BRCA1-related. Mismatch repair somatic analyses in the second family demonstrated that the endocervical cancers of patient 2 and of her sister are MSH2-related. These data confirm the relationship between the pathogenesis of endocervical cancer and the presence of germline BRCA1 and MSH2 mutations. CONCLUSIONS: Our study confirms that gynecologic cancers including rare entities such as non-human papillomavirus-related endocervical cancer (NHPVA) are sentinels for inherited cancer syndromes. Endocervical cancer NHPVAs might be considered for cancer genetic counseling in order to improve cancer prevention. For this reason, the role of pathologists is particularly important for the correct identification of the cervical tumor site.

5.
Int J Gynecol Cancer ; 30(1): 56-61, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31780564

RESUMO

OBJECTIVE: Women with Lynch syndrome have a risk up to 40-60% of developing endometrial cancer, which is higher than their risk of developing colorectal or ovarian cancer. To date, no data on the outcomes of patients with Lynch syndrome diagnosed with non-endometrioid endometrial cancer are available. The goal of this study was to evaluate the outcome of patients with Lynch syndrome diagnosed with non-endometrioid endometrial cancer. METHODS: Data from consecutive patients diagnosed with Lynch syndrome and with a histological diagnosis of non-endometrioid endometrial cancer were retrospectively collected in two referral institutes in Italy. A case-control comparison (applying a propensity matching algorithm) was performed in order to compare patients with proven Lynch syndrome and controls. Inclusion criteria were: (a) histologically-proven endometrial cancer; (b) detection of a germline pathogenic variant in one of the MMR genes; (c) adequate follow-up. Only carriers of pathogenic or likely pathogenic variants (ie, class 5 and 4 according to the InSiGHT classification) were included in the study. Survival outcomes were assessed using KaplanMeier and Cox models. RESULTS: Overall, 137 patients with Lynch syndrome were collected. Mean patient age was 49.2 (10.9) years. Genes involved in the Lynch syndrome included MLH1, MSH2, and MSH6 in 43%, 39%, and 18% of cases, respectively. The study population included 27 patients with non-endometrioid endometrial cancer, who were matched 1:2 with patients with sporadic cancers using a propensity matching algorithm. After a median follow-up of 134 months (range 1-295), 2 (7.4%) of the 27 patients developed recurrent disease (3 and 36 months) and subsequently died of disease (7 and 91 months). Patients diagnosed with Lynch syndrome experienced better disease-free survival (HR 7.86 (95% CI 1.79 to 34.5); p=0.006) and overall survival (HR 5.33 (95% CI 1.18 to 23.9); p=0.029) than controls. CONCLUSIONS: Non-endometrioid endometrial cancer occurring in patients with Lynch syndrome might be associated with improved oncologic outcomes compared with controls. Genetic/molecular profiling should be investigated in order to better understand the mechanism underlying the prognosis.


Assuntos
Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Adulto , Idoso , Carcinoma Endometrioide/cirurgia , Estudos de Casos e Controles , Estudos de Coortes , Neoplasias Colorretais Hereditárias sem Polipose/cirurgia , Proteínas de Ligação a DNA/genética , Intervalo Livre de Doença , Neoplasias do Endométrio/cirurgia , Feminino , Humanos , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Estudos Retrospectivos , Taxa de Sobrevida
6.
Cancer Res ; 80(3): 624-638, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31723001

