Amelia Buffone

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.

Vemurafenib and panitumumab combination tailored therapy in BRAF-mutated metastatic colorectal cancer: A case report

As the knowledge on cancer genetic alterations progresses, it fosters the need for more personalized therapeutic intervention in modern cancer management. Recently, mutations in KRAS, BRAF, and PIK3CA genes have emerged as important mechanisms of resistance to EGFR-targeted therapy in metastatic colorectal cancer (mCRC). Here we report the first case of a mCRC patient whose disease had progressed on standard lines of treatment and for which we devised a personalized therapeutic approach consisting of vemurafenib (ZelborafTM) and panitumumab (VectibixTM), based on the following molecular profile: BRAFV600E-mutant, amplified EGFR (double positive) and WT KRAS, WT PIK3CA, not-amplified HER2 (triple negative). This new combination therapy was well tolerated and resulted in a strong control of the disease. In particular, the vemurafenib-panitumumab combination appears to limit the typical toxicity of single agents, since no cutaneous toxic effects typically associated with vemurafenib were observed. Here we report the first clinical evidence that the combination of an anti-EGFR (panitumumab) and an inhibitor of BRAFV600E (vemurafenib) is well tolerated and results in a strong disease control in an extensively pretreated mCRC patient.

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.

PRDX1 and PRDX6 are repressed in papillary thyroid carcinomas via BRAF V600E-dependent and -independent mechanisms

Many clinical studies highlight the dichotomous role of PRDXs in human cancers, where they can exhibit strong tumor-suppressive or tumor-promoting functions. Recent evidence suggests that lower expression of PRDXs correlates with cancer progression in colorectal cancer (CRC) or in esophageal squamous carcinoma. In the thyroid, increased levels of PRDX1 has been described in follicular adenomas and carcinomas, as well as in thyroiditis, while reduced levels of PRDX6 has been found in follicular adenomas. We studied the expression of PRDX1 and PRDX6, in a series of thyroid tissue samples, covering different thyroid diseases, including 13 papillary thyroid carcinomas (PTCs). Our results show that PRDX1 and PRDX6 are significantly reduced in all PTCs compared to normal tissues, to non-neoplastic tissue (MNG) or follicular neoplasms. This reduction is strongly evident in PTCs harboring BRAF V600E (31% of our cases). Using TPC-1 and BCPAP and FRTL-5 cell lines, we demonstrate for the first time that the presence of BRAF V600E is responsible of the hypoexpression of PRDX1 and PRDX6 both at mRNA and protein levels. Finally, independently of BRAF status, we observe an interesting correlation between the tumor size, the presence of lymph node metastasis and the lowest PRDX1 and PRDX6 levels. Therefore, these data indicate that PRDX1 and PRDX6 expression not only may play a key role in papillary thyroid carcinogenesis via a BRAF V600E-dependent mechanism, but their determination could be considered as potential tumor marker for indicating tumor progression in PTCs, independently of BRAF status.

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.

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

AbstractFanconi 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.

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.