Long Non-Coding RNA Landscape in Prostate Cancer Molecular Subtypes: A Feature Selection Approach.

Prostate cancer is one of the most common malignancies in men. It is characterized by a high molecular genomic heterogeneity and, thus, molecular subtypes, that, to date, have not been used in clinical practice. In the present paper, we aimed to better stratify prostate cancer patients through the selection of robust long non-coding RNAs. To fulfill the purpose of the study, a bioinformatic approach focused on feature selection applied to a TCGA dataset was used. In such a way, LINC00668 and long non-coding(lnc)-SAYSD1-1, able to discriminate ERG/not-ERG subtypes, were demonstrated to be positive prognostic biomarkers in ERG-positive patients. Furthermore, we performed a comparison between mutated prostate cancer, identified as "classified", and a group of patients with no peculiar genomic alteration, named "not-classified". Moreover, LINC00920 lncRNA overexpression has been linked to a better outcome of the hormone regimen. Through the feature selection approach, it was found that the overexpression of lnc-ZMAT3-3 is related to low-grade patients, and three lncRNAs: lnc-SNX10-87, lnc-AP1S2-2, and ADPGK-AS1 showed, through a co-expression analysis, significant correlation values with potentially druggable pathways. In conclusion, the data mining of publicly available data and robust bioinformatic analyses are able to explore the unknown biology of malignancies.

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions.

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5'UTR and 3'UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband's group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3'UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.

A Promising Role of TGF-ß Pathway in Response to Regorafenib in Metastatic Colorectal Cancer: A Case Report.

Colorectal cancer (CRC) is one of the most common cancer types around the world. The prognosis of patients with advanced diseases is still poor in spite of currently available therapeutic options. Regorafenib is an oral tyrosine kinase inhibitor (TKI) approved to treat refractory metastatic colorectal cancer (mCRC). We investigated Somatic mutations in several genes involved in immunological response and cancer progression in both long/short responder mCRC patients who underwent third-line therapy with regorafenib to identify predictive biomarkers of response using Ion Torrent PGM sequencing and bioinformatic tools. We found Somatic mutations in TGFBR1, TGFBR2, and TGFBR3 genes in primary tumor and metastases samples of long-responder patients. Furthermore, our bioinformatic results show that they were mainly enriched in immune response, cell junction, and cell adhesion in long responder patients, particularly in primary tumor and metastatic sites. These data suggest that the TGF-b pattern could be the leading actor of a prolonged response to this drug.

Standardization of CTC AR-V7 PCR assay and evaluation of its role in castration resistant prostate cancer progression.

BACKGROUND: Castration resistant prostate cancer (CRPC) represents the most aggressive status of this neoplastic disease, also characterized by the absence of biomarkers predictive of clinical outcome. New drugs as abiraterone or enzalutamide, affecting androgen receptor pathway at different levels, inhibit the proliferative advantage of prostate cancer cells with important long term benefits. Despite the advantages of this second-generation androgen deprivation therapy (ADT), resistance mechanisms, primitive or acquired, often develop. The existence of androgen receptor (AR) splice variants (AR-Vs), in particular AR-V7 expression detected in circulating tumor cells (CTCs), represents an example of acquired resistance, as evidenced in preclinical and clinical studies. Recent studies also have suggested the role of AR-V7 as a prognostic biomarker in mCRPC. In this field, hot topics are the methodology used to isolate CTC and the assay for AR-V7 measurement. Our study aims to develop a standardized operating procedure (SOP) to evaluate AR-V7 in CRPC. METHOD: The application of a realized cell based Reference Sample as Standardized Quality Control tool for CTC-AR-V7 assay has been shown. Then the development, the performance evaluation and contextualization in a clinical setting of this standardized operating procedure (SOP) have been reported to evaluate the prognostic biomarker AR-V7 in metastatic prostate cancer. RESULTS AND CONCLUSIONS: The standardized procedure has high sensitivity and specificity and enables the detection and quantification of the spliced variant with respect to the full length AR (AR-FL) mRNA in CTC DNA purified from the blood of patients with CRPC. This procedure has been further validated in a consecutive series of patients with mCRPC, confirming its role as prognostic biomarker.

BRCA1-2 diagnostic workflow from next-generation sequencing technologies to variant identification and final report.