RESUMO

Pathogenic sequence variants (PSV) in BRCA1 or BRCA2 (BRCA1/2) are associated with increased risk and severity of prostate cancer. We evaluated whether PSVs in BRCA1/2 were associated with risk of overall prostate cancer or high grade (Gleason 8+) prostate cancer using an international sample of 65 BRCA1 and 171 BRCA2 male PSV carriers with prostate cancer, and 3,388 BRCA1 and 2,880 BRCA2 male PSV carriers without prostate cancer. PSVs in the 3' region of BRCA2 (c.7914+) were significantly associated with elevated risk of prostate cancer compared with reference bin c.1001-c.7913 [HR = 1.78; 95% confidence interval (CI), 1.25-2.52; P = 0.001], as well as elevated risk of Gleason 8+ prostate cancer (HR = 3.11; 95% CI, 1.63-5.95; P = 0.001). c.756-c.1000 was also associated with elevated prostate cancer risk (HR = 2.83; 95% CI, 1.71-4.68; P = 0.00004) and elevated risk of Gleason 8+ prostate cancer (HR = 4.95; 95% CI, 2.12-11.54; P = 0.0002). No genotype-phenotype associations were detected for PSVs in BRCA1. These results demonstrate that specific BRCA2 PSVs may be associated with elevated risk of developing aggressive prostate cancer. SIGNIFICANCE: Aggressive prostate cancer risk in BRCA2 mutation carriers may vary according to the specific BRCA2 mutation inherited by the at-risk individual.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Predisposição Genética para Doença , Genômica/métodos , Mutação , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Associação Genética , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Risco , Adulto Jovem
7.
Fam Pract ; 37(1): 43-48, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31536618

RESUMO

BACKGROUND: The hereditary cancer syndromes represent overall <10% of all cancers. These syndromes are not irrelevant for public health because all the cancers typical of these syndromes affected young people and many members of the same family and the cancers are more aggressive than the sporadic ones and need specific surgery and medical therapy. We developed a new family assessment tool: STELO designed for family physicians to identify patients could benefit from Cancer Genetic Counselling. OBJECTIVE: Test the sensitivity and specificity of a new assessment tool for the correct identification of inherited cancer syndromes. METHODS: Retrospectively we tested the new tool on a subset of patients who had already undergone genetic counselling at the Cancer Genetic Counselling Service of ASST (Azienda Socio Sanitaria Territoriale) Settelaghi Varese, to investigate sensitivity, specificity and applicability of this new tool in routine genetic screening. STELO responses were matched against the opinion of two cancer geneticists (i.e. gold standard) who blinded each other decided if the history of these patients was properly suspected as a hereditary cancer syndrome. RESULTS: The Genetic Counselling Service followed 546 subjects from 2014 to 2015. STELO tool was tested retrospectively on these clinical records and resulted positive in 418 cases, out of 546 (76.5%). STELO reported, towards the gold standard, 88.5% and 52.3% of sensitivity and specificity, respectively. CONCLUSIONS: STELO has demonstrated to have a good sensitivity. The specificity was expectedly low given that STELO has been developed for general medicine, so it needs to be simple, practical, of rapid consultation and effectively used in clinical practice.

8.
Front Microbiol ; 10: 1746, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31417532

RESUMO

Lynch syndrome (LS) is a dominantly inherited condition with incomplete penetrance, characterized by high predisposition to colorectal cancer (CRC), endometrial and ovarian cancers, as well as to other tumors. LS is associated with constitutive DNA mismatch repair (MMR) gene defects, and carriers of the same pathogenic variants can show great phenotypic heterogeneity in terms of cancer spectrum. In the last years, human gut microbiota got a foothold among risk factors responsible for the onset and evolution of sporadic CRC, but its possible involvement in the modulation of LS patients' phenotype still needs to be investigated. In this pilot study, we performed 16S rRNA gene sequencing of bacterial DNA extracted from fecal samples of 10 postoperative LS female patients who had developed colonic lesions (L-CRC) or gynecological cancers (L-GC). Our preliminary data show no differences between microbial communities of L-CRC and L-GC patients, but they plant the seed of the possible existence of a fecal microbiota pattern associated with LS genetic background, with Faecalibacterium prausnitzii, Parabacteroides distasonis, Ruminococcus bromii, Bacteroides plebeius, Bacteroides fragilis and Bacteroides uniformis species being the most significantly over-represented in LS patients (comprising both L-CRC and L-GC groups) compared to healthy subjects.

