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1.
Fam Pract ; 2019 Sep 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.

2.
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.

3.
Tumori ; : 300891618792460, 2018 Aug 17.
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.

4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
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
13.
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
14.
Hum Pathol ; 39(10): 1483-94, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18619649

RESUMO

The aim of the study was to identify the impact on prognosis of hypoxia-inducible factor-1 genetic program in colorectal carcinomas and to develop an experimental procedure that would allow a reliable quantitative gene expression analysis in formalin-fixed and paraffin-embedded tissue. The expression of hypoxia-inducible factor-1alpha and 13 hypoxia-inducible factor-1 target genes (AMF, CAIX, VEGF, VEGFR1, VEGFR2, HGF, MET, TGFalpha, EGFR, IGF2, MMP2, PLAUR, NIX) was quantified by real-time polymerase chain reaction in 18 colorectal, poorly differentiated neuroendocrine carcinomas and in 60 invasive colorectal carcinomas. Moreover, hypoxia-inducible factor-1alpha protein expression was evaluated by immunohistochemistry. High levels of hypoxia-inducible factor-1alpha were positively associated with poorly differentiated neuroendocrine carcinoma histology (P < .005), poor differentiation (P < .025), presence of necrosis, and presence of microsatellite instability (P < .05). AMF, TGFalpha, IGF2, NIX, VEGF, and VEGFR2 transcripts were significantly higher in the very aggressive poorly differentiated neuroendocrine carcinomas than in exocrine colorectal carcinomas and TGFalpha expression was significantly associated with presence of lymph nodal metastases (P < .05). High levels of TGFalpha and NIX were significantly associated with decreased overall survival (P < .001; P < .01). The multivariate analysis showed that advanced stage, presence of lymph node metastases, and high TGFalpha expression had an independent effect on survival (P < .006; P < .01; P < .0006). Our study suggests an up-regulation of the hypoxia-inducible factor-1 transcriptional pathway in colorectal carcinomas. hypoxia-inducible factor-1alpha overexpression alone, has no impact on the prognosis of colorectal carcinomas likely because the consequences of hypoxia-inducible factor-1alpha expression/stabilization strongly depend on the genetic background of the transformed cells. Mechanisms leading to increased synthesis of hypoxia-inducible factor-1alpha mRNA via autocrine growth factor loops may play a crucial role in invasive growth in this site.


Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Carcinoma Neuroendócrino/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/secundário , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma Neuroendócrino/mortalidade , Carcinoma Neuroendócrino/secundário , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Linfonodos/patologia , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Necrose , Prognóstico , RNA Mensageiro/metabolismo , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Transcrição Genética , Regulação para Cima
15.
Surg Oncol ; 16 Suppl 1: S25-7, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18023174

RESUMO

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates gene expression in critical pathways involved in tumor growth and metastases. In our study we evaluated the expression of HIF-1alpha transcript and protein by quantitative real time PCR and by immunohistochemistry in a total of 78 colorectal carcinomas (CRCs) and in 8 samples of normal colorectal mucosa in order to identify the impact on prognosis of HIF-1alpha overexpression in CRCs. Our study demonstrates a significant up-regulation of HIF-1alpha mRNA as well as a high frequency of the protein immunoreactivity in colorectal cancers compared to normal samples. However, no significant correlation was found between HIF-1alpha immunohistochemical expression and any of the clinico-pathological parameters evaluated, with the only exception of the positive association between HIF-1alpha immunoreactivity and the presence of tumor necrosis. Analogously, high levels of HIF-1alpha mRNA were found to be correlated with a poor grade of tumor differentiation but no other significant association was observed. Despite the careful selection of the tumor samples, our findings do not appear to confirm, for colorectal cancers, the significant association between HIF-1alpha overexpression and tumor aggressiveness or unfavorable prognosis demonstrated for cancers of other sites. The lack of prognostic impact of HIF-1 in this site could be explained by an intricate interaction between the survival program mediated by HIF-1 and the genetic background of tumors cells in which its activation occurs.


Assuntos
Neoplasias Colorretais/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Regulação para Cima , Neoplasias Colorretais/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imuno-Histoquímica , RNA Mensageiro/metabolismo
16.
Virchows Arch ; 451(3): 649-57, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17653761

