Novel and recurrent BRCA2 mutations in Italian breast/ovarian cancer families widen the ovarian cancer cluster region boundaries to exons 13 and 14.

Hereditary breast and ovarian cancer are mainly linked to mutations in BRCA1 and BRCA2 genes which confer a similar cumulative risk of developing breast cancer. Importantly, while BRCA2 mutation carriers generally have a lower cumulative risk for ovarian cancer, mutations clustered in the central portion of BRCA2 are associated with a higher proportion of ovarian compared with breast cancer cases. The boundaries of this ovarian cancer cluster region (OCCR) have been tentatively defined within a 3.3 kb region of BRCA2 exon 11, and herein, we reassessed these boundaries using our series of Italian breast/ovarian cancer families. We used direct sequencing to investigate BRCA mutations in 367 breast/ovarian cancer families. We also studied the association between the location of the mutations and the ovarian cancer phenotype in our cohort of BRCA2-mutated families. We observed the novel c.7309_7309delA frameshift mutation and the c.7007G>A deleterious mutation in BRCA2 exons 14 and 13, respectively, in five independent Italian families characterized by a high proportion of ovarian cancer cases. Of note, a significantly higher proportion of ovarian versus breast cancer cases was associated not only with mutations in the previously defined OCCR (OR = 5.91; p = 0.004), but also with the exon 13-14 region (OR = 7.37; p = 0.001) in our BRCA2-mutated families. Our data provide initial evidence for a novel putative OCCR in BRCA2 exons 13-14.

Next-generation sequencing of BRCA1 and BRCA2 genes for rapid detection of germline mutations in hereditary breast/ovarian cancer.

BACKGROUND: Conventional methods used to identify BRCA1 and BRCA2 germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment. METHODS: We first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set). RESULTS: By minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel BRCA2 pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis. DISCUSSION: Our experience strongly supports that the Ion Torrent PGM technology in BRCA1 and BRCA2 germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches.

Identification of novel BRCA1 large genomic rearrangements by a computational algorithm of amplicon-based Next-Generation Sequencing data.

BACKGROUND: Genetic testing for BRCA1/2 germline mutations in hereditary breast/ovarian cancer patients requires screening for single nucleotide variants, small insertions/deletions and large genomic rearrangements (LGRs). These studies have long been run by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The recent introduction of next-generation sequencing (NGS) platforms dramatically improved the speed and the efficiency of DNA testing for nucleotide variants, while the possibility to correctly detect LGRs by this mean is still debated. The purpose of this study was to establish whether and to which extent the development of an analytical algorithm could help us translating NGS sequencing via an Ion Torrent PGM platform into a tool suitable to identify LGRs in hereditary breast-ovarian cancer patients. METHODS: We first used NGS data of a group of three patients (training set), previously screened in our laboratory by conventional methods, to develop an algorithm for the calculation of the dosage quotient (DQ) to be compared with the Ion Reporter (IR) analysis. Then, we tested the optimized pipeline with a consecutive cohort of 85 uncharacterized probands (validation set) also subjected to MLPA analysis. Characterization of the breakpoints of three novel BRCA1 LGRs was obtained via long-range PCR and direct sequencing of the DNA products. RESULTS: In our cohort, the newly defined DQ-based algorithm detected 3/3 BRCA1 LGRs, demonstrating 100% sensitivity and 100% negative predictive value (NPV) (95% CI [87.6-99.9]) compared to 2/3 cases detected by IR (66.7% sensitivity and 98.2% NPV (95% CI [85.6-99.9])). Interestingly, DQ and IR shared 12 positive results, but exons deletion calls matched only in five cases, two of which confirmed by MLPA. The breakpoints of the 3 novel BRCA1 deletions, involving exons 16-17, 21-22 and 20, have been characterized. CONCLUSIONS: Our study defined a DQ-based algorithm to identify BRCA1 LGRs using NGS data. Whether confirmed on larger data sets, this tool could guide the selection of samples to be subjected to MLPA analysis, leading to significant savings in time and money.

Coexistence of three EGFR mutations in an NSCLC patient: A brief report.

