Molecular characterization of diffuse large B-cell lymphomas associated with hepatitis C virus infection.

Hepatitis C virus (HCV)-associated diffuse large B-cell lymphoma (DLBCL) displays peculiar clinicopathological characteristics, but its molecular landscape is not fully elucidated. In this study, we investigated the clinicopathological and molecular features of 54 patients with HCV-associated DLBCL. The median age was 71 years. An underlying marginal zone lymphoma component was detected in 14.8% of cases. FISH analysis showed rearrangements involving BCL6 in 50.9% of cases, MYC in 11.3% and BCL2 in 3.7%. Lymph2Cx-based assay was successful in 38 cases, recognizing 16 cases (42.1%) as ABC and 16 cases as GCB subtypes, while six resulted unclassified. ABC cases exhibited a higher lymphoma-related mortality (LRM). Next-generation sequencing analysis showed mutations in 158/184 evaluated genes. The most frequently mutated genes were KMT2D (42.6%), SETD1B (33.3%), RERE (29.4%), FAS and PIM1 (27.8%) and TBL1XR1 (25.9%). A mutation in the NOTCH pathway was detected in 25.9% of cases and was associated with worst LRM. Cluster analysis by LymphGen classified 29/54 cases within definite groups, including BN2 in 14 (48.2%), ST2 in seven (24.2%) and MCD and EZB in four each (13.8%). Overall, these results indicate a preferential marginal zone origin for a consistent subgroup of HCV-associated DLBCL cases and suggest potential implications for molecularly targeted therapies.

Relationship between clone metrics and clinical outcome in clonal cytopenia.

Clonal cytopenia of undetermined significance (CCUS) is associated with an increased risk of developing a myeloid neoplasm with myelodysplasia (MN). To identify the features of the mutant clone(s) that is associated with clinical phenotype and progression, we studied the following cohorts of individuals: 311 patients with idiopathic cytopenia of undetermined significance (ICUS), 532 community-dwelling individuals without hematologic phenotype (n = 355) or with unexplained anemia (n = 177), and 592 patients with overt MN. Ninety-two of 311 (30%) patients with ICUS carried a somatic genetic lesion that signaled CCUS. Clonal hematopoiesis (CH) was detected in 19.7% and 27.7% of nonanemic and anemic community-dwelling individuals, respectively. Different mutation patterns and variant allele frequencies (VAFs) (clone metrics parameters) were observed in the conditions studied. Recurrent mutation patterns exhibited different VAFs associated with marrow dysplasia (0.17-0.48), indicating variable clinical expressivity of mutant clones. Unsupervised clustering analysis based on mutation profiles identified 2 major clusters, characterized by isolated DNMT3A mutations (CH-like cluster) or combinatorial mutation patterns (MN-like cluster), and showing different overall survival (HR, 1.8). In patients with CCUS, the 2 clusters had different risk of progression to MN (HR, 2.7). Within the MN-like cluster, distinct subsets with different risk of progression to MN were identified based on clone metrics. These findings unveil marked variability in the clinical expressivity of myeloid driver genes and underline the limitations of morphologic dysplasia for clinical staging of mutant hematopoietic clones. Clone metrics appears to be critical for informing clinical decision-making in patients with clonal cytopenia.

Rare variants in Toll-like receptor 7 results in functional impairment and downregulation of cytokine-mediated signaling in COVID-19 patients.

Toll-like receptors (TLR) are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the production of pro-inflammatory cytokines and type I and II interferons, which are responsible for innate antiviral responses. Among the different TLRs, TLR7 recognizes several single-stranded RNA viruses including SARS-CoV-2. We and others identified rare loss-of-function variants in X-chromosomal TLR7 in young men with severe COVID-19 and with no prior history of major chronic diseases, that were associated with impaired TLR7 signaling as well as type I and II IFN responses. Here, we performed RNA sequencing to investigate transcriptome variations following imiquimod stimulation of peripheral blood mononuclear cells isolated from patients carrying previously identified hypomorphic, hypofunctional, and loss-of-function TLR7 variants. Our investigation revealed a profound impairment of the TLR7 pathway in patients carrying loss-of-function variants. Of note, a failure in IFNγ upregulation following stimulation was also observed in cells harboring the hypofunctional and hypomorphic variants. We also identified new TLR7 variants in severely affected male patients for which a functional characterization of the TLR7 pathway was performed demonstrating a decrease in mRNA levels in the IFNα, IFNγ, RSAD2, ACOD1, IFIT2, and CXCL10 genes.

B-cell activating factor (BAFF), BAFF promoter and BAFF receptor allelic variants in hepatitis C virus related Cryoglobulinemic Vasculitis and Non-Hodgkin's Lymphoma.

