Molecular Signature in HCV-Positive Lymphomas

Hepatitis C virus (HCV) is a positive, single-stranded RNA virus, which has been associated to different subtypes of B-cell non-Hodgkin lymphoma (B-NHL). Cumulative evidence suggests an HCV-related antigen driven process in the B-NHL development. The underlying molecular signature associated to HCV-related B-NHL has to date remained obscure. In this review, we discuss the recent developments in this field with a special mention to different sets of genes whose expression is associated with BCR coupled to Blys signaling which in turn was found to be linked to B-cell maturation stages and NF-κb transcription factor. Even if recent progress on HCV-B-NHL signature has been made, the precise relationship between HCV and lymphoma development and phenotype signature remain to be clarified

HLA-G 3′UTR Polymorphisms Predict Drug-Induced G3-4 Toxicity Related to Folinic Acid/5-Fluorouracil/Oxaliplatin (FOLFOX4) Chemotherapy in Non-Metastatic Colorectal Cancer

Polymorphisms in drug-metabolizing enzymes might not completely explain inter-individual differences in toxicity profiles of patients with colorectal cancer (CRC) that receive folinic acid/5-fluorouracil/oxaliplatin (FOLFOX4). Recent data indicate that the immune system could contribute to FOLFOX4 outcomes. In light of the immune inhibitory nature of human leukocyte antigen-G (HLA-G), a non-classical major histocompatibility complex (MHC) class I molecule, we aimed to identify novel genomic markers of grades 3 and 4 (G3-4) toxicity related to FOLFOX4 therapy in patients with CRC. We retrospectively analyzed data for 144 patients with stages II-III CRC to identify HLA-G 3′ untranslated region (3′UTR) polymorphisms and related haplotypes and evaluate their impact on the risk of developing G3-4 toxicities (i.e., neutropenia, hematological/non-hematological toxicity, neurotoxicity) with logistic regression. The rs1610696-G/G polymorphism was associated with increased risk of G3-4 neutropenia (OR = 3.76, p = 0.015) and neurotoxicity (OR = 8.78, p = 0.016); rs371194629-Ins/Ins was associated with increased risk of neurotoxicity (OR = 5.49, p = 0.027). HLA-G 3′UTR-2, which contains rs1610696-G/G and rs371194629-Ins/Ins polymorphisms, was associated with increased risk of G3-4 neutropenia (OR = 3.92, p = 0.017) and neurotoxicity (OR = 11.29, p = 0.009). A bootstrap analysis confirmed the predictive value of rs1610696 and rs371194629, but the UTR-2 haplotype was validated only for neurotoxicity. This exploratory study identified new HLA-G 3′UTR polymorphisms/haplotypes as potential predictive markers of G3-4 toxicities in CRC

Identificazione e caratterizzazione di varianti germinali del gene CDH1 nel carcinoma gastrico sporadico e in individui a rischio di carcinoma gastrico