The BRCA1-BRCA2 genes predispose to hereditary breast and ovarian cancer, and the germline and mutational status of these genes defines a target population that can benefit from PARP inhibitor treatments. To respond to the increasing number of BRCA1-BRCA2 tests, it is necessary to shift to high-throughput technologies that are reliable and less time consuming. Different methodological platforms are dedicated to this purpose with different approaches and algorithms for analysis. Our aim was to set up a cost-effective and low time-consuming BRCA1-BRCA2 mutation detection workflow using the Ion Torrent PGM technology. A retrospective cohort of 40 patients with familial breast/ovarian cancer previously tested by Sanger sequencing and a prospective cohort of 72 patients (validation set) were analyzed. The validation set included 64 patients affected by familial breast/ovarian cancer and eight sporadic ovarian cancer cases, who are potential candidates for PARPi treatments. A complete and standardized workflow easily usable and suitable in a certified laboratory has been proved and validated. This includes all steps from library preparation to the final report. The use of next-generation sequencing will be of benefit for patients enrolled in the genetic counseling process and, moreover, will enhance the process of selecting patients eligible for personalized treatments. © 2016 Wiley Periodicals, Inc.

Combined microRNA and ER expression: a new classifier for familial and sporadic breast cancer patients.

BACKGROUND: The role of miRNAs in familial breast cancer (fBC) is poorly investigated as also in the BRCA-like tumors. To identify a specific miRNA expression pattern which could allow a better fBC classification not only based on clinico-pathological and immunophenotypical parameters we analyzed miRNA profile in familial and sporadic samples. Moreover since BRCA1 tumors and sporadic triple negative (TN) breast tumors share similarities regarding clinical outcomes and some histological characteristics, we focused on TN and not TN cases. METHODS: The sample set included fresh frozen tissue samples, including 39 female fBCs (19 BRCA-related and 20 BRCAX) and 12 male fBC (BRCAX). Moreover, we considered TN and non TN (NTN), 21 BRCA-related and 27 sporadic BCs. MiRNA profiling was performed through GeneChip miRNA v.1.0 Array (Affymetrix). ANOVA, hierarchical and consensus clustering analyses allowed identification of pattern of expression of miRNAs and pathway enrichment analysis, considering validated target genes, was carried out to achieve a deeper biological understanding. RESULTS: ANOVA test led to the identification of 53 deregulated miRNAs; hierarchical and consensus clustering of female fBCs (fFBCs) and male fBCs (fMBCs) highlighted the presence of 3 sample clusters named FBC1, FBC2 and FBC3. We found a correlation between ER-status and the three sample clusters. The three clusters are distinct by a different expression of two clusters of miRNAs (CLU1 and CLU2), which resulted to be different in targeted pathways. In particular, CLU1 targets cellular pathways and CLU2 is involved in epigenetic activities. Considering TN and NTN BRCA-related and sporadic tumors, a hierarchical clustering identified two clusters of miRNAs, which were not so different from CLU1 and CLU2, both in miRNA content and targeted pathways. CONCLUSIONS: Our results highlighted the importance of miRNA regulation to better clarify similarities and differences between familial and sporadic BC groups.

Genetic risk transmission in a family affected by familial breast cancer.

Breast Cancer is the most common malignancy among women. Family history is the strongest single predictor of breast cancer risk, and thus great attention has been focused on BRCA1 and BRCA2 genes whose mutations lead to a high risk of developing this disease. Today, only 25% of high- and moderate-risk genes are known, suggesting the importance of the discovery of new risk modifiers. Therefore, the investigation of new polygenic alterations is of great importance, especially if considered high- and moderate-risk variants. In this study, the transmission of BRCA1-2 polymorphisms in association with the transmission of polymorphisms in the genes NUMA1, CCND1, COX11, FGFR2, TNRC9 and SLC4A7 were examined in all members of a family with the BRCA2 c.6447_6448dup mutation. This is the first study about the transmission of high-risk polygenic variants in all members of a family with a strong history of breast cancer. The results about the possible polygenic variant associations that could increase and modify the risk suggested the importance to search new variants to better manage patients and their family members.

Case report: Germline POT1 mutation in a patient with GIST and lung adenocarcinoma.

