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Le risorse genetiche costituiscono il patrimonio genetico presente nelle piante, animali e microrganismi di attuale o potenziale valore per l'intera umanità. In particolare, le risorse genetiche vegetali (RGV) comprendono tutte le specie coltivate, i progenitori selvatici, le specie affini sia coltivate che spontanee. A partire dai primi del '900 è iniziata la raccolta sistematica delle RGV, ma loro conservazione (ex situ) nelle banche del germoplasma o "genebanks", è partita solo dopo la prima metà dello stesso secolo. La salvaguardia delle RGV è diventata nel tempo sempre più importante poiché le RGV costituiscono una riserva di geni potenzialmente utili per combattere patogeni e parassiti. Inoltre si preservano specie che pur non avendo oggi nessun valore economico, potrebbero rivelarsi in futuro, date le crescenti necessità alimentari e non del genere umano, fondamentali nella risoluzione di particolari problemi. Pertanto nelle banche dei semi è importante non solo conservare i semi in maniera corretta ma anche classificarli in modo inequivocabile.

A genomic DNA library enriched with GA/TC repeats from Camelina sativa variety Calena has been analysed. After sequencing of about 200 randomly selected clones, approximately 60% of them showed to contain simple or compound microsatellites with a high number of repeats. Among all microsatellite markers analysed 15 primer pairs amplified polymorphic fragments. Forty C. sativa accessions of different origin were genotyped with 15 microsatellite markers that generated 134 alleles with an average of 8.93 alleles per locus. The observed heterozygosity (Ho) among the accessions ranged from 0.0 to 0.15 with an average of 0.0370, whereas the average of expected heterozygosity (He) among accessions was 0.2769. The analysis of the average total heterozygosity (HT = 0.651), the intrapopulation genetic diversity (HS = 0.260), the interpopulation genetic diversity (DST = 0.391) and the coefficient of genetic differentiation among populations (GST = 0.574) demonstrated that 57.4% of the genetic diversity is among the accessions, while 42.6% resides within them. Phylogenetic tree of the 40 C. sativa accessions was constructed based on Nei's genetic distance. The UPGMA (Unweighted Pair Group Method with Arithmetic Mean) dendrogram shows, except for CAM108 and CAM170, a clear discrimination among C. sativa accessions grouping them in five subgroups. ANOVA analysis indicates significant differences in some biochemical and agronomic parameters among the C. sativa accessions grouped according to Nei's genetic distance. The result of the Tukey HSD test demonstrated that the A4 subgroup showed a significant higher TWS and linoleic acid (LA) content, while the subgroup A1 showed a significant higher linolenic and lower linoleic acid content compared to the remaining groups.

Purpose The prevalence of BRAF, NRAS, and p16CDKN2A mutations during melanoma progression remains inconclusive. We investigated the prevalence and distribution of mutations in these genes in different melanoma tissues. Patients and Methods In all, 291 tumor tissues from 132 patients with melanoma were screened. Paired samples of primary melanomas (n = 102) and synchronous or asynchronous metastases from the same patients (n = 165) were included. Tissue samples underwent mutation analysis (automated DNA sequencing). Secondary lesions included lymph nodes (n = 84), and skin (n = 36), visceral (n = 25), and brain (n = 44) sites. Results BRAF/NRAS mutations were identified in 58% of primary melanomas (43% BRAF; 15% NRAS); 62% in lymph nodes, 61% subcutaneous, 56% visceral, and 70% in brain sites. Mutations were observed in 63% of metastases (48% BRAF; 15% NRAS), a nonsignificant increase in mutation frequency after progression from primary melanoma. Of the paired samples, lymph nodes (93% consistency) and visceral metastases (96% consistency) presented a highly similar distribution of BRAF/NRAS mutations versus primary melanomas, with a significantly less consistent pattern in brain (80%) and skin metastases (75%). This suggests that independent subclones are generated in some patients. p16CDKN2A mutations were identified in 7% and 14% of primary melanomas and metastases, with a low consistency (31%) between secondary and primary tumor samples. Conclusion In the era of targeted therapies, assessment of the spectrum and distribution of alterations in molecular targets among patients with melanoma is needed. Our findings about the prevalence of BRAF/NRAS/p16CDKN2A mutations in paired tumor lesions from patients with melanoma may be useful in the management of this disease.