2011/2012Abstract Gastric cancer (GC) remains a leading cause of cancer death worldwide. A hallmark of this type of tumour is the poor prognosis of patients. Identification of subjects with an increased risk of developing GC and the early detection of GC, are promising approaches to reduce the morbidity and mortality of this malignancy. CDH1 gene encodes E-cadherin, a known tumour suppressor protein expressed in epithelial cells that plays a major role in cellular adhesion and tissue architecture maintenance. CDH1 gene mutations are thought to affect protein function and lead to cell-cell adhesion deregulation in epithelial tissues and to enhance metastatic tumour potential. Germline mutations in CDH1 gene are implicated in hereditary diffuse gastric cancer (HDGC) syndrome and in early onset gastric cancer (EOGC), but the role of these variants in sporadic GC and in subjects at risk to develop GC, is poorly investigated. Most studies limit the screening of CDH1 mutations to patients with HDGC, familial GC or GC patients at very early onset through genetic counselling, but only few studies investigated the CDH1 the contribution of these mutations in sporadic GC cases without familial aggregation or in subjects at risk of developing GC. Moreover, the potential functional effects of CDH1 variants are not always explored. In our study, screening of the entire coding region and all exon flanking sequences of the CDH1 gene was performed by direct sequencing in a series of random, consecutive sporadic GC cases. We extended the analysis of CDH1 germline mutations in a collection of individuals at risk for GC: 59 subjects who reported an ascertained case of GC among parents, children, siblings or offspring’s, named first degree GC-Relatives (FDR), and 20 autoimmune metaplastic atrophic gastritis (AMAG) patients. In the study 52 healthy blood donors (BD) were also included in the CDH1 analyses. The aim of this PhD project was to evaluate CDH1 mutations in these individuals to identify a possible hereditable marker that might improve the early detection of GC, stratifying high risk subjects in association with other known risk factors for GC diagnosis. All the participants were voluntarily subjected to a gastrointestinal endoscopy with biopsy in our institution. This is the first study in which CDH1 screening was investigated in AMAG patients. CDH1 germline mutations found were characterized by means of structural modelling and bioinformatic tools, immunohistochemical (IHC) stains for evaluation of E-cadherin and β-catenin proteins expression and subcellular localization, and E-cadherin mRNA level by real-time polymerase chain reaction (PCR). The functional impact of splicing was assessed for non-polymorphic intronic variants. Among the 59 GCs, the lower portion (antrum) of the stomach was the prevalent anatomic site (p< 0.05) for the tumours detected by endoscopy. No statistically differences were found comparing associations between gender, histotype, tumour location, H. Pylori infection, and TNM staging. A complex panel of germline variants was detected for CDH1 gene. In GC series, we found six different alterations in nine distinct GC patients (15.2%): 67% of the missense type (p.G274S; p.A298T; p.T470I: p.A592T) and 33% of non-missense type, one in the promoter region (5’UTR-71C>G) and one in the intronic region close to exon 13 (IVS12 c.1937-13T>C). All the missense substitutions are localized in the extracellular functional E-cadherin domain which is directly implied in cell-cell adhesion mechanisms. Of note the germline missense (p.G274S) was a new CDH1 mutation, while the p.A298T was already found only in HDGC syndrome and it was described as pathogenetic. Our molecular modelling studies highlighted that p.G274S change in mature protein is not dramatic for local structure but the serine residue represents a potential site for post translation modifications, such as phosphorylation or glycosylation. However, data from our functional in vitro assays did not support an effect of this mutation on adhesion mobility of cells. Moreover, E-cadherin was not under expressed in the tumour gastric tissue of patient harbouring the p.G274S, even if a reduction in β-catenin expression was observed in the same sample. Therefore, the pathogenic effect of p.G274S change by our in silico and in vitro approaches seems to be unlikely, so we cannot conclude the significance of this mutation. Modelling and SIFT results confirmed that the p.A298T substitution affects protein function, while the p.T470I change is tolerated. Through the structural analysis for p.T470I, we hypothesized a protective role that may favour protein-protein interactions in the extracellular medium. The p.A592T was detected in the four studied groups, suggesting that it is a polymorphic variant (frequence >1%). Modelling suggested a non-pathogenic role as already confirmed by published in vitro and in silico studies. The C-to-G change before the start codon (-71C>G), represented the most common variant associated with GC in our series, occurring in three (5.1%) out of 59 GC patients. Due to a lack of tumour material, an evaluation or a correlation between CDH1 hypermethylation and E-cadherin expression was not performed, so the predicted pathogenicity of this promoter variant still remains to be elucidated. The intronic IVS12 c.1937-13T>C variant was found in two GC patients and in one BD, but it was already described in HDGC and EOGC subjects. Our assays demonstrated for the first time that this substitution leads to an aberrant CDH1 transcript harbouring a deletion of exon 11 with the formation of a premature stop codon in the extracellular E-cadherin domain. The translated protein will lack part of the extracellular domain, and completely loose the transmembrane domain and the cytoplasmic tail of E-cadherin, which is involved in β-catenin binding. Moreover, IHC stains showed a reduction in the expression of E-cadherin and β-catenin in the gastric tumour tissues of patients harbouring this intronic mutation. Evaluation by real time-PCR of E-cadherin expression from the EBV immortalized B-lymphocytes from one GC patient carrying this mutation showed a strong (60%) and significantly reduction (p<0.01) compared with the control without CDH1 alterations, and the BD with the same CDH1 alteration. In FDR