Cryoglobulinemic Vasculitis (CV) is an autoimmune/lymphoproliferative disorder associated with HCV infection that in 5%-10% of cases evolves into a B cell Non-Hodgkin's Lymphoma (NHL). B-cell activating factor (BAFF) is a key regulator in B-cell development and survival. Particular genetic variants are responsible for BAFF signaling impairment in autoimmune and neoplastic diseases. We evaluated BAFF and BAFF-receptor (BAFF-R) polymorphisms in order to determine if they predispose to HCV-related CV and NHL. The analysis was performed on 416 HCV-chronically infected patients: 136 HCV without signs/symptoms of lymphoproliferations/autoimmunity (HCV), 166 HCV with CV (HCV-CV) and 114 HCV with NHL (HCV-NHL). Rs9514828 SNP on BAFF promoter, rs61756766 on BAFF-R and rs12428930 on the BAFF gene were evaluated by Real-Time PCR. Concerning rs9514828, the frequency of C/T genotype was significantly higher in HCV-CV than in HCV. The difference in the distribution of the T/T mutant genotype in HCV-CV compared to HCV was significant as well as the distribution of C/T and T/T genotype in HCV-NHL versus HCV. T minor allele was more frequent in HCV-NHL and HCV-CV than in HCV. The distribution of C/T + T/T (for the dominant model of penetrance C/T + T/T vs. C/C) was significantly higher in HCV-CV and HCV-NHL than in HCV. Genotyping of rs61756766 on BAFF-R coding gene, revealed C/T heterozygosis at a frequency of 11% in HCV-NHL versus 3% in HCV. The T minor allele frequency was higher in HCV-NHL than in HCV. No differences emerged by genotyping rs12428930 SNP on BAFF coding gene. Our results reinforce the hypothesis that BAFF/BAFF-R genetic pattern has a role in the pathogenesis of HCV-related lymphoproliferations. BAFF/BAFF-R variants could identify a risk haplotype for HCV related CV and NHL and a BAFF/BAFF-R genetic profile assessment could potentially contribute to tailoring anti-BAFF therapy by identifying patients with BAFF alterations in which the treatment could be more beneficial.

Targeted next-generation sequencing reveals molecular heterogeneity in non-chronic lymphocytic leukemia clonal B-cell lymphocytosis.

Non-chronic lymphocytic leukemia (non-CLL) clonal B-cell lymphocytosis (CBL) encompasses a heterogeneous group of hematologic disorders that are still poorly understood. To shed light on their biological aspects, we retrospectively analyzed a highly selected series of 28 patients, who had a clonal B-cell population in the peripheral blood and in the bone marrow, without evidence of lymphoma. Extended targeted next-generation sequencing revealed wide molecular heterogeneity with MYD88 (14%), PDE4DIP (14%), BIRC3 (11%), CCND3 (11%), NOTCH1 (11%), and TNFAIP3 (11%) as the most mutated genes. Mutations of MYD88 were "nonclassic" in most cases. Although some genetic lesions were overlapping with indolent lymphomas, mainly splenic B-cell lymphomas of marginal zone origin and splenic diffuse red pulp small B-cell lymphoma, the genetic profile of our non-CLL CBL series seemed to suggest that various pathways could be involved in the pathogenesis of these disorders, not mirroring any specific lymphoma entity. These data better enlighten the molecular characteristics of non-CLL CBL; however, more efforts are needed in order to improve the diagnostic process, prognostication, and clinical management.

A risk-stratification model based on the initial concentration of the serum monoclonal protein and MYD88 mutation status identifies a subset of patients with IgM monoclonal gammopathy of undetermined significance at high risk of progression to Waldenström macroglobulinaemia or other lymphoproliferative disorders.

IgM monoclonal gammopathies of undetermined significance (IgM MGUS) are associated with a risk of progression to Waldenström macroglobulinaemia (WM) or other lymphoproliferative disorders (LPD) of 1-2% per year. We analysed 176 consecutive patients with IgM MGUS to evaluate risk factors for progression. With a median follow-up of 83 months (1214 person-years), 15 patients (8·5%) progressed to WM (n = 14) or marginal zone lymphoma (n = 1). The rate of progression was 1·32% per year (95% confidence interval [CI] 0·80-2·20). The serum monoclonal protein concentration and the MYD88 mutation were independent risk factors for progression (Hazard ratio [HR] 23·3, 95% CI 2·0-273·3, P = 0·012 and HR 24·4, 95% CI 2·2-275·3, P = 0·010, respectively). The cumulative incidence of progression, while considering death as a competing event, was 11·6% at 5 years and 38·0% at 10 years in MYD88-mutated patients with a serum monoclonal protein of 10 g/l or higher, as compared with 0% at 5 years and 1·1% at 10 years for patients with none or one risk factor. This risk-stratification model is able to identify a subset of patients with IgM MGUS at high risk of progression to WM or LPD who deserve a lifelong follow-up.