The gene protection of telomere 1 (POT1) is involved in telomere maintenance and stability and plays a crucial role in the preservation of genomic stability. POT1 is considered a high-penetrance melanoma susceptibility gene; however, the number of cancer types associated with the pathogenic germline variants of POT1 is gradually increasing, including chronic lymphocytic leukemia (CLL), angiosarcomas, and gliomas, even though many associations are still elusive. Here, we reported a case of a 60-year-old man who showed early-onset multiple neoplasms, including multiple melanomas, gastrointestinal stromal tumor (GIST), and lung adenocarcinoma. Next-generation sequencing (NGS) analyses revealed a germline heterozygous pathogenic variant in the POT1 gene. Notably, GIST and lung adenocarcinoma were not previously reported in association with the POT1 germline variant. Lung cancer susceptibility syndrome is very rare and the actual knowledge is limited to a few genes although major genetic factors are unidentified. Recently, genome-wide association studies (GWAS) have pointed out an association between POT1 variants and lung cancer. This case report highlights the clinical relevance of POT1 alterations, particularly their potential involvement in lung cancer. It also suggests that POT1 testing may be warranted in patients with familial cancer syndrome, particularly those with a history of melanoma and other solid tumors.

Different methylation and microRNA expression pattern in male and female familial breast cancer.

Epigenetic regulation, has been very scarcely explored in familial breast cancer (BC). In the present study RASSF1A and RAR beta promoter methylation and miR17, miR21, miR 124, and let-7a expression were investigated to highlight possible differences of epigenetic regulation between male and female familial BC, also in comparison with sporadic BC. These epigenetic alterations were studied in 56 familial BC patients (27 males and 29 females) and in 16 female sporadic cases. RASSF1A resulted more frequently methylated in men than women (76% vs. 28%, respectively, P = 0.0001), while miR17 and let-7a expression frequency was higher in women than in men (miR17: 66% in women vs. 41% in men, P < 0.05; let-7a: 45% in women vs. 15% in men, P = 0.015). RASSF1A methylation affected 27.6% of familial BC while 83% of familial cases showed high expression of the gene (P = 0.025); on the contrary, only 17% of familial BC presented RAR beta methylation and 55% of familial cases overexpressed this gene (P = 0.005). Moreover, miR17, miR21, and let-7a resulted significantly overexpressed in familial compared to sporadic BC. RASSF1A overexpression (86% vs. 65%, P = 0.13) and RAR beta overexpression (57% vs. 32%, P = 0.11) were higher in BRCA1/2 carriers even if not statistical significance was reached. BRCA mutation carriers also demonstrated significant overexpression of: miR17 (93% vs. 35%, P = 0.0001), let-7a (64% vs. 16%, P = 0.002), and of miR21 (100% vs. 65%, P = 0.008). In conclusion, the present data suggest the involvement of RASSF1A in familial male BC, while miR17 and let-7a seem to be implied in familial female BC.

HOX gene methylation status analysis in patients with hereditary breast cancer.

Cancer development is related not only to genetic alterations but also to aberrant epigenetic changes that could lead to heritable gene patterns critical for neoplastic initiation and progression. Knowledge of epigenetic regulation in cancer cells is useful for both the understanding of carcinogenesis and for the possibility of using epigenetic drugs. HOX genes deregulation have a crucial role in oncogenesis process and tumor suppression. In this report, the methylation of HOXA1, HOXA9, HOXA10, HOXB13, HNF1B, OTX1, TLX1 genes have been analyzed in patients with hereditary breast cancer. This is the first study analyzing BRCA mutational status of patients with respect to methylation of HOX genes. HOXA10 has been found to be methylated in all patients analyzed but never in healthy subjects. With respect to clinical pathological information, hypermethylation of all studied genes, with the exception of OTX1, was significantly associated with absence of HER2 neu expression (P<0.05). Moreover, hypermethylation of HOXB13, HOXA10 and HOXA1 was associated with a high proliferation index (Mib1≥10%, P<0.05) and hypermethylation of HOXB13 and HOXA10 also with high expression of estrogen and progesterone receptors. These preliminary data suggest a possible involvement of HOX genes in familial breast cancer as marker helpful to identify high-risk patients.

The ERRα-VDR axis promotes calcitriol degradation and estrogen signaling in breast cancer cells, while VDR-CYP24A1-ERRα overexpression correlates with poor prognosis in patients with basal-like breast cancer.

