Homologous recombination DNA repair gene alterations identify a subset of pancreatic cancers potentially responding to platinum based therapy

Il sequenziamento massivo dell’intero genoma di un gran numero di cancri del pancreas da parte del consorzio internazionale per il genoma del cancro (ICGC) ha identificato una media di 26 mutazioni per singolo tumore. Le mutazioni di KRAS sono l’impronta di questi tumori, seguite dalla inattivazione di TP53, SMAD4 e CDKN2A. Accanto a queste alterazioni sono state riscontrate mutazioni in diversi geni che insistono in 10 pathways molecolari, una delle quali è la pathway-BRCA coinvolta nella riparazione del DNA via ricombinazione omologa. Lo scopo di questa tesi è di utilizzare i dati dell’ICGC focalizzandosi su tale pathway, in quanto i geni che a questa partecipano sono coinvolti nella predisposizione ereditaria ai tumori e sono bersaglio di terapie specifiche quali i sali del platino e gli inibitori della poli-ADP-riboso polimerasi. Lo studio qui presentato ha visto la produzione di 100 xenotrapianti in topo immunodeficiente di cancri del pancreas da pazienti (PDX), per avere a disposizione un modello in vivo da utilizzare sia per la caratterizzazione molecolare che per la sperimentazione terapeutica. I 100 PDX e i rispettivi 100 tumori primitivi sono stati oggetto di analisi mutazionale dei geni più comunemente alterati nel cancro del pancreas e dei geni della pathway-BRCA. KRAS era mutato nel 96% dei casi; TP53 nel 66%, SMAD4 nel 16%, e CDKN2A nel 13%. Mutazioni pathogeniche dei geni della pathway-BRCA sono state rilevate nel 13% dei casi: ATM (1%), BARD1 (1%), BRCA1 (1%), BRCA2 (8%), REV3L (1%), e STK11 (1%). Tali mutazioni erano mutualmente esclusive. Con l’eccezione di due mutazioni in STK11 e REV3L, tutte le mutazioni erano germinali. Un ulteriore 13% di casi presentava varianti di significato sconosciuto in diversi geni di questa pathway. La concordanza fra i tumori primitivi e gli xenotrapianti è stata riscontrata nel 94% dei casi.L’esistenza di un sottogruppo significativo (13%) di cancri del pancreas con mutazioni germinali identifica pazienti che possono beneficiare di terapie mirate, e famiglie che possono rientrare in programmi di screening. Inoltre, questo studio ha identificato una serie di varianti di significato patogenico sconosciuto, che possono essere valutate per la potenziale risposta a terapia utilizzando i modelli PDX sviluppati. I PDX, infatti, rappresentano un modello prezioso che rispecchia fedelmente gli assetti genetici della malattia primitiva.Background: The International Cancer Genome Consortium (ICGC) whole genome sequencing effort identified an average of 26 mutations per pancreatic ductal adenocarcinoma (PDAC). KRAS mutations are the hallmark, followed by TP53, SMAD4 and CDKN2A inactivation. A dominating tail of decreasingly mutated genes follows, but individual pathogenic gene alterations aggregate into ten core molecular pathways, one of which is the homologous recombination (HR) DNA repair genes pathway. Aim: Within this framework, the aim of this thesis is to avail of ICGC data and focus on the HR DNA damage repair pathway, as genes in this pathway are involved in cancer predisposition and are targets of specific therapies such as platinum salts and innovative PARP inhibitors. The study also envisaged the creation of patient PDAC xenografts (PDX) as a model for primary cancers in molecular stratification and drug validation. Materials and methods: 100 PDAC and matched PDXs were analysed using targeted next generation sequencing to investigate variants in the genes commonly altered in PDAC and in the homologous recombination (HR) pathway genes.Results: KRAS was mutated in 96% of cases; TP53 in (66%), SMAD4 in 16%, and CDKN2A in 13%. Pathogenic HR mutations were found in 13% of cases: ATM (1%), BARD1 (1%), BRCA1 (1%), BRCA2 (8%), REV3L (1%), and STK11 (1%). These mutations were mutually exclusive. All but those in STK11 and REV3L were germ-line. An additional 13% of cases had variants of unknown significance (VUS) in genes of this pathway. Concordance between PDAC and PDX was found in 94% of cases.Conclusion: The finding of a significant PDAC subgroup (13%) with germ-line HR gene mutations identifies a group of patients that could profit from existing and novel target therapies as well as screening programs for family members. This study also identifies VUS that may be tested for potential response to therapy availing of the in vivo PDX avatars developed herein. PDX in fact, represent a valuable model that faithfully recapitulates the main genetic feature of primary diseases that may be used for novel diagnostics to predict drug responses as well as enable identification of effective therapeutic schemes