To elucidate the function of the FMR1 gene, we applied RNA in situ hybridization to cryosections of mice from different developmental stages. The murine Fmr-1 was found transcribed in a ubiquitous manner with an expression pattern similar to glyceraldehyd phosphate dehydrogenase, Gapdh, which was used as a control gene. A significant difference in the Fmr-1 expression pattern, however, was markedly enhanced expression specifically confined to the testis and the fetal ovary. In the immature and mature testis an elevated level of Fmr-1 expression is found in type A1 spermatogonia. Expression in the testis is observed in fetal life, reaches the highest level in the immature testis, and declines early in adult life. In the mature ovary no specific Fmr-1 expression signal was found but enhanced levels were seen in the fetal ovary. At this developmental stage proliferation of oogonia takes place. It is suggested that FMR1 serves a special function during germ cell proliferation in males and females. These findings are discussed in the light of the current observation that fragile X patients produce only sperm with a premutation sized allele. Two hypotheses are put forward: (1) In males lack of FMR1 function results in a premeiotic defect preventing spermatogonia with a full mutation to reach meiosis. A fragile X mutation can be passed on to offsprings only as a premutation (selection hypothesis). (2) Transition of a premutation allele to full mutation occurs in a postzygotic stage after separation of the germ line and is restricted to soma cells (restriction hypothesis). Expression of FMR1 in proliferating germ cells is in line with both hypothesis.

As an essential step towards an exhaustive analysis of the coding potential of large regions of the genome, we have developed a protocol allowing the rapid isolation of transcripts defined by overlapping clone libraries. The method is based on the hybridisation of cDNA inserts, which had been amplified by PCR from cDNA libraries, to biotinylated DNA from cosmids or cosmid pools. Nonspecific hybrids are then removed, the selected cDNAs are eluted and reamplified by PCR. Using a cosmid containing part of the FMR-1 gene as test, we were able to demonstrate an eighty thousand fold enrichment of cDNAs for this gene after two rounds of selection-amplification. The technique was applied to the analysis of transcripts from two cosmid contigs, together encompassing a region of 900 kb in Xq28. These experiments have thus far resulted in the identification of 81 cDNA clones, of which 54 clones were mapped back to the cosmid contigs. Of the 54 clones placed on the contig maps, 12 cDNA clones can be shown to belong to two genes which have been previously reported (L1CAM and QM).

Summary Background The chromosome 9p21 and its CDKN locus, with the p16 tumour suppressor gene (CDKN2A), are recognized as the genomic regions involved in the pathogenesis of melanoma. Objectives To elucidate further the role of such regions during the different phases of melanocytic tumorigenesis. Methods Tissue sections from naevi, primary and metastatic melanomas were investigated by fluorescence in situ hybridization for allelic loss at the 9p21 chromosome and by immunochemistry for p16CDKN2A expression. Results Dysplastic naevi and primary or secondary melanomas were found to carry hemizygous deletions within the entire 9p21 region at similar frequencies (varying from 55% to 62%). Allelic deletion spanning the CDKN locus was observed at significantly increased rates moving from early (7%) to advanced (28%) primary melanomas and to secondary melanoma lesions (37%) (P = 0·018). Also, inactivation of the p16 gene (CDKN2A) was absent in naevi and present at steadily increasing rates moving from primary melanomas (7% early lesions to 17% advanced lesions) to melanoma metastases (62%) (P = 0·004). Conclusions Our findings indicate that, in a model of sequential accumulation of genetic alterations, 9p21 deletions may play a role in melanocytic transformation and tumour initiation whereas rearrangements at the CDKN locus, and p16 gene (CDKN2A) inactivation may contribute to tumour progression.