Vitamin D is used to reduce cancer risk and improve the outcome of cancer patients, but the vitamin D receptor (VDR; also known as the calcitriol receptor) pathway needs to be functionally intact to ensure the biological effects of circulating calcitriol, the active form of vitamin D. Besides estrogen receptor alpha (ERα), estrogen-related receptor alpha (ERRα) has also been shown to interfere with the VDR pathway, but its role in the antitumor and transactivation activity of calcitriol is completely unknown in breast cancer (BC). We observed that ERRα functionally supported the proliferation of BC cell lines and acted as a calcitriol-induced regulator of VDR. As such, ERRα deregulated the calcitriol-VDR transcription by enhancing the expression of CYP24A1 as well as of both ERα and aromatase (CYP19A1) in calcitriol-treated cells. ERRα knockdown limited the effect of calcitriol by reducing calcitriol-induced G0/G1 phase cell cycle arrest and by affecting the expression of cyclin D1 and p21/Waf. The interactome analysis suggested that Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-α (PGC-1α) and Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) are key players in the genomic actions of the calcitriol-VDR-ERRα axis. Evaluation of patient outcomes in The Cancer Genome Atlas (TCGA) dataset showed the translational significance of the biological effects of the VDR-ERRα axis, highlighting that VDR, CYP24A1, and ERRα overexpression correlates with poor prognosis in basal-like BC.

Oncosuppressor methylation: a possible key role in colon metastatic progression.

K-RAS and BRAF gene mutations are mandatory to set anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients. Due to the relationship of these mutations with tumor epigenotype, we hypothesized the potential role of oncosuppressor methylation of genes involved in K-RAS/BRAF pathway (CDKN2A, RASSF1A, and RARbeta suppressor genes) in inhibiting EGFR signaling cascade. Primary tumor and synchronous liver metastatic tissues of 75 mCRC patients were characterized for promoter methylation by QMSP and for K-RAS and BRAF mutations. RARbeta, RASSF1A, and CDKN2A genes were methylated in 82%, 35%, and 26% of primary tumors, respectively. RASSF1A resulted significantly more frequently methylated in liver metastasis than in primary site (P=0.015), while RARbeta was significantly lower methylated in distant metastasis (P=1.2 × 10(-6)). As regards methylation content, RASSF1A methylation status was significantly higher in liver metastasis with respect to primary tumor (P=0.000) underlying the role of this gene in liver metastatic progression. In our series K-RAS and BRAF were mutated in 39% and 4% of cases, respectively. Methylation frequencies seemed to be unrelated to gene mutations; on the other hand, RASSF1A mean content methylation resulted significantly higher in liver than in primary tumor (288.78 vs. 56.23, respectively, P=0.05) only in K-RAS wild-type cases sustaining a specific role of this gene in metastatic site thus supporting its function in strengthening the apoptotic role of K-RAS. These evidences held the role of oncosuppressor methylation in both colon tumorigenesis and progression and suggested that epigenetic events should be taken into account when biological therapies in mCRC patients have to be set.

Mutations and polymorphic BRCA variants transmission in breast cancer familial members.

We previously showed that about 80% of breast cancer patients at high risk to carry mutation in BRCA genes presented at least one polymorphism in these genes which resulted potentially harmful by in silico analysis. In the present paper, the genealogic transmission of those polymorphic coding and noncoding variants of BRCA genes in family's members has been investigated. Thirty families, enrolled within the Genetic Counselling Program of our Institute, with probands and at least one-first degree relative (n = 67 family members) available, have been studied for both BRCA1 and BRCA2 pathological mutation and polymorphic variants' transmission. Ten and 6 probands carried Mendelian transmitted mutations in BRCA1 and BRCA2, respectively. Polymorphic coding and noncoding variants were transmitted in each family's relatives with a frequency ranging from 42 to 100%, with similar rate for each SNP in mutated and nonmutated families with the only exception of BRCA1 K1183R significantly more frequent in mutated families (P = 0.004); conversely, this SNP and BRCA2 N372H, were more frequently present in breast cancer relatives belonging to families in which pathological BRCA mutations were not present. Furthermore, specific haplotypes were transmitted in all relatives as BRCA1 871Leu-1038Gly, present in both BRCA mutated and nonmutated families, while BRCA2 289His-991Asp-IVS14+53 C>T present only in BRCAX families suggesting the harmful role of these SNPs. In conclusion, analysis of SNPs maps and modality of their transmission could identify further susceptibility markers and provide a basis for a better DNA-based cancer classification.