high risk group, we found one substitution in the 5’UTR promoter region (-54G>C) already described as a rare variant able to decrease the transcriptional activity of CDH1, one 5’ near gene variant (-176C>T) with unknown significance, and an intronic mutation close to exon 5, IVS4 c.532- 18C>T, already reported in EOGC cases. This last one showed no effects in producing CDH1 aberrant splicing, as the new germline mutation in the intronic region close to exon 1 (IVS1 c.48+7C>T) found in one AMAG patient. Other variants in the AMAGs were polymorphic, and thus we excluded a potential effect of these on E-cadherin function. In conclusion, our results show that pathogenic CDH1 germline variants (p.A298T) can also be detected in sporadic GC patients without fulfilling the strict criteria for HDGC. We also report the finding, in a sporadic GC, of a new missense germline mutation (p.G274S) with an uncertain significance. Furthermore, we demonstrate for the first time a potential deleterious effect on splicing and a decreased E-cadherin expression for the intronic IVS12 c.1937-13T>C mutation. Our detection of the same genetic alteration and splicing effect in one BD, but with a limited effect on E-cadherin mRNA level, is intriguing and deserves further studies. Performing CDH1 screening in individuals at risk for GC, we detected the IVS4 c.532-18C>T variant already reported in EOGC cases, and a new IVS1 intronic mutation in one AMAG patient, although there was no influence on CDH1 splicing in both cases. The overall percentage of the missense mutations (67%) in our series is predominantly of the non-missense type, suggesting that the North-East of Italy should be considered as a middle-to-high-risk area for the incidence of E-cadherin mutations and the risk of GC. Moreover, variants identified in the FDR and AMAG subjects that, until now, have only been described for GC patients, and recent findings of novel mutations in sporadic GC patients, encourage us to continue to screen for CDH1 genetic alterations that, in addition to other risk factors, could be used to define a high-risk group of patients that would benefit from an early GC diagnosis.Riassunto Il cancro gastrico (GC) rimane una delle maggiori cause di mortalità dovuta al cancro a livello mondiale. Un elemento caratteristico di questo tipo di tumore è la scarsa prognosi dei pazienti. L’identificazione dei soggetti maggiormente a rischio per lo sviluppo di GC e la diagnosi precoce per questo tipo di tumore, sono approcci promettenti per ridurre la morbidità e la mortalità di tale patologia. Il gene CDH1 codifica per E-caderina, una nota proteina oncosoppressore espressa nelle cellule epiteliali che gioca un ruolo prominente nell’adesione cellulare e nel mantenimento dell’architettura tissutale. Si ritiene che mutazioni germinali a carico del gene CDH1 siano in grado di influenzare la funzione della proteina e compromettere l’adesività cellulare nei tessuti epiteliali, aumentando il potenziale metastatico del tumore. Mutazioni germinali a carico del gene CDH1 sono implicate nella sindrome del carcinoma gastrico di tipo diffuso ereditario (HDGC) e nel carcinoma gastrico ad insorgenza precoce (EOGC), ma il ruolo di tali varianti nel GC di tipo sporadico e nei soggetti a rischio di sviluppare GC sono scarsamente indagate. La maggior parte degli studi limita lo screening di mutazioni del gene CDH1 nei pazienti con HDGC, forme familiari di GC o pazienti con GC in forma precoce attraverso consulenza genetica, ma solamente un numero limitato di studi ha valutato il contributo di queste mutazioni nel gene CDH1 nella casistica sporadica di GC priva di familiarità o in soggetti ad elevato rischio di sviluppare questo tumore. Inoltre, i potenziali effetti funzionali delle varianti nel gene CDH1 spesso non sono approfonditi. Nel nostro studio, lo screening dell’intera regione codificante e delle sequenze fiancheggianti gli esoni del gene CDH1 è stato condotto attraverso sequenziamento diretto in una serie casuale e consecutiva di pazienti con GC sporadico. Abbiamo esteso l’analisi per la ricerca di mutazioni germinali di CDH1 in un gruppo di individui a rischio per GC: 59 soggetti che hanno riportato un caso accertato di GC tra genitori, figli, fratelli o cugini, denominati parenti di primo grado-GC (FDR), e 20 pazienti affetti da gastrite atrofica metaplasica autoimmunitaria (AMAG). Nello studio sono stati inclusi anche 52 donatori di sangue sani (BD). Lo scopo di questo progetto di dottorato era valutare le mutazioni a carico del gene CDH1 in tali individui per identificare un possibile marker ereditario che possa migliorare la diagnosi precoce del GC, con la prospettiva di andare a stratificare i pazienti ad alto rischio in associazione con altri marcatori di rischio già noti. Tutti i partecipanti sono stati volontariamente sottoposti ad una endoscopia gastrointestinale con biopsia nel nostro istituto. Questo è il primo studio nel quale lo screening per il gene CDH1 è stato valutato nei pazienti con AMAG. Le mutazioni germinali trovate nel CDH1 sono state caratterizzate attraverso l’uso del modelling strutturale e di strumenti bioinformatici, colorazioni immunoistochimiche (IHC) per valutare sia l’espressione che la localizzazione cellulare delle proteine E-caderina e β-catenina, e il livello di mRNA per E-caderina con la tecnica di real-time PCR. L’impatto funzionale nello splicing è stato determinato per le varianti introniche non polimorfiche. Tra i 59 GC, la porzione inferiore (l’antro) dello stomaco era la sede anatomica prevalente (p< 0.05) dei tumori rilevati mediante endoscopia. Non sono state trovate differenze statistiche confrontando associazioni tra sesso, istotipo e localizzazione del tumore, infezione da Helicobacter Pylori e stadi TNM. Un complesso pannello di varianti germinali è stato riscontrato per CDH1 nella nostra casistica. Nella serie di pazienti con GC, abbiamo trovato sei differenti alterazioni genetiche in nove distinti pazienti (15.2%): 67% del tipo missenso (p.G274S; p.A298T; p.T470I: p.A592T) e 33% non missenso, una nella regione del promotore (5’UTR-71C>G) e una nella regione intronica in prossimità dell’esone 13 (IVS12 c.1937-13T>C). Tutte le sostituzioni missenso sono localizzate nel dominio extracellulare di E-caderina che è direttamente coinvolto nel processo di adesività