Mutational profiling in melanocytic tumors: multiple somatic mutations and clinical implications.

In this study, we analyzed multiple somatic mutations in 10 genes relevant in melanoma tumorigenesis and targeted therapies. Overall, 45% of the tumors showed mutations and, in particular, 33% had multiple mutations. Based on our results, we conclude that the assessment of mutation status of multiple genes, including CDKN2A, could provide a genetic profile that can be useful as a prognostic and therapeutic marker in melanocytic tumors.

SULT1A1 gene deletion in BRCA2-associated male breast cancer: a link between genes and environmental exposures?

SULT1A1, a member of sulfotransferase superfamily, is a drug and hormone metabolizing enzyme involved in the metabolism of a variety of potential mammary carcinogens of endogenous and exogenous origin. Interestingly, the metabolic activity of SULT1A1 can be affected by variations in gene copy number. Male Breast Cancer (MBC) is a rare disease and less investigated disease compared to female BC (FBC). As in FBC, the concurrent effects of genetic risk factors, particularly BRCA2 mutations, increased exposure to estrogens and environmental carcinogens play a relevant role in MBC. By quantitative real-time PCR with TaqMan probes, we investigated the presence of SULT1A1 gene copy number variations (CNVs) in a series of 72 MBCs. SULT1A1 gene deletion was observed in 10 of the 72 MBCs (13.9%). In a multivariate analysis association between BRCA2 mutation and SULT1A1 gene deletion emerged (p = 0.0005). Based on the evidence that the level of SULT1A1 enzyme activity is correlated with CNV, our data suggest that in male breast tumors SULT1A1 activity may be decreased. Thus, it can be hypothesized that in a proportion of MBCs, particularly in BRCA2-associated MBCs, the level of estrogens and environmental carcinogens exposure might be increased suggesting a link between gene and environmental exposure in the pathogenesis of MBC.

[Medical research through historical resources. Talking objects: a case of a parasitic perineal monstrosity]. / La ricerca medica attraverso la ricerca storica: mostri e mostruosità. Di una mostruosità parassitaria felicemente risolta con operazione chirurgica.

A case of a parasitic perineal monstrosity from the collection of the Pathology Museum of the University of Florence, is described on the basis of the original medical records and illustrations. The surgeon Giorgio Pellizzari (1814-1894) first reported this extraordinary case of sacrococcygeal teratoma containing a rudimentary inferior limb. Reader of Descriptive Anatomy, Pellizzari was a well-known Anatomy Dissector and Curator of the Physiological Museum of the Regio Arcispedale di Santa Maria Nuova in Florence. This report underlines the importance of studying the archive material in order to thoroughly comprehend a single museum talking object. This handling of matters will help to turn anatomical collections into a unique teaching toolfor modern medical practice and a noteworthy documentation of scientific, artistic and historical value. Through analysis of the original catalogue and investigation by means of modern scientific techniques, discovering the story behind the object becomes afeasible challenge.

[Fire worship on Soratte: form prehistory to present days]. / IL culto del fuoco sul Soratte: dalla preistoria ai nostri giorni.

Fire has always been involved in human thoughts, believes and actions. For many psychological reasons fire has been considered as living power: red as the human blood, warm as the human body, clearly shines in the night. Fire worship is very common, especially in regions where terrestrial fire is believed to be the image of the heavenly fire. Fire worship has represented the base for symbolic systems involved in the cultural-anthropological evolution of populations settled close to mount Soratte. Fire cults in this region have originated in independent and long-time separated contexts, according to available knowledge. Hirpi Sorani, ancient inhabitants of mount Soratte territory, celebrated Sorano Apollo by a famous fire walking ceremony, with a likely cathartic and apotropaic meaning, as reported by ancient Roman writers. The victory of Christianity over paganism caused the decline of these religious practices. The beginning of the XIX century witnessed the establishment ofa new kind of devotion to the Virgin Mary at the mount Soratte. Today, this religious piety-linked identity has weakened due to changes in lifestyles,from an agricultural to one based on outside home employment, and has been largely shifted on cultural and anthropological bases. In conclusion, fire worship at mount Soratte has evolved because of external influences and local inhabitants have reacted by asserting their own identity.

