Paolo Aretini

Angiogenesis and VEGF expression in pre-invasive lesions of the human breast.

Angiogenesis (as microvascular density—MVD) and vascular endothelial growth factor (VEGF) expression were evaluated by immunohistochemistry in all types of human pre‐invasive breast lesion, un‐associated with invasive carcinoma, including florid ductal hyperplasia of usual type (FDHUT, 40 cases), atypical ductal hyperplasia (ADH, 10), well‐differentiated intraductal carcinoma (WDIC, 16), intermediately differentiated intraductal carcinoma (IDIC, 25), poorly differentiated intraductal carcinoma (PDIC, 20), atypical lobular hyperplasia (ALH, 13), and lobular carcinoma in situ (LCIS, 12). Both parameters were also studied in normal glandular structures obtained from normal breasts or from breasts containing pre‐invasive lesions. Increased vascularization was present in all lesion types (MVD mean values (expressed as vessel number/mm2): 115 ± 8 in normal lobules, 146 ± 26 in lesions; p < 0.05) and increased with lesion severity. In ductal lesions, MVDs were significantly higher in PDIC (190 ± 65) and IDIC (167 ± 61) than in FDHUT (123 ± 40) and ADH (122 ± 47); MVD was much higher in PDIC than in WDIC (p < 0.001). WDIC showed peculiar features, with a degree of vascularization closer to hyperplasia than to the other histological types of in situ ductal cancer; this observation is in line with the hypothesis that IDIC and PDIC may originate ‘de novo’, without a mandatory transition through WDIC. LCIS was more vascularized than ALH (168 ± 50 and 125 ± 40, respectively; p < 0.05), showing MVD values similar to those of PDIC and IDIC. The vascularization of normal lobules was constant, regardless of their association with lesions. VEGF expression in normal glandular structures was lower than in lesions, with the highest levels found in ductal lesions when compared with lobular lesions. No correlation was found between VEGF expression and the degree and/or type of vascularization. Copyright

Penetrances of breast and ovarian cancer in a large series of families tested for BRCA1/2 mutations.

Accurate estimates of breast and ovarian cancer penetrance in BRCA1/2 mutation carriers are crucial in genetic counseling. Estimation is difficult because of the low frequency of mutated alleles and the often-uncertain mechanisms of family ascertainment. We estimated the penetrances of breast and ovarian cancers in carriers of BRCA1/2 mutations by maximizing the retrospective likelihood of the genetic model, given the observed test results, in 568 Italian families screened for germline mutations. The software BRCAPRO was used as a probability calculation tool in a Markov Chain Monte Carlo approach. Breast cancer penetrances were 27% (95% CI 20–34%) at age 50 years and 39% (27–52%) at age 70 in BRCA1 carriers, and 26% (0.18–0.34%) at age 50 and 44% (29–58%) at age 70 in BRCA2 carriers, and ovarian cancer penetrances were 14% (7–22%) at age 50 and 43% (21–66%) at age 70 in BRCA1 carriers and 3% (0–7%) at age 50 and 15% (4–26%) at age 70 in BRCA2 carriers. The new model gave a better fit than the current default in BRCAPRO, the likelihood being 70 log units greater; in addition, the observed numbers of mutations in families stratified by gene and by cancer profile were not significantly different from those expected. Our new penetrance functions are appropriate for predicting breast cancer risk, and for determining the probability of carrying BRCA1/2 mutations, in people who are presently referred to genetic counseling in Italy. Our approach could lead to country-customized versions of the BRCAPRO software by providing appropriate population-specific estimates.

Evaluation of widely used models for predicting BRCA1 and BRCA2 mutations

Deleterious mutations of the BRCA1 and BRCA2 genes are a major risk factor for the development of breast and ovarian cancers.1–4 Mutation tests for these two genes commonly are now offered in specialised clinics.5,6 As a result, a large number of women with personal or family histories of breast or ovarian cancer seek genetic counselling. Accurate evaluation of the probability that a woman carries a germline pathogenic mutation at BRCA1 or BRCA2 therefore is essential to help counsellors and those being counselled to decide whether testing is appropriate. In this context, the questions of practical interest are: Given the pedigree, what is the chance of a mutation being present? and What is the chance of the DNA laboratory finding a mutation? After testing became available, several models were developed to assess the pre-test probability of identifying carriers of mutations. Broadly speaking, two different approaches have been used to develop predictive models: the “empirical approach” and the “Mendelian approach”.7 In empirical models, families are stratified according to variables that describe their family history; regression or other approaches are used to predict the results of Mendelian testing. In some cases, this approach simply consists of observing the proportion of mutations found in different strata. Mendelian models, in contrast, address the probability that a proband is a mutation carrier on the basis of explicit assumptions about the genetic parameters (allele frequencies and cancer penetrances in carriers and non-carriers) and the Mendelian rules of gene transmission. A consequence of the two different strategies is that the Mendelian models evaluate the probability that a proband is a gene carrier, whereas the empirical models evaluate the probability of identifying a mutation. The main purpose of this study was to compare the performances of published models in predicting mutation test results in …

