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.

A TGF-ß associated genetic score to define prognosis and platinum sensitivity in advanced epithelial ovarian cancer.

OBJECTIVE: Epithelial ovarian cancer (EOC) is usually diagnosed at advanced stages with highly variable clinical outcomes, even among patients with similar clinical characteristics and treatments. Host immune system plays a pivotal role in EOC pathogenesis and progression. Here, we assessed the clinical significance of 192 single nucleotide polymorphisms (SNPs) on 34 immune-system related genes in EOC patients. METHODS: Two hundred and thirty advanced EOC patients treated with platinum-based chemotherapy were included. Germ-line DNA was analyzed with Illumina GoldenGate Genotyping Assay. RESULTS: Nineteen polymorphisms were significantly associated with overall survival (OS), 17 with progression free survival (PFS) and 20 with platinum-free interval (PFI). Of the 8 polymorphisms associated with all three outcomes, 7 SNPs belonged to genes involved in the TGF-ß pathway. A genetic score was built considering the unfavourable genotypes (UGs) of these 7 polymorphisms (group 0-2 UGs: presence of 0, 1, or 2 UGs; group 3-4 UGs: 3 or 4 UGs; group 5-7: 5, 6, or 7 UGs). According to this score, OS decreased as the number of UGs increased (median OS: 0-2 UGs = not reached, 3-4 UGs = 44.6 and 5-7 UGs = 19.3 months, p < 0.0001). The same trend was observed also for PFS (median PFS: 0-2 UGs = 21.5, 3-4 UGs = 17.3 and 5-7 UGs = 11 months, p < 0.0001) and PFI (median PFI: 0-2 UGs = 16.6, 3-4 UGs = 9.8 and 5-7 UGs = 3.8 months, p < 0.0001). The score was validated by permutation analysis. CONCLUSIONS: The proposed TGF-ß pathway score could be useful to define prognosis and platinum sensitivity of advanced EOC patients.

Clonal Evolution of TP53 c.375+1G>A Mutation in Pre- and Post- Neo-Adjuvant Chemotherapy (NACT) Tumor Samples in High-Grade Serous Ovarian Cancer (HGSOC).

Carboplatin/paclitaxel is the reference regimen in the treatment of advanced high-grade serous ovarian cancer (HGSOC) in neo-adjuvant chemotherapy (NACT) before interval debulking surgery (IDS). To identify new genetic markers of platinum-resistance, next-generation sequencing (NGS) analysis of 26 cancer-genes was performed on paired matched pre- and post-NACT tumor and blood samples in a patient with stage IV HGSOC treated with NACT-IDS, showing platinum-refractory/resistance and poor prognosis. Only the TP53 c.375+1G>A somatic mutation was identified in both tumor samples. This variant, associated with aberrant splicing, was in trans configuration with the 72Arg allele of the known germline polymorphism TP53 c.215C>G (p. Pro72Arg). In the post-NACT tumor sample we observed the complete expansion of the TP53 c.375+1G>A driver mutant clone with somatic loss of the treatment-sensitive 72Arg allele. NGS results were confirmed with Sanger method and immunostaining for p53, BRCA1, p16, WT1, and Ki-67 markers were evaluated. This study showed that (i) the splice mutation in TP53 was present as an early driver mutation at diagnosis; (ii) the mutational profile was shared in pre- and post-NACT tumor samples; (iii) the complete expansion of a single dominant mutant clone through loss of heterozygosity (LOH) had occurred, suggesting a possible mechanism of platinum-resistance in HGSOC under the pressure of NACT.

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.

The Genotype for DPYD Risk Variants in Patients With Colorectal Cancer and the Related Toxicity Management Costs in Clinical Practice.

Lack of information on the clinical utility of preemptive DPYD screening before fluoropyrimidine treatment is a major barrier preventing its use in clinical practice. This study aimed to define the association between DPYD variants and fluoropyrimidine-related toxicity management costs. A cost analysis was conducted on the toxicities experienced by 550 patients with colorectal cancer treated with fluoropyrimidine-based chemotherapy. Genotyping for DPYD*2A, DPYD*13, DPYDc. 2846A>T, DPYD-HapB3, and UGT1A1*28 was done retrospectively and did not affect patients' treatments. Carriers of at least one DPYD variant experienced higher toxicity management costs (€2,972; 95% confidence interval (CI), €2,456-€3,505) than noncarriers (€825; 95% CI, €785-€864) (P < 0.0001) and had a higher risk for toxicity requiring hospitalization (odds ratio, 4.14; 95% CI, 1.87-9.14). In patients receiving fluoropyrimidine/irinotecan, the incremental cost between DPYD variant and UGT1A1*28/*28 carriers and noncarriers was €2,975. This study suggests that the toxicity management costs during fluoropyrimidine-based therapy are associated with DPYD and UGT1A1*28 variants and supports the utility of genotyping.

