Loredana Bruno

Hereditary ovarian cancer

At least 10% of ovarian tumors are hereditary and associated with highly penetrant, autosomal, dominant genetic predisposition. Three clinical manifestations of hereditary ovarian cancer have been identified: site-specific ovarian cancer, hereditary breast and/or ovarian cancer (HBOC) and hereditary non-polyposis colorectal cancer (HNPCC) syndromes. BRCA germline mutations account for more than 90% of all hereditary epithelial ovarian tumors whereas most of the remaining 10% are caused by MLH1 and MSH2 mutations, which are susceptibility genes of HNPCC. Genetic testing is available for each of the three hereditary syndromes above mentioned. The recommendations for OC surveillance in high-risk women having a strong family history or BRCA mutation carriers include transvaginal pelvic ultrasound with color Doppler and serum CA125 every 6 months. Bilateral salpingo-oophorectomy appears to be effective to reduce the risk of ovarian cancer in BRCA mutation carriers. Hysterosalpingo-oophorectomy should be considered in HNPCC women who undergo surgery for colorectal carcinoma.

TP53 and p16INK4A, but not H-KI-Ras, are involved in tumorigenesis and progression of pleomorphic adenomas.

The putative role of TP53 and p16INK4A tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex‐PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific‐PCR (MS‐PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H‐Ras and K‐Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K‐Ras. p16INK4A promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16INK4A promoter methylation, but not alterations in the H‐Ras and K‐Ras genes, might be involved in the malignant progression of PA into carcinoma.

Role of S128R polymorphism of E-selectin in colon metastasis formation

The extravasation of cancer cells is a key step of the metastatic cascade. Polymorphisms in genes encoding adhesion molecules can facilitate metastasis by increasing the strength of interaction between tumor and endothelial cells as well as impacting other properties of cancer cells. We investigated the Ser128Arg (a561c at the nucleotide level) polymorphism in the E‐selectin gene in patients with metastatic colon cancer and its functional significance. Genotyping for a561c polymorphism was performed on 172 cancer patients and on an age‐matched control population. The colon cancer group was divided into groups with (M+) and without observable metastasis (M−). For in vitro functional assays, Huvec transfected cells expressing wild‐type (WT) or the S128R variant of E‐selectin were established to study in vitro binding ability and signal transduction processes of T84 colon cancer cell line. Our results demonstrated that the Arginine128 allele was more prevalent in the M+ group than in the M− group or normal controls (p < 0.005; odds ratio, 1.56; 95% confidence interval (CI) 1.16–1.92; p < 0.001, odds ratio = 1.65; CI = 1.24–1.99, respectively). In vitro, S128R E‐selectin transfected Huvec cells, supported increased adhesion as well as increased cellular signaling of T84 cancer cells compared to WT E‐selectin and mock‐transfected Huvec cells. These findings suggest that the E‐selectin S128R polymorphism can functionally affect tumor‐endothelial interactions as well as motility and signaling properties of neoplastic cells that may modulate the metastatic phenotype.

The Clinical Significance of Unknown Sequence Variants in BRCA Genes

Germline mutations in BRCA1/2 genes are responsible for a large proportion of hereditary breast and/or ovarian cancers. Many highly penetrant predisposition alleles have been identified and include frameshift or nonsense mutations that lead to the translation of a truncated protein. Other alleles contain missense mutations, which result in amino acid substitution and intronic variants with splicing effect. The discovery of variants of uncertain/unclassified significance (VUS) is a result that can complicate rather than improve the risk assessment process. VUSs are mainly missense mutations, but also include a number of intronic variants and in-frame deletions and insertions. Over 2,000 unique BRCA1 and BRCA2 missense variants have been identified, located throughout the whole gene (Breast Cancer Information Core Database (BIC database)). Up to 10–20% of the BRCA tests report the identification of a variant of uncertain significance. There are many methods to discriminate deleterious/high-risk from neutral/low-risk unclassified variants (i.e., analysis of the cosegregation in families of the VUS, measure of the influence of the VUSs on the wild-type protein activity, comparison of sequence conservation across multiple species), but only an integrated analysis of these methods can contribute to a real interpretation of the functional and clinical role of the discussed variants. The aim of our manuscript is to review the studies on BRCA VUS in order to clarify their clinical relevance.

TP53 Mutations and S-Phase Fraction but not DNA-Ploidy Are Independent Prognostic Indicators in Laryngeal Squamous Cell Carcinoma

To prospectively evaluate the prognostic significance of TP53, H‐, K‐, and N‐Ras mutations, DNA‐ploidy and S‐phase fraction (SPF) in patients affected by locally advanced laryngeal squamous cell carcinoma (LSCC). Eight‐one patients (median follow‐up was 71 months) who underwent resective surgery for primary operable locally advanced LSCC were analyzed. Tumor DNA was screened for mutational analysis by PCR/SSCP and sequencing. DNA‐ploidy and SPF were performed by flow cytometric analyses. Thirty‐six patients (44%) had, at least, a mutation in the TP53 gene. Of them, 22% (8/36) had double mutations and 3% (1/36) had triple mutations. In total, 46 TP53 mutations were observed. The majority (41%) of these occur in exon 5 (19/46), while the mutations in exons 6, 7, and 8 were represented in 14, 7, and 6 patients, respectively (31%, 15%, and 16%). Five LSCC patients (6%) showed a mutation in H‐Ras gene. Sixty‐three percent of the cases (51/81) were DNA aneuploidy, 14% of these (7/51) were multiclonal. Thirty‐nine patients (48%) had an high SPF value. At Univariate analysis, the DNA aneuploidy, high SPF (>15.1%), TP53 mutations and, in particular, the mutations that occur in exons 5 and 8 were significantly related to quicker disease relapse and short OS. At Multivariate analysis, the major significant predictors for both disease relapse and death were high SPF and any TP53 mutations. While histological grade G3 was an independent factor only for relapse. In conclusions, any TP53 mutations and high SPF are important biological indicators to predict the outcome of LSCC patients.

