Cristina D'Amico

Subcellular localization of the BRCA1 gene product in mitotic cells

The product of the hereditary breast cancer susceptibility gene BRCA1 is a multifunctional protein involved in the maintenance of genomic integrity, in transcriptional coactivation, and in the control of cell growth. BRCA1‐deficient cells manifest chromosomal instability. During mitosis, BRCA1 is known to interact with γ‐tubulin in the centrosomes, key elements of the mitotic spindle. Using confocal microscopy and immunogold electron microscopy, we investigated the distribution of endogenous BRCA1 relative to mitotic spindle markers in breast cancer cells. By confocal analysis, BRCA1 and β‐tubulin colocalized to microtubules of the mitotic spindle and to the centrosomes. Immunogold electron microscopy confirmed these results and further revealed that BRCA1 and α‐tubulin codistributed to the walls of the centrioles and to pericentriolar fibers at centrosomes. During chromatid segregation, codistribution was also detected along individual spindle microtubules and at sites of insertion of microtubules on chromosomes. At cytokinesis, BRCA1 and α‐tubulin codistributed to the midbody. Coimmunoprecipitation supported the association of full‐length BRCA1 with α‐ and β‐tubulin. These results are consistent with an involvement of BRCA1 in the dynamics of the mitotic spindle and in the segregation of duplicated chromosomes.

Mutations at coding mononucleotide repeats in gastric cancer with the microsatellite mutator phenotype

We analysed 50 gastric carcinomas (GCs) to verify whether mutations at coding repeats were associated with microsatellite instability (MSI). The tumors included: ten cases with no MSI, 14 cases with MSI=1 locus, 13 cases with MSI=two loci and 13 cases with MSI⩾3 loci. We investigated coding repeats within the TGF-β RII, IGFIIR, BAX, hMSH6, hMSH3 and BRCA2 genes. The TGF-β RII, IGFIIR, BAX, hMSH6 and hMSH3 repeats were altered in 11 (22%), five (10%), four (8%), 16 (32%) and five (10%) cases respectively. Mutations occurred only in MSI-positive (MSI+) tumors and correlated with increasing MSI levels. No alterations of the BRCA2 repeat were found. Mutations in genes other than hMSH6 were strongly associated to hMSH6 mutations, suggesting a key role of this gene. The non-coding BAT-26 and E-Cadherin 3′ UTR poly(A)8/(T)15 repeats were analysed in 44 of the 50 cases. Novel tumor-associated alleles were observed only in MSI-positive GCs and were in most cases associated with mutations at coding repeats. Further investigations with BAT-40 confirmed that four cases manifested mononucleotide repeat alterations restricted to hMSH6 and one case to TGF-β RII. A subset of tumors with MSI at two or more dinucleotide loci resulted negative for mutations at coding and non-coding mononucleotide repeats.

BRCA1 and BRCA2 mutations in central and southern Italian patients

Statement of findingsProtein truncation test (PTT) and single-strand conformation polymorphism (SSCP) assay were used to scan the BRCA1 and BRCA2 genes in 136 unrelated Italian breast/ovarian cancer patients. In the sample tested, BRCA1 and BRCA2 equally contributed to site-specific breast cancer patients who reported one to two breast cancer-affected first-/ second-degree relative(s) or who were diagnosed before age 40 years in the absence of a family history of breast/ovarian cancer. BRCA1 and BRCA2 mutations were mostly found in patients with disease diagnosis before and after age 50 years, respectively. Moreover, in cases with familial clustering of site-specific breast cancer, BRCA1 mostly accounted for tumours diagnosed before age 40 years and BRCA2 for tumours diagnosed after age 50 years. The BRCA1 and BRCA2 mutation spectrum was consistent with a lack of significant founder effects in the sample of patients studied.