9.
Hum Mutat ; 40(9): 1557-1578, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31131967

RESUMO

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Biologia Computacional/métodos , Mutação de Sentido Incorreto , Neoplasias/diagnóstico , Processamento Alternativo , Detecção Precoce de Câncer , Feminino , Predisposição Genética para Doença , Humanos , Funções Verossimilhança , Masculino , Herança Multifatorial , Neoplasias/genética
10.
Tumori ; 105(1): 76-83, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30117378

RESUMO

OBJECTIVE:: To investigate the performance of tumor testing approaches in the identification of Lynch syndrome (LS) in a single-center cohort of people with colorectal cancer (CRC). METHODS:: A retrospective analysis of data stored in a dedicated database was carried out to identify patients with CRC suspected for LS who were referred to Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy, between 1999 and 2014. The sensitivity and specificity of immunohistochemistry (IHC) for mismatch repair (MMR) proteins and microsatellite instability (MSI) analysis (alone or combined) were calculated with respect to the presence of causative MMR germline variants. RESULTS:: A total of 683 patients with CRC suspected for LS were identified. IHC results of MMR protein analysis and MSI were assessed in 593 and 525 CRCs, respectively, while germline analysis was performed in 418 patients based on the IHC or MSI test result and/or clinical features. Univariate and multivariate analysis revealed a significant correlation of pathogenic MMR germline variants with all clinicopathologic features including Amsterdam criteria, presence of endometrial cancer, CRC site, age at onset, stage, and grade. The highest odds ratio values were observed for IHC and MSI (17.1 and 8.8, respectively). The receiver operating characteristic curve and area under the curve values demonstrated that IHC alone or combined with other clinicopathologic parameters was an excellent test for LS identification. CONCLUSIONS:: This study confirms the effectiveness of tumor testing to identify LS among patients with CRC. Although IHC and MSI analysis were similarly effective, IHC could be a better strategy for LS identification as it is less expensive and more feasible.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Neoplasias do Endométrio/genética , Feminino , Testes Genéticos/métodos , Mutação em Linhagem Germinativa/genética , Humanos , Imuno-Histoquímica/métodos , Itália , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Estudos Retrospectivos
11.
Int J Gynecol Cancer ; 27(7): 1543-1549, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28471861

RESUMO

OBJECTIVE: Recent data from the literature indicate gynecological cancers (GCs) as sentinel cancers for a diagnosis of Lynch syndrome (LS). Clinical approaches to identifying LS have low sensitivity, whereas somatic tests on GCs may be a more sensitive and cost-effective strategy. METHODS: A series of 78 GCs belonging to 74 patients sent to the Genetic Counselling Service were investigated using microsatellite instability, immunohistochemical expression of mismatch repair (MMR) genes, and MLH1 promoter methylation. RESULTS: The presence of microsatellite instability was observed in 67.5% of GCs, and the absence of immunohistochemical expression of at least 1 of the 4 MMR proteins was observed in 71.4% of GCs, showing 96.1% concordance between the methods. Methylation analysis using methylation specific multiplex ligation-dependent probe amplification performed on 35 samples revealed MLH1 promoter hypermethylation in 18 cases (54%). Molecular analysis identified 36 LS carriers of MMR variants (27 pathogenetic and 9 variants of uncertain significance), and, interestingly, 3 LS patients had MLH1 methylated GC.With regard to histological features, LS-related GCs included endocervical cancers and also histological types different from the endometrioid cancers. The presence of peritumoral lymphocytes in GCs was statistically associated with LS tumors. CONCLUSIONS: Somatic analysis is a useful strategy to distinguish sporadic from LS GC. Our data allow the identification of a subset of LS patients otherwise unrecognized on the basis of clinical or family history alone. In addition, our results indicate that some clinicopathological features including age of GC diagnosis; presence of peritumoral lymphocytes; isthmic, endocervical sites, and body mass index value could be useful criteria to select patients for genetic counseling.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias dos Genitais Femininos/diagnóstico , Adulto , Idoso , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Metilação de DNA , Enzimas Reparadoras do DNA/biossíntese , Enzimas Reparadoras do DNA/genética , Feminino , Neoplasias dos Genitais Femininos/genética , Neoplasias dos Genitais Femininos/metabolismo , Humanos , Imuno-Histoquímica , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/biossíntese , Proteína 1 Homóloga a MutL/genética , Regiões Promotoras Genéticas
12.
J Clin Pathol ; 70(9): 792-797, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28416640