Clinical and pathologic characteristics of BRCA-positive and BRCA-negative male breast cancer patients: results from a collaborative multicenter study in Italy.

Recently, the number of studies on male breast cancer (MBC) has been increasing. However, as MBC is a rare disease there are difficulties to undertake studies to identify specific MBC subgroups. At present, it is still largely unknown whether BRCA-related breast cancer (BC) in men may display specific characteristics as it is for BRCA-related BC in women. To investigate the clinical-pathologic features of MBC in association with BRCA mutations we established a collaborative Italian Multicenter Study on MBC with the aim to recruit a large series of MBCs. A total of 382 MBCs, including 50 BRCA carriers, were collected from ten Italian Investigation Centres covering the whole country. In MBC patients, BRCA2 mutations were associated with family history of breast/ovarian cancer (p<0.0001), personal history of other cancers (p=0.044) and contralateral BC (p=0.001). BRCA2-associated MBCs presented with high tumor grade (p=0.001), PR-(p=0.026) and HER2+ (p=0.001) status. In a multivariate logistic model BRCA2 mutations showed positive association with personal history of other cancers (OR 11.42, 95% CI 1.79-73.08) and high tumor grade (OR 4.93, 95% CI 1.02-23.88) and inverse association with PR+ status (OR 0.19, 95% CI 0.04-0.92). Based on immunohistochemical (IHC) profile, four molecular subtypes of MBC were identified. Luminal A was the most common subtype (67.7%), luminal B was observed in 26.5% of the cases and HER2 positive and triple negative were represented by 2.1% and 3.7% of tumors, respectively. Intriguingly, we found that both luminal B and HER2 positive subtypes were associated with high tumor grade (p=0.003 and 0.006, respectively) and with BRCA2 mutations (p=0.016 and 0.001, respectively). In conclusion, our findings indicate that BRCA2-related MBCs represent a subgroup of tumors with a peculiar phenotype characterized by aggressive behavior. The identification of a BRCA2-associated phenotype might define a subset of MBC patients eligible for personalized clinical management.

PAI-1 4G/5G repeat is a target in gastric carcinomas with microsatellite instability.

BACKGROUND: The plasminogen activator inhibitor-1 (PAI-1) plays an important role in the pathogenesis of cancer. The 4G/5G promoter polymorphism of PAI-1 is potentially involved in regulating gene transcription. AIMS: To explore the role of the PAI-1 4G/5G repeat as target of microsatellite instability (MSI), 50 gastric carcinomas (GCs), characterized for MSI, were screened. METHODS: PAI-1 4G/5G was analysed by direct sequencing. RESULTS: Allelic imbalance was observed in 5 of the 50 (10%) GCs. Specifically, 2 cases (40%) harboured a G deletion and 3 (60%) a G insertion in tumour compared to normal DNA. These five cases were included in the subgroup of 20 GCs (25%) with high level of MSI (MSI-H). A statistically significant association emerged between PAI-1 mutations and MSI-H status (p=0.0073). The frequency of PAI-1 mutations was also evaluated, together with other known target genes, by analysing MSI-H GCs for mutations at selected coding repeats within genes controlling cell growth, apoptosis and DNA repair. Overall, mutation frequency ranged from 56.3% to 5.3%. CONCLUSION: The frequency of PAI-1 mutations here reported in MSI-H GCs is, accordingly, comparable with values obtained for real targets. The relatively high incidence of PAI-1 mutations is suggestive of a positive pressure towards selection in MSI-H GCs.

Mutation analysis of BRIP1 in male breast cancer cases: a population-based study in Central Italy.

Breast cancer (BC) in men is rare compared with BC in women, but its incidence is increasing along with attention toward this uncommon disease. Although with some differences, male and female BC share similar genetic predisposition factors, including BRCA1/2, CHEK2, and PALB2 mutations. As other BRCA1/2 functionally related DNA repair genes, such as CHEK2 and PALB2, BRIP1 is considered a moderate-penetrance BC susceptibility gene. At present, the role of BRIP1 on BC susceptibility in men is unknown. In this study, we aimed to assess whether BRIP1 variants may contribute to male BC (MBC) risk, by screening 97 MBC cases, all negative for BRCA1/2, CHEK2, and PALB2 mutations, selected from a population-based series of 126 MBCs from Central Italy. A total of five BRIP1 germ-line sequence alterations, three coding, and two non-coding variants, were detected in our series. The two non-coding variants IVS4-28G > A and 3'UTR 4049C > T were classified as neutral by in silico analysis. Of the three coding variants, one was a silent variant (E879E) and two resulted in amino acid substitution (R264W and P919S) showing a putative pathogenic role by in silico analysis. However, further analysis of tumor-associated loss of heterozygosity and the frequency of variant alleles, tested in 203 male population controls, suggested a neutral effect for both of these variants. Overall, our results indicate that BRIP1 variants may not play a relevant role in MBC predisposition.