Vinblastine (VBL) was tested in the mouse for induction of chromosome malsegregation in bone marrow cells. The occurence of aneuploidy and polyploidy was correlated with cell-cycle kinetics measured by DNA labelling with bromodeoxyuridine (BrdUrd). Sister-chromatid exchanges (SCE) were also detected. A dose-dependent lengthening of the cell cycle was induced in the dose range of 0.9–4.5 mg/kg body weight, up to a complete inhibition of cell-cycle progression (100% of metaphases were arrested before completion of the first mitotic division following a recovery time of 18 h, compared with 8% in the controls). Both aneuploidy and polyploidy were induced. Aneuploid metaphases were grouped into 2 classes, those with no more than 2 extra chromosomes and those with 3–10 extra chromosomes. The frequencies of cells with severe aneuploidy and polyploidy increased considerably when second-generation cells were sampled at a recovery time of 24 h. This observation suggested that gross chromosome imbalances occur preferentially after a period of mitotic arrest, probably as a consequence of multipolar spindles or failure of proper spindle assembly. Non-disjunction of single chromosomes arises independently of the mitotic block. A slight increase in SCE frequency was observed only at a recovery time of 18 h. This study may provide information on the kinetics and mechanisms of origin of VBL-induced numerical aberrations in vivo.

Chromosome 10q25-q26 has been strongly correlated to endometrial tumorigenesis. A novel human gene, CASC2, has previously been identified at chromosome 10q26. One out of the three alternative transcripted forms, CASC2a, has been demonstrated to be mutated at a low frequency in endometrial cancer (EC). In this study, the role of the CASC2a gene in cancer has been further defined.Tumour and corresponding normal tissues were analysed for CASC2a mRNA expression by real-time RT-PCR and mutation status by PCR-based approaches.A significantly decreased level of CASC2a transcripts was observed in 13/17 (76%) EC tissues, as well as in 6/9 (67%) colorectal cancers. Exogenous expression of CASC2a in undifferentiated AN3CA endometrial cancer cells inhibited cellular growth in anchorage-independent growth assays. Finally, infrequent CASC2a mutations were able to impair the gene function.Altogether, our findings strongly suggest that CASC2a may act as a tumour suppressor gene, with both epigenetic and genetic alterations concurring to gene inactivation. Down-regulation of CASC2a may provide a growth advantage in EC cells.

Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression.

Defective DNA mismatch repair and nonfunctional mechanisms controlling the proper progression of the cell cycle have been proposed as being responsible for the genomic instability and accumulation of karyotypic alterations in endometrial cancer (EC). To assess whether numerical chromosomal anomalies (aneuploidy) and microsatellite instability (MSI) might be representative of distinctive tumour behaviour, paraffin-embedded tissue samples from 86 patients with sporadic EC were evaluated by both fluorescence in situ hybridisation (FISH) and microsatellite analysis, using free nuclei and genomic DNAs (respectively). Approximately one-third of the tumours analysed (24/74; 32%) exhibited MSI, whereas 38/86 (44%) of the EC samples displayed aneuploidy. The majority of the unstable cases (15/24; 63%) were from advanced-stage patients. Conversely, 23 (61%) out of the 38 tumours with aneuploidy were from early-stage patients. No apparent correlation was found between MSI and aneuploidy, whereas the immunohistochemical (IHC) analysis revealed that inactivation of the MLH1 mismatch repair gene may be involved in the majority of the MSI+ sporadic ECs. No genetic or cytogenetic alteration analysed here seems to add any significant predictive value to the stage of disease. © 2002 Elsevier Science Ltd. All rights reserved.

The distal long arm of chromosome 10 harbors genes of biomedical interest such as MXI1, a putative tumor suppressor gene, and those encoding the adrenergic receptors alpha2A (ADRA2A) and beta1 (ADRB1). As part of a physical and genetic study of this genomic region, we constructed a 1.5-Mb YAC contig mapping to 10q25 that contains MXI1 and ADRA2A as well as a number of STSs. Rare cutting restriction site analysis of overlapping YACs allowed fine mapping of these genes and markers along the contig and revealed the presence of four CpG islands. MXI1 and ADRA2A appear to be about 600 kb apart, whereas ADRB1 is separated from ADRA2A by a distance larger than previously reported.

