Breast cancer: the first comparative evaluation of oncobiome composition between males and females.

BACKGROUND: Emerging evidence suggests that breast microbiota dysbiosis contributes to cancer initiation, progression, prognosis and treatment efficacy. Anyway, available data are referred only to female patients, and studies on males are completely missing. Male breast cancer (MBC) is 70-100 times less frequent, but the mortality rate adjusted to incidence is higher in men than in females. Currently, MBC diagnostic approaches and treatments have generally been extrapolated from the clinical experience gained in women, while few studies focus on characterizing male cancer biology. Taking into account the rising importance of the oncobiome field and the need of MBC targeted studies, we explored the breast cancer oncobiome of male and female patients. METHODS: 16S rRNA gene sequencing was performed in 20 tumor and 20 non-pathological adjacent FFPE breast tissues from male and female patients. RESULTS: We documented, for the first time, the presence of a sexually dimorphic breast-associated microbiota, here defined as "breast microgenderome". Moreover, the paired analysis of tumor and non-pathological adjacent tissues suggests the presence of a cancer-associated dysbiosis in male patients, with surrounding tissue conserving a healthier microbiome, whereas in female patients, the entire breast tissue is predisposed to cancer development. Finally, the phylum Tenericutes, especially the genera Mesoplasma and Mycobacterium, could to be involved in breast carcinogenesis, in both sexes, deserving further investigation, not only for its role in cancer development but even as potential prognostic biomarker. CONCLUSIONS: Breast microbiota characterization can enhance the understanding of male breast cancer pathogenesis, being useful for detection of new prognostic biomarkers and development of innovative personalized therapies, remarking the relevant gender differences.

Breast tissue can become inhabited by microbes through different pathways, and an uneven distribution of these microorganisms could potentially contribute to the development, prognosis, and treatment response of breast cancer. However, the current available data primarily focus on female patients, with a significant dearth of studies on males. To address this gap, the present study investigates the microbiota composition of both tumorous and healthy breast tissue samples from both male and female patients.The findings of this research highlight a disparity in the types of bacteria present in male and female breast tissue. Specifically, it shows that male patients with breast cancer have a higher imbalance of bacteria in the cancerous area compared to the surrounding healthy tissue. In contrast, in females the dysbiosis extend to the whole breast tissue.Moreover, the study identifies specific strains of bacteria that might potentially be involved in the development of breast cancer in both males and females.In conclusion, this study underscores the significance of microbial colonization in breast tissue and its potential influence on breast cancer in both males and females. By expanding our understanding of the microbial composition in breast cancer, we can pave the way for innovative diagnostic methods and treatment approaches for male breast cancer, while simultaneously advancing our knowledge of this complex disease.

Epigenomic, genomic, and transcriptomic landscape of schwannomatosis.

Schwannomatosis (SWNTS) is a genetic cancer predisposition syndrome that manifests as multiple and often painful neuronal tumors called schwannomas (SWNs). While germline mutations in SMARCB1 or LZTR1, plus somatic mutations in NF2 and loss of heterozygosity in chromosome 22q have been identified in a subset of patients, little is known about the epigenomic and genomic alterations that drive SWNTS-related SWNs (SWNTS-SWNs) in a majority of the cases. We performed multiplatform genomic analysis and established the molecular signature of SWNTS-SWNs. We show that SWNTS-SWNs harbor distinct genomic features relative to the histologically identical non-syndromic sporadic SWNs (NS-SWNS). We demonstrate the existence of four distinct DNA methylation subgroups of SWNTS-SWNs that are associated with specific transcriptional programs and tumor location. We show several novel recurrent non-22q deletions and structural rearrangements. We detected the SH3PXD2A-HTRA1 gene fusion in SWNTS-SWNs, with predominance in LZTR1-mutant tumors. In addition, we identified specific genetic, epigenetic, and actionable transcriptional programs associated with painful SWNTS-SWNs including PIGF, VEGF, MEK, and MTOR pathways, which may be harnessed for management of this syndrome.

