Predictors of germline status for hereditary melanoma: 5 years of multi-gene panel testing within the Italian Melanoma Intergroup.

BACKGROUND: The incidence of cutaneous melanoma is increasing in Italy, in parallel with the implementation of gene panels. Therefore, a revision of national genetic assessment criteria for hereditary melanoma may be needed. The aim of this study was to identify predictors of susceptibility variants in the largest prospective cohort of Italian high-risk melanoma cases studied to date. MATERIALS AND METHODS: From 25 Italian centers, we recruited 1044 family members and germline sequenced 940 cutaneous melanoma index cases through a shared gene panel, which included the following genes: CDKN2A, CDK4, BAP1, POT1, ACD, TERF2IP, MITF and ATM. We assessed detection rate according to familial status, region of origin, number of melanomas and presence and type of non-melanoma tumors. RESULTS: The overall detection rate was 9.47% (5.53% analyzing CDKN2A alone), ranging from 5.14% in sporadic multiple melanoma cases (spoMPM) with two cutaneous melanomas to 13.9% in familial cases with at least three affected members. Three or more cutaneous melanomas in spoMPM cases, pancreatic cancer and region of origin predicted germline status [odds ratio (OR) = 3.23, 3.15, 2.43, P < 0.05]. Conversely, age > 60 years was a negative independent predictor (OR = 0.13, P = 0.008), and was the age category with the lowest detection rate, especially for CDKN2A. Detection rate was 19% when cutaneous melanoma and pancreatic cancer clustered together. CONCLUSIONS: Gene panel doubled the detection rate given by CDKN2A alone. National genetic testing criteria may need a revision, especially regarding age cut-off (60) in the absence of strong family history, pancreatic cancer and/or a high number of cutaneous melanomas.

Impact of deleterious variants in other genes beyond BRCA1/2 detected in breast/ovarian and pancreatic cancer patients by NGS-based multi-gene panel testing: looking over the hedge.

BACKGROUND: Hereditary breast cancer (BC), ovarian cancer (OC), and pancreatic cancer (PC) are the major BRCA-associated tumours. However, some BRCA1/2-wild-type (wt) patients with a strong personal and/or family history of cancer need a further genetic testing through a multi-gene panel containing other high- and moderate-risk susceptibility genes. PATIENTS AND METHODS: Our study was aimed to assess if some BC, OC, or PC patients should be offered multi-gene panel testing, based on well-defined criteria concerning their personal and/or family history of cancer, such as earliness of cancer onset, occurrence of multiple tumours, or presence of at least two or more affected first-degree relatives. For this purpose, 205 out of 915 BC, OC, or PC patients, resulted negative for BRCA1/2 and with significant personal and/or family history of cancer, were genetically tested for germline pathogenic or likely pathogenic variants (PVs/LPVs) in genes different from BRCA1/2. RESULTS: Our investigation revealed that 31 (15.1%) out of 205 patients harboured germline PVs/LPVs in no-BRCA genes, including PALB2, CHEK2, ATM, MUTYH, MSH2, and RAD51C. Interestingly, in the absence of an analysis conducted through multi-gene panel, a considerable percentage (15.1%) of PVs/LPVs would have been lost. CONCLUSIONS: Providing a multi-gene panel testing to BRCA1/2-wt BC/OC/PC patients with a strong personal and/or family history of cancer could significantly increase the detection rates of germline PVs/LPVs in other cancer predisposition genes beyond BRCA1/2. The use of a multi-gene panel testing could improve the inherited cancer risk estimation and clinical management of patients and unaffected family members.

Risk Perception and Psychological Distress in Genetic Counselling for Hereditary Breast and/or Ovarian Cancer.

Oncological Genetic Counselling (CGO) allows the identification of a genetic component that increases the risk of developing a cancer. Individuals' psychological reactions are influenced by both the content of the received information and the subjective perception of their own risk of becoming ill or being a carrier of a genetic mutation. This study included 120 participants who underwent genetic counselling for breast and/or ovarian cancer. The aim of the study was to examine the relation between their cancer risk perception and the genetic risk during CGO before receiving genetic test results, considering the influence of some psychological variables, in particular distress, anxiety and depression. Participants completed the following tools during a psychological interview: a socio-demographic form, Cancer Risk Perception (CRP) and Genetic Risk Perception (GRP), Hospital Anxiety and Depression Scale (HADS) and Distress Thermometer (DT). The data seem to confirm our hypothesis. Positive and significant correlations were found between the observed variables. Moreover, genetic risk perception determined an increase in depressive symptomatology and cancer risk perception led to an increase in anxious symptomatology, specifically in participants during cancer treatment. The present results suggest the importance of assessing genetic and cancer risk perception in individuals who undergo CGO, to identify those who are at risk of a decrease in psychological well-being and of developing greater psychological distress.