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.

Polymer-Mediated Delivery of siRNAs to Hepatocellular Carcinoma: Variables Affecting Specificity and Effectiveness.

Despite the advances in anticancer therapies, their effectiveness for many human tumors is still far from being optimal. Significant improvements in treatment efficacy can come from the enhancement of drug specificity. This goal may be achieved by combining the use of therapeutic molecules with tumor specific effects and delivery carriers with tumor targeting ability. In this regard, nucleic acid-based drug (NABD) and particularly small interfering RNAs (siRNAs), are attractive molecules due to the possibility to be engineered to target specific tumor genes. On the other hand, polymeric-based delivery systems are emerging as versatile carriers to generate tumor-targeted delivery systems. Here we will focus on the most recent findings in the selection of siRNA/polymeric targeted delivery systems for hepatocellular carcinoma (HCC), a human tumor for which currently available therapeutic approaches are poorly effective. In addition, we will discuss the most attracting and, in our opinion, promising siRNA-polymer combinations for HCC in relation to the biological features of HCC tissue. Attention will be also put on the mathematical description of the mechanisms ruling siRNA-carrier delivery, this being an important aspect to improve effectiveness reducing the experimental work.

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.

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.

Strategies to optimize siRNA delivery to hepatocellular carcinoma cells.

INTRODUCTION: hepatocellular carcinoma (hcc) is the predominant form of primary liver cancer and the second leading cause of cancer-associated mortality worldwide. available therapies for hcc have limited efficacy due to often late diagnosis and the general resistance of hcc to anti-cancer agents; therefore, the development of novel therapeutics is urgently required. small-interfering rna (sirna) molecules are short, double-stranded rnas that specifically recognize and bind the mrna of a target gene to inhibit gene expression. despite the great therapeutic potential of sirnas towards many human tumors including hcc, their use is limited by suboptimal delivery. Areas covered: In this review, we outline the current data regarding the therapeutic potential of siRNAs in HCC and describe the development of effective siRNA delivery systems. We detail the key problems associated with siRNA delivery and discuss the possible solutions. Finally, we provide examples of the various siRNA delivery strategies that have been employed in animal models of HCC and in human patients enrolled in clinical trials. Expert opinion: Despite the existing difficulties in siRNA delivery for HCC, the increasing scientific attention and breakthrough studies in this field is facilitating the design of novel and efficient technical solutions that may soon find practical applications.

HLA-G 3'UTR Polymorphisms Impact the Prognosis of Stage II-III CRC Patients in Fluoropyrimidine-Based Treatment.

An important hallmark of CRC is the evasion of immune surveillance. HLA-G is a negative regulator of host's immune response. Overexpression of HLA-G protein in primary tumour CRC tissues has already been associated to worse prognosis; however a definition of the role of immunogenetic host background is still lacking. Germline polymorphisms in the 3'UTR region of HLA-G influence the magnitude of the protein by modulating HLA-G mRNA stability. Soluble HLA-G has been associated to 3'UTR +2960 Ins/Ins and +3035 C/T (lower levels) and +3187 G/G (high levels) genotypes. HLA-G 3'UTR SNPs have never been explored in CRC outcome. The purpose of this study was to investigate if common HLA-G 3'UTR polymorphisms have an impact on DFS and OS of 253 stage II-III CRC patients, after primary surgery and ADJ-CT based on FL. The 3'UTR was sequenced and SNPs were analyzed for their association with survival by Kaplan-Meier and multivariate Cox models; results underwent internal validation using a resampling method (bootstrap analysis). In a multivariate analysis, we estimated an association with improved DFS in Ins allele (Ins/Del +Ins/Ins) carriers (HR 0.60, 95% CI 0.38-0.93, P = 0.023) and in patients with +3035 C/T genotype (HR 0.51, 95% CI 0.26-0.99, P = 0.045). The +3187 G/G mutated carriers (G/G vs A/A+A/G) were associated to a worst prognosis in both DFS (HR 2.46, 95% CI 1.19-5.05, P = 0.015) and OS (HR 2.71, 95% CI 1.16-6.63, P = 0.022). Our study shows a prognostic and independent role of 3 HLA-G 3'UTR SNPs, +2960 14-bp INDEL, +3035 C>T, and +3187 A>G.