KLF4 is a Novel Candidate Tumor Suppressor Gene in Pancreatic Ductal Carcinoma

Ductal pancreatic carcinoma (DPC) is a deadly disease with an incidence of 9 cases in 100,000 people per year and a mortality rate close to 100%. Allelic losses in the long arm of chromosome 9 are commonly encountered in many human malignancies but no data are yet available about DPC. We screened 40 laser-microdissected DPC samples and 6 pre-invasive lesions for 9 microsatellite mapping markers of region 9q21.3 through 9q34.2. A small overlapping region of deletion, spanning 8 million base pairs, was identified between D9S127 and D9S105. Two genes, RSG3 and KLF4, mapped to 9q31.1 through 9q32, were further investigated. A highly significant association was found between KLF4 gene expression levels and genomic status. Similarly, absence of immunohistochemical expression of KLF4 protein was found in 86.8% cases of DPC (33/38). Overexpression of KLF4 in a human pancreatic carcinoma cell line induced a significant decrease in the proliferation associated with up-regulation of p21 and the down-regulation of cyclin D1. In conclusion, we identified a novel oncosuppressor region located at the 9q 31.1-3 locus that is lost in DPC at high frequency. Loss of KLF4 expression is closely related to the genomic loss, and its restoration inhibits cancer cell proliferation, suggesting a key suppressor role in pancreatic tumorigenesis.

High level of messenger RNA for BRMS1 in primary breast carcinomas is associated with poor prognosis.

BRMS1 is regarded as a metastasis suppressor gene for its ability to reduce metastatic potential of human and murine breast cancer cells as well as human melanoma cells. However, BRMS1 association to human tumor progression is not clearly understood. In the present study we analyzed BRMS1 mRNA expression in tumor progression and its potential prognostic value for breast carcinoma. BRMS1 mRNA expression level was quantified by real‐time PCR in 47 tumoral, in 14 peritumoral and in 15 metastatic microdissected cellular populations from 47 breast cancer patients with 10‐year follow up. We found BRMS1 expression to be higher in carcinoma cells than in matching normal epithelial cell populations in 10 out of 14 cases (p = 0.0005), while lymph‐nodal carcinoma cells showed lower BRMS1 expression in 9 out of 15 cases (p = 0.001). Using both in vivo (human mammary breast carcinomas) and in vitro systems (breast cancer cell lines) we were able to demonstrate that BRMS1 overexpression was not a bias effect induced by cell proliferation rate. BRMS1 expression levels did not correlate with standard breast cancer prognostic factors but BRMS1 higher expression was associated with patient shorter disease‐free and overall survival. Our findings are apparently inconsistent with the concept of BRMS1 as a metastasis suppressor gene. One possible explanation is that epithelial cells increase their BRMS1 expression as a compensatory response to tumor formation or metastasis progression, which is elevated in proportion to tumor aggressiveness, whereas those cells of the primary tumor that cannot upregulate BRMS1 escape to form metastasis.

Papillary lesions of the breast: a molecular progression?

SummaryIntroduction. Breast papillary lesions represent a heterogeneous group of tumors ranging from benign to malignant, including several intermediate forms. Malignant papillary tumors are rare and their molecular characterization is still limited. A few studies pointed to the presence of specific genetic alterations that could be relevant both for diagnostic purposes and to elucidate tumour development and progression. In order to look into the issue, we compared LOH relative frequencies of four microsatellite markers located on chromosome 16 in a set of morphologically different papillary breast lesions. LOH at TP53 locus was also analyzed throughout lesions. Materials and methods. Fifteen cases were analyzed. Sections including a malignant papillary lesion, a benign lesion (when available), and normal breast tissue were selected. Fifteen malignant and twelve benign areas were microdissected using the Leica laser microdissection system (AS LMD). After DNA extraction samples were tested for the following markers: TP53, D16S423, D16S310, DS163210 and D16S476, and analyzed on ABI PRISM 3100 (Applied Biosystems, Foster city CA). Results. Fourteen malignant lesions and twelve paired benign areas appeared to be informative for at least one of the four markers on chromosome 16. In particular, LOH at loci 16p13 and 16q21 was detected in both benign and malignant lesions, whereas LOH at locus 16q23 was limited to malignant lesions. Nine malignant and seven benign lesions were informative for LOH at TP53 locus, that was found to be significantly associated (p=0.01) with the malignant phenotype. Conclusions. Our data suggest an involvement of chromosome 16 mutations in the early steps of breast papillary tumorigenesis. TP53 deletion and possibly LOH at 16q23 appear to play a role as progression factors, being they significantly associated with malignant transformation of breast papilloma.