Early-onset malignant phyllodes breast tumor in a patient with germline pathogenic variants in NF1 and BRCA1 genes.

We present a 24-year-old female patient affected by neurofibromatosis type 1 (NF1) who developed a malignant phyllodes tumor of the breast. The molecular studies showed that the patient carried a heterozygous inactivating deleterious variant in BRCA1 inherited from the father associated with a germline de novo pathogenic alteration in NF1; the tumor presented a biallelic inactivation of both genes. Therefore, tumor analyses helped to establish that the germline NF1 and BRCA1 variants were in cis on the paternal chromosome. This last information is important to provide adequate genetic counselling regarding the risk of recurrence in the offspring, as well as opportunity for early intervention. In conclusion, we present the first case of a malignant phyllodes tumor of the breast in patient carrying pathogenic variants in NF1 and BRCA1. Further studies will be necessary to understand if the phyllodes histotype represents a very rare component of NF1-associated breast cancer.

The Spectrum of FANCM Protein Truncating Variants in European Breast Cancer Cases.

Germline protein truncating variants (PTVs) in the FANCM gene have been associated with a 2-4-fold increased breast cancer risk in case-control studies conducted in different European populations. However, the distribution and the frequency of FANCM PTVs in Europe have never been investigated. In the present study, we collected the data of 114 European female breast cancer cases with FANCM PTVs ascertained in 20 centers from 13 European countries. We identified 27 different FANCM PTVs. The p.Gln1701* PTV is the most common PTV in Northern Europe with a maximum frequency in Finland and a lower relative frequency in Southern Europe. On the contrary, p.Arg1931* seems to be the most common PTV in Southern Europe. We also showed that p.Arg658*, the third most common PTV, is more frequent in Central Europe, and p.Gln498Thrfs*7 is probably a founder variant from Lithuania. Of the 23 rare or unique FANCM PTVs, 15 have not been previously reported. We provide here the initial spectrum of FANCM PTVs in European breast cancer cases.

Evaluation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Mutation Detection.

The efficiency of a novel targeted next-generation sequencing (NGS) test, the Devyser BRCA kit, for a comprehensive analysis of all 48 coding exons of the high-risk breast/ovarian cancer susceptibility genes BRCA1 and BRCA2 has been assessed. The new assay intended to detect nucleotide substitutions, small deletions/insertions, and large deletions/duplications. To document the false-negative and false-positive rates of the NGS assay in the hands of end users, 48 samples with previously identified 444 small variants and seven gross rearrangements were analyzed, showing 100% concordance with gold standards. Furthermore, all other 43 variants (42 single-nucleotide variation or insertion/deletion variation and one copy number variation, whose significance is or may be of clinical value), which were called by the NGS assay in a prospectively analyzed 179-sample set, were confirmed by Sanger sequencing or multiplex ligation probe amplification, according to their nature. We conclude that the Devyser BRCA kit performed satisfactorily for use in a clinical laboratory.

Multiple primary melanomas (MPMs) and criteria for genetic assessment: MultiMEL, a multicenter study of the Italian Melanoma Intergroup.

BACKGROUND: Multiple primary melanoma (MPM), in concert with a positive family history, is a predictor of cyclin-dependent kinase (CDK) inhibitor 2A (CDKN2A) germline mutations. A rule regarding the presence of either 2 or 3 or more cancer events (melanoma and pancreatic cancer) in low or high melanoma incidence populations, respectively, has been established to select patients for genetic referral. OBJECTIVE: We sought to determine the CDKN2A/CDK4/microphthalmia-associated transcription factor mutation rate among Italian patients with MPM to appropriately direct genetic counseling regardless of family history. METHODS: In all, 587 patients with MPM and an equal number with single primary melanomas and control subjects were consecutively enrolled at the participating centers and tested for CDKN2A, CDK4, and microphthalmia-associated transcription factor. RESULTS: CDKN2A germline mutations were found in 19% of patients with MPM versus 4.4% of patients with single primary melanoma. In familial MPM cases the mutation rate varied from 36.6% to 58.8%, whereas in sporadic MPM cases it varied from 8.2% to 17.6% in patients with 2 and 3 or more melanomas, respectively. The microphthalmia-associated transcription factor E318K mutation accounted for 3% of MPM cases altogether. LIMITATIONS: The study was hospital based, not population based. Rare novel susceptibility genes were not tested. CONCLUSION: Italian patients who developed 2 melanomas, even in situ, should be referred for genetic counseling even in the absence of family history.

