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Background: Parkinson's disease (PD) is a chronic, progressive neurodegenerative condition that gradually worsens motor function and leads to postural instability and, eventually, falls. Several factors may influence the frequency of future falls, such as slowness, freezing of gait, loss of balance, and mobility problems, cognitive impairments, and the number of previous falls. The TED bracelet is an advanced technological wearable device able to predict falls. Aims: This principal aim is to investigate the feasibility of a full-scale research project that uses the TED bracelet to identify whether individuals with PD are at risk of falling. Methods: This study will involve a pilot prospective observational study design; the subjects will include 26 patients suffering from mild PD and 26 others with no PD and no gait problems. Data will be collected from the TED bracelet and then compared to a paper-based fall diary. The enrolled participants will have a scheduled outpatient evaluation to collect both clinical and instrumental data as well as biological samples. Discussion: This pilot study could then be implemented in a larger form to further evaluate the effectiveness of the TED device. Finally, it will help further develop gait monitoring systems for people with Parkinson's disease and other neurodegenerative diseases that can affect physical function and mobility, such as dementia and Alzheimer's. Conclusions