Clinical significance of somatic mutation in unexplained blood cytopenia.

Unexplained blood cytopenias, in particular anemia, are often found in older persons. The relationship between these cytopenias and myeloid neoplasms like myelodysplastic syndromes is currently poorly defined. We studied a prospective cohort of patients with unexplained cytopenia with the aim to estimate the predictive value of somatic mutations for identifying subjects with, or at risk of, developing a myeloid neoplasm. The study included a learning cohort of 683 consecutive patients investigated for unexplained cytopenia, and a validation cohort of 190 patients referred for suspected myeloid neoplasm. Using granulocyte DNA, we looked for somatic mutations in 40 genes that are recurrently mutated in myeloid malignancies. Overall, 435/683 patients carried a somatic mutation in at least 1 of these genes. Carrying a somatic mutation with a variant allele frequency ≥0.10, or carrying 2 or more mutations, had a positive predictive value for diagnosis of myeloid neoplasm equal to 0.86 and 0.88, respectively. Spliceosome gene mutations and comutation patterns involving TET2, DNMT3A, or ASXL1 had positive predictive values for myeloid neoplasm ranging from 0.86 to 1.0. Within subjects with inconclusive diagnostic findings, carrying 1 or more somatic mutations was associated with a high probability of developing a myeloid neoplasm during follow-up (hazard ratio = 13.9, P < .001). The predictive values of mutation analysis were confirmed in the independent validation cohort. The findings of this study indicate that mutation analysis on peripheral blood granulocytes may significantly improve the current diagnostic approach to unexplained cytopenia and more generally the diagnostic accuracy of myeloid neoplasms.

Clinical and molecular characteristics of lymphoplasmacytic lymphoma not associated with an IgM monoclonal protein: A multicentric study of the Rete Ematologica Lombarda (REL) network.

Lymphoplasmacytic lymphoma (LPL) is usually associated with a serum IgM paraprotein, corresponding to Waldenström's Macroglobulinemia (WM). Cases presenting with IgG or IgA, or without a monoclonal protein are extremely rare. We analyzed clinical characteristics, frontline treatment, and the outcome of 45 patients with non-IgM LPL, and compared them with a control group of WM patients. The median age was similar, with significantly higher prevalence of females in non-IgM LPL, than in WM patients (60% vs 39%, P = .016). Patients with non-IgM LPL more frequently presented with lymphadenopathies (53% vs 15%, P < .001), splenomegaly (22% vs 8%, P = .015) or extranodal involvement (20% vs 8%, P = .05). In non-IgM LPL a serum monoclonal protein and bone marrow infiltration were less common than in WM patients (69% and 84% of cases respectively, P < .001 for both comparisons). The MYD88 (L265P) mutation was found in 8/19 patients using allele-specific polymerase chain reaction. A CXCR4 mutation was found in 4/17 cases using Sanger. In 16 patients we performed targeted next-generation sequencing of genes MYD88, CXCR4, ARID1-A, KMT2D, NOTCH2, TP53, PRDM1, CD79B, TRAF3, MYBBP1A, TNFAIP3. Seven patients (44%) had a MYD88 mutation (S219C in one), four (25%) a CXCR4 mutation, three (19%) a KMT2D mutation, one (6%) a TP53 mutation and one (6%) a TRAF3 mutation. With a median follow-up of 55.7 months, 36 non-IgM LPL patients (80%) were treated. Non-IgM LPL patients received more frequently anthracycline-containing regimens, as compared with WM patients, who mainly received alkylating-based therapies. Five-year overall survival (OS) was 84%, similar to that of WM patients.

