Virginia Valentini

Novel and known genetic variants for male breast cancer risk at 8q24.21, 9p21.3, 11q13.3 and 14q24.1: Results from a multicenter study in Italy

Increasing evidence indicates that common genetic variants may contribute to the heritable risk of breast cancer (BC). In this study, we investigated whether single nucleotide polymorphisms (SNPs), within the 8q24.21 multi-cancer susceptibility region and within BC-associated loci widespread in the genome, may influence the risk of BC in men, and whether they may be associated with specific clinical-pathologic characteristics of male BC (MBC). In the frame of the ongoing Italian Multicenter Study on MBC, we performed a case-control study on 386 MBC cases, including 50 BRCA1/2 mutation carriers, and 1105 healthy male controls, including 197 unaffected BRCA1/2 mutation carriers. All 1491 subjects were genotyped by Sequenom iPLEX technology for a total of 29 susceptibility SNPs. By logistic regression models, we found a significant association with MBC risk for five SNPs: rs1562430 (p=0.002) and rs445114 (p=0.026) both within the 8q24.21 region; rs1011970/9p21.3 (p=0.011), rs614367/11q13.3 (p=0.016) and rs1314913/14q24.1 (p<0.0001). Differences in the distribution of rs614367/11q13.3 genotypes according to oestrogen receptor (ER) status (p=0.006), and of rs1011970/9p21.3 genotypes according to human epidermal growth factor receptor 2 (HER2) status (p=0.002) emerged. Association of rs1011970/9p21.3 risk genotype with HER2+MBC was confirmed by a multivariate analysis. rs1314913/14q24.1 was associated with increased MBC risk in analyses restricted to male BRCA1/2 mutation carriers (p=0.041). In conclusion, we provided the first evidence that the 8q24.21 region is associated with MBC risk. Furthermore, we showed that the SNPs rs1562430/8q24.21 and rs1314913/14q24.1 strongly influence BC risk in men and suggested that the SNP rs1314913/14q24.1 may act as a risk modifier locus in male BRCA1/2 mutation carriers.

Whole-exome sequencing and targeted gene sequencing provide insights into the role of PALB2 as a male breast cancer susceptibility gene

Male breast cancer (MBC) is a rare disease whose etiology appears to be largely associated with genetic factors. BRCA1 and BRCA2 mutations account for about 10% of all MBC cases. Thus, a fraction of MBC cases are expected to be due to genetic factors not yet identified. To further explain the genetic susceptibility for MBC, whole‐exome sequencing (WES) and targeted gene sequencing were applied to high‐risk, BRCA1/2 mutation–negative MBC cases.

Somatic alterations of targetable oncogenes are frequently observed in BRCA1/2 mutation negative male breast cancers

Male breast cancer (MBC) is a rare disease. Due to its rarity, MBC research and clinical approach are mostly based upon data derived from its largely known female counterpart. We aimed at investigating whether MBC cases harbor somatic alterations of genes known as prognostic biomarkers and molecular therapeutic targets in female breast cancer. We examined 103 MBC cases, all characterized for germ-line BRCA1/2 mutations, for somatic alterations in PIK3CA, EGFR, ESR1 and CCND1 genes. Pathogenic mutations of PIK3CA were detected in 2% of MBCs. No pathogenic mutations were identified in ESR1 and EGFR. Gene copy number variations (CNVs) analysis showed amplification of PIK3CA in 8.1%, EGFR in 6.8% and CCND1 in 16% of MBCs, whereas deletion of ESR1 was detected in 15% of MBCs. Somatic mutations and gene amplification were found only in BRCA1/2 mutation negative MBCs. Significant associations emerged between EGFR amplification and large tumor size (T4), ER-negative and HER2-positive status, between CCND1 amplification and HER2-positive and MIB1-positive status, and between ESR1 deletion and ER-negative status. Our results show that amplification of targetable oncogenes is frequent in BRCA1/2 mutation negative MBCs and may identify MBC subsets characterized by aggressive phenotype that may benefit from potential targeted therapeutic approaches.