MLH1 constitutional and somatic methylation in patients with MLH1 negative tumors fulfilling the revised Bethesda criteria.

Lynch syndrome (LS) is a tumor predisposing condition caused by constitutional defects in genes coding for components of the mismatch repair (MMR) apparatus. While hypermethylation of the promoter of the MMR gene MLH1 occurs in about 15% of colorectal cancer samples, it has also been observed as a constitutional alteration, in the absence of DNA sequence mutations, in a small number of LS patients. In order to obtain further insights on the phenotypic characteristics of MLH1 epimutation carriers, we investigated the somatic and constitutional MLH1 methylation status of 14 unrelated subjects with a suspicion of LS who were negative for MMR gene constitutional mutations and whose tumors did not express the MLH1 protein. A novel case of constitutional MLH1 epimutation was identified. This patient was affected with multiple primary tumors, including breast cancer, diagnosed starting from the age of 55 y. Investigation of her offspring by allele specific expression revealed that the epimutation was not stable across generations. We also found MLH1 hypermethylation in cancer samples from 4 additional patients who did not have evidence of constitutional defects. These patients had some characteristics of LS, namely early age at onset and/or positive family history, raising the possibility of genetic influences in the establishment of somatic MLH1 methylation.

Application of COLD-PCR for improved detection of NF2 mosaic mutations.

Somatic mosaicism represents the coexistence of two or more cell populations with different genotypes in one person, and it is involved in >30 monogenic disorders. Somatic mosaicism characterizes approximately 25% to 33% of patients with de novo neurofibromatosis type 2 (NF2). The identification of mosaicism is crucial to patients and their families because the clinical course of the disease and its transmission risk is influenced by the degree and distribution of mutated cells. Moreover, in NF2, the capability of discriminating patients with mosaicism is especially important to make differential diagnosis with schwannomatosis. However, the identification of mosaic variants is considerably difficult, and the development of specific molecular techniques to detect low levels of unknown molecular alterations is required. Co-amplification at lower denaturation temperature (COLD)-PCR has been described as a powerful method to selectively amplify minority alleles from mixtures of wild-type and mutation-containing sequences. Here, we applied COLD-PCR to molecular analysis of patients with NF2 mosaicism. With the use of COLD-PCR, followed by direct sequencing, we were able to detect NF2 mutations in blood DNA of three patients with NF2 mosaicism. Our study has shown the capability of COLD-PCR in enriching low-represented mutated allele in blood DNA sample, making it usable for molecular diagnosis of patients with mosaicism.

Clinical genetic testing for familial melanoma in Italy: a cooperative study.

BACKGROUND: The Italian Society of Human Genetics' (SIGU) recommendations on genetic counseling and testing for hereditary melanoma state that clinical genetic testing can be offered to Italian melanoma families with at least two affected members. OBJECTIVE: In the framework of a cooperative study, we sought to establish the frequency of cyclin-dependent kinase inhibitor 2A mutations in melanoma families that underwent clinical genetic counseling and testing in accordance with the SIGU recommendations at 9 centers in different Italian regions. METHODS: Cyclin-dependent kinase inhibitor 2A testing was conducted by direct sequencing and multiplex ligation-dependent probe amplification analysis in melanoma families with at least two affected members. RESULTS: A total of 33% (68/204) of the families harbored cyclin-dependent kinase inhibitor 2A mutations. In the 145 families with two affected members the mutation frequency was 25%. Three novel mutations, L94P, A86T, and c.407dupG, were identified among the cases and not in 200 controls. LIMITATIONS: We were unable to perform separate analyses for individual centers, as in some cases the number of families was too small. CONCLUSIONS: The availability of clinical genetic testing for melanoma to families with just two affected members in the same branch is justified in Italy in terms of the likelihood of identifying a mutation.