A complex pattern of mutations and abnormal splicing of Smad4 is present in thyroid tumours

Sensitivity to transforming growth factor-β is impaired in thyroid tumours. Similar to Mad – Mother Against Decapentaplegic-(Smad)4 is frequently altered in cancers, but its involvement in this system is unknown. We analysed 56 thyroid tumours of various histotypes for Smad4 mutations by PCR-SSCP and sequencing, linking them to Smad4 reactivity as examined by immunohistochemistry (IHC), and 29 of them also for abnormalities in RNA expression due to alternative splicing. In all, 15/56 cases (27%), both benign and malignant lesions, harbour alterations of Smad4 coding sequence. We found several novel intragenic mutations (13 missense, two silent, one frameshift and one large insertion-deletion), with high incidence in the linker region. A subset of mutated tumours failed to express Smad4 protein by IHC. We have also detected four alternatively spliced tumour-associated Smad4 isoforms, lacking portions of the linker region, and three more due to unreported internal exon–exon rearrangements. Smad4 is both frequently mutated and deregulated by aberrant splicing in thyroid tumours and these alterations may contribute as an early event to thyroid tumorigenesis.

Association between the BRCA2 N372H variant and male breast cancer risk: a population-based case-control study in Tuscany, Central Italy

BackgroundMale breast cancer (MBC) is a rare disease and little is known about its aetiology. Germ-line mutations of BRCA2 and, at lower frequency, of BRCA1 are implicated in a relatively small proportion of MBC cases. Common polymorphic variants in BRCA1 and BRCA2 genes may represent breast cancer (BC) susceptibility alleles and could be associated with a modestly increased risk of MBC at population level. Considering the relevant role of BRCA2 in MBC, we investigated whether the BRCA2 N372H variant, representing the only common non-synonymous polymorphism in BRCA2, might modulate the risk of BC in male populations.MethodsA case-control study was performed comparing a population-based series of 99 MBC cases, characterized for BRCA1 and BRCA2 mutations, with 261 male population controls, all residing in Tuscany, Central Italy. All MBC cases and controls were genotyped for the BRCA2 N372H allele by TaqMan allelic discrimination assays. To evaluate the genotype specific risk of the BRCA2 N372H variant, MBC carriers of germ-line BRCA1/2 mutations were excluded from the analyses.ResultsNo association emerged in univariate and age-adjusted analyses. Age-specific analyses suggested an increased risk for the HH homozygous genotype in subjects younger than 60 years. A statistically significant interaction emerged between this genotype and age (p = 0.032). When analyses were restricted to MBC cases enrolled in the first 4 years following diagnosis, a recessive model showed a significantly increased risk of MBC in HH subjects younger than 60 years (OR = 5.63; 95% CI = 1.70;18.61).ConclusionOverall, our findings, although based on a relatively small series, suggest that the BRCA2 HH homozygous genotype might be positively associated with an increased risk of MBC in men younger than 60 years.

p53 expression is decreased in primary breast carcinomas with microsatellite instability

Abstractp53 and p185 expression in primary breast cancer with microsatellite instability (MSI) is still largely unexplored. To investigate the relationship between these oncoproteins and the pathways of genomic instability, we examined 52 primary invasive breast cancers stratified by the presence and absence of MSI. We determined the status of eight microsatellite loci using radioactive and silver staining methods, and evaluated the immunohistochemical expression of p53 and p185 in a consecutive series of Italian cancer patients characterized by clinical-pathological and biological parameters. Nineteen cases (36.5%) were MSI-positive in at least two loci. p53 was expressed in 15 cases (28.8%) and p185 in eight (15.4%). MSI-positive tumors were inversely correlated with p53 expression ( p = 0.0007); in addition, the percent of p53-expressing cells decreased as the number of MSI-positive loci increased. MSI-positive tumors were correlated with a larger tumor size ( p = 0.04), lymph-node metastasis ( p = 0.001), and advanced clinical stage ( p = 0.0006). These data demonstrate the existence of two subsets of primary breast cancers: one characterized by MSI, the other by p53 expression. MSI-positive patients had a more advanced and/or aggressive disease.