PIK3CA in Breast Carcinoma: A Mutational Analysis of Sporadic and Hereditary Cases

The PI3K-Akt cascade is a key signaling pathway involved in cell proliferation, survival, and growth. Activating PIK3CA mutations have been reported in breast carcinoma (BC). The aim of this study was to characterize the PIK3CA mutations at exons 9 and 20 in a series of 176 sporadic and 22 hereditary BCs and to correlate the results with clinicopathologic parameters and survival. In sporadic BC, 68 missense mutations were detected. PIK3CA mutations were significantly associated with ER+ in HER2-negative cases. A higher frequency of PIK3CA mutations was present in lobular carcinoma compared with ductal carcinoma (50% vs. 35%). There was no association between the survival and PIK3CA mutational status. In hereditary BC, PIK3CA mutations were found only in the BRCA2 group. The PIK3CA mutation seems to characterize the luminal-type BC, in both sporadic and BRCA2 mutated forms, and is absent in the basal-type BC, in both the sporadic and BRCA1 mutated forms.

BRCA1 and BRCA2 germline mutations in Moroccan breast/ovarian cancer families: Novel mutations and unclassified variants

OBJECTIVE Breast cancer is the most common female cancer in Morocco. About 5 to 10% are due to hereditary predisposition and mutations in BRCA1 and BRCA2 genes are responsible for an important proportion of high-risk breast/ovarian cancer families. The relevance of BRCA1/2 mutations in the Moroccan population was not studied. The main objective of this study is to investigate the spectrum of BRCA1 and BRCA2 germline mutations in early onset and familial breast/ovarian cancer among Moroccan women. METHODS We screened the entire coding sequences and intron/exon boundaries of BRCA1 and BRCA2 genes in 40 patients by direct sequencing. RESULTS Nine pathogenic mutations were detected in ten unrelated families, five deleterious mutations in BRCA1 gene and four mutations in BRCA2 gene. Four novel mutations were found: one in BRCA1 (c.2805delA/2924delA) and three in BRCA2 (c.3381delT/3609delT; c.7110delA/7338delA and c.7235insG/7463insG). We also identified 51 distinct polymorphisms and unclassified variants (three described for the first time). CONCLUSIONS Our data suggest that BRCA1 and BRCA2 mutations are responsible for a significant proportion of familial breast cancer in Moroccan patients. Therefore full BRCA1/2 screening should be offered to patients with a family history of breast/ovarian cancer.

A yeast recombination assay to characterize human BRCA1 missense variants of unknown pathological significance.

The BRCA1 tumor suppressor gene is found mutated in familial breast cancer. Although many of the mutations are clearly pathological because they give rise to truncated proteins, several missense variants of uncertain pathological consequences have been identified. A novel functional assay to screen for BRCA1 missense variants in a simple genetic system could be very useful for the identification of potentially deleterious mutations. By using two prediction computer programs, Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen), seven nonsynonymous missense BRCA1 variants likely disrupting the gene function were selected as potentially deleterious. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) was used to test these cancer‐related missense mutations for their ability to affect cell growth and homologous recombination (HR) at the HIS3 and ADE2 loci. The variants localized in the BRCA1 C‐Terminus (BRCT) domain did not show any growth inhibition when overexpressed in agreement with previous results. Overexpression of either wild‐type BRCA1 or two neutral missense variants did not increase yeast HR but when cancer‐related variants were overexpressed a significant increase in recombination was observed. Results clearly showed that this genetic system can be useful to discriminate between neutral and deleterious BRCA1 missense variants. Hum Mutat 0, 1–11, 2008.

p53 inactivation is a rare event in familial breast tumors negative for BRCA1 and BRCA2 mutations

Germline mutations at BRCA1 or BRCA2 genes result in susceptibility to breast and ovarian cancers. BRCA1- and BRCA2-associated tumors have distinct histologic and molecular phenotypes, as compared to sporadic breast tumors. Typically, a higher grade of malignancy is observed in BRCA-associated cancers. A number of studies have suggested that BRCA1 and BRCA2 proteins are of importance in DNA repair and maintenance of genome integrity, bringing about molecular models of tumor pathogenesis. In particular, alterations at p53 gene have been suggested to be a necessary step in the tumorigenesis of BRCA-associated carcinomas. In fact, BRCA-associated breast cancers have higher p53 mutation frequencies than sporadic ones. At present, very little is known regarding BRCA non-associated familial tumors (termed BRCAx tumors). To our knowledge no data is available on p53 alterations in this sub-group of familial tumors. In this study p53 alteration frequencies were evaluated in 13 BRCA1, 11 BRCA2 and 55 BRCAx breast tumors. Tumor samples were analyzed for p53 gene mutations by PCR-SSCP/direct sequencing, and for p53 protein overexpression by immunohistochemistry (IHC). Altogether, p53 alterations were detected in 54% of BRCA1 tumors compared with 5% of BRCAx tumors. No p53 alteration was found in BRCA2 tumors. While loss of p53 checkpoint control is likely to be an important step in the molecular pathogenesis of BRCA1-associated cancers, our data seem to indicate a p53-independent molecular mechanism underlying BRCAx neoplastic transformation.