cellulare. Rilevante è stata l’identificazione di una nuova mutazione germinale missenso di CDH1 (p.G274S), mentre la mutazione p.A298T era stata precedentemente documentata solo nella sindrome HDGC e descritta come patogenetica. I nostri studi di modelling strutturale hanno evidenziato che la sostituzione p.G274S nella proteina matura non sembra avere un impatto notevole nel perturbare la struttura locale, ma il residuo di serina rappresenta un potenziale sito per modificazioni post traduzionali, come la fosforilazione o la glicosilazione. Comunque, dati ottenuti dai nostri esperimenti funzionali condotti in vitro sembrano non supportare alcun tipo di effetto da parte di tale mutazione sulla mobilità e adesività cellulare. Inoltre, E-caderina non era ipoespressa nel tessuto tumorale gastrico del paziente con la variante p.G274S, dove però è stata osservata una riduzione nell’espressione di β-catenina. Quindi, grazie alla valutazione sia in silico che in vitro, il ruolo patogenetico della mutazione p.G274S sembra essere improbabile, e il reale significato di questa alterazione risulta comunque incerto. Il modelling e i risultati ottenuti con lo strumento bioinformatico SIFT confermano che la sostituzione p.A298T è in grado di interferire sulla funzionalità della proteina, che invece non è influenzata dalla mutazione p.T470I la quale sembra essere tollerata. Attraverso l’analisi strutturale abbiamo ipotizzato un ruolo protettivo per la variante p.T470I che sembra favorire le interazioni proteina-proteina nel contesto dello spazio extracellulare. La sostituzione p.A592T è stata riscontrata in tutti i quattro gruppi di soggetti studiati, suggerendo che si tratta di una variante polimorfica (frequenza >1%). L’approccio condotto con il modelling suggerisce un ruolo non patogenetico come già confermato e descritto da studi pubblicati condotti sia in vitro che in silico. La sostituzione da C a G prima del codone di inizio (-71C>G), rappresenta la variante più comune rilevata nella nostra casistica, in quanto trovata in tre (5.1%) dei 59 pazienti con carcinoma gastrico. Purtroppo, a causa della mancanza di campione biologico tumorale non è stato possibile valutare una correlazione tra un eventuale stato di ipermetilazione a carico del promotore di CDH1 e l’espressione della E-caderina, perciò l’effettivo contributo di tale variante sulla trascrizione del gene rimane ancora da definire. La variante intronica IVS12 c.1937-13T>C è stata trovata in due pazienti con GC e in un BD, ma era già stata precedentemente descritta nella sindrome ereditaria HDGC e in soggetti affetti da EOGC. I nostri esperimenti hanno dimostrato per la prima volta che questa mutazione produce un trascritto aberrante di CDH1 con delezione dell’esone 11 e la formazione di un codone di stop prematuro a livello del dominio extracellulare di E-caderina. La proteina tradotta risulterà priva di gran parte del dominio extracellulare, e perderà completamente il dominio trans membrana e la coda citoplasmatica che è direttamente coinvolta nel legame con la β-catenina. Inoltre, la valutazione mediante IHC ha evidenziato una riduzione nell’espressione di E-caderina e β-catenina nei tessuti tumorali gastrici dei due pazienti che presentavano tale variante intronica. La valutazione mediante real time-PCR dell’espressione di E-caderina nei linfociti B immortalizzati con il virus EBV, in uno dei due pazienti GC con tale mutazione, ha mostrato una forte (60%) e significativa (p<0.01) riduzione rispetto sia al donatore sano senza alterazioni germinali di CDH1, sia nel donatore con la stessa variante intronica. Nel gruppo ad alto rischio FDR, abbiamo trovato una sostituzione nella regione promotoriale 5’UTR (-54G>C) già descritta come una variante rara in grado di ridurre l’attività trascrizionale di CDH1, un’altra nella regione vicina al gene in 5’ (-176C>T) con significato ignoto, e una mutazione intronica vicina all’esone 5, IVS4 c.532-18C>T, riportata nella casistica EOGC. Quest’ultima, non ha dimostrato effetti rilevanti nello splicing di CDH1, come la nuova mutazione germinale nella regione intronica a valle dell’esone 1 (IVS1 c.48+7C>T) trovata in un paziente affetto da AMAG. Altre varianti di tipo polimorfico prive di un potenziale effetto sulle funzioni della proteina E-caderina, sono state rilevate nella serie costituita da pazienti con AMAG. Concludendo, i nostri risultati evidenziano che mutazioni germinali a carico del gene CDH1, già descritte come patogenetiche (p.A298T) possono essere rilevate anche in pazienti con GC di tipo sporadico, senza particolare familiarità o non afferenti ai severi criteri descritti per il HDGC. Riportiamo inoltre la scoperta, in un paziente con GC di tipo sporadico, di una nuova mutazione germinale missenso (p.274S) di significato incerto. Inoltre, per la prima volta, sono stati dimostrati un potenziale effetto deleterio nello splicing di CDH1 e una riduzione nell’espressione di E-caderina per la mutazione intronica IVS12 c.1937-13T>C. Il rilevamento della stessa mutazione intronica IVS12, e dell’analogo impatto nello splicing nel donatore sano, ma con un limitato effetto sul livello di mRNA per E-caderina, è interessante e necessita di ulteriori approfondimenti. Estendendo lo screening per il gene CDH1 negli individui a rischio per il GC, abbiamo potuto trovare la mutazione IVS4 c.532-18C>T già descritta nei pazienti EOGC, e la nuova variante intronica IVS1 in un paziente AMAG, sebbene una conseguenza nello splicing non sia stata riscontrata per entrambi i casi. La percentuale complessiva delle mutazioni missenso (67%) nella nostra casistica è predominante rispetto a quella relativa alle mutazioni di tipo non missenso, e ciò suggerisce che l’area del Nord Est d’Italia dovrebbe essere considerata come una zona a medio-alto rischio per l’incidenza delle mutazioni relative a CDH1 e per il rischio di GC. Inoltre, le varianti trovate nei soggetti FDR e nei pazienti AMAG, che sino ad ora erano state descritte solo nei pazienti con carcinoma gastrico, e recenti scoperte di nuove mutazioni in pazienti con GC sporadico, ci incoraggiano a continuare nello screening germinale del gene CDH1 che, insieme ad altri fattori di rischio, potrebbe essere utilizzato per definire un gruppo di pazienti ad alto rischio per GC il quale potrebbe beneficiare di una diagnosi precoce.XXV Ciclo197