RESUMO

Microsatellite instability (MSI) testing is tricky in gynaecological cancers (GC). Thus, we aimed to describe the instability patterns to improve MSI test interpretation in sporadic and hereditary GCs. Ninety-five cases, including uterine and ovarian cancers, with known genetic and immunohistochemical (IHC) features, were analysed for MSI by a mononucleotide repeats pentaplex (MRP). We identified 13 ambiguous cases that did not fully meet MSI criteria ('borderline' cases, B-MSI), which were mainly represented by MSH2/MSH6-deficient and Lynch syndrome cases. Also, we evaluated nine additional loci of candidate MSI markers that did not improve the detection of MSI cases, but might be useful for discordant or borderline samples. In conclusion, although MSI and IHC test are highly concordant, a subset of ambiguous MSI cases deserves a careful interpretation in particular when MSH2/MSH6 are involved. RPL22 and SRPR testing may be useful to integrate MRP panel for the analysis of critical cases.


Assuntos
Biomarcadores Tumorais/genética , Instabilidade de Microssatélites , Repetições de Microssatélites , Neoplasias Ovarianas/genética , Neoplasias Uterinas/genética , Biomarcadores Tumorais/análise , Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/genética , Feminino , Predisposição Genética para Doença , Hereditariedade , Humanos , Imuno-Histoquímica , Técnicas de Diagnóstico Molecular , Neoplasias Ovarianas/química , Neoplasias Ovarianas/patologia , Fenótipo , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Neoplasias Uterinas/química , Neoplasias Uterinas/patologia
13.
Int J Gynecol Pathol ; 36(1): 64-70, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27167672

RESUMO

Ovarian carcinosarcomas (OCS), also known as malignant mixed mesodermal/Müllerian tumors, are rare neoplasms (1%-4% of all malignant ovarian tumors) composed of high-grade malignant epithelial and mesenchymal elements. OCS occurs in older women. It is associated with a poor outcome and is usually not involved in inherited cancer syndromes. We present 2 cases of OCS; one arising in a patient with a pathogenetic BRCA1 mutation and the other in a woman affected by Lynch Syndrome (LS) carrying a MSH6 germline mutation. To the best of our knowledge, this is the first time that this second type of case has been reported. In this study, we investigated somatic impairment of the wild-type BRCA1 and MSH6 alleles in the OCS of these 2 patients. We also explored in both OCS, the occurrence of TP53 loss of function, which is a genetic alteration known to occur in BRCA-linked ovarian tumorigenesis but not in LS tumors. Moreover, we also provide further data about the histogenesis of OCS.


Assuntos
Proteína BRCA1/genética , Carcinossarcoma/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteínas de Ligação a DNA/genética , Síndromes Neoplásicas Hereditárias/genética , Neoplasias Ovarianas/genética , Adulto , Carcinossarcoma/diagnóstico , Carcinossarcoma/patologia , Neoplasias Colorretais Hereditárias sem Polipose/complicações , Feminino , Aconselhamento Genético , Mutação em Linhagem Germinativa , Humanos , Pessoa de Meia-Idade , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/patologia , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/patologia , Ovário/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
14.
Cancer Genet ; 209(3): 107-11, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26797314