Inherited and acquired alterations in development of breast cancer.

Breast cancer is the most common cancer among women, accounting for about 30% of all cancers. In contrast, breast cancer is a rare disease in men, accounting for less than 1% of all cancers. Up to 10% of all breast cancers are hereditary forms, caused by inherited germ-line mutations in "high-penetrance," "moderate-penetrance," and "low-penetrance" breast cancer susceptibility genes. The remaining 90% of breast cancers are due to acquired somatic genetic and epigenetic alterations. A heterogeneous set of somatic alterations, including mutations and gene amplification, are reported to be involved in the etiology of breast cancer. Promoter hypermethylation of genes involved in DNA repair and hormone-mediated cell signaling, as well as altered expression of micro RNAs predicted to regulate key breast cancer genes, play an equally important role as genetic factors in development of breast cancer. Elucidation of the inherited and acquired genetic and epigenetic alterations involved in breast cancer may not only clarify molecular pathways involved in the development and progression of breast cancer itself, but may also have an important clinical and therapeutic impact on improving the management of patients with the disease.

[Water cults on Soratte Mount]. / Il culto dell'acqua sul Soratte: dalla preistoria ai nostri giorni.

Mount Soratte is a limestone ridge that rises on a lonely plateau of Pliocene tuff on the right of the Tiber, about forty kilometers North of Rome. Studies related to human settlements during prehistory in this territory have been sporadic and occasional. The first evidence of prehistoric cults on mount Soratte has been found in the early Fifties when ajar, dating back to Neolithic times, was discovered in the cave of the Meri. The jar was placed in a position to be always filled of water and indicates the existence of ancient practices of worship linked to groundwater. In the Middle Ages, although caves became a step towards the Hell, dripping caves were often associated with the magical-religious and therapeutic aspects of water linked to fertility in the popular imagination. In the cave church of the Saint Romana, on the eastern slope of Mount Soratte close to Meri, there is a small marble basin near the altar and the water drips from the rock above it. This water is taken out for devotion and drunk by mothers who did not get milk from their breasts. Recently, the water of the Saint Romana would have drained as a result of an act of sacrilege, albeit unintentionally, as reported in a oral testimony. Overall, the territory of Mount Soratte is characterized by a sharp and clear karst. This causes the water, that collects on the inside, coming out in many springs all around the valley. This water is collected to supply fountains used years ago by farmers and livestock and nowadays may represent a cultural space of social life with the aim to build a strong link with the territory and a new awareness of the past and history of the countryside around Mount Soratte.

Gene-environment interactions in the pre-Industrial Era: the cancer of King Ferrante I of Aragon (1431-1494).

King Ferrante I of Aragon, leading figure of the Italian Renaissance, died in 1494. The autopsy of his mummy revealed a tumor infiltrating the small pelvis. We examined the histologic and molecular features of this ancient tumor to investigate its primary origin. Hematoxylin-eosin, Van Gieson, and Alcian Blue staining showed neoplastic cells infiltrating muscular fibers and forming pseudo-glandular lumina disseminated in fibrous stroma with scarce mucus. A strong immunoreactivity of the neoplastic cells was shown for pancytokeratins and proliferating cell nuclear antigen. Molecular fingerprints were investigated by examining K-ras, BRAF, and microsatellite instability in ancient tumor DNA. Sequencing analysis showed G-to-A transition in codon 12 of K-ras. BRAF mutations and microsatellite instability were not observed. Because the presence of K-ras codon 12 mutation could be associated with exposure to chemical carcinogens, possibly present in some food items, paleodietary reconstruction of the King Ferrante I was carried out by carbon (δ(13)C ) and nitrogen (δ(15)N) stable isotopes analysis. δ(13)C and δ(15)N values found in bone collagen of the King were consistent with a massive intake of animal proteins. Overall, our data show that the tumor of Ferrante I was a mucinous adenocarcinoma with molecular fingerprints characteristic of colorectal carcinogenesis linked to K-ras pathway. Paleodietary reconstruction and historical chronicles indicate a strong consumption of meat by the King. The possible abundance of dietary carcinogens, related to meat consumption, could explain the K-ras mutation causing the colorectal tumor that killed Ferrante I more than 5 centuries ago.