Prognostic Role of Serum Antibody Immunity to p53 Oncogenic Protein in Ovarian Cancer: A Systematic Review and a Meta-Analysis.

OBJECTIVE: Serum p53 autoantibodies (p53-AAbs) are the product of an endogenous immune response against p53 overexpression driven by the ovarian tumour. The p53-AAbs are detectable only in a subset of patients. To date, the evidence of an association between the presence of p53-AAbs and ovarian cancer outcomes has been poorly investigated. METHODS: A systematic literature search was performed to identify eligible studies investigating the association of serum p53-AAbs and overall survival (OS) and disease free survival (DFS). Associations between presence of serum p53-AAbs and baseline tumour characteristics were also evaluated. Pooled hazard ratios (HRs) and corresponding 95% confidence intervals (CI) were computed to estimate the prognostic impact of serum p53-AAbs. Heterogeneity between studies was assessed. RESULTS: A total of 583 patients (7 studies) for OS and 356 patients (4 studies) for DFS were included in the meta-analysis. Presence of p53-AAbs was not associated to OS (pooled uni- multivariate HR = 1.09; 95% CI: 0.55-2.16), and a large heterogeneity was found. When only multivariate HRs were pooled together (4 studies), presence of p53-AAbs was significantly associated to a better OS (pooled HR = 0.57; 95% CI: 0.40-0.81), and no significant heterogeneity was observed. A reduced DFS was associated to p53-AAbs (pooled uni- multivariate HR = 1.37; 95% CI: 0.83-2.25), though not significantly and with a moderate heterogeneity. CONCLUSIONS: The prognostic significance of serum p53-AAbs in ovarian cancer was diverging according to uni or multivariate models used. Since the results of this work were based on only few investigations, large prospective studies are needed to better define the role of antibody immunity against p53.

Inhibition of host immune response in colorectal cancer: human leukocyte antigen-G and beyond.

Colorectal cancer (CRC) is one of the most diffuse cancers worldwide and is still a clinical burden. Increasing evidences associate CRC clinical outcome to immune contexture represented by adaptive immune cells. Their type, density and location are summarized in the Immune Score that has been shown to improve prognostic prediction of CRC patients. The non-classical MHC class I human leukocyte antigen-G (HLA-G), is a crucial tumor-driven immune escape molecule involved in immune tolerance. HLA-G and soluble counterparts are able to exert inhibitory functions by direct interactions with inhibitory receptors present on both innate cells such as natural killer cells, and adaptive immune cells as cytotoxic T and B lymphocytes. HLA-G may play a prominent role in CRC strategies to avoid host immunosurveillance. This review highlights the current knowledge on HLA-G contribution in CRC, in related inflammatory diseases and in other type of cancers and disorders. HLA-G genetic setting (specific haplotypes, genotypes and alleles frequencies) and association with circulating/soluble profiles was highlighted. HLA G prognostic and predictive value in CRC was investigated in order to define a novel prognostic immune biomarker in CRC.

Identification and characterization of CDH1 germline variants in sporadic gastric cancer patients and in individuals at risk of gastric cancer.