e20013 Background: Genetic factors predisposing to melanoma at germline level have been demonstrated to be geographically heterogeneous. We here evaluated the spectrum of NRAS and BRAF mutations at somatic level, in a large subset of melanoma tissues from patients originating from different Italian geographical areas: Sardinia, whose population is genetically homogeneous, and Middle-South Italy, with a genetically heterogeneous population. Methods: Patients were enrolled consecutively between June 2008 and December 2012. Genomic DNA was isolated from tumor tissues [primary melanomas (N=439) and melanoma metastases (N=269)] or melanoma cell lines (N=32). Paired samples of primary melanomas (n=140) and synchronous or asynchronous metastases from the same patients (n=203) were included. The full coding sequences and splice junctions of NRAS (exons 2-3) and BRAF (exon 15) genes were screened for mutations through automated sequencing. Results: BRAF/NRAS mutations were identified in 63% of primary melanomas (48% BRAF; 15% NRAS), 67% melanoma metastases (51% BRAF; 16% NRAS), and 68% melanoma cell lines (56% BRAF; 12% NRAS). A non-significant increase in mutation frequency after progression from primary melanoma was observed. However, distribution of BRAF/NRAS mutations varied between in vivo tumors and melanoma cell lines, suggesting a preponderant role for BRAF activation in highly proliferating cultured melanoma cells. Among paired samples, consistency of BRAF/NRAS mutation patterns between metastatic and primary melanomas ranged from 71% (skin metastases) to about 90% (lymph node and visceral metastases). A significant inverse distribution of BRAF/NRAS mutation rates was observed in our series on the basis of the geographical origin of patients: for BRAF, 58% Sardinian vs. 42% non-Sardinian cases (p=0.045); for NRAS, 2% Sardinian vs. 21% non-Sardinian cases (p

Background: Mutations of NRAS and BRAF genes have been identified with high frequency in nevi, cutaneous melanomas, and melanoma metastases. Prevalence of such mutations during the disease progression phases and among the different types of metastasis still remains inconclusive. Methods: A total of 275 tumour tissues from 116 melanoma patients were screened for mutations; among them, paired samples of microdissected primary melanomas (N=92) and synchronous or asynchronous metastases (N=156) from same patients were included. Tissue samples underwent mutation analysis by automated DNA sequencing. Secondary lesions were from: lymph nodes (LM; N=77), skin (SM; N=36), visceral (VM; N=23) and brain (BM; N= 44) sites. The full coding sequences and splice junctions of p16CDKN2A (exons 1, 2, and 3) and NRAS (exons 2 and 3) genes as well as the entire sequence at the exon 15 of the BRAF gene were screened for mutations by direct sequencing on automated fluorescence-cycle sequencer (ABIPRISM 3130, Applied Biosystems, Foster City, CA). Results: Overall, mutations were identified in 56/95 (59%) primary melanomas [43% BRAF - 16% NRAS], 49/77 (64%) lymph nodes [48% BRAF - 16% NRAS], 22/36 (61%) subcutaneous metastases [53% BRAF - 8% NRAS], 13/23 (57%) visceral metastases [43% BRAF - 13% NRAS], and 31/44 (70%) brain metastases [48% BRAF - 23% NRAS]. Overall, a slight and not significant increase in mutation frequency after progression from primary melanoma was observed in our series: 115/180 (64%) mutated metastases [48% BRAF - 16% NRAS]. Considering the paired samples from the same patients, LM (92% consistency) and VM (96% consistency) presented a highly similar prevalence and distribution of BRAF/NRAS mutations in comparison with primary melanomas, whereas a discontinuous pattern of mutations was detected in BM (80% consistency) or, mostly, SM (75% consistency). Occurrence of distinct mutation distribution between primary melanomas and correspondent metastases suggests that independent subclones have been generated in a limited fraction of patients. The rate of mutations in p16CDKN2A gene was found to steadily increase moving from primary melanomas (5/69; 7%) to melanoma metastases (21/144; 15%), with the highest prevalence of p16CDKN2A alterations (18/29; 62%) in melanoma cell lines Conclusions: Our results provide further clues about the impact of NRAS and BRAF mutations among the different stages of melanoma progression. Moreover, we confirmed that p16CDKN2A mutation rates are increasing during disease progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3677. doi:1538-7445.AM2012-3677

8574 Background: Mutations of NRAS and BRAF genes have been identified with high frequency in nevi, cutaneous melanomas, and melanoma metastases. Prevalence of such mutations during the disease pro...