Genomic rearrangements of the CDKN2A locus are infrequent in Italian malignant melanoma families without evidence of CDKN2A/CDK4 point mutations.

Predisposition to familial cutaneous malignant melanoma has been associated with mutations in the CDKN2A and CDK4 genes. However, only a small subgroup of melanoma pedigrees harbour CDKN2A or CDK4 germline mutations. It is possible that other types of CDKN2A rearrangements, not detectable by routine PCR-based approaches, are involved in a fraction of melanoma cases negative for point sequence changes. In order to gain insights on the possible role of CDKN2A large deletions or duplications in melanoma susceptibility in the Italian population, we screened a series of 124 cutaneous malignant melanoma families referred to five national medical/cancer genetics centres. All probands were negative for point mutations in CDKN2A and CDK4. All samples were tested by MLPA (multiplex ligation-dependent probe amplification), and the results were confirmed by real-time quantitative PCR in a subset of 53 cases. No genomic rearrangements were detected in this series, one of the largest so far investigated. These data suggest that large deletions/duplications in the CDKN2A locus are infrequently involved in the development of familial melanoma in the Italian population. Based on these results, routine search for these rearrangements in CDKN2A- and CDK4-mutation negative melanoma families is not warranted, although it would be reasonable to pursue it in selected cases with very strong family history and/or showing linkage to 9p21.

The p.G23S CDKN2A founder mutation in high-risk melanoma families from Central Italy.

We have investigated the frequency and spectrum of CDKN2A/CDK4 mutations in 23 cutaneous melanoma families from Central Italy (Tuscany). Three distinct mutations were identified in five families. One mutation, p.G23S, was present in three families. Several lines of evidence indicate that p.G23S is a pathogenic mutation: it is located in the functionally important first ankyrinic domain of p16, it was not detected in a sample of 100 control individuals, and it was present in all tested affected individuals from the three families. Haplotype analysis showed a common ancestral origin of the p.G23S mutation. Our data show that the p.G23S mutation is an important cause of hereditary melanoma in Tuscany.

Impact of E27X, a novel CDKN2A germ line mutation, on p16 and p14ARF expression in Italian melanoma families displaying pancreatic cancer and neuroblastoma.

Mutations in the CDKN2A gene underlie melanoma susceptibility in as many as 50% of melanoma kindreds in selected populations, and several CDKN2A founder mutations have been described. Inherited mutations in CDKN2A have been found to be associated with other, non-melanoma cancers including pancreatic cancer (PC) and neural system tumors (NST). Here we report a novel germline mutation in exon 1 of the CDKN2A gene, E27X, which we first detected in melanoma patients living in or originally from a small geographic area bordering Liguria in north-western Italy. A subset of melanoma kindreds positive for this mutation displayed PC and neuroblastoma. E27X generates a premature stop codon, leading to dramatically reduced protein levels of p16 and leaving p14ARF unaltered. As PC and NSTs have been postulated to be preferentially associated with CDKN2A mutations located in exon 2 and/or affecting p14ARF alone, the position of E27X in exon 1alpha provides interesting insights towards clarifying the mechanisms by which the CDKN2A/ARF locus is involved in cancer predisposition.

A kindred with MYH-associated polyposis and pilomatricomas.

MYH-associated polyposis (MAP) is a recently described autosomal recessive form of familial adenomatous polyposis (FAP) associated with susceptibility to colorectal carcinoma (CRC). MAP is caused by biallelic inactivating mutations of the MYH gene, a component of the base excision repair (BER) machinery, whose dysfunction leads to an increase in the rate of G > T transversions following DNA oxidative damage. MAP patients can present with either classic or attenuated polyposis. However, the MAP colonic and extracolonic phenotype has yet to be defined. We report on two siblings, born from consanguineous parents, who were found to be homozygotes for an MYH frameshift mutation. The propositus presented with a low number of colonic lesions and an early-onset CRC. Both siblings had a history of pilomatricomas, benign tumors derived from hair follicles, in childhood. The findings presented provide further evidence of phenotypic variability in MAP, and suggest that multiple pilomatricomas may be a useful cutaneous marker of MAP.