Hypoxic Modulation of HLA-G Expression through the Metabolic Sensor HIF-1 in Human Cancer Cells

The human leukocyte antigen-G (HLA-G) is considered an immune checkpoint molecule involved in tumor immune evasion. Hypoxia and the metabolic sensor hypoxia-inducible factor 1 (HIF-1) are hallmarks of metastasization, angiogenesis, and intense tumor metabolic activity. The purpose of this review was to examine original in vitro studies carried out in human cancer cell lines, which reported data about HLA-G expression and HIF-1 mediated-HLA-G expression in response to hypoxia. The impact of HLA-G genomic variability on the hypoxia responsive elements (HREs) specific for HIF-1 binding was also discussed. Under hypoxia, HLA-G-negative cell lines might transcribe HLA-G without translation of the protein while in contrast, HLA-G-positive cell lines, showed a reduced HLA-G transcriptional activity and protein level. HIF-1 modulation of HLA-G expression induced by hypoxia was demonstrated in different cell lines. HLA-G SNPs rs1632947 and rs41551813 located in distinct HREs demonstrated a prominent role of HIF-1 binding by DNA looping. Our research revealed a fine regulation of HLA-G in hypoxic conditions through HIF-1, depending on the cellular type and HLA-G genomic variability. Specifically, SNPs found in HREs should be considered in future investigations as markers with potential clinical value especially in metastatic malignancies