The FMR1 gene, associated with fragile X syndrome, has recently been cloned and the sequence of partial cDNA clones is known. We have determined additional cDNA sequences both at the 5' and 3' end. We have characterized the expressed gene by means of RT-PCR in various tissues and have found that alternative splicing takes place in the FMR1 gene, which does not seem to be tissue specific. When the different alternative spiking events are combined, 12 distinct mRNA products could result from FMR1 expression in each tested tissue. In all these transcripts the open reading frame is maintained until the same stop codon. At the 3' end alternative use of polyadenylation signals is found. The alternative splicing allows functional diversity of the FMR-1 gene. Whether all the possible proteins will be synthesized and whether they will be functionally active has to be determined.

Background: Arginine deiminase (ADI), an enzyme that catalize the conversion of arginine to citrulline, has been used in clinical trials for the treatment of tumors which have been shown to be sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). We here evaluated the effect of ADI treatment on ASS expression in a series of melanoma cell lines. Methods: Twenty-five melanoma cell lines (6 derived from primary melanomas and 19 from MM metastases) and normal fibroblast cell as control underwent cell proliferation assays in presence or absence of ADI. The percentage of cell proliferation was estimated on day 6 of the ADI treatment by a colorimetric assay; the IC50 value was assessed for each cell line. Western blot analysis on lysates from treated and untreated cells were performed for evaluating the expression levels of both the ASS and the ornithine carbamoyl transferase (OTC) proteins. A subset of tissue sections from primary melanomas (N=10) and hepatocellular carcinomas (N=20) was investigated by immunohistochemistry for ASS expression. Results: Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition with a median IC50 value of 1.3 mU/mL (range, 0.4–2.0); growth of the remaining 4 (16%) MM cell lines was not inhibited and the IC50 was not reached. As expected, proliferation of the control fibroblasts was not inhibited. Expression analysis revealed that none of the 21 ADI-responsive cell lines presented detectable levels of the ASS protein before the ADI treatment; conversely, the 4 melanoma cell lines and the normal fibroblasts, which did not reached an IC50 value, showed a constitutive ASS expression in both treated and untreated cells. After treatment with ADI at a concentration corresponding to the IC50 values, 7/21 (33%) melanoma cell lines presented a markedly increased expression levels of the ASS protein; interestingly, six of them were from paired primary and secondary lesions of three patients. The median value of IC50 in this subset was of 1.8 mU/mL (range, 1.2–2.0). Nearly all samples (13/14; 93%) with absence of the ASS expression in both treated and untreated cells originated from MM metastases; among them, three cell lines were from paired primary and secondary lesions of the same patient. The median value of IC50 in this subset was of 1.2 mU/mL (range, 0.4–1.7). A constitutive expression of the OTC protein was found in all melanoma cell lines and no change in OTC expression was observed after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens (10 melanomas and 20 hepatocellular carcinomas). Conclusions: In our series, the most effective cell growth inhibition was observed in melanoma cells with absence of ASS expression either before or after the ADI treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A79.

8522 Background: Cytogenetic and molecular studies indicated the chromosome 9p21 and its CDKN locus, with the p16/CDKN2A tumor suppressor genes, as the genomic regions involved into the pathogenesis of malignant melanoma (MM). To further elucidate the role of such regions in melanoma, we evaluated the 9p21-linked molecular alterations during the different phases of melanocytic tumorigenesis. Methods: Paraffin-embedded tissue sections from common nevi, dysplastic nevi, and melanomas, including primary and metastatic MM lesion, as well as melanoma cell lines were evaluated by both fluorescence in situ hybridization (FISH) using probes spanning the entire 9p21 region and immunohistochemistry (IHC) for p16/CDKN2A expression. Results: Allelic deletion at the CDKN locus and p16/CDKN2A gene silencing were both observed at increased rates moving from nevi to early or advanced primary melanomas and to correspondent metastases. Dysplastic nevi, primary and secondary melanomas were instead found to be heterozygously deleted at one or more loci within the 9p21 chromosome in a quite similar fraction (55–62%) of cases. Inactivation of the p16/CDKN2A gene was found at rates higher in melanoma cell lines (67%) than in vivo melanomas (62% secondary and 12.5% primary melanomas), with a normal p16/CDKN2A IHC staining in all analyzed nevi. Conclusions: Our findings indicate a model of sequential accumulation of genetic alterations from normal melanocytes to metastatic melanoma, with a) 9p21 allelic loss playing a role in melanocytic transformation and tumor initiation; b) deletions at CDKN locus and p16/ CDKN2A gene inactivation participating to tumor dissemination. Presence of such alterations could be further evaluated as markers of the different phases of melanoma progression. No significant financial relationships to disclose.