RESUMO

Turcot syndrome (TS) refers to the combination of colorectal polyps and primary tumours of the central nervous system. TS is a heterogeneous genetic condition due to APC and/or mismatch repair germline mutations. When APC is involved the vast majority of mutations are truncating, but in approximately 20%-30% of patients with familial polyposis no germline mutation can be found. A 30-year-old Caucasian woman with a positive pedigree for TS was referred to our Genetic Counselling Service. She was negative for APC and MUTYH but showed a reciprocal balanced translocation t(5;7)(q22;p15) at chromosome analysis. FISH analysis using specific BAC probes demonstrated that 5q22 breakpoint disrupted the APC gene. Transcript analysis by MLPA and digital PCR revealed that the cytogenetic rearrangement involving the 3' end of the APC gene caused a defective expression of a truncated transcript. This result allowed cytogenetic analysis to be offered to all the other family members and segregation analysis clearly demonstrated that all the carriers were affected, whereas non-carriers did not have the polyposis. A cytogenetic approach permitted the identification of the mutation-causing disease in this family, and the segregation analysis together with the transcript study supported the pathogenetic role of this mutation. Karyotype analysis was used as a predictive test in all members of this family. This family suggests that clinically positive TS and FAP cases, which test negative with standard molecular analysis, could be easily and cost-effectively resolved by a classical and molecular cytogenetic approach.


Assuntos
Neoplasias Encefálicas/genética , Cromossomos Humanos Par 5 , Cromossomos Humanos Par 7 , Neoplasias Colorretais/genética , Genes APC , Síndromes Neoplásicas Hereditárias/genética , Translocação Genética , Adulto , Feminino , Humanos , Cariotipagem
15.
Carcinogenesis ; 36(4): 452-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25742745

RESUMO

Lynch syndrome (LS) is an inherited predisposition cancer syndrome, typically caused by germline mutations in the mismatch repair genes MLH1, MSH2, MSH6 and PMS2. In the last years, a role for epimutations of the same genes has also been reported. MLH1 promoter methylation is a well known mechanism of somatic inactivation in tumors, and more recently, several cases of constitutional methylation have been identified. In four subjects affected by multiple tumors and belonging to a suspected LS family, we detected a novel secondary MLH1 gene epimutation. The methylation of MLH1 promoter was always linked in cis with a 997 bp-deletion (c.-168_c.116+713del), that removed exon 1 and partially involved the promoter of the same gene. Differently from cases with constitutional primary MLH1 inactivation, this secondary methylation was allele-specific and CpGs of the residual promoter region were totally methylated, leading to complete allele silencing. In the colon tumor of the proband, MLH1 and PMS2 expression was completely lost as a consequence of a pathogenic somatic point mutation (MLH1 c.199G>A, p.Gly67Arg) that also abrogated local methylation by destroying a CpG site. The evidences obtained highlight how MLH1 mutations and epimutations can reciprocally influence each other and suggest that an altered structure of the MLH1 locus results in epigenetic alteration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Trifosfatases/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Metilação de DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Adenosina Trifosfatases/biossíntese , Sequência de Bases , Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/biossíntese , Proteínas de Ligação a DNA/biossíntese , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteínas Nucleares/biossíntese , Análise de Sequência de DNA , Deleção de Sequência/genética
16.
Int J Cancer ; 132(5): 1060-9, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22865608

RESUMO

MUTYH variants are differently distributed in geographical areas of the world. In MUTYH-associated polyposis (MAP) patients from North-Eastern Italy, c.933+3A>C (IVS10+3A>C), a transversion causing an aberrant splicing process, accounts for nearly 1/5 of all mutations. The aim of this study was to verify whether its high frequency in North-Eastern Italy is due to a founder effect and to clarify its impact on MUTYH transcripts and protein. Haplotype analysis and age estimate performed on members of eleven Italian MAP families and cancer-free controls provided evidence that c.933+3A>C is a founder mutation originated about 83 generations ago. In addition, the Italian haplotype associated with the c.933+3A>C was also found in German families segregating the same mutation, indicating it had a common origin in Western Europe. Altogether c.933+3A>C and the two common Caucasian mutations p.Tyr179Cys and p.Gly396Asp represent about 60% of MUTYH alterations in MAP patients from North-Eastern Italy, suggesting the opportunity to perform targeted molecular screening for these variants in the diagnostic setting. Expression analyses performed on lymphoblastoid cell lines supported the notion that MUTYH c.933+3A>C alters splicing causing the synthesis of a non functional protein. However, some primary transcripts escape aberrant splicing, producing traces of full-length transcript and wild-type protein in a homozygote; this is in agreement with clinical findings that suggest a relatively mild phenotypic effect for this mutation. Overall, these data, that demonstrate a founder effect and further elucidate the splicing alterations caused by the MUTYH c.933+3A>C mutation, have important implications for genetic counseling and molecular diagnosis of MAP.