Soluble HLA-G expression levels and HLA-G/irinotecan association in metastatic colorectal cancer treated with irinotecan-based strategy

We here explore the soluble Human Leukocyte Antigen-G (sHLA-G) expression level as clinical biomarker in metastatic colorectal cancer (mCRC). To this aim the sHLA-G protein was measured in plasma samples of 40 patients with mCRC treated with the FOLFIRI (irinotecan (CPT-11) plus 5-fluorouracil (5-FU) and leucovorin (LV)) regimen. The results suggest a link between HLA-G levels and irinotecan (CPT-11) pharmacokinetic, leading to hypothesize a molecular interaction between sHLA-G and CPT-11. This interaction was confirmed experimentally by fluorescence spectroscopy. HLA-G is known to exist in a number of polymorphs that affect both the protein expression levels and its peptide-binding cleft. The interaction between HLA-G polymorphs and CPT-11 was explored by means of computational modelling, confirming the hypothesis that CPT-11 could actually target the peptide binding cleft of the most common HLA-G polymorphs

New Challenges in Tumor Mutation Heterogeneity in Advanced Ovarian Cancer by a Targeted Next-Generation Sequencing (NGS) Approach

Next-generation sequencing (NGS) technology has advanced knowledge of the genomic landscape of ovarian cancer, leading to an innovative molecular classification of the disease. However, patient survival and response to platinum-based treatments are still not predictable based on the tumor genetic profile. This retrospective study characterized the repertoire of somatic mutations in advanced ovarian cancer to identify tumor genetic markers predictive of platinum chemo-resistance and prognosis. Using targeted NGS, 79 primary advanced (III-IV stage, tumor grade G2-3) ovarian cancer tumors, including 64 high-grade serous ovarian cancers (HGSOCs), were screened with a 26 cancer-genes panel. Patients, enrolled between 1995 and 2011, underwent primary debulking surgery (PDS) with optimal residual disease (RD < 1 cm) and platinum-based chemotherapy as first-line treatment. We found a heterogeneous mutational landscape in some uncommon ovarian histotypes and in HGSOC tumor samples with relevance in predicting platinum sensitivity. In particular, we identified a poor prognostic signature in patients with HGSOC harboring concurrent mutations in two driver actionable genes of the panel. The tumor heterogeneity described, sheds light on the translational potential of targeted NGS approach for the identification of subgroups of patients with distinct therapeutic vulnerabilities, that are modulated by the specific mutational profile expressed by the ovarian tumor

Common mutations in immunoglobulin VK3 light chain sequences from B-cells of HCV-related disorders