Smoking and FGFR2 rs2981582 variant independently modulate male breast cancer survival: A population-based study in Tuscany, Italy

AIM Male breast cancer (MBC) is a rare disease and recommendations for its clinical management are often extrapolated from those for female breast cancer, even if breast cancer (BC) has different characteristics in the two sexes. The purpose of this study was to assess the influence of several individual characteristics including clinico-pathological, lifestyle and genetic factors on overall survival (OS) of a relatively large and well characterized population-based series of 166 MBCs enrolled in Tuscany. METHODS We genotyped MBC cases at BRCA1/2 genes and at 9 candidate BC susceptibility SNPs. Kaplan-Meier method and multivariate Cox regression, adjusted for several individual characteristics were used. To reduce a possible selection bias related to the interval between diagnosis and enrolment of MBC cases into the study, we used the date of blood donation as the date of the start of observation for survival analysis. RESULTS Only smoking habits had a significant effect on OS at 10 years (for current smokers, HR: 3.34; 95% CI 1.45-7.68; p = 0.004), while lymph node status fell short of reaching statistical significance (for pN positive, HR: 2.07; 95% CI 0.93-4.55; p = 0.07). In the same multivariate analysis we found a significantly higher OS in cases with FGFR2 rs2981582 variant in the dominant transmission model (HR: 0.29; 95% CI: 0.13-0.62; p = 0.028). A sensitivity analysis with left truncation showed similar results. CONCLUSIONS Our results may contribute to shed light on factors influencing MBC survival suggesting an important role for cigarette smoking and FGFR2 rs2981582 variant, and provide clues for better patient management.

Gene-specific methylation profiles in BRCA -mutation positive and BRCA -mutation negative male breast cancers

Male breast cancer (MBC) is a rare disease. Due to its rarity, MBC research and clinical approach are mostly based upon data derived from female breast cancer (FBC). Increasing evidence indicate that on molecular level MBC may be an heterogeneous disease different from FBC. In order to investigate whether epigenetic signatures could define molecular subgroups of MBCs, we performed promoter methylation analysis of genes involved in signal transduction and hormone signalling in BRCA1/2 mutation-positive and -negative MBCs. We examined 69 MBCs, paired blood samples, and 15 normal tissues for promoter methylation of hTERT, ESR1, RASSF1, AR, MYC and WNT1 genes. MBCs showed higher gene promoter methylation levels compared to paired blood and normal breast samples. Significantly higher RASSF1 methylation levels were observed in association with BRCA1/2 mutations, HER2 expression and high tumor grade. Significantly higher AR methylation levels were observed in BRCA1/2 wild-type cases and higher WNT1 methylation levels in PR negative cases. Overall, our results indicate that alterations in gene methylation profiles are common in MBC and that methylation pattern of tumor-associated genes may allow for the identification of MBC molecular subgroups, that could have implications in clinical management of MBC patients.

Metastases risk in thin cutaneous melanoma: prognostic value of clinical-pathologic characteristics and mutation profile

Background A high percentage of patients with thin melanoma (TM), defined as lesions with Breslow thickness ≤1 mm, presents excellent long-term survival, however, some patients develop metastases. Existing prognostic factors cannot reliably differentiate TM patients at risk for metastases. Objective We aimed at characterizing the clinical-pathologic and mutation profile of metastatic and not-metastatic TM in order to distinguish lesions at risk of metastases. Methods Clinical-pathologic characteristics were recorded for the TM cases analyzed. We used a Next Generation Sequencing (NGS) multi-gene panel to characterize TM for multiple somatic mutations. Results A statistically significant association emerged between the presence of metastases and Breslow thickness ≥0.6 mm (p=0.003). None of TM with lymph-node involvement had Breslow thickness <0.6 mm. Somatic mutations were identified in 19 of 21 TM analyzed (90.5%). No mutations were observed in two not-metastatic cases with the lowest Breslow thickness (≤0.4 mm), whereas mutations in more than one gene were detected in one metastatic case with the highest Breslow thickness (1.00 mm). Conclusion Our study indicates Breslow thickness ≥0.6 mm as a valid prognostic factor to distinguish TM at risk for metastases.