Assuntos
Polipose Adenomatosa do Colo/genética , DNA Glicosilases/genética , Grupo com Ancestrais do Continente Europeu/genética , Mutação , Polipose Adenomatosa do Colo/metabolismo , Estudos de Casos e Controles , Linhagem Celular , DNA Glicosilases/biossíntese , Expressão Gênica , Predisposição Genética para Doença , Haplótipos , Humanos , Itália
17.
Virchows Arch ; 462(1): 47-56, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23224118

RESUMO

Methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) is a fast, new, inexpensive method that has rarely been exploited in DNA methylation profiling of colorectal cancers (CRCs). The aim of this study was to test the diagnostic utility of MS-MLPA to evaluate the methylation status of 34 genes in normal colonic mucosa samples and in a well-characterized series of 83 adenocarcinomas and 21 neuroendocrine carcinomas of colon-rectum. Two commercial MS-MLPA kits (SALSA MS-MLPA ME001-C1 Tumor suppressor-1 Kit and SALSA MS-MLPA ME002-B1 Tumor suppressor-2 Kit) were used to perform promoter methylation analysis on formalin-fixed and paraffin-embedded tissues. MS-MLPA analysis was validated by bisulfite pyrosequencing, bisulfite cycle sequencing, and methylation-specific PCR. MS-MLPA analysis identified a subset of 27 CRCs (26 % of cases) showing high levels of gene methylation involving a mean percentage of 34 % of the promoters examined. These tumors exhibited all the main clinicopathological and genetic features described for CRCs with CpG island Methylator Phenotype-High. High levels of methylation were observed with similar frequency in adenocarcinomas and in neuroendocrine carcinomas (25 % versus 29 %, respectively), but different methylation profiles were observed in the two tumor types. In both groups, tumors with microsatellite instability and widespread methylation represented a homogeneous clinicopathological entity. MS-MLPA assay is an easy and reliable system for epigenetic characterization of tumor tissues and leads to a rapid identification of CRCs with the highest levels of gene methylation. Aberrant gene methylation is a common abnormality in CRC initiation and may be observed in tumors with very different genetic and clinicopathological profiles.


Assuntos
Adenocarcinoma/genética , Carcinoma Neuroendócrino/genética , Neoplasias Colorretais/genética , Metilação de DNA , DNA de Neoplasias/genética , Perfilação da Expressão Gênica , Adenocarcinoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Neuroendócrino/diagnóstico , Neoplasias Colorretais/diagnóstico , Ilhas de CpG/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Técnicas de Amplificação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Supressoras de Tumor/genética
18.
Mod Pathol ; 24(1): 126-37, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20852594

RESUMO

This work has evaluated the potential superiority of a morphomolecular classification based on the combination of clinicopathologic and molecular features of colorectal cancers. A cohort of 126 colorectal carcinomas was investigated by unsupervised hierarchical clustering analysis to combine 13 routinely assessed clinicopathologic features and all five molecular markers recently suggested by Jass' classification to distinguish four molecular subtypes of sporadic colorectal carcinomas. Survival analysis was assessed by a Cox proportional hazards model. A clear separation into three prognostically significant groups was identified: cluster A and cluster C were associated with good prognosis and cluster B with poor prognosis (P=0.006). Clinicopathologic and molecular features of cluster A and cluster B tumors were strongly concordant with colorectal cancer profiles characterized by microsatellite instability or by chromosomal instability, respectively. The clinicopathologic features of cluster C tumors were suggestive of a less aggressive disease than cluster B tumors. Genetically, they appeared intermediate between cluster A and cluster B tumors, as they were mainly microsatellite stable tumors showing high levels of both MGMT methylation and loss of heterozygosity. Chromosomal instability was significantly lower in cluster C than in cluster B tumors. A more accurate tumor classification should combine the prognostic power of clinicopathologic parameters with molecular biomarkers that provide information regarding the natural history of the cancer. Hierarchical clustering seems to be a useful, promising and powerful tool for further translational studies and should lead us to define a diagnostic and prognostic signature for different carcinomas.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Instabilidade Cromossômica , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Instabilidade de Microssatélites , Adenocarcinoma/mortalidade , Idoso , Biomarcadores Tumorais/genética , Análise por Conglomerados , Estudos de Coortes , Neoplasias Colorretais/mortalidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Taxa de Sobrevida
19.
J Histochem Cytochem ; 58(10): 881-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20566755