RESUMO

Prognostic factors for ampullary carcinomas (ACs) are poorly defined. Fifty three resected ACs were analyzed for CDX2, MUC1, MUC5AC, MUC6, MUC2, and for mismatch repair proteins (hMLH1, hMSH2, PMS2, hMSH6) using immunohistochemistry. Microsatellite instability (MSI) status was evaluated by fluorescently labeled PCR using an automated sequencer. Univariate and multivariate analysis was performed for clinicopathological, immunohistochemical and molecular parameters. CDX2 was found in 32 out of 53 (60%) ACs with a significantly higher frequency among intestinal ACs compared with biliopancreatic (BP) ACs. The MUC1, MUC5AC, MUC6, MUC2 apomucins were expressed in 75, 43, 39, and 28% of ACs, respectively, with a significantly higher coexpression of MUC1/MUC5AC in BP ACs. MSI and loss of expression of hMLH1/PMS2 or hMSH2/hMSH6 proteins were observed only in intestinal ACs. Factors significantly correlated with improved survival in the univariate analysis were: low stage, absence of lymph nodes metastases, negative surgical margins (R0 status), and presence of MSI. In the multivariate analysis, stage was the only independent prognostic factor of survival. We conclude that stage is the only independent prognostic factor of survival in the multivariate analysis, whereas histological criteria and the immunohistochemical expression of apomucins and CDX2 are helpful in the classification and understanding of the histogenesis of ACs.


Assuntos
Adenocarcinoma/patologia , Ampola Hepatopancreática , Neoplasias do Ducto Colédoco/patologia , Instabilidade de Microssatélites , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Fator de Transcrição CDX2 , Neoplasias do Ducto Colédoco/genética , Neoplasias do Ducto Colédoco/mortalidade , Feminino , Proteínas de Homeodomínio/análise , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mucinas/análise , Estadiamento de Neoplasias , Prognóstico , Taxa de Sobrevida
17.
Clin Cancer Res ; 12(11 Pt 1): 3329-36, 2006 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-16740754

RESUMO

PURPOSE: The methylation status of hMLH1, CDKN2A, and MGMT was investigated in a panel of synchronous cancers of the ovary and endometrium, fulfilling the clinicopathologic criteria for independent primary tumors to define the possible role of epigenetic mechanisms in the development of these cancers. EXPERIMENTAL DESIGN: Bisulfite-converted DNA from 31 tumors (13 endometrial and 18 ovarian carcinomas) and from matched normal tissue of 13 patients was analyzed by a methylation-specific PCR assay at the CpG-rich 5' regions of all three genes. In all tumors, we also investigated the presence of microsatellite instability and hMLH1 immunohistochemical expression in relation to hMLH1 hypermethylation status. RESULTS: Methylation of hMLH1, CDKN2A, and MGMT was detected in 39%, 41%, and 48% of endometrial and ovarian tumors, respectively. hMLH1 hypermethylation was observed in all tumors of five patients, and it was invariably associated with loss of hMLH1 protein and presence of microsatellite instability. CDKN2A and MGMT methylation was randomly detected among both endometrial (45% and 24% of cases, respectively) and ovarian carcinomas (39% and 39% of cases, respectively). Concordant methylation at two or three genes was observed in 35% of cases. CONCLUSIONS: Epigenetic inactivation of hMLH1, CDKN2A, and MGMT may be a common and early event in the development of synchronous primary endometrial and ovarian carcinomas and may qualify as a marker of a field cancerization encompassing the ovary and endometrium. Detection of MGMT hypermethylation may be useful to define a set of gynecologic malignancies with a specific sensitivity to alkylating chemotherapy.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma/genética , Metilação de DNA , Neoplasias do Endométrio/genética , Neoplasias Primárias Múltiplas/genética , Neoplasias Ovarianas/genética , Regiões Promotoras Genéticas , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Carcinoma/diagnóstico , Carcinoma/terapia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/terapia , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Estadiamento de Neoplasias , Neoplasias Primárias Múltiplas/diagnóstico , Neoplasias Primárias Múltiplas/terapia , Proteínas Nucleares/genética , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/terapia , Reação em Cadeia da Polimerase/métodos , Proteínas Supressoras de Tumor/genética
18.
J Histochem Cytochem ; 54(8): 863-75, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16517981

RESUMO

Histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (v-MAT2) are involved in the biosynthesis and storage of histamine. DOPA decarboxylase (DDC) is involved in the biosynthesis of a variety of amines and shares a high degree of homology with HDC. HDC and v-MAT2 immunoreactivities (IR) have recently been detected in well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroendocrine carcinomas (PDNECs) of various sites and have been proposed as general endocrine markers. We evaluated HDC and v-MAT2 IR in a series of 117 WDNETs and PDNECs from different sites. Western blotting analysis was performed to verify the specificity of anti-DDC and anti-HDC antibodies. Real-time RT-PCR was performed using specific probes for HDC and DDC on 42 cases, examined also for DDC IR. HDC and v-MAT2 IR were observed in the majority of WDNETs and PDNECs of all sites and HDC-IR cases were always also DDC-IR. In contrast, high levels of HDC mRNA were detected only in the gastroenteropancreatic WDNETs, which did not show increased DDC mRNA levels. On the other hand, bronchial carcinoids and lung PDNECs showed high DDC mRNA levels, but nearly undetectable HDC mRNA levels. Western blotting analysis showed a cross-reaction between anti-HDC and anti-DDC antibodies. HDC should not be considered as a general endocrine marker and HDC IR in bronchial carcinoids and PDNECs of the lung can probably be attributed to a cross-reaction with DDC.