Spectrum of Germline Pathogenic Variants in BRCA1/2 Genes in the Apulian Southern Italy Population: Geographic Distribution and Evidence for Targeted Genetic Testing.

BRCA1/2-associated hereditary breast and ovarian cancer is the most common form of hereditary breast and ovarian cancer and occurs in all ethnicities and racial populations. Different BRCA1/BRCA2 pathogenic variants (PVs) have been reported with a wide variety among populations. In this study, we retrospectively analyzed prevalence and geographic distribution of pathogenic germline BRCA1/2 variants in families from Apulia in southern Italy and evaluated the genotype-phenotype correlations. Data were collected from Oncogenetic Services present in Apulian hospitals and a shared database was built containing Apulian native probands (n = 2026) that had undergone genetic testing from 2004 to 2019. PVs were detected in 499 of 2026 (24.6%) probands and 68.5% of them (342 of 499) were in the BRCA1 gene. We found 65 different PVs in BRCA1 and 46 in BRCA2. There were 10 most recurrent PVs and their geographical distribution appears to be significantly specific for each province. We have assumed that these PVs are related to the historical and geopolitical changes that occurred in Apulia over time and/or to a "founder effect". Broader knowledge of BRCA1/2 prevalence and recurring PVs in specific geographic areas could help establish more flexible genetic testing strategies that may enhance our ability to detect high-risk subjects.

Lifestyle Characteristics in Women Carriers of BRCA Mutations: Results From an Italian Trial Cohort.

BACKGROUND: Women with deleterious mutations in BRCA1/2 have a high lifetime penetrance of developing breast cancer and/or ovarian cancer. Genetic and/or environmental factors may influence BRCA penetrance, and identifying modifiable exposures might be valuable for prevention. PATIENTS AND METHODS: We implemented a multicenter prospective 2-arm (1:1) randomized controlled trial to investigate whether a Mediterranean dietary intervention with moderate protein restriction would reduce potential modulators of BRCA penetrance such as insulin-like growth factor 1 (IGF-1), body weight, and metabolic risk factors. We studied the baseline characteristics of women with BRCA-positive disease who joined the trial cohort, focusing on the relationships between selected lifestyle exposures, metabolic/anthropometric parameters, and BRCA-related cancer. RESULTS: A total of 502 women (304 with a previous diagnosis of breast cancer and/or ovarian cancer and 198 unaffected) with deleterious BRCA mutations, with or without a previous cancer, aged 18 to 70 years and without metastases were included. Late age at menarche and pregnancy were negatively associated with BRCA-related cancer, especially in women with BRCA1-positive disease. Higher fat mass and the presence of 4 or 5 metabolic risk factors were significantly associated with BRCA-related cancer (hazard ratio, 1.87, 95% confidence interval, 1.21-2.88; and hazard ratio, 1.87, 95% confidence interval, 1.11-3.19, respectively), with greater effect in BRCA2-positive women. CONCLUSIONS: Our findings confirm previous observations about reproductive factors in women with BRCA disease and suggest a potential impact of metabolic factors in BRCA-related cancer. The prospective follow-up of the trial cohort will enable us to study the environmental modulators of BRCA penetrance and their impact in relation to the history of BRCA-related cancer. [ClinicalTrials.gov NCT03066856].

Maternal and paternal lineage double heterozygosity alteration in familial breast cancer: a first case report.

Hereditary breast cancer syndrome was firstly associated with BRCA1 and BRCA2 genes the mutations of which confer high risk to develop breast and/or ovarian cancer. Double heterozygosity is a rare condition in which both BRCA1 and BRCA2 mutations are present in a family at the same time. In the current study, a family with double heterozygosity has been reported. Furthermore, for the first time a molecular analysis in both proband lineages, maternal and paternal, has been reported to understand the provenience of both germinal mutations.The case regards a woman who developed breast and ovarian cancer with liver metastasis which presented two mutations, each in the two genes, transmitted from her mother and her father, respectively. In this family all available members have been investigated. The concomitant presence of these peculiar mutations was never reported before suggesting a link with Caucasian population from Southern Italy.

Somatic BRCA Mutation in a Cholangiocarcinoma Patient for HBOC Syndrome Detection.