Copper binding to naturally occurring, lactam form of angiogenin differs from that to recombinant protein, affecting their activity.

Angiogenin is a member of the ribonuclease family and a normal constituent of human plasma. It is one of the most potent angiogenic factors known and is overexpressed in different types of cancers. Copper is also an essential cofactor in angiogenesis and, during this process, it is mobilized from inside to outside of the cell. To date, contrasting results have been reported about copper(ii) influencing angiogenin activity. However, in these studies, the recombinant form of the protein was used. Unlike recombinant angiogenin, that contains an extra methionine with a free terminal amino group, the naturally occurring protein present in human plasma starts with a glutamine residue that spontaneously cyclizes to pyroglutamate, a lactam derivative. Herein, we report spectroscopic evidence indicating that copper(ii) experiences different coordination environments in the two protein isoforms, and affects their RNase and angiogenic activity differently. These results show how relatively small differences between recombinant and wild type proteins can result in markedly different behaviours.

High prevalence of BRCA1 deletions in BRCAPRO-positive patients with high carrier probability.

Mutation screening of the BRCA1 and BRCA2 genes in probands with familial breast/ovarian cancer has been greatly improved by the multiplex ligation-dependent probe amplification (MLPA) assay able to evidence gene rearrangements not detectable by standard screening methods. However, no criteria for selection of cases to be submitted to the MLPA test have been reported yet. We used the BRCAPro software for the selection of familial breast/ovarian cancer probands investigated with the MLPA approach after negative BRCA1/2 conventional mutation screening. One hundred and seventy-seven probands were investigated for germline BRCA1/2 mutations after assessment of genetic risk using BRCAPro. Probands were classified as BRCAPro positive (n = 67) when the carrier probability (CP) was >10% and as BRCAPro negative (n = 110), when the CP was <10%. Conventional mutational analyses of the BRCA1/2 genes and, in one case, of p53 identified 22 pathogenetic germline mutations, 12 in BRCA1, 9 in BRCA2 and 1 in p53, in 22/177 (12.4%) probands. All the mutations except one were detected in BRCAPro-positive patients. In the 46 BRCAPro-positive cases that resulted negative by BRCA1/2 mutation, screening analysis of rearrangements within BRCA1/2 by MLPA was carried out. Three patients with a very high CP showed BRCA1 deletions, consisting of deletions of exons 1-2 in two probands and of exon 24 in the third proband. In one case, the exons 1-2 deletion was shown to cosegregate with disease in the family. No BRCA2 rearrangements were detected, but one patient showed the 1100delC of the CHEK2 gene, whose probe is present in the BRCA2 kit. In our series, the highest carrier detection rate of mutation screening plus MLPA analysis (52.3%) was in patients with a BRCAPro CP >50%.

Founder mutations in BRCA1 and BRCA2 genes.

BRCA1 and BRCA2 germline mutations contribute to a significant number of familial and hereditary breast and/or ovarian cancers. The proportion of high-risk families with breast and/or ovarian cancer cases due to mutations in these tumor suppressor genes varies widely among populations. In some population, a wide spectrum of different mutations in both genes are present, whereas in other groups specific mutations in BRCA1 and BRCA2 have been reported with high frequency. Most of these mutations are prevalent in restricted populations as consequence of a founder effect. The comparison of haplotypes between families with the same mutation can distinguish whether high-frequency alleles derive from an older or more recent single mutational event or whether they have arisen independently more than once. Here, we review some of the most well-known and significant examples of founder mutations in BRCA genes found in European and non-European populations. In conclusion, the identification of the ethnic group of families undergoing genetic counseling enables the geneticist and oncologist to make more specific choices, leading to simplify the clinical approach to genetic testing carried out on members of high-risk families. Futhermore, the high frequency of founder mutations, allowing to analyze a large number of cases, might provide accurate information regarding their penetrance.

4843delC of the BRCA1 gene is a possible founder mutation in Southern Italy (Sicily).