RESUMO

Carboxyl ester lipase (CEL) is an enzyme that hydrolyzes a wide variety of lipid substrates, including ceramides, which are known to show inhibitory regulation of pituitary hormone secretion in experimental models. Because no studies on CEL expression in human pituitary and pituitary adenomas have been reported in the literature, we investigated CEL expression in 10 normal pituitary glands and 86 well-characterized pituitary adenomas [12 FSH/LH cell, 17 α-subunit/null cell, 6 TSH cell, 21 ACTH cell, 11 prolactin (PRL) cell, and 19 GH cell adenomas] using IHC, immunoelectron microscopy, Western blotting, and quantitative RT-PCR. In normal adenohypophysis, CEL was localized in GH, ACTH, and TSH cells. In adenomas, it was mainly found in functioning GH, ACTH, and TSH tumors, whereas its expression was poor in the corresponding silent adenomas and was lacking in FSH/LH cell, null cell, and PRL cell adenomas. Ultrastructurally, CEL was localized in secretory granules close to their membranes. This is the first study demonstrating CEL expression in normal human pituitary glands and in functioning GH, ACTH, and TSH adenomas. Considering that CEL hydrolyzes ceramides, inactivating their inhibitory function on pituitary hormone secretion, our findings suggest a possible role of CEL in the regulation of hormone secretion in both normal and adenomatous pituitary cells.


Assuntos
Adenoma/enzimologia , Lipase/biossíntese , Hipófise/enzimologia , Neoplasias Hipofisárias/enzimologia , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Microscopia Imunoeletrônica , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Fam Cancer ; 9(3): 275-82, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20041308

RESUMO

In this work we report for the first time a family in Italy with the BRCA1 ins6kbEx13 mutation, a recurrent founder mutation originating from northern Britain. After the initial identification of exon 13 duplication by multiplex ligation-dependent probe amplification (MLPA assay), we confirmed the identity of the alteration with the previously published BRCA1 ins6kbEx13 mutation, by mutation specific PCR and RT-PCR assays and by haplotype analysis. As rarely reported previously, the MLPA assay was also used to examine DNA from formalin fixed paraffin embedded (FFPE) normal tissues of other affected subjects in the family and it was the only effective method to perform a complete segregation analysis of the BRCA1 ins6kbEx13 mutation in the family. A combination of different approaches including MLPA analysis, haplotype analysis and LOH study on tumor samples of all the affected members allowed to reassess the maternal transmission of the mutation expected by the pedigree analysis. Moreover, detailed morphological analysis of breast cancers of BRCA1 ins6kbEx13 mutation carriers demonstrated a rare histological variant of breast carcinomas that has never been described in patients carrying BRCA1 mutations. Our study confirms the MLPA technique as a reliable and effective method for a primary screening for BRCA1 rearrangements also by using FFPE tissues and strongly suggests that histo-pathological, immunonohistochemical and molecular information from FFPE tumor tissues should be more often considered and integrated into routine diagnostic practice of hereditary tumors.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Genes BRCA1 , Mutação em Linhagem Germinativa , Éxons/genética , Família , Feminino , Duplicação Gênica , Predisposição Genética para Doença , Haplótipos , Humanos , Imuno-Histoquímica , Itália , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Linhagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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