The mutant p53-driven secretome has oncogenic functions in pancreatic ductal adenocarcinoma cells

The cancer secretome is a rich repository of useful information for both cancer biology and clinical oncology. A better understanding of cancer secretome is particularly relevant for pancreatic ductal adenocarcinoma (PDAC), whose extremely high mortality rate is mainly due to early metastasis, resistance to conventional treatments, lack of recognizable symptoms, and assays for early detection. TP53 gene is a master transcriptional regulator controlling several key cellular pathways and it is mutated in ~75% of PDACs. We report the functional effect of the hot-spot p53 mutant isoforms R175H and R273H on cancer cell secretome, showing their influence on proliferation, chemoresistance, apoptosis, and autophagy, as well as cell migration and epithelial-mesenchymal transition. We compared the secretome of p53-null AsPC-1 PDAC cells after ectopic over-expression of R175H-mutp53 or R273H-mutp53 to identify the differentially secreted proteins by mutant p53. By using high-resolution SWATH-MS technology, we found a great number of differentially secreted proteins by the two p53 mutants, 15 of which are common to both mutants. Most of these secreted proteins are reported to promote cancer progression and epithelial-mesenchymal transition and might constitute a biomarker secreted signature that is driven by the hot-spot p53 mutants in PDAC

IDH-wild type glioblastomas featuring at least 30% giant cells are characterized by frequent RB1 and NF1 alterations and hypermutation

: Giant cell glioblastoma (GC-GBM) is a rare variant of IDH-wt GBM histologically characterized by the presence of numerous multinucleated giant cells and molecularly considered a hybrid between IDH-wt and IDH-mutant GBM. The lack of an objective definition, specifying the percentage of giant cells required for this diagnosis, may account for the absence of a definite molecular profile of this variant. This study aimed to clarify the molecular landscape of GC-GBM, exploring the mutations and copy number variations of 458 cancer-related genes, tumor mutational burden (TMB), and microsatellite instability (MSI) in 39 GBMs dichotomized into having 30-49% (15 cases) or\u2009 65\u200950% (24 cases) GCs. The type and prevalence of the genetic alterations in this series was not associated with the GCs content (<\u200950% or 65\u200950%). Most cases (82% and 51.2%) had impairment in TP53/MDM2 and PTEN/PI3K pathways, but a high proportion also featured TERT promoter mutations (61.5%) and RB1 (25.6%) or NF1 (25.6%) alterations. EGFR amplification was detected in 18% cases in association with a shorter overall survival (P\u2009=\u20090.004). Sixteen (41%) cases had a TMB\u2009>\u200910 mut/Mb, including two (5%) that harbored MSI and one with a POLE mutation. The frequency of RB1 and NF1 alterations and TMB counts were significantly higher compared to 567 IDH wild type (P\u2009<\u20090.0001; P\u2009=\u20090.0003; P\u2009<\u20090.0001) and 26 IDH-mutant (P\u2009<\u20090.0001; P\u2009=\u20090.0227; P\u2009<\u20090.0001) GBMs in the TCGA PanCancer Atlas cohort. These findings demonstrate that the molecular landscape of GBMs with at least 30% giant cells is dominated by the impairment of TP53/MDM2 and PTEN/PI3K pathways, and additionally characterized by frequent RB1 alterations and hypermutation and by EGFR amplification in more aggressive cases. The high frequency of hypermutated cases suggests that GC-GBMs might be candidates for immune check-point inhibitors clinical trials

SLC22A3 polymorphisms do not modify pancreatic cancer risk, but may influence overall patient survival

Expression of the solute carrier (SLC) transporter SLC22A3 gene is associated with overall survival of pancreatic cancer patients. This study tested whether genetic variability in SLC22A3 associates with pancreatic cancer risk and prognosis. Twenty four single nucleotide polymorphisms (SNPs) tagging the SLC22A3 gene sequence and regulatory elements were selected for analysis. Of these, 22 were successfully evaluated in the discovery phase while six significant or suggestive variants entered the validation phase, comprising a total study number of 1,518 cases and 3,908 controls. In the discovery phase, rs2504938, rs9364554, and rs2457571 SNPs were significantly associated with pancreatic cancer risk. Moreover, rs7758229 associated with the presence of distant metastases, while rs512077 and rs2504956 correlated with overall survival of patients. Although replicated, the association for rs9364554 did not pass multiple testing corrections in the validation phase. Contrary to the discovery stage, rs2504938 associated with survival in the validation cohort, which was more pronounced in stage IV patients. In conclusion, common variation in the SLC22A3 gene is unlikely to significantly contribute to pancreatic cancer risk. The rs2504938 SNP in SLC22A3 significantly associates with an unfavorable prognosis of pancreatic cancer patients. Further investigation of this SNP effect on the molecular and clinical phenotype is warranted

Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors owing to its robust desmoplasia, low immunogenicity, and recruitment of cancer-conditioned, immunoregulatory myeloid cells. These features strongly limit the success of immunotherapy as a single agent, thereby suggesting the need for the development of a multitargeted approach. The goal is to foster T lymphocyte infiltration within the tumor landscape and neutralize cancer-triggered immune suppression, to enhance the therapeutic effectiveness of immune-based treatments, such as anticancer adoptive cell therapy (ACT). METHODS: We examined the contribution of immunosuppressive myeloid cells expressing arginase 1 and nitric oxide synthase 2 in building up a reactive nitrogen species (RNS)-dependent chemical barrier and shaping the PDAC immune landscape. We examined the impact of pharmacological RNS interference on overcoming the recruitment and immunosuppressive activity of tumor-expanded myeloid cells, which render pancreatic cancers resistant to immunotherapy. RESULTS: PDAC progression is marked by a stepwise infiltration of myeloid cells, which enforces a highly immunosuppressive microenvironment through the uncontrolled metabolism of L-arginine by arginase 1 and inducible nitric oxide synthase activity, resulting in the production of large amounts of reactive oxygen and nitrogen species. The extensive accumulation of myeloid suppressing cells and nitrated tyrosines (nitrotyrosine, N-Ty) establishes an RNS-dependent chemical barrier that impairs tumor infiltration by T lymphocytes and restricts the efficacy of adoptive immunotherapy. A pharmacological treatment with AT38 ([3-(aminocarbonyl)furoxan-4-yl]methyl salicylate) reprograms the tumor microenvironment from protumoral to antitumoral, which supports T lymphocyte entrance within the tumor core and aids the efficacy of ACT with telomerase-specific cytotoxic T lymphocytes. CONCLUSIONS: Tumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance

The trans-ancestral genomic architecture of glycemic traits

Glycemic traits are used to diagnose and monitor type 2 diabetes and cardiometabolic health. To date, most genetic studies of glycemic traits have focused on individuals of European ancestry. Here we aggregated genome-wide association studies comprising up to 281,416 individuals without diabetes (30% non-European ancestry) for whom fasting glucose, 2-h glucose after an oral glucose challenge, glycated hemoglobin and fasting insulin data were available. Trans-ancestry and single-ancestry meta-analyses identified 242 loci (99 novel; P < 5 x 10(-8)), 80% of which had no significant evidence of between-ancestry heterogeneity. Analyses restricted to individuals of European ancestry with equivalent sample size would have led to 24 fewer new loci. Compared with single-ancestry analyses, equivalent-sized trans-ancestry fine-mapping reduced the number of estimated variants in 99% credible sets by a median of 37.5%. Genomic-feature, gene-expression and gene-set analyses revealed distinct biological signatures for each trait, highlighting different underlying biological pathways. Our results increase our understanding of diabetes pathophysiology by using trans-ancestry studies for improved power and resolution. A trans-ancestry meta-analysis of GWAS of glycemic traits in up to 281,416 individuals identifies 99 novel loci, of which one quarter was found due to the multi-ancestry approach, which also improves fine-mapping of credible variant sets.Peer reviewe

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Multiancestry analysis of the HLA locus in Alzheimer’s and Parkinson’s diseases uncovers a shared adaptive immune response mediated by HLA-DRB1*04 subtypes

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson’s disease (PD) and Alzheimer’s disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1*04 subtypes best accounted for the association, strongest with HLA-DRB1*04:04 and HLA-DRB1*04:07, and intermediary with HLA-DRB1*04:01 and HLA-DRB1*04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1*04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1*04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues

New genomic landscapes and therapeutic targets for biliary tract cancers

Biliary tract cancers (BTCs) are a heterogeneous group of neoplasms characterized by a dismal prognosis. At variance with most solid tumors, no effective molecular targeted agent has been currently approved for BTCs treatment and their molecular landscape has only been recently investigated. Comprehensive mutational profiling studies identified IDH1/2 and BAP1 as characteristic of intrahepatic cholangiocarcinomas, while extrahepatic cholangiocarcinomas and gallbladder carcinomas were characterized by frequent KRAS and TP53 alterations. Moreover, targeted next-generation sequencing has uncovered alterations in several key cellular pathways. BTC-specific alterations include disorders of major regulators of cell cycle and chromatin remodeling processes, as well as deregulation of the mTOR-, TGF-beta/Smad- and receptor tyrosine kinases signaling. The next step will be the correlation of these findings with clinical trials to identify predictive biomarkers for the development of personalized therapies. This will permit early access for BTC patients to innovative drugs