Integrating clinical features and genetic lesions in the risk assessment of patients with chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm with variable clinical course. To predict the clinical outcome, we previously developed a CMML-specific prognostic scoring system (CPSS) based on clinical parameters and cytogenetics. In this work, we tested the hypothesis that accounting for gene mutations would further improve risk stratification of CMML patients. We therefore sequenced 38 genes to explore the role of somatic mutations in disease phenotype and clinical outcome. Overall, 199 of 214 (93%) CMML patients carried at least 1 somatic mutation. Stepwise linear regression models showed that these mutations accounted for 15% to 24% of variability of clinical phenotype. Based on multivariable Cox regression analyses, cytogenetic abnormalities and mutations in RUNX1, NRAS, SETBP1, and ASXL1 were independently associated with overall survival (OS). Using these parameters, we defined a genetic score that identified 4 categories with significantly different OS and cumulative incidence of leukemic evolution. In multivariable analyses, genetic score, red blood cell transfusion dependency, white blood cell count, and marrow blasts retained independent prognostic value. These parameters were included into a clinical/molecular CPSS (CPSS-Mol) model that identified 4 risk groups with markedly different median OS (from >144 to 18 months, hazard ratio [HR] = 2.69) and cumulative incidence of leukemic evolution (from 0% to 48% at 4 years, HR = 3.84) (P < .001). The CPSS-Mol fully retained its ability to risk stratify in an independent validation cohort of 260 CMML patients. In conclusion, integrating conventional parameters and gene mutations significantly improves risk stratification of CMML patients, providing a robust basis for clinical decision-making and a reliable tool for clinical trials.

Pattern of somatic mutations in patients with Waldenström macroglobulinemia or IgM monoclonal gammopathy of undetermined significance.

We analyzed MYD88 and CXCR4 mutation status of 260 patients with Waldenström macroglobulinemia or IgM monoclonal gammopathy of undetermined significance using allele-specific real time quantitative polymerase chain reaction and Sanger sequencing, respectively. A subgroup of 119 patients was further studied with next-generation sequencing of 11 target genes (MYD88, CXCR4, ARID1A, KMT2D, NOTCH2, TP53, PRDM1, CD79B, TRAF3, MYBBP1A, and TNFAIP3). MYD88 (L265P) was found at diagnosis in 91% of patients with Waldenström macroglobulinemia and in 60% of patients with IgM monoclonal gammopathy of undetermined significance using allele-specific polymerase chain reaction analysis. MYD88 mutations other than the classical L265P (V217F, S219C and M232T) were found in four cases by next-generation sequencing. Waldenström macroglobulinemia patients with wild-type MYD88 had a distinct clinical phenotype characterized by less bone marrow infiltration (P=0.01) and more frequent extramedullary involvement (P=0.001) compared to patients with mutated MYD88 Patients with wild-type MYD88 did not show additional mutations in the other target genes. CXCR4 mutations were found by Sanger sequencing in 22% of patients with Waldenström macroglobulinemia. With next-generation sequencing, a CXCR4 mutation was detected in 23% of patients with Waldenström macroglobulinemia and 9% of those with IgM monoclonal gammopathy of undetermined significance. Asymptomatic Waldenström macroglobulinemia patients harboring a CXCR4 mutation had a shorter treatment-free survival (51 months) than that of patients with wild-type CXCR4 (median not reached) (P=0.007). Analysis of variant allele frequencies indicated that CXCR4 mutations were present in the dominant clone in the majority of cases. Recurrent somatic mutations of KMT2D were found in 24% of patients with Waldenström macroglobulinemia and 5% of patients with IgM monoclonal gammopathy of undetermined significance and were primarily subclonal.

Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstrom's macroglobulinemia and related lymphoid neoplasms.

A study has shown that MYD88 (L265P) is a recurring somatic mutation in Waldenström's macroglobulinemia (WM). We developed an allele-specific polymerase chain reaction (PCR) for this mutation, and analyzed bone marrow or peripheral blood samples from 58 patients with WM, 77 with IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), 84 with splenic marginal zone lymphoma (SMZL), and 52 with B-cell chronic lymphoproliferative disorders (B-CLPD). MYD88 (L265P) was detected in 58/58 (100%) patients with WM, 36/77 (47%) with IgM-MGUS, 5/84 (6%) with SMZL, and 3/52 (4%) with B-CLPD. Compared to IgM-MGUS patients with wild-type MYD88, those carrying MYD88 (L265P) showed significantly higher levels of IgM (P < .0001) and presented Bence-Jones proteinuria more frequently at diagnosis (P = .002). During follow-up, 9 patients with IgM-MGUS progressed to WM or to marginal zone lymphoma. Using a case-control approach, the risk of evolution of patients carrying MYD88 (L265P) was significantly higher than that of patients with wild-type MYD88 (odds ratio 4.7, 95% confidence interval 0.8 to 48.7, P = .047). These findings indicate that the allele-specific PCR we developed is a useful diagnostic tool for patients with WM or IgM-MGUS. In this latter condition, MYD88 (L265P) is associated with greater disease burden and higher risk of disease progression, and the mutation may therefore also represent a useful prognostic marker.

The BRAF V600E mutation in hairy cell leukemia and other mature B-cell neoplasms.