OBJECTIVE: To screen and characterize germline variants for E-cadherin (CDH1) in non-hereditary gastric cancer (GC) patients and in subjects at risk of GC. METHODS: 59 GCs, 59 first degree relatives (FDRs) of GC, 20 autoimmune metaplastic atrophic gastritis (AMAGs) and 52 blood donors (BDs) were analyzed for CDH1 by direct sequencing, structural modelling and bioinformatics. Functional impact on splicing was assessed for intronic mutations. E-cadherin/ß-catenin immunohistochemical staining and E-cadherin mRNA quantification using RT-PCR were performed. RESULTS: In GCs, 4 missense variants (p.G274S; p.A298T; p.T470I; p.A592T), 1 mutation in the 5'UTR (-71C>G) and 1 mutation in the intronic IVS12 (c.1937-13T>C) region were found. First pathogenic effect of p.A298T mutation was predicted by protein 3D modelling. The novel p.G274S mutation showed a no clear functional significance. Moreover, first, intronic IVS12 (c.1937-13T>C) mutation was demonstrated to lead to an aberrant CDH1 transcript with exon 11 deletion. This mutation was found in 2 GCs and in 1 BD. In FDRs, we identified 4 variants: the polymorphic (p.A592T) and 3 mutations in untranslated regions with unidentified functional role except for the 5'UTR (-54G>C) that had been found to decrease CDH1 transcription. In AMAGs, we detected 2 alterations: 1 missense (p.A592T) and 1 novel variant (IVS1 (c.48+7C>T)) without effect on CDH1 splicing. Several silent and polymorphic substitutions were found in all the groups studied. CONCLUSIONS: Overall our study improves upon the current characterization of CDH1 mutations and their functional role in GC and in individuals at risk of GC. Mutations found in untranslated regions and data on splicing effects deserve a particular attention like associated with a reduced E-cadherin amount. The utility of CDH1 screening, in addition to the identification of other risk factors, could be useful for the early detection of GC in subjects at risk (i.e. FDRs and AMAGs), and warrants further study.

A novel CDH1 germline missense mutation in a sporadic gastric cancer patient in north-east of Italy.

It is well documented that germline mutations in the E-cadherin (CDH1) gene are linked to hereditary diffuse gastric cancer (HDGC). Despite the known molecular genetic causes, most gastric cancers are sporadic and poorly investigated for susceptibility genes. We report the finding of a novel germline missense mutation in exon 6, c. 820 G > A (p.G274S) in one sporadic gastric cancer patient. This new variant does not affect cryptic splicing of CDH1 as demonstrated by molecular assay. Immunohistochemical analysis shows a mixed pattern of E-cadherin staining (membranous and cytoplasmic) in the intestinal component, while in the diffuse counterpart, the membranous staining was prevalent and a reduced membranous expression of ß-catenin was observed. In vitro assays suggest that the mutant G274S does not affect the E-cadherin protein function, its expression pattern or subcellular localization. This new variant is present in EC2 extracellular domain of the protein (p.G120S in mature protein). The molecular modelling shows that this point mutation is not dramatic for local structure. However, p.S120 is located on the surface of the protein close to the functional calcium sites and in the region of interaction with EC1 domain of another E-cadherin molecule involved in the formation of the intercellular junction. Moreover, p.S120 residue could be involved in posttranslational modifications, such as phosphorylation or glycosylation, with possible effects on stability and integrity of adhesive properties of E-cadherin. In conclusion, the pathogenicity of this mutation is unlikely; probably we found a new germline CDH1 missense mutation with potential impact, however, of uncertain significance.

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.

Evaluation of the suitability of archival Bouin-fixed paraffin-embedded tissue specimens to proteomic investigation.

Bouin's solution has been used for over a century as a common fixative in several pathology laboratories worldwide. Therefore, a considerable number of Bouin-fixed paraffin-embedded (BFPE) tumor samples of various origin are available in hospital repositories as a powerful information mine for clinical investigations. To date, however, such archived tissues have not been subjected to a systematic study aimed to evaluate their potential use in proteomics. In this report, we investigated whether archival BFPE tissue specimens could be exploited for proteomic studies, upon application of protein extraction and proteomic analysis methods previously optimized for formalin-fixed samples. As a result, gastric BFPE protein extracts exhibited poor suitability for 2D-PAGE analysis, whereas over 300 unique proteins could be successfully detected when extracts were subjected to SDS-PAGE followed by LC-MS/MS (GeLC-MS/MS). Among these, several known markers for gastric cancer and normal gastric functionality were identified, indicative of biological and clinical significance of proteomic data mined from BFPE tissues. A quantitative and qualitative comparison of FFPE and BFPE tissue proteomes was also performed, and results are reported. In conclusion, we demonstrated that BFPE specimens can be analyzed by means of a proteomic approach such as GeLC-MS/MS. Although considerable molecular biases and technical constraints exist, BFPE tissue archives can be fruitfully exploited for gathering proteomic data from particularly precious samples.

Characterization of antibodies directed against the immunoglobulin light kappa 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 kappa (VK)3-20/15 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.