PO-076 Molecular analysis of BRCA-negative breast and/or ovarian cancer families by multigene panel testing

Introduction About 5%–10% of the hereditary breast and/or ovarian cancer (BC/BOC) is associated with an autosomal dominant genetic susceptibility due to highly penetrant mutations of the BRCA1/2 genes. In particular, BRCA1/2 gene mutations are found in 25%–30% of the BC families subjected to genetic testing. These numbers suggest the possible involvement of other genes in BC/BOC genetic predisposition and a fraction of these cases remains to be assigned to specific genetic factors. Here we report on the application of the NGS multigene panel to a group of BRCA1/2 mutation negative BC/BOC cases, in order to identify germline mutations that could further explain BC/BOC genetic susceptibility. Material and methods We selected a group of 27 BRCA1/2 negative BC and BOC families on the basis of a clear dominant inheritance pattern and/or a moderate/high BRCAPro score. We performed a genomic screening by a comprehensive multi-gene custom panel of 29 cancer-related genes, using Ion Torrent platform (Thermo Fisher Scientific). Results and discussions In three cases (11%) we found mutations described as pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/) in ATM, MUTYH and PALB2 genes. In the series analysed, the most frequently altered genes were APC and ATM (15%) but were also identified mutations in MSH6 and TP53 (11%), MUTYH and RAD51B (7%), MRE11, EPCAM, BRIP1, CHEK2, PALB2, BARD1, STK11 and RAD50 (4%). In particular, we found six genomic variants of uncertain significance (VUS) in MSH6, ATM, BRIP1, RAD50 and APC genes; nine genomic variants of conflicting interpretations of pathogenicity in MUTYH, MRE11, TP53, APC, MSH6, CHEK2, EPCAM and ATM genes and eight genomic variants not reported in ClinVar in APC, RAD51B, STK11, TP53, ATM and BARD1 genes predicted deleterious by in silico analysis. Their biological significance and involvement in the development of the pathology is still unknown today. Only six patients were negative for the presence of mutations in the 29 genes analysed. Conclusion Preliminary results of this study suggest that NGS could offer a great contribution to identify the genetic component of susceptibility to BC/BOC and could potentially be used with implications for clinical management and counselling of patients and their families. Moreover, our results suggest that multigene testing approach may benefit appropriately selected patients, especially those with increased risk of BC/BOC development

MRE11 inhibition highlights a replication stress-dependent vulnerability of MYCN-driven tumors

MRE11 is a component of the MRE11/RAD50/NBS1 (MRN) complex, whose activity is essential to control faithful DNA replication and to prevent accumulation of deleterious DNA double-strand breaks. In humans, hypomorphic mutations in these genes lead to DNA damage response (DDR)-defective and cancer-prone syndromes. Moreover, MRN complex dysfunction dramatically affects the nervous system, where MRE11 is required to restrain MYCN-dependent replication stress, during the rapid expansion of progenitor cells. MYCN activation, often due to genetic amplification, represents the driving oncogenic event for a number of human tumors, conferring bad prognosis and predicting very poor responses even to the most aggressive therapeutic protocols. This is prototypically exemplified by neuroblastoma, where MYCN amplification occurs in about 25% of the cases. Intriguingly, MRE11 is highly expressed and predicts bad prognosis in MYCN-amplified neuroblastoma. Due to the lack of direct means to target MYCN, we explored the possibility to trigger intolerable levels of replication stress-dependent DNA damage, by inhibiting MRE11 in MYCN-amplified preclinical models. Indeed, either MRE11 knockdown or its pharmacological inhibitor mirin induce accumulation of replication stress and DNA damage biomarkers in MYCN-amplified cells. The consequent DDR recruits p53 and promotes a p53-dependent cell death, as indicated by p53 loss- and gain-of-function experiments. Encapsulation of mirin in nanoparticles allowed its use on MYCN-amplified neuroblastoma xenografts in vivo, which resulted in a sharp impairment of tumor growth, associated with DDR activation, p53 accumulation, and cell death. Therefore, we propose that MRE11 inhibition might be an effective strategy to treat MYCN-amplified and p53 wild-type neuroblastoma, and suggest that targeting replication stress with appropriate tools should be further exploited to tackle MYCN-driven tumors

A simplified genomic profiling approach predicts outcome in metastatic colorectal cancer