BRCA-associated hereditary breast and ovarian cancer syndrome (HBOC) is characterized by an increased risk of developing other malignancies including cholangiocarcinoma (CCA). Somatic BRCA mutations have been reported in CCA, but they have yet to be utilized in a proband case to identify HBOC in families. Two healthy daughters of a deceased female patient who had had metachronous breast cancer and CCA received genetic counseling to assess their cancer risk. Somatic BRCA1/2 mutation analysis was performed by next-generation sequencing on the DNA extracted from a formalin-fixed, paraffin-embedded CCA biopsy specimen of their mother. A pathogenic variant was identified (c.6468_6469delTC in a BRCA2 gene mutation). Germline BRCA mutation analysis of the two daughters detected the same pathogenic variant in one of them. For the first time, a CCA somatic BRCA mutation has been used to identify a family with HBOC.

The Role of Circulating Adiponectin and SNP276G>T at ADIPOQ Gene in BRCA-mutant Women.

BACKGROUND: Environmental factors may influence the lifetime risk of cancer (penetrance) in women with a BRCA mutation. MATERIALS AND METHODS: In 89 BRCA-mutant women, affected or unaffected by breast/ovarian cancer, we explored serum levels of adipokines and their relation with the polymorphism SNP276G>T as modulators of BRCA penetrance. RESULTS: Affected women had significantly lower adiponectin than healthy women. Affected women with rs1501299 TT had significantly lower adiponectin and higher leptin than GT and GG genotypes. GT genotype was significantly associated with the disease status [odds ratio (OR)=3.24, 95% confidence interval (95% CI)=1.03-10.17]. Women in the lower tertile of serum adiponectin had a RR of BRCA-associated cancer of 2.80, 95% CI=1.1-7.1 (p for trend=0.03) compared with women in the higher tertile. CONCLUSION: In the SNP rs1501299 the T allele was significantly associated with lower serum levels of adiponectin in affected women, suggesting that the T allele might be related to cancer.

Molecular and in silico analysis of BRCA1 and BRCA2 variants.

Germline mutations of high penetrant BRCA1 and BRCA2 genes have been associated to hereditary breast cancer risk, while polymorphic variants of the two genes still have an unknown role in breast pathogenesis. The aim of our study was to characterize BRCA1 and BRCA2 genes polymorphic variants in familial breast cancer. 110 patients affected by familial breast and/or ovarian cancer have been consecutively enrolled according to family history and BRCA mutation risk. All of them have been screened for BRCA1 and BRCA2 pathogenetic mutations, SNPs and intronic variants. In silico analysis have been also performed using different computational methods to individualize genetic variations that can alter the two genes expression and function. BRCA1 resulted mutated in 14% while BRCA2 in 3% of cases, while 80% of patients presented at least one polymorphism. A neural network splicing prediction model individualized one BRCA1 and one BRCA2 intronic variants able to determine alternative splicing. Furthermore, Q356R BRCA1 and N289H BRCA2 appear to show a possible harmful role also due to their location in functional regions of the two genes. However, in silico data are not always consistent with biological evidences. In conclusion, SNPs profile provides a basis for DNA-based cancer risk classification and help to define the gene alterations that could influence biochemistry activity protein or could modify drug sensitivity.

Impact of sample preparation in peptide/protein profiling in human serum by MALDI-TOF mass spectrometry.

The low molecular weight (LMW) serum proteome (<15 kDa) is the most generally informative from a medical point of view. Different sample pre-treatment approaches and devices for serum depletion in high-abundant proteins were tested in order to analyze, by MALDI-TOF-MS (both in "linear" and "reflectron" acquisition mode), the serum low molecular weight proteins/peptides. The best results in terms of detected ions number and abundance were obtained by using ultrafiltration of serum on 30 kDa molecular weight cut off membranes followed by miniaturized reverse-phase solid-phase extraction (mu-SPE) as sample pre-treatment; this procedure yielded also satisfactory within-sample and sample-to-sample repeatability (on both m/z values and peak intensity of the main observable ions). The procedure was finally applied to serum samples of breast cancer patients, and the relevant results compared to "normal" samples seem to be promising for the individuation of different profiles ("linear" and "reflectron" mode) and/or peptides capable of differentiating for malignancies ("reflectron" mode).