Impaired fibrinolysis in retinal vein occlusion: a role for genetic determinants of PAI-1 levels.

Few and contrasting data are available on the presence of a thrombophilic state in patients with retinal vein occlusion (RVO), and we have previously demonstrated a role of elevated PAI-1 activity as a risk factor for this condition. The present study was undertaken to investigate whether PAI 4G/5G and ACE I/D polymorphisms are independent risk factors for RVO and whether they account for elevated PAI-1 activity levels. We studied 112 RVO patients (52 males and 60 females; range 18-83 years; median age 60 years) and 112 healthy subjects (52 males and 60 females; range 20-84 years; median age 57 years). PAI-1 activity was determined by a chromogenic assay and ACE I/D and PAI-1 4G/5G polymorphisms by polymerase chain reaction (PCR) and restriction length fragment polymorphism (RLFP) methods. Elevated PAI-1 activity (above 95(th) percentile of the controls) was significantly associated with RVO at multivariate analysis after adjustment for age, sex, traditional cardiovascular risk factors and haemostasis-related risk factors (OR = 4.93, 95% CI 1.70-14.30; p = 0.003). The homozygosity for ACE DD was found to be an independent risk factor for RVO at multivariate analysis (OR = 1.98, 95% CI 1.01-3.83; p = 0.049), whereas no significant association between homozygosity for PAI-1 4G4G and risk of RVO was observed. Subjects carrying both ACE DD genotype and PAI-1 4G4G genotype showed an increased risk for RVO at multivariate analysis (OR = 4.82, 95% CI 1.89-12.29; p = 0.001). In 45/112 patients without the established risk factors for RVO (hyper-tension, hypercholesterolemia and diabetes) or characteristics known to be associated to increased PAI-1 activity (overweight, hypertriglyceridemia, and smoking habit) the contemporary presence of ACE DD and PAI-1 4G4G genotype was significantly associated with a risk for RVO (OR = 8.26, 95% CI 1.18-57.92; p = 0.034). In conclusion, in our study: 1-indicates that ACE DD genotype is a risk factor for RVO in the whole group of patients, and in the subgroup of patients without the established risk factors for RVO or characteristics influencing the PAI-1 activity, when associated to PAI-1 4G4G genotype, and 2-confirms the role of hypofibrinolysis, documented by high levels of PAI-1 activity, in the occurrence of patients with RVO.

Hereditary nonpolyposis colorectal cancer and related conditions.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a cancer-predisposing condition caused by inactivating mutations in at least four genes (MSH2, MLH1, MSH6, and PMS2) belonging to the mismatch repair system. At present, availability of the microsatellite instability (MSI) test allows screening of a relevant fraction of patients with a constellation of features suggestive of HNPCC. By analogy with several other genetic disorders, it is clearly emerging that the term HNPCC encompasses a wide spectrum of different clinical presentations, including Muir-Torre syndrome, Turcot syndrome, and the neurofibromatosis-hematological malignancy association. Notwithstanding the remarkable genetic and allelic heterogeneity, a few consistent phenotype-genotype associations can be recognized. Mutations in the MSH2 gene entail higher risks of developing cancer, including extraintestinal ones, than MLH1 alterations. MSH2 also accounts for most cases of Muir-Torre syndrome, which is characterized by the presence of sebaceous skin tumors. The few known PMS2 mutations show a striking association with the presence of gliomas, which are the hallmark of the Turcot variant of HNPCC. Homozygotes for mismatch repair gene mutations present with stigmata of neurofibromatosis 1 and usually die in childhood due to a variety of leukemias and lymphomas. While such correlations are being defined, the underlying reasons have only partially been elucidated, and may include heterogeneous gene functions and properties; types of mutation, some of which may exert dominant negative effects; and genetic and environmental modifiers.