The BRCAPRO 5.0 model is a useful tool in genetic counseling and clinical management of male breast cancer cases.

No study has evaluated the performance of BRCA1/2 mutations prediction models in male breast cancer (MBC) series. Although rare, MBC deserves attention because male and female breast cancers share many characteristics, including the involvement of genetic predisposition factors such as BRCA1/BRCA2 mutations. Indeed, the occurrence of MBC is a commonly used criterion to select families for BRCA mutation testing. We evaluated the performance and clinical effectiveness of four different predictive models in a population-based series of 102 Italian MBC patients characterized for BRCA1/2 mutations. Sensitivity, specificity, and positive and negative predictive values (PPV, NPV) were calculated for each risk model at the 10% threshold. The area under the ROC (AUC) curves and its corresponding asymptotic 95% CIs were calculated as a measure of the accuracy. In our study, the BRCAPRO version 5.0 had the highest combination of sensitivity, specificity, NPV and PPV for the combined probability and for the discrimination of BRCA2 mutations. In individuals with negative breast-ovarian cancer family history, BRCAPRO 5.0 reached a high discriminatory capacity (AUC=0.92) in predicting BRCA2 mutations and showed values of sensitivity, specificity, NPV and PPV of 0.5, 0.98, 0.97 and 0.67, respectively, for the combined probability. BRCAPRO version 5.0 can be particularly useful in dealing with non-familial MBC, a circumstance that often represents a challenging situation in genetic counseling.

[When history meets molecular medicine: molecular history of human tuberculosis]. / Quando la storia incontra la medicina molecolare: storia molecolare della tubercolosi umana.

Tuberculosis represents one of the humankind's most socially devastating diseases. Despite a long history of medical research and the development of effective therapies, this disease remains a global health danger even in the 21st century. Tuberculosis may cause death but infected people with effective immunity may remain healthy for years, suggesting long-term host-pathogen co-existence. Because of its antiquity, a supposed association with human settlements and the tendency to leave typical lesions on skeletal and mummified remains, tuberculosis has been the object of intensive multidisciplinary studies, including paleo-pathological research. During the past 10 years molecular paleo-pathology developed as a new scientific discipline allowing the study of ancient pathogens by direct detection of their DNA. In this work, we reviewed evidences for tuberculosis in ancient human remains, current methods for identifying ancient mycobacterial DNA and explored current theories of Mycobacterium tuberculosis evolution and their implications in the global development of tuberculosis looking into the past and present at the same time.

Down-regulation of epidermal growth factor receptor by selective expansion of a 5'-end regulatory dinucleotide repeat in colon cancer with microsatellite instability.

PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed in several tumor types, and its expression is influenced by the length of a 5'-end microsatellite repeat (CA)n: the longer the repeat, the lower the expression. Dinucleotide repeats accumulate insertion/deletion types of mutations in tumors with microsatellite instability. We designed this study to estimate the occurrence of these mutations in EGFR(CA)n and their relevance in carcinogenesis of microsatellite instability-positive colon and gastric tumors. EXPERIMENTAL DESIGN: We analyzed the frequency of EGFR(CA)n mutations in vivo in 55 colorectal and 14 gastric microsatellite instability-positive cancers, and in vitro in single-cell clone cultures of microsatellite instability-positive colon tumor cell line LS174. Single-cell clone cultures with different repeat lengths were analyzed by fluorescent-activated cell sorter for EGFR cell-surface expression. A correlation analysis was done between EGFR(CA)n mutations and mutations in KRAS, BRAF, and p53. RESULTS: Unlike single-cell clone cultures, which exhibited higher rate of deletions compared with insertions, most of EGFR(CA)n mutations in colon and gastric tumors were insertions. Longer EGFR(CA)n correlated with lower EGFR cell-surface expression in single-cell clone cultures. In colon cancers, the elongation of the repeat was associated negatively with mutations in KRAS and BRAF, but not in p53. CONCLUSIONS: The EGFR(CA)n elongation observed in tumors cannot be explained by an intrinsic property of this repeat favoring insertions versus deletions. Instead, a selection for repeat elongation occurs in microsatellite instability-positive tumors, leading to EGFR down-regulation. These findings suggest that in microsatellite instability-positive tumors current therapies targeting EGFR overexpression may have either no effect or an opposite to the expected effect.