It has been proposed that persistent HCV infection drives the proliferation of B cells through an antigen-selective stimulation, mediated by B-cell receptor (BCR) involvement that preferentially includes a IgL Vk-3 chain. The aim of this study is to characterize IgL Vk rearrangements from different setting of HCV-related disorders to investigate if there is a preferential subset of BCR repertoire among 5 clinical groups. 26 HCV+ve patients (HBV-ve; HIV-ve) were studied: 2 MC, 12 NHL without clinical manifestation of MC, 6 HCV spontaneously resolved (SR), 6 HCV chronic infected (CE). Six blood donors (BD) were used as controls. VK3 gene region was amplified using VK3 specific and a mixture of JK primers. In the present study VK3-20, VK3-15 and VK3-11 subfamilies were the most represented (>90% of total clones). IGKV3-15 gene was over represented in both NHL and MC groups (67%) vs CE+SR+BD (22%, p<0.001). IGKV3-20 gene was the most represented gene in BD and IGKV3-11 in CE, SR. An higher mutation frequency was detected both in BD (media of mutations 2,62%) and NHL (2,41%) with respect to CE (2,04%), MC (1,75%) and SR (1,55%) groups. The higher and the lower levels of R mutations were detected in SR, respectively in FR1 region (1,52%) and in the CDR2 (0,06%). Both in FR1 and FR2, the R:S ratio of SR was the highest (35%), in CDR2 was the lowest (1,5%). In FR3 and in CDR1 the R:S ratio was similar between groups, while in CDR2 BD had the highest value (10,5%). Multiple alignments of amino acid sequences obtained (472 clones) were carried to produce a consensus sequence Data highlighted that FR2 and CDR2 VK3-regions were conserved in all the 5 groups. FR1, CDR1 and CDR3 were commonly shared by CE, SR and BD. FR3 distinguish SR from these other groups in the in the first residue (Thr vs Asn). MC and NHL shared a consensus sequence among them that was more similar to VK3-15 chain mainly in the hypervariable CDR3 (Asn93), FR1 (Val13) and FR3 (Thr54, Glu71; Gln80 and Ser81) regions. In addition, Asn residue in pos32 in MC and in pos33 in NHL, distinguishes MC from NHL in CDR1. A 3D structural modelling is now in course to evaluate the potential impact on antibody-HCV antigen interactions (Leukemia, 2006). In conclusion we described common mutations in HCV-related lymphoproliferative diseases that should be useful to corroborate the molecular insights of HCV outcomes and to better define targets for potential anti-idiotype therapeutic approaches

Identification of Novel Somatic TP53 Mutations in Patients with High-Grade Serous Ovarian Cancer (HGSOC) Using Next-Generation Sequencing (NGS)

Somatic mutations in TP53 are a hallmark of high-grade serous ovarian cancer (HGSOC), although their prognostic and predictive value as markers is not well defined. Next-generation sequencing (NGS) can identify novel mutations with high sensitivity, that may be repurposed as potential druggable anti-cancer targets and aid in therapeutic decisions. Here, a commercial NGS cancer panel comprising 26 genes, including TP53, was used to identify new genetic markers of platinum resistance and patient prognosis in a retrospective set of patients diagnosed with epithelial ovarian cancer. Six novel TP53 somatic mutations in untreated tumors from six distinct patients diagnosed with HGSOC were identified: TP53 c.728_739delTGGGCGGCATGA (p.Met243_Met247del, in-frame insertion or deletion (INDEL); TP53 c.795_809delGGGACGGAACAGCTT (p.Gly266_Phe270del, in-frame INDEL); TP53 c.826_827GC>AT (p.Ala276Ile, missense); TP53 c.1022insT (p.Arg342Profs*5, frameshift INDEL); TP53 c.1180delT (p.Ter394Aspfs*28, frameshift INDEL); and TP53 c.573insT (p.Gln192Serfs*17, frameshift INDEL). Novel TP53 variants were validated by classical sequencing methods and their impact on protein expression in tumors explored by immunohistochemistry. Further insights into the potential functional effect of the mutations were obtained by different in silico approaches, bioinformatics tools, and structural modeling. This discovery of previously unreported TP53 somatic mutations provides an opportunity to translate NGS technology into personalized medicine and identify new potential targets for therapeutic applications

Characterization of antibodies directed against the immunoglobulin light κ chain variable chain region (VK) of hepatitis C virus-related type-II mixed cryoglobulinemia and B-cell proliferations

Autoimmune type-II cryoglobulinemia (II-MC) is sustained by hepatitis C virus (HCV) infection and B-cell (oligo)clones. This is the reason why the disease may be considered an "indolent B-cell lymphoma (NHL)." B clones show a restricted use of immunoglobulin variable genes (BCR), in particular in the use of the variable κ (VK)3-2015 light chain, and show a homology between their BCR functional regions and those of autoimmune rheumatoid factors. We underlined the BCR unique repertoire with frequent rheumatoid factor activity also observed in other autoimmune disorders associated with NHL. The immunoglobulin idiotype is a clonal B-cell marker and an ideal target for immunotherapy. Five monoclonal antibodies were produced in our laboratory toward the VK3-20 of a subject with HCV infection and a II-MC-associated NHL. Epitope determination was performed using the epitope excision approach. Monoclonal antibody reactivity was tested in vitro in ELISA, Western blot, and cytofluorimetry. Data confirmed that a panel of antibodies, reactive against shared idiotypes, can be produced from patients with HCV-associated B-cell lymphoproliferative diseases, thus obviating the need to produce an anti-idiotype antibody for each patient