BACKGROUND: The epidermal growth factor receptor (EGFR) represents a molecular target for tyrosine kinase inhibitors for non-small cell lung cancer (NSCLC) patients with a mutation in the EGFR gene. Mutations of the EGFR gene that occur at a single position in NSCLC tissue are found as single, whereas two or more mutations on the same allele are poorly detected and investigated. PATIENT AND METHODS: We investigated the presence of the EGFR gene mutations in tumor tissue by Sanger sequencing and ion torrent sequencing in an NSCLC patient at Stage IV of disease. RESULTS: We found the presence of three coexisting mutations on the EGFR gene-two of which on exon 21 are present on the same allele, and the third, on exon 20, was analyzed by Sanger sequencing of the peripheral blood lymphocytes. The patient staged as cT4N0M1c (Stage IV) and started afatinib 40 mg daily 8 months ago, showing a clinical benefit. CONCLUSION: In this report we describe the case of an NSCLC patient harboring three coexisting mutations on the EGFR gene, two of which are present on the same allele. This mutation pattern may represent, for patient progeny, a genetic risk of cancer development. Therefore it should be possible to obtain screening guidelines to improve the risk calculation for lung cancer susceptibility in the future.

Optimizing the identification of risk-relevant mutations by multigene panel testing in selected hereditary breast/ovarian cancer families.

The introduction of multigene panel testing for hereditary breast/ovarian cancer screening has greatly improved efficiency, speed, and costs. However, its clinical utility is still debated, mostly due to the lack of conclusive evidences on the impact of newly discovered genetic variants on cancer risk and lack of evidence-based guidelines for the clinical management of their carriers. In this pilot study, we aimed to test whether a systematic and multiparametric characterization of newly discovered mutations could enhance the clinical utility of multigene panel sequencing. Out of a pool of 367 breast/ovarian cancer families Sanger-sequenced for BRCA1 and BRCA2 gene mutations, we selected a cohort of 20 BRCA1/2-negative families to be subjected to the BROCA-Cancer Risk Panel massive parallel sequencing. As a strategy for the systematic characterization of newly discovered genetic variants, we collected blood and cancer tissue samples and established lymphoblastoid cell lines from all available individuals in these families, to perform segregation analysis, loss-of-heterozygosity and further molecular studies. We identified loss-of-function mutations in 6 out 20 high-risk families, 5 of which occurred on BRCA1, CHEK2 and ATM and are esteemed to be risk-relevant. In contrast, a novel RAD50 truncating mutation is most likely unrelated to breast cancer. Our data suggest that integrating multigene panel testing with a pre-organized, multiparametric characterization of newly discovered genetic variants improves the identification of risk-relevant alleles impacting on the clinical management of their carriers.

Improving the accuracy of BRCA1/2 mutation prediction: validation of the novel country-customized IC software.

Inherited mutations of the BRCA1/2 genes confer a significantly increased risk for breast and/or ovarian cancer development. Several models were elaborated to help genetic counsellors in selecting individuals with high probability of being mutation carriers. The IC software, a country-customized version of the Brcapro model, was recently shown to be particularly accurate in the prediction of carrier probability status in the Italian population. Here, we used our independent series of 70 breast/ovarian cancer families to analyze the performances of the IC software and compare it to widely used models, such as Brcapro and the Myriad mutation prevalence tables. Analysis of the areas under the receiver operator characteristics (ROC) curves indicated that overall the models performed well. However, the IC software and Myriad tables were more efficient in predicting mutated cases, showing a higher sensitivity (94 and 88%, respectively) and negative predictive value (NPV, 94 and 92%, respectively) compared to Brcapro (sensitivity 71 and NPV 83%). IC software also appeared particularly accurate in the identification of families belonging the low mutation risk group (<10%). Finally, most Brcapro failures occurred in the hereditary breast cancer (HBC) family subset, and in 75% of the cases, the IC software corrected them. Our data suggest that the country-customized implementation operated on the Brcapro software generated a more accurate tool for the prediction of BRCA1/2 gene mutation. Whether the IC or other country-customized models might improve BRCA1/2 mutation prediction also in non-Italian families needs to be further explored.