Assuntos
Carcinoma Neuroendócrino/metabolismo , Dopa Descarboxilase/biossíntese , Histidina Descarboxilase/biossíntese , Proteínas Vesiculares de Transporte de Monoamina/biossíntese , Especificidade de Anticorpos , Western Blotting , Reações Cruzadas , Dopa Descarboxilase/genética , Dopa Descarboxilase/imunologia , Perfilação da Expressão Gênica , Histidina Descarboxilase/genética , Histidina Descarboxilase/imunologia , Humanos , Imuno-Histoquímica , Especificidade de Órgãos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Cancer Epidemiol Biomarkers Prev ; 14(8): 2049-52, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16103460

RESUMO

Familial adenomatous polyposis (FAP) is an autosomal condition caused by inherited mutations in the adenomatous polyposis coli (APC) or in the MYH genes. Clinical trials have established that nonsteroidal anti-inflammatory drugs (NSAID) are effective in preventing the development as well as reducing the size and decreasing the number of adenomas in FAP patients. Our aim was to evaluate the cyclooxygenase-2 (COX-2) expression in surgical specimens from patients with no evidence of germ line APC mutations but carrying germ line MYH mutations. COX-2 expression was evaluated through immunohistochemical and mRNA analysis in carcinomas, adenomas, and healthy mucosa from six patients carrying germ line biallelic MYH mutations. A modulation of COX-2 expression from adenoma (lower level) to carcinoma (higher level) was observed in all patients by both immunohistochemical and mRNA analysis. Moreover, patients with MYH mutations showed a weak COX-2 expression in the whole colorectal mucosa, as for classic FAP patients carrying germ line APC mutations. All together, our data suggest that biallelic MYH patients might benefit from NSAID treatment, because in these patients COX-2 is overexpressed in the whole colorectal mucosa, a finding possibly related to the interplay between COX-2 and APC protein being the APC gene a common target of mutations in MYH patients.


Assuntos
Adenocarcinoma/genética , Polipose Adenomatosa do Colo/genética , DNA Glicosilases/genética , Mutação em Linhagem Germinativa/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Adenocarcinoma/patologia , Polipose Adenomatosa do Colo/tratamento farmacológico , Polipose Adenomatosa do Colo/patologia , Adulto , Anti-Inflamatórios não Esteroides/uso terapêutico , Ciclo-Oxigenase 2 , Feminino , Humanos , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade
20.
Cancer Res ; 65(6): 2321-9, 2005 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15781646

RESUMO

Although in vitro establishment of new colorectal carcinoma (CRC) cell lines is an infrequent event, we have observed that primary cultures of CRC can be repeatedly and reproducibly initiated following in vitro plating of tumor-derived epithelial cells. These cultures, however, usually display a short life span as they undergo a limited number of cell passages before entering a state of irreversible growth arrest. In this study, we show that short-lived CRC primary cultures lack constitutive telomerase activity and undergo a senescence process characterized by progressive telomere shortening. Moreover, transduction of these cells with a retroviral vector encoding human telomerase reverse transcriptase (hTERT) is sufficient to reconstitute telomerase activity and allow immortalization. Detailed molecular characterization of hTERT-immortalized CRC cell lines confirms their individual tumor origin by showing expression of colonic epithelial differentiation markers, such as cytokeratin-20 (CK20), full match with class I and class II human leukocyte antigen genotyping of autologous B-lymphoblastoid cells, and presence of somatic mutations in key cancer genes (KRAS2, APC) identical to those of the corresponding autologous original tumor tissues. Moreover, functional characterization of hTERT-immortalized CRC cell lines shows that they have a transformed phenotype, being able to form colonies in soft agar and tumors in severe combined immunodeficient mice. Most interestingly, immunohistochemical analysis of original tumor tissues indicates that short-lived CRC primary cultures, although hTERT-negative in vitro, derive from hTERT-positive tumors. Taken together, our data show that, in a least subset of CRC, biochemical pathways involved in maintenance of telomere length, such as telomerase, are not activated in a constitutive way in all tumor cells.


Assuntos
Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Telomerase/biossíntese , Animais , Senescência Celular/fisiologia , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA , Feminino , Vetores Genéticos , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Retroviridae/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/fisiologia , Transdução Genética , Transplante Heterólogo , Células Tumorais Cultivadas
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