BACKGROUND: The frequency and the type of BRCA1 mutations vary widely and might have different geographic and ethnic distribution. Most of these alterations are generally found in isolated populations as a consequence of the founder effect. The object of this study was to determine whether 4843delC, a deleterious mutation of the BRCA1 gene, might be due to a founder effect originating in the Sicilian region of Italy. This mutation was described by us for the first time and identified in two unrelated Sicilian families with hereditary breast/ovarian cancer. The two families were from the same geographical area (south-western area of Palermo, Sicily). The homogeneity of the ethnic group of the two families and the Single Nucleotide Polymorphism (SNPs) analysis of probands led us to perform a study of the allelotype of the various members. PATIENTS AND METHODS: The analysis of the haplotype of the probands and of several family members was conducted by means of a study of the highly polymorphic microsatellites within or flanking the BRCA1 gene. RESULTS: This analysis revealed the presence of a common allele associated with the mutation. CONCLUSIONS: We therefore conclude that 4843delC of the BRCA1 gene is a possible founder mutation in the Sicilian population.

Significance of P16INK4A hypermethylation gene in primary head/neck and colorectal tumors: it is a specific tissue event? Results of a 3-year GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study.

BACKGROUND: Methylation of the p16 promoter is one of the most frequent mechanisms of gene inactivation; its incidence is extremely variable according to the type of tumor involved. Our purpose was to analyze the hypermethylation of the p16 promoter in laryngeal squamous cell carcinomas (LSCC), salivary gland (SG) tumors and in colorectal cancer (CRC), to detect any possible association with the clinicopathological features and to determine the prognostic significance of the p16 gene in the tumors analyzed. PATIENTS AND METHODS: The hypermethylation of the p16 promoter was prospectively analyzed, by MSP, in a consecutive series of 64 locally advanced LSCC patients, in a consecutive series of 33 SG tumor patients and in a consecutive series of 66 sporadic CRC patients. RESULTS: Hypermethylation was observed in 9% of the LSCC cases, in all cases of SG cancer and in 21% of the CRC cases. No significant association was observed between p16 hypermethylation and clinicopathological variables in all the tissue samples analyzed. Moreover at univariate analysis p16 mutations were not independently related at disease relapse and death in LSCC and CRC. CONCLUSIONS: The results of this study suggest that the lack of p16 function could happen in advanced stage of SG tumors.

Y179C, F486L and N550H are BRCA1 variants that may be associated with breast cancer in a Sicilian family: results of a 5-year GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study.

BACKGROUND: Over 600 different pathogenic mutations have been identified in the BRCA1 gene. Nevertheless, numerous missense mutations of unknown biological function still exist. Understanding of biological significance of these mutations should help in genetic counselling to carriers and their families. PATIENTS AND METHODS: A total of 104 patients with breast and/or ovarian cancer whose genetic counselling answered the criteria of the American Society of Clinical Oncology (ASCO 2003), were prospectively screened for mutations in all coding exons of the BRCA1 gene by automatic direct sequencing. RESULTS: During these mutational screening procedures one case presented three mutations classified in the Breast Cancer Information Core Database as unknown variants. These were 655A/G found in exon 8 of BRCA1, 1575T/C and 1767A/C found in exon 11 of the same gene. The identification of the three unknown variants in the proband (16SIRIO) and in her mother and sister indicates that such alterations exist in cis. CONCLUSIONS: Our results suggest that the charge and stechiometry variations determined by the changes in the amino acids Y179C, F486L and N550H might produce an effect on the conformation of the protein and, consequently, on its function.

Detection and quantification of mammaglobin in the blood of breast cancer patients: can it be useful as a potential clinical marker? Preliminary results of a GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study.

BACKGROUND: Mammaglobin is expressed mainly in mammary tissue, overexpressed in breast cancer (BC) and rarely in other tissue. The aim of this study was to assess the sensitivity and specificity of transcript MGB1 detection and to evaluate the role of MGB1 as potential clinical marker for the detection of disseminated cancer cells in the blood of BC patients. PATIENTS AND METHODS: A consecutive series of 23 BC tissues, 36 peripheral blood BC samples and 35 healthy peripheral blood samples was prospectively recruited to investigate MGB1 expression by means of a quantitative Real Time RT-PCR assay. RESULTS: MGB1 overexpression in tissue samples of BC patients is significantly associated only with high level of Ki67 (P <0.05). None of the samples from peripheral blood of 35 healthy female individuals were positive for MGB1 transcript. In contrast MGB1 mRNA expression was detected in three of 36 (8%) peripheral blood of BC patients. CONCLUSIONS: Our preliminary results demonstrate that the detection of MGB1 transcript in peripheral blood of BC patients was specific but with low sensitivity. MGB1 overexpression by itself or in combination with Ki67 might be considered an index of BC progression.