Cloning of the mouse insulin receptor substrate-3 (mIRS-3) promoter, and its regulation by p53.

The insulin receptor susbtrate-3 (IRS-3) is a member of a family of intermediate adapter proteins that function as major intracellular targets for phosphorylation by the activated insulin and IGF-I receptors. Among the four IRS proteins identified so far, IRS-3 exhibits a rather peculiar expression pattern during both the embryonic development and adult life, suggesting a different mechanism of regulation of its expression. In this study, we cloned the 5' flanking region of the mIRS-3 gene and analyzed its promoter activity. The mIRS-3 promoter is inhibited by wild-type p53, and this effect is completely abolished by cotransfection of a dominant negative p53. Tumor-derived p53 mutants show variable, but lower suppressing capability than wt p53. In addition, treatment with doxorubicin inhibits endogenous expression of mIRS-3 mRNA in C2C12 and 3T3-L1 cells. The DNA region spanning from nucleotides -287 and -178 in the mIRS-3 promoter is responsible for a 32.2% reduction of the mouse double minute 2 (MDM2) promoter activity, suggesting its involvement in the p53-mediated inhibitory effect. In conclusion, our study demonstrates that the mIRS-3 promoter is regulated by p53 at the transcriptional level. The inhibition of mIRS-3 promoter by wild-type p53, and its de-repression by tumor-derived p53 mutants, appears to be similar to that previously reported for the IGF-I receptor promoter, suggesting a common role of these two genes in p53-mediated cell growth and differentiation.

Validation of the Ion Torrent PGM sequencing for the prospective routine molecular diagnostic of colorectal cancer.

OBJECTIVES: Treatment individualization based on specific molecular biomarkers is becoming increasingly important in oncology. In colorectal cancer (CRC), the molecular characterization of RAS and BRAF mutation status for prognostic and predictive purposes is commonly performed by different validated methods. However, as the number of clinically relevant mutations to be analyzed increases, the definition of new approaches for more sensitive, rapid and economic patient selection urges. To this aim, we evaluated the Ion Semiconductor sequencing using the Ion Torrent Personal Genome Machine (IT-PGM) in our routine molecular diagnostics for CRC in comparison with the gold standard direct Sanger sequencing. DESIGN AND METHODS: Formalin-fixed and paraffin-embedded tumor tissues obtained by surgery or biopsy of 66 CRCs were collected. DNA was extracted and sequenced by IT-PGM and Sanger method. RESULTS: The proposed IT-PGM sequencing strategy exceeded the 500 reads of coverage for all clinically relevant RAS/BRAF amplicons in most samples and thus guaranteed optimal determination. Indeed, the frequencies and the mutational spectrum of RAS and BRAF mutations were in agreement with literature data and revealed 100% concordance between the IT-PGM and routine Sanger sequencing approaches. Turnaround time and cost evaluation indicate that the IT-PGM sequencing permits the characterization of the clinically relevant mutational spots at lower cost and turnaround time compared to Sanger sequencing and allows inclusion of additional amplicons whose characterization may acquire significance in the very next future. CONCLUSION: The IT-PGM is a valid, flexible, sensitive and economical method alternative to the Sanger sequencing in routine diagnostics to select patients for anti-epidermal growth factor receptor therapy for metastatic CRC.

Characterization of medulloblastoma in Fanconi Anemia: a novel mutation in the BRCA2 gene and SHH molecular subgroup.

Fanconi Anemia (FA) is an inherited disorder characterized by the variable presence of multiple congenital somatic abnormalities, bone marrow failure and cancer susceptibility. Medulloblastoma (MB) has been described only in few cases of FA with biallelic inactivation in the tumor suppressor gene BRCA2/FANCD1 or its associated gene PALB2/FANCN. We report the case of a patient affected by Fanconi Anemia with Wilms tumor and unusual presentation of two medulloblastomas (MB1 and MB2). We identified a new pathogenetic germline BRCA2 mutation: c.2944_2944delA. Molecular analysis of MBs allowed us to define new features of MB in FA. MBs were found to belong to the Sonic Hedgehog (SHH) molecular subgroup with some differences between MB1 and MB2. We highlighted that MB in FA could share molecular aspects and hemispheric localization with sporadic adult SHH-MB. Our report provides new findings that shed new light on the genetic and molecular pathogenesis of MB in FA patients with implications in the disease management.