3629 Background: Fanconi Anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and high incidence of leukemia and solid tumors. Eight FA associated genes have been cloned, and their products are thought to function in an integrated pathway which includes BRCA genes and maintains genomic stability. A molecular mechanism involved in the development and progression of some human malignancies, including colorectal cancer, is represented by a defective replication fidelity. Here we tested the role of the main FA genes in colorectal cancer (CRC). Methods: One hundred consecutively-collected patients with histologically-proven diagnosis of CRC and no mutation in the main mismatch repair genes (MLH1 and MSH2) were included into the study. Genomics DNA was screened using DHPLC for FANC-C/G/E/F genes and using MPLA for FANC-A/D2 genes. All PCR products with abnormal DHPLC profiles were sequenced on an automated sequencer. For 28 cases, tumor tissue samples were also analyzed. To date, total RNA was isolated from paired normal and tumor samples of 12 CRC patients. Gene expression was quantified by real-time RT-PCR using the Taqman approach. Results: At germline level, 2 cases presented FANC-C sequence variations (Asp267Ser, Val449Ser) and 5 cases showed a exon 26 deletion in FANC-A as detected by MLPA analysis. Among the 28 tumor samples available from patients negative for mutations in germline DNA, 2 (7%) cases presented a FANC-E mutation (Ala552Thr). Moreover, one of such somatic samples presented a BRCA2–8765delAG mutation (BRCA2 is also recognized as FANC-D1 gene). Assuming as significant difference in gene expression the presence of a variation of more than 50% in mRNA levels between normal and corresponding tumor samples, all tested FA genes (FANC-C/G/E/F) were found overexpressed in 5/12 (42%) tumor samples. Conclusions: Although preliminary, our findings seem to identify a subset of CRC patients with alterations in the FA/BRCA pathway which is required for the activation of DNA repair. Ongoing molecular analyses will better clarify the real frequency of each alteration in FA genes and their relationship with the histological and/or clinical features in colorectal cancer. No significant financial relationships to disclose.

Background: In our previous experience (JCO 2012;30:2522-9), we demonstrated a high consistency of BRAF and NRAS mutations between primary tumors and lymph node metastases in melanoma patients. A subset of paired samples of multiple asynchronous lymph node metastases from that series was here screened mainly using NGS technologies in order to better elucidate the existence of a real genetic homogeneity during clonal expansion to lymph node sites. Methods: Genomic DNA was isolated from frozen tissues of paired asynchronous lymph nodes, ascertained for presence of melanoma metastases (at least 80% of metastatic cells), using standard methods. Specimens from 12 patients were analyzed for mutations in 50 most common cancer genes with the Ion Torrent AmpliSeq Cancer Panel HotSpot.V2 (CHPv2) on the Ion PGMsequencer. All variants detected by NGS were confirmed through PCR-based Sanger sequencing. Paraffinembedded tumor tissues from the same series were evaluated by fluorescence in situ hybridization (FISH) assays, using probes specific for CyclinD1/EGFR genes or 9p21 locus and correspondent control centromeres. Results: Overall, only one (8%) of the 12 analyzed patients presented discrepancies in mutation patterns between the two distinct lymph nodal metastases developed in different times. In particular, the differences were represented by the BRAF-V600E and the CDKN2A-R80X mutations, suggesting that changes, when occur (though in a limited fraction of cases), they affect the main genes controlling cell proliferation and survival involved in melanomagenesis. No discrepancy was observed by FISH analysis into the same series. Considering the total number of analyzed samples, we detected the following mutations: BRAF-V600E (54% of cases), KDR-Q472H (50%), TP53-P72R (42%), cKIT-M541L (25%), NRAS-Q61R/K (25%). Conclusions: Our findings indicated a very low genetic heterogeneity during lymph node dissemination in melanoma patients, confirming the previously published data. Mutations in BRAF, NRAS, and cKIT genes were further confirmed to play a predominant role in melanoma pathogenesis.