Founder mutations account for the majority of BRCA1-attributable hereditary breast/ovarian cancer cases in a population from Tuscany, Central Italy.

BACKGROUND: Germline mutations in the BRCA1 and BRCA2 tumour-suppressor genes predispose to early-onset breast and ovarian cancer. Although both genes display a highly heterogeneous mutation spectrum, a number of alterations recur in some populations. Only a limited number of founder mutations have been identified in the Italian population so far. OBJECTIVE: To investigate the spectrum of BRCA1/BRCA2 mutations in a set of families originary from the Central-Eastern part of Tuscany and to ascertain the presence of founder effects. We also wanted to approximate the age of the most frequent BRCA1 founder mutation. RESULTS: Overall, four distinct BRCA1 mutations accounted for a large fraction (72.7%) of BRCA1-attributable hereditary breast/ovarian cancer in families originary from this area. We identified common haplotypes for two newly recognised recurrent BRCA1 mutations, c.3228_3229delAG and c.3285delA. The c.3228_3229delAG mutation was estimated to have originated about 129 generations ago. Interestingly, male breast cancer cases were present in 3 out of 11 families with the c.3228_3229delAG mutation. CONCLUSIONS: The observation that a high proportion of families with BRCA1 alterations from Central-Eastern Tuscany harbours a limited number of founder mutations can have significant impact on clinical management of at risk subjects from this area. In addition, the identification of a large set of families carrying an identical mutation that predisposes to breast and ovarian cancer provides unique opportunities to study the effect of other genetic and environmental factors on penetrance and disease phenotype.

BRCA1/BRCA2 mutation status and clinical-pathologic features of 108 male breast cancer cases from Tuscany: a population-based study in central Italy.

BACKGROUND: Male breast cancer (MBC) is a rare and scarcely investigated disease. The strongest genetic risk factor for MBC is represented by inherited BRCA2 mutations, whereas the association between MBC and BRCA1 mutations is less clear. MBC appears to be biologically similar to breast cancer in females, however the phenotypic characteristics of BRCA1/2-related MBCs are not yet well elucidated. OBJECTIVE: To investigate the genetic and phenotypic characteristics of MBC in a large and well-characterized population-based series of 108 MBCs from Tuscany (Central Italy) and to evaluate associations between BRCA1/BRCA2 mutation status and clinical-pathological features including breast/ovarian cancer first-degree family history, tumor histology and grade, proliferative activity, estrogen/progesterone receptors (ER/PR) and epidermal growth factor receptor 2 (HER2) expression. Results BRCA1/BRCA2 mutations were identified in ten MBCs, in particular, two cases (1.9%) carried BRCA1 and eight cases (7.4%) carried BRCA2 mutations. The same BRCA1 mutation (3347delAG) was detected in two unrelated MBC cases. Three novel BRCA2 pathogenic mutations were found. Statistically significant associations emerged between BRCA2-related tumors and absence of PR expression (P = 0.008), HER2 over-expression (P = 0.002) and high tumor grade (P = 0.005). Conclusions Here, we (i) reported that in our population about 9% of MBC cases are accounted for by BRCA1/BRCA2 mutations; (ii) enlarged the BRCA2 mutational spectrum and (iii) characterized a specific phenotype associated with BRCA2-related MBCs suggestive of aggressive behavior. Overall, our results may have important implications on clinical management for this rare disease.

Genome-wide expression profile of sporadic gastric cancers with microsatellite instability.