A novel human Smad4 mutation is involved in papillary thyroid carcinoma progression.

Smad proteins are the key effectors of the transforming growth factor ß (TGFß) signaling pathway in mammalian cells. Smad4 plays an important role in human physiology, and its mutations were found with high frequency in wide range of human cancer. In this study, we have functionally characterized Smad4 C324Y mutation, isolated from a nodal metastasis of papillary thyroid carcinoma. We demonstrated that the stable expression of Smad4 C324Y in FRTL-5 cells caused a significant activation of TGFß signaling, responsible for the acquisition of transformed phenotype and invasive behavior. The coexpression of Smad4 C324Y with Smad4 wild-type determined an increase of homo-oligomerization of Smad4 with receptor-regulated Smads and a lengthening of nuclear localization. FRTL-5 clones overexpressing Smad4 C324Y showed a strong reduction of response to antiproliferative action of TGFß1, acquired the ability to grow in anchorage-independent conditions, showed a fibroblast-like appearance and a strong reduction of the level of E-cadherin, one crucial event of the epithelial-mesenchymal transition process. The acquisition of a mesenchymal phenotype gave the characteristics of increased cellular motility and a significant reduction in adhesion to substrates such as fibronectin and laminin. Overall, our results demonstrate that the Smad4 C324Y mutation plays an important role in thyroid carcinogenesis and can be considered as a new prognostic and therapeutic target for thyroid cancer.

Induction of mitochondrial biogenesis is a maladaptive mechanism in mitochondrial cardiomyopathies.

OBJECTIVES: The purpose of this study was to clarify the molecular mechanisms linking human mitochondrial deoxyribonucleic acid (mtDNA) dysfunction to cardiac remodeling. BACKGROUND: Defects of the mitochondrial genome cause a heterogeneous group of clinical disorders, including mitochondrial cardiomyopathies (MIC). The molecular events linking mtDNA defects to cardiac remodeling are unknown. Energy derangements and increase of mitochondrial-derived reactive oxygen species (ROS) could both play a role in the development of cardiac dysfunction in MIC. In addition, mitochondrial proliferation could interfere with sarcomere alignment and contraction. METHODS: We performed a detailed morphologic and molecular analysis on failing hearts from 3 patients with MIC, failing human hearts due to ischemic heart disease (IHD) or dilated cardiomyopathies (DCM), and nonfailing hearts. RESULTS: The MIC hearts showed marked mitochondrial proliferation with myofibril displacement. Consistent with morphologic features, increase in mtDNA content per cell and induction of genes involved in mitochondrial biogenesis, fatty acid metabolism, and glucose transport were observed. Down-regulation of these genes characterized DCM and IHD hearts. A pronounced increase in mitochondrial-derived ROS was observed in MIC hearts compared with failing hearts due to other causes. This was paralleled by up-regulation of genes encoding for uncoupling proteins and antioxidant enzymes. However, there was not a significant increase in antioxidant enzyme activity. CONCLUSIONS: Our results suggest that besides energy deficiency, mitochondrial biogenesis per se is a maladaptive response in MIC and, possibly, in other metabolic cardiomyopathies.

Prevalence of BRCA1 and BRCA2 genomic rearrangements in a cohort of consecutive Italian breast and/or ovarian cancer families.

Germline point mutations in BRCA1 and BRCA2 genes account for about 30% of the inherited breast and ovarian cancers. Germline genomic rearrangements have been found in both BRCA1 and BRCA2 genes, but the extent to which these alterations might contribute to increasing the actual mutation detection rate is still debated. Here we screened a cohort of 112 consecutive Italian families at moderate-to-high risk for breast and/or ovarian cancer for BRCA1 and BRCA2 point mutations and genomic rearrangements. Of the 83 point mutation negative probands, two (2.4%) showed BRCA1 rearrangements, accounting for 10.5% of the BRCA1 mutations. BRCA1 del18-19 has been previously described in another Italian family, while the molecular characterization of the BRCA1 del23-24 is given here for the first time. Conversely, we failed to identify any BRCA2 rearrangements even in the hereditary breast cancer families, where we detected an higher prevalence of BRCA2 compared to BRCA1 point mutations. Our results support the idea that search for BRCA1 rearrangements should be included in the genetic screening of even moderate risk breast/ovarian cancer families. In contrast, they suggest BRCA2 rearrangements might be very rare out of the high risk families including a male breast cancer.