Molecular detection of TP53, Ki-Ras and p16INK4A promoter methylation in plasma of patients with colorectal cancer and its association with prognosis. Results of a 3-year GOIM (Gruppo Oncologico dell'Italia Meridionale) prospective study.

BACKGROUND: Despite the improvement in detection and surgical therapy in the last years, the outcome of patients affected by colorectal carcinoma (CRC) remains limited by metastatic relapse. The aim of this study was to investigate the presence of free tumor DNA in the plasma of CRC patients in order to understand its possible prognostic role. PATIENTS AND METHODS: Ki-Ras, TP53 mutations and p16(INK4A) methylation status were prospectively evaluated in tumor tissues and plasma of 66 CRC patients. RESULTS: In 50 of the 66 primitive tumor cases (76%) at least one significant alteration was identified in Ki-Ras and/or TP53 and/or p16(INK4A) genes. Eighteen of the 50 patients presented the same alteration both in the plasma and in the tumor tissue. At univariate analysis, Ki-Ras mutations proved to be significantly related to quicker relapse (P <0.01), whereas only a trend towards statistical significance (P = 0.083) was observed for the TP53 mutations CONCLUSIONS: Detection of Ki-Ras and TP53 mutation in plasma should be significantly related to disease recurrence. These data suggest that patients with a high risk of recurrence can be identified by means of the analysis of tumor-derived plasma DNA with the use of fairly non-invasive techniques.

Specific TP53 and/or Ki-ras mutations as independent predictors of clinical outcome in sporadic colorectal adenocarcinomas: results of a 5-year Gruppo Oncologico dell'Italia Meridionale (GOIM) prospective study.

BACKGROUND: Although Ki-ras and TP53 mutations have probably been the genetic abnormalities most exhaustively implicated and studied in colorectal cancer (CRC) progression, their significance in terms of disease relapse and overall survival has not yet clearly been established. PATIENTS AND METHODS: A prospective study was carried out on paired tumor and normal colon tissue samples from a consecutive series of 160 previously-untreated patients, undergoing resective surgery for primary operable sporadic CRC. Mutations within the TP53 (exons 5-8) and Ki-ras (exon 2) genes were detected by PCR-SSCP analyses following sequencing. RESULTS: Mutation analyses of exons 5 to 8 of the TP53 gene showed mutations in 43% (68/160) of the cases, while mutation analyses of exon 2 of the Ki-ras gene showed mutations in 46% (74/160) of the cases. Multivariate analyses showed that clinical outcome were strongly associated with the presence of specific TP53 mutations in L3 domain alone (only in DFS) or in combination with specific Ki-ras mutations at codon 13. CONCLUSION: Specific TP53 mutations in L3 domain alone (only in DFS) or in combination with specific Ki-ras mutations at codon 13 are associated with a worse prognosis in sporadic CRC.

DNA ploidy and S-phase fraction, but not p53 or NM23-H1 expression, predict outcome in colorectal cancer patients. Result of a 5-year prospective study.

PURPOSE: The aim of this study was to determine TP53 and NM23-H1 immunoreactivity, DNA ploidy, and S-phase fraction (SPF) in a series of 160 patients undergoing resective surgery for primary operable colorectal cancer (CRC) and to establish whether these alterations have any clinical value in predicting CRC patients' prognosis. METHODS: TP53 and NM23-H1 expressions were evaluated on paraffin-embedded tissue by immunohistochemistry and DNA-ploidy and SPF on frozen tissue by flow-cytometric analysis. RESULTS: The median follow-up time in our study group was 71 months (range 34-115 months). P53 protein expression was associated with distal tumors (P<0.05) and DNA aneuploid tumors (P<0.05) tumors. DNA-aneuploidy was associated with distal tumors (P<0.01), histological grade (G3) (P<0.05), advanced Dukes' stage (C and D) (P<0.01), lymph node metastases (P<0.01) and high SPF (>18.3%) (P<0.01). The major significant predictors for both disease relapse and death were advanced Dukes' stage, DNA-aneuploidy, and high SPF, while lymphohematic invasion was the only independent factor for relapse and non-curative resection for death. CONCLUSIONS: Our results indicate that DNA aneuploidy and high SPF are associated in CRC with a poor clinical 5-year outcome, while in contrast the prognostic role of TP53 and NM23-H1 expression is still to be clarified.