The response of metastatic colorectal cancer (mCRC) to the first-line conventional combination therapy is highly variable, reflecting the elevated heterogeneity of the disease. The genetic alterations underlying this heterogeneity have been thoroughly characterized through omic approaches requiring elevated efforts and costs. In order to translate the knowledge of CRC molecular heterogeneity into a practical clinical approach, we utilized a simplified Next Generation Sequencing (NGS) based platform to screen a cohort of 77 patients treated with first-line conventional therapy. Samples were sequenced using a panel of hotspots and targeted regions of 22 genes commonly involved in CRC. This revealed 51 patients carrying actionable gene mutations, 22 of which carried druggable alterations. These mutations were frequently associated with additional genetic alterations. To take into account this molecular complexity and assisted by an unbiased bioinformatic analysis, we defined three subgroups of patients carrying distinct molecular patterns. We demonstrated these three molecular subgroups are associated with a different response to first-line conventional combination therapies. The best outcome was achieved in patients exclusively carrying mutations on TP53 and/or RAS genes. By contrast, in patients carrying mutations in any of the other genes, alone or associated with mutations of TP53/RAS, the expected response is much worse compared to patients with exclusive TP53/RAS mutations. Additionally, our data indicate that the standard approach has limited efficacy in patients without any mutations in the genes included in the panel. In conclusion, we identified a reliable and easy-to-use approach for a simplified molecular-based stratification of mCRC patients that predicts the efficacy of the first-line conventional combination therapy

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

Flow Cytometry in Formamide Treated Cells

The use of formamide for the study in flow cytometry of cell cycle phases, by DNA content measurement in human cancer cell lines, was recently published. In this manuscript we verify the possibility of extending the procedure to simultaneous analysis of other parameters. The results obtained, here reported, show that the treatment of samples by formamide is compatible with the simultaneous detection of DNA content and surface phenotypes, with quantification of replicating DNA and with measurement of cells with fractional content of DNA. For each of these three applications we have adapted the procedure in order to gain simple, reproducible and above all advantageous protocols. Regarding the simultaneous analysis of DNA content and phenotyping the use of formamide achieves optimal DNA stoichiometric staining (C.V.<3; G2/G1 ratio = 2 ± 0.05) and sufficient maintenance of physical parameters and membrane fluorescence. In the study of duplicating DNA labeled with click chemistry, our procedure eliminates paraformaldehyde (PFA) fixation improving the DNA stoichiometric staining and allows the use of 7-aminoactinomycin D (7-AAD) preserving the Alexa Fluor 488 quantum efficiency. Concerning the detection of cells with fractional content of DNA, permeabilization and fixation by formamide gives the advantage of resolve on linear scale sub-G1 cells from debris and to allow optimal sample recovery (>90%) which is essential in the study of cell necrobiology. Cells treatment by formamide, suitably modified for different applications, can be used to prepare cell samples for flow cytometry analyses that go far beyond of stoichiometric staining of DNA

Sistema stabile di coltura in vitro di cellule precursori granulari cerebellari (GCP), metodo stabile per la coltura in vitro di dette cellule e usi di detto sistema o metodo per la coltura in vitro

La presente invenzione si riferisce ad un sistema di coltura stabile in vitro di cellule precursori dei granuli cerebellari (GCP). Il suddetto sistema di coltura mantiene nei GCP un’elevata espressione della via di Sonic Hedgehog e un illimitato potere proliferativo garantito dagli alti livelli delle proteine staminali Sox2 e Nestin. Per tali motivi il sopra citato sistema di coltura può rappresentare un valido modello in vitro per lo studio della fisiopatologia dei granuli cerebellari, per lo studio di malattie cerebellari conseguenti a danno o a neurodegenerazione, e potenzialmente per il loro trattamento mediante approcci di terapia genica

Effective treatment of a platinum-resistant cutaneous squamous cell carcinoma case by EGFR pathway inhibition

Cutaneous squamous cell carcinoma (cSCC) is the second most common type of non-melanoma skin cancer. Platinum-based regimens have been an integral part of palliative care for patients with locally advanced or metastatic disease. There is no evidence of efficacy for later lines of chemotherapy and no targeted therapy has been introduced as 'standard of care'. Here we report on the case of an elderly cSCC patient, resistant to conventional therapy, however successfully treated with anti-epidermal growth factor receptor (EGFR) agent (Cetuximab) in addition to a daily dose of Curcumin phospholipid. The patient responded to treatment and experienced no recurrence for 11 months with only minor skin-related toxicity. To our knowledge, this is the first report of clinical evidence that an anti EGFR targeted therapy with a daily oral dose of Curcumin phospholipid is well tolerated and results in a highly effective disease control in a heavily pretreated cSCC patient

New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screens

The tetracycline repressor (tetR)-regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line, which allows for the inducible expression of an EGFP fusion of the TAR DNA-binding protein 43 (TDP-43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP-43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA-approved drugs. While the primary screen identified several compounds that prevented TDP-43 toxicity, further experiments revealed that these chemicals abrogated the doxycycline-dependent TDP-43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet-On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome-wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase and TRIM28 as potential modifiers of TDP-43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline-dependent expression of TDP-43. In summary, we have identified new chemical and genetic regulators of the tetR system, thereby raising awareness of the limitations of this approach to conduct chemical or genetic screening in mammalian cells

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.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

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