Gastric cancers with mismatch repair (MMR) inactivation are characterised by microsatellite instability (MSI). In this study, the transcriptional profile of 38 gastric cancers with and without MSI was analysed. Unsupervised analysis showed that the immune and apoptotic gene networks efficiently discriminated these two cancer types. Hierarchical clustering analysis revealed numerous gene expression changes associated with the MSI phenotype. Amongst these, the p53-responsive genes maspin and 14-3-3 sigma were significantly more expressed in tumours with than without MSI. A tight immunosurveillance coupled with a functional p53 gene response is consistent with the better prognosis of MSI cancers. Frequent silencing of MLH1 and downregulation of MMR target genes, such as MRE11 and MBD4, characterised MSI tumours. The downregulation of SMUG1 was also a typical feature of these tumours. The DNA repair gene expression profile of gastric cancer with MSI is of relevance for therapy response.

Gastric cancer with high-level microsatellite instability: target gene mutations, clinicopathologic features, and long-term survival.

Gastric cancer is one of the leading causes of cancer death worldwide, and although the incidence has decreased in Western countries, specific high-risk areas are present in Italy. Gastric cancer with high-level microsatellite instability (MSI-H) represents a well-defined subset of carcinomas showing distinctive clinicopathologic features. We examined clinicopathologic associations and long-term survival in a series of 159 gastric cancer cases from a high-risk population in Tuscany (central Italy). MSI-H was associated with antral location of the tumor (P = .001), intestinal type according to Lauren classification (P = .002), expanding type according to Ming classification (P = .0001), and mucinous histologic type according to the Japanese Research Society for Gastric Cancer classification (P = .002). In addition, MSI-H was strongly associated with a higher survival at 15 years (P = .01) and with loss of hMLH1 expression, evaluated by immunohistochemistry (P = .001). Multivariate analyses showed a significant association between the absence of hMLH1 reactivity and the expanding tumor type (P = .002). We also investigated the MSI-H-related genetic changes by analyzing coding repeats within target genes involved in pathways that control cell growth (TGFbetaRII, IGFIIR, RIZ, TCF4, DP2), apoptosis (BAX, BCL10, FAS, CASPASE5, APAF1), and DNA repair genes (hMSH6, hMSH3, MED1, RAD50, BLM, ATR, BRCA2, MRE11). Gastric cancer cases with MSI-H were found to accumulate heterozygous mutations affecting multiple molecular pathways and multiple genes within each pathway. Intriguingly, in this subset, TGFbetaRII mutations appeared to be inversely related to BLM mutations (P = .006), whereas RAD50 mutation carriers showed significantly reduced survival (P = .03).

Histological heterogeneity and somatic mtDNA mutations in gastric intraepithelial neoplasia.

Somatic mutations of mitochondrial DNA (mtDNA) are associated with various types of human cancer. To elucidate their role in gastric carcinogenesis, we analyzed mutations in the displacement loop region of mtDNA in 24 paraffin-embedded gastric intraepithelial neoplasias (formerly dysplasia) from a high gastric cancer risk area in northern Italy. Helicobacter pylori infection was assessed by histological examination (Giemsa staining). Gastritis was classified according to the guidelines of the Updated Sydney System. The mtDNA displacement loop region was amplified and sequenced from gastric intraepithelial neoplasia samples and adjacent non-neoplastic gastric mucosa. The gastric intraepithelial neoplasias were divided into two groups by their association with H. pylori gastritis. Group A with lesions arising on a background of H. pylori-positive gastritis contained 7 patients, and group B with lesions associated with H. pylori-negative gastritis contained 17 patients. Group A had a larger proportion of high-grade lesions than group B and showed a foveolar phenotype (type II dysplasia). Group B had a larger proportion of cases with mtDNA displacement loop region mutations than group A (P=0.004, Fisher's exact test) and exhibited an intestinal phenotype. No evidence of heteroplasmic variants in the mtDNA displacement loop, suggestive of mutations, was detected in gastric biopsies from 25 H. pylori-negative subjects and 60 cancer-unaffected H. pylori-positive patients. These results provide further evidence for the morphologic and mtDNA biomolecular differences of gastric intraepithelial neoplasias, and suggest the existence of two distinct pathways to gastric cancer--corpus-dominant H. pylori gastritis and the atrophy-metaplasia pathway.