Glaucoma is a group of ocular diseases characterized by an optic neuropathy in which degeneration of retinal ganglion cells leads to a characteristic excavation of the optic nerve head. Primary open-angle glaucoma (POAG) can be subdivided into two groups according to age of onset:- 1. the more common middle- to late-age onset, chronic open-angle glaucoma (COAG) diagnosed after the age of 40 years; 2. the rarer juvenile open-angle glaucoma (JOAG), which is diagnosed between the age of 3 years and early adulthood. Recently, the gene coding for the trabecular meshwork-induced glucocorticoid response protein (TIGR), located in chromosome 1 (1q23-25), was found mutated in patients affected by POAG. In this work we describe the clinical and molecular genetic features of several Italian families affected by autosomal dominant POAG, collected in various regions of Italy.

During the last few years, the incidence and mortality of human melanoma have rapidly increased. Metastatic spread of malignant melanoma is often associated with cancer progression with poor prognosis and survival. These processes are controlled by dynamic interactions between tumor melanocytes and neighboring stromal cells, whose deregulation leads to the acquisition of cell proliferation capabilities and invasiveness. It is increasingly clear that a key role in carcinogenesis is played by secreted molecules either by tumor and surrounding stromal cells. To address the issue of the proteins secreted during cancer progression, the proteomic profiling of secretomes of cancer cell lines from different melanoma metastases of the same patient (PE-MEL-41, PE-MEL-47, and PE-MEL-43) was performed by applying a shotgun LC-MS/MS-based approach. The results provide a list of candidate proteins associated with the metastatic potential of PE-MEL melanoma cell lines. Among them, several matricellular proteins previously reported as involved in melanoma aggressiveness were identified (i.e., SPARC, osteopontin). In addition, the extracellular matrix protein 1 that stimulates proliferation and angiogenesis of endothelial cells as well as the fibronectin, involved in cell adhesion and motility, were identified. The present work provides the basis to clarify the complex extracellular protein networks implicated in human melanoma cell invasion, migration, and motility.

Background A series of patients with multiple primary melanoma (MPM) has been studied for assessing frequency and distribution of alterations in candidate genes involved in susceptibility (CDKN2A) and pathogenesis (BRAF, cKIT, CyclinD1) of such a disease. Methods Two hundred and twenty-seven genomic DNA samples from paired synchronous and/or asynchronous tumour tissues of 106 MPM patients (92 cases with two, 13 with three, and 1 with four primary melanomas) were screened for somatic mutations in BRAF gene and FISHbased amplifications in cKIT and CyclynD1 genes. For 87 (84%) MPM patients from our series, peripheral blood was available and germline DNAs was analyzed for mutations in p16CDKN2A and p14CDKN2A genes. All mutation analyses we performed by direct automated DNA sequencing. Family history for melanoma was defined according to standardized criteria. Results At somatic level, BRAF mutations were identified in 108/ 227 (48%) primary melanoma tissues, whereas amplification of cKIT and CyclinD1 genes was observed in 8/208 (4%) and 28/204 (14%) analyzed tissue samples, respectively. Considering all types of genetic events, paired samples presented a poorly consistent distribution of somatic alterations in same patients [55/106 (52%) discrepant cases). Among them, 35/106 (33%) patients presented discrepant MPM lesions according to the BRAF mutation status. Among the 87 MPM patients whose germline DNA was available, 8 (9%) of them showed different CDKN2A germline mutations: 7 in p16CDKN2A and 1 in p14CDKN2A. Assessment of family history for melanoma revealed that 13/87 (15%) patients presented at least one additional family member affected; a total of ≥ 3 melanomas in family was observed in 20/87 (23%) cases of our series. The CDKN2A germline mutations were found significantly more frequent in patients with familial history of melanoma (7/13; 54%) compared with patients without (1/74; 1.4%) (P