The somatically acquired V600E mutation of the BRAF gene has been recently described as a molecular marker of hairy cell leukemia (HCL). We developed an allele-specific PCR for this mutation and studied 62 patients with HCL, 1 with HCL variant, 91 with splenic marginal zone lymphoma, 29 with Waldenström macroglobulinemia, and 57 with B-cell chronic lymphoproliferative disorders. The BRAF V600E mutation was detected in all HCL cases and in only 2 of the remaining 178 patients. These 2 subjects had B-cell chronic lymphoproliferative disorders that did not fulfill the diagnostic criteria for HCL. Despite the positive PCR finding, the mutation could not be detected by Sanger sequencing in these 2 cases, suggesting that it was associated with a small subclone. We conclude that the BRAF V600E mutation is present in all patients with HCL and that, in combination with clinical and morphologic features, represents a reliable molecular marker for this condition.

Constant activation of the RAF-MEK-ERK pathway as a diagnostic and therapeutic target in hairy cell leukemia.

The BRAF-V600E mutation defines genetically hairy cell leukemia among B-cell leukemias and lymphomas. In solid tumors, BRAF-V600E is known to aberrantly activate the oncogenic MEK-ERK pathway, and targeted BRAF and/or MEK inhibitors have shown remarkable efficacy in clinical trials in melanoma patients. However, the MEK-ERK pathway status in hairy cell leukemia has not been thoroughly investigated. We assessed phospho-ERK expression in 37 patients with hairy cell leukemia and 44 patients with neoplasms mimicking hairy cell leukemia (40 splenic marginal zone lymphoma, 2 hairy cell leukemia-variant and 2 splenic lymphoma/leukemia unclassifiable) using immunohistochemistry on routine biopsies and/or Western blotting on purified leukemic cells, and correlated the phospho-ERK status with the BRAF-V600E mutation status. Besides confirming the constant presence of BRAF-V600E in all patients with hairy cell leukemia, we observed ubiquitous phospho-ERK expression in this malignancy. Conversely, all 44 cases with neoplasms mimicking hairy cell leukemia were devoid of BRAF-V600E and none expressed phospho-ERK. Furthermore, the two exceptionally rare cases of non-hairy cell leukemia unclassifiable chronic B-cell neoplasms previously reported to be BRAF-V600E(+) on allele-specific polymerase chain reaction lacked phospho-ERK expression as well, suggesting the presence of the mutation in only a small part of the leukemic clone in these cases. In conclusion, our findings support the use of phospho-ERK immunohistochemistry in the differential diagnosis between hairy cell leukemia and its mimics, and establish the MEK-ERK pathway as a rational therapeutic target in this malignancy.

Chronic lymphocytic leukemia with del13q14 as the sole abnormality: dynamic prognostic estimate by interphase-FISH.

This study analyzed 140 patients with isolated del13q14 on interphase FISH (I-FISH), to identify subsets with a different progression risk and to assess the acquisition of additional chromosomal abnormalities (clonal evolution) in treatment-naïve del13q14 patients. A monoallelic deletion (del13qx1) was detected in 123 cases (88%), a biallelic deletion (del13qx2) in eight and a mosaic of monoallelic and biallelic deletions (del13qx1/del13qx2) in nine. In 33% of cases, deletion encompassed the Rb1 locus The median percentage of abnormal nuclei was 50% (15%-96%), and it was higher in patients with a biallelic/mosaic pattern in comparison with patients with monoallelic deletion. Sixty two patients (44%) have been treated; 5-year treatment free survival rate was 56% and the median treatment free survival was 65 months. The baseline percentage of deleted nuclei, as a continuous variable, was related to progression (HR: 1.02; p = 0.001). According to deletion burden, three groups were identified: 64 cases (46%) had <50% deleted nuclei, 47 (33%) had 50-69% deleted nuclei, and 29 (21%) had ≥70% deleted nuclei. The 5-year untreated rate was 70.5% , 52.6% and 28.7% (p < 0.0001), respectively. In multivariate analysis using IGHV mutational status, presence of a nullisomic clone, CD38 expression and percentage of deleted nuclei as covariates, only IGHV mutational status and the percentage of deleted nuclei were independent risk factors for treatment. In 103 patients serially monitored by I-FISH before starting any treatment, we observed a significant increase in the proportion of del13q14 cells, and this increase affected the risk of subsequent treatment requirement (HR 2.54, p = 0.001). The appearance of a new clone was detected in 16 patients (15.5%) and chromosome 13 was involved in 14 of them. I-FISH monitoring proves worthwhile for a dynamic risk stratification and for planning clinical surveillance.