Novel BRCA1 and BRCA2 germline mutations and assessment of mutation spectrum and prevalence in Italian breast and/or ovarian cancer families.

Familial aggregations of breast/ovarian cancer cases frequently depend on BRCA1/2 pathogenic mutations. Here we counselled 120 Italian breast/ovarian cancer families and selected 73 probands for BRCA1/2 mutation screening. Through this analysis we defined the prevalence of BRCA1/2 pathogenic mutations occurring in Italian breast/ovarian cancer families, enlarged the spectrum of Italian BRCA1/2 mutations by 15% and report on the identification of 13 novel variants, including two deleterious truncating mutations and two potentially pathogenic missense mutations, on the BRCA1 and BRCA2 genes. Finally in hereditary breast cancer families with three or more female breast cancer cases we observed a low mutation prevalence and a significant association with BRCA2 mutations.

EGF- and cell-cycle-regulated STAG1/PMEPA1/ERG1.2 belongs to a conserved gene family and is overexpressed and amplified in breast and ovarian cancer.

The abnormal activation of the epidermal growth factor (EGF) pathway is one of the most common findings in human cancer, and a number of molecular devices of laboratory and clinical relevance have been designed to block this transduction pathway. Because of the large number of cellular events that might be regulated through the activation of the four EGF receptor family members, it is possible that screening methodologies for the identification of new molecular targets working downstream of these pathways may provide new tools for cancer diagnosis and potentially prevention and therapy. In searching for EGF target genes, we have identified ERG1.2, the mouse homolog of the solid tumor-associated gene STAG1. Both in humans and in mice, it belongs to a new gene family that can give origin to several protein isoforms through alternative splicing and/or multiple translation starts. Sequence analysis and experimental data suggest that ERG1.2 is likely to function as a membrane-bound protein interacting with downstream signaling molecules through WW- and SH3-binding domains. ERG1.2 is a cell-cycle-regulated gene, and both ERG1.2 and STAG1 are induced by EGF and other growth factors at the transcript and protein levels. Finally, we have demonstrated that, besides prostate cancer and renal cell carcinoma, STAG1 was also overexpressed in breast and ovarian cancer cell lines and in breast primary tumors. Although in most cases STAG1 overexpression is probably due to the abnormal activation of the EGF pathway, we have also demonstrated genetic amplification and rearrangement of its locus in one breast cancer cell line and one primary ovarian cancer, suggesting that STAG1 might be a direct molecular target in the carcinogenetic process. Thus its overexpression might be regarded not only as a tumor marker but also as a potentially pathogenetic event.

Human MRE11 is inactivated in mismatch repair-deficient cancers.

Mutations of the ATM and NBS1 genes are responsible for the inherited Ataxia-Telangiectasia and Nijmegen Breakage Syndrome, both of which are associated with a predisposition to cancer. A related syndrome, the Ataxia-Telangiectasia-like disorder, is due to mutations of the MRE11 gene. However, the role of this gene in cancer development has not been established. Here we describe an often homozygous mutation of the poly(T)11 repeat within human MRE11 intron 4 that leads to aberrant splicing, impairment of wild-type MRE11 expression and generation of a truncated protein. This mutation is present in mismatch repair-deficient, but not proficient, colorectal cancer cell lines and primary tumours and is associated with reduced expression of the MRE11--NBS1--RAD50 complex, an impaired S-phase checkpoint and abrogation of MRE11 and NBS1 ionizing radiation-induced nuclear foci. Our findings identify MRE11 as a novel and major target for inactivation in mismatch repair-defective cells and suggest its impairment may contribute to the development of colorectal cancer.