Transient disappearance of RAS mutant clones in plasma: A counterintuitive clinical use of EGFR inhibitors in RAS mutant metastatic colorectal cancer

Genomic studies performed through liquid biopsies widely elucidated the evolutionary trajectory of RAS mutant clones under the selective pressure of EGFR inhibitors in patients with wild type RAS primary colorectal tumors. Similarly, the disappearance of RAS mutant clones in plasma has been more recently reported in some patients with primary RAS mutant cancers, supporting for the first time an unexpected negative selection of RAS mutations during the clonal evolution of mCRC. To date, the extent of conversion to RAS wild type disease at the time of progression has not been clarified yet. As a proof of concept, we prospectively enrolled mCRC patients progressing under anti-VEGF based treatments. Idylla™system was used to screen RAS mutations in plasma and the wild type status of RAS was further confirmed through IT-PGM (Ion Torrent Personal Genome Machine) sequencing. RAS was found mutant in 55% of cases, retaining the same plasma mutation as in the primary tumor at diagnosis, while it was found wild-type in 45%. Four patients testing negative for RAS mutations in plasma at the time of progression of disease (PD) were considered eligible for treatment with EGFR inhibitors and treated accordingly, achieving a clinical benefit. We here propose a hypothetical algorithm that accounts for the transient disappearance of RAS mutant clones over time, which might extend the continuum of care of mutant RAS colorectal cancer patients through the delivery of a further line of therapy

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

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

Obinutuzumab-mediated high-affinity ligation of FcγRIIIA/CD16 primes NK cells for IFNγ production

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses. Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood. Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcεRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcεRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli. These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional progra

A novel BRCA2 splice variant identified in a young woman

Background: BRCA1/2 VUSs represent an important clinical issue in risk assessment for the breast/ovarian cancer families (HBOC) families. Among them, some occurring within the intron-exon boundary may lead to aberrant splicing process by altering or creating de novo splicing regulatory elements or unmasking cryptic splice site. Defining the impact of these potential splice variants at functional level is important to establish their pathogenic role. Methods: Genomic DNA was extracted from peripheral blood sample of a young woman affected with breast cancer belonging to a HBOC family and the entire coding regions of the BRCA1 and BRCA2 genes were amplified using the Ion AmpliSeq BRCA1 and BRCA2 Panel. The BRCA2 c.682-2delA variant has been characterized by RT-PCR analysis performed on mRNA extracted from blood and lymphoblastoid cell line. Results: We demonstrated that a novel BRCA2 c.682-2delA variant at the highly conserved splice consensus site in intron 8 disrupts the canonical splice acceptor site generating a truncated protein as predicted by several bioinformatics tools. Segregations analysis in the family and LOH performed on proband breast cancer tissue further confirmed its classification as pathogenic variant. Conclusion: Combining different methodologies, we characterized this new BRCA2 variant and provided findings of clinical utility for its classification as pathogenic variant

Evaluation of polygenic determinants of non-alcoholic fatty liver disease (NAFLD) by a candidate genes resequencing strategy

NAFLD is a polygenic condition but the individual and cumulative contribution of identified genes remains to be established. To get additional insight into the genetic architecture of NAFLD, GWAS-identified GCKR, PPP1R3B, NCAN, LYPLAL1 and TM6SF2 genes were resequenced by next generation sequencing in a cohort of 218 NAFLD subjects and 227 controls, where PNPLA3 rs738409 and MBOAT7 rs641738 genotypes were also obtained. A total of 168 sequence variants were detected and 47 were annotated as functional. When all functional variants within each gene were considered, only those in TM6SF2 accumulate in NAFLD subjects compared to controls (P = 0.04). Among individual variants, rs1260326 in GCKR and rs641738 in MBOAT7 (recessive), rs58542926 in TM6SF2 and rs738409 in PNPLA3 (dominant) emerged as associated to NAFLD, with PNPLA3 rs738409 being the strongest predictor (OR 3.12, 95% CI, 1.8-5.5, P 0.28 was associated with a 3-fold increased risk of NAFLD. Interestingly, rs61756425 in PPP1R3B and rs641738 in MBOAT7 genes were predictors of NAFLD severity. Overall, TM6SF2, GCKR, PNPLA3 and MBOAT7 were confirmed to be associated with NAFLD and a score based on these genes was highly predictive of this condition. In addition, PPP1R3B and MBOAT7 might influence NAFLD severity

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

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