Is BRCA1-5083del19, identified in breast cancer patients of Sicilian origin, a Calabrian founder mutation?

Various studies have been published in Italy regarding the different BRCA1 mutations, but only the BRCA1-5083del19 mutation is recurrent and specific to individuals of Italian descent with a founder effect on the Calabrian population. In our previous study, BRCA1-5083del19 mutation carriers were found in four index cases of 106 Sicilian patients selected for familial and/or hereditary breast/ovarian cancers. The high frequency rate of this mutation identified in the Sicilian population led us to perform haplotype analysis in all family carriers. Five highly polymorphic microsatellite markers were used (D17S1320, D17S932, D17S1323, D17S1326, D17S1325) to establish whether or not all these families had a common ancestor. This analysis showed that all mutation carriers of these families had a common allele. None of the non-carriers of the mutation or of the 50 healthy Sicilian controls showed this haplotype. This allelotype analysis highlighted the presence of a common allele (ancestor), thus suggesting the presence of a founder effect in the Sicilian population. Our results are in contrast with other studies but only the allelotype analysis of all the BRCA1-5083del19 mutation carriers of two neighboring regions of the south of Italy (Calabria and Sicily) will make it possible to identify the real ancestor of this mutation.

Abstract 448: Molecular analysis of BRAF gene and PTEN gene expression in metastatic colorectal cancer patients: Feasibility study

Introduction There are numerous causes triggering CRC. 25-80% of CRC shown a deregulation in Epidermal Growth Factor Receptor (EGFR) pathway. Two signaling pathways downstream of the EGFR are dysregulated in CRC the mitogen-activated protein kinase (MAPK) and the phosphoinositide-3-kinase (PI3K) pathway. Activating mutations in KRAS and BRAF (MAPK pathway) and PIK3CA affect prognosis and/or response to anti-EGFR MoAb. PTEN is a downstream effector of EGFR pathway and is involved in PI3K pathway. Loss of PTEN protein expression can occur through epigenetic silencing and mutation or allelic loss. Immunohistochemistry (IHC) is the most effective way to assay for loss of PTEN expression. IHC lack of reproducibility and this has lead to discordant results regarding the concordant in paired CRC metastases and primary tumors.The aim of the work was to asses the feasibility of PTEN gene expression analysis from FFPE tissue in CRC samples. Materials and methods We selected a series of 33 patients, already tested for KRAS mutational status and resulted wild-type. At the time of the study mutational status of NRAS gene has not a role as predictive marker, so was not performed. A retrospective analysis on CRC FFPE tissue was performed. As control samples we have selected specimen of colorectal surgery not tumor-associated (diverticulitis). On these samples BRAF mutational analysis and PTEN gene expression was performed. For BRAF analysis, DNA was extracted by means of QIAamp DNA FFPE tissue kit (Qiagen, USA). BRAF mutational status was assed through direct automated sequencing (3100 Genetic Analyzer, Applied Biosystems) and TaqMan mutation detection assay (ABI PRIM 7900HT, Applied Biosystems). For PTEN gene expression, total RNA was obtained through the miRNeasy FFPE tissue kit (Qiagen, USA), according to manufacturer instructions, and reverse transcribed. PTEN expression was assessed by means of TaqMan probes. Results According to BRAF mutational status we have found 10/33 (30%) mutated samples. PTEN expression levels were evaluated by the comparison of tumor samples vs diverticula samples, the data obtained were then integrated with those from BRAF mutational analysis. Interestingly we found that all the BRAF mutated samples present also a down-regulation of PTEN. Statistical analyses were conducted through the X 2 test with the Yatesha correction (p=0.00023). The p-value was also calculated Exact Fisher Test (p= 0.00021) to confirm the results. Conclusion The determination of PTEN gene expression level is feasible with real-time PCR using TaqMan probe technology. BRAF analysis might be used as prognostic and/or predictive marker in CRC patients’ treatment. The identification of a validated method for PTEN analysis would establish it as a molecular marker meriting further investigation in large numbers of available primary tumors from patients with CRC. Further analyses are requested to confirm these data. Citation Format: Christian D. Rolfo, Marta Castiglia, Daniela Cabibi, Valentina Calo9, Florinda Di Piazza, Viviana Bazan, Fabio Brocco, Stefano Caruso, Loredana Bruno, Konstantinos Papadimitriou, Francesco Passiglia, Marc Peeters, Patrick Pauwels, Antonio Russo. Molecular analysis of BRAF gene and PTEN gene expression in metastatic colorectal cancer patients: Feasibility study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 448. doi:10.1158/1538-7445.AM2014-448