PIK3CA c.3140A>G mutation in a patient with suspected Proteus Syndrome: a case report

We present a patient with suspected Proteus Syndrome, an overgrowth disorder associated with AKT1c.49G>A mutation. NGS analysis detected PIK3CAc.3140A>G mutation in the patients affected tissue allowing for PROS (PIK3CA‐related overgrowth spectrum) diagnosis. The overlapping clinical features in overgrowth disorders highlight the importance of molecular testing for a correct diagnosis.

Abstract 4775: Gene-specific methylation profiles in male breast cancer

Background: Male breast cancer (MBC) is rare, but despite the rarity, morbidity and mortality in MBC patients is a serious concern. The role of the genetic factors in MBC pathogenesis is now well known. Mutations of BRCA1 and, mainly, BRCA2 have been found to significantly increase the risk of MBC. Conversely, the knowledge on molecular profiles of MBC is still incomplete. There is growing evidence that methylation plays an important role in breast cancer (BC) development and that characterization of tumor-specific methylation profiles may allow the identification of specific BC subtypes. Significant differences in tumor-associated gene methylation patterns have been related to receptor status (ER, PR and HER2) and different genome-wide DNA methylation profiles emerged between sporadic and BRCA1/2-associated BC. The contribution of DNA methylation in MBC pathogenesis have not yet well investigated. Using candidate-gene approach, we aimed to perform methylation analysis of a panel of BC-related genes, in order to identify biomarkers that could define MBC subgroups and elucidate pathways underlying MBC development. Materials and methods: Promoter methylation analysis of hTERT (human Telomerase reverse transcriptase), ESR1(Estrogen receptor 1), RASSF1(Ras association domain-containing protein 1), AR (Androgen receptor), MYC (v-myc avian myelocytomatosis viral oncogene homolog) and WNT1 (wingless-type MMTV integration site family, member 1) genes was performed in 69 male breast tumors and in 7 normal male breast tissues by Pyrosequencing, a highly sensitive and reproducible method, which provides absolute quantitative information on bases at each CpG site analyzed. Methylation levels were determined by calculating the average of methylation for each gene. Kruskal-Wallis test was used to identify significant differences in methylation levels among tumors stratified according to relevant clinical-pathologic features, including BRCA1/2 mutation. Results: Compared with normal tissues, hTERT, AR and RASSF1 showed higher methylation levels in tumors, whereas ESR1 showed lower methylation levels. No differences were observed in the methylation levels of MYC and WNT1. Higher methylation levels of RASSF1 were significantly associated with BRCA1/2 mutations (p = 0.008), HER2+ status (p = 0.01) and high tumor grade (G3) (p = 0.007). Higher methylation level of AR was significantly associated with BRCA1/2 wild-type status (p = 0.016) and lower methylation level of WNT1 was significantly associated with PR+ status (p = 0.014). Conclusions: Our results indicate that tumor-associated gene methylation patterns may identify specific MBC subgroups possibly related to BRCA1/2 mutation status. In particular, methylation of RASSF1, a tumor suppressor gene that mediates apoptosis, may identify a subgroup of MBC with an aggressive phenotype. Study supported by AIRC (IG 12780) to L.O. Citation Format: Piera Rizzolo, Valentina Silvestri, AnnaSara Navazio, Virginia Valentini, Veronica Zelli, Mario Falchetti, Ines Zanna, Simonetta Bianchi, Domenico Palli, Laura Ottini. Gene-specific methylation profiles in male breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4775. doi:10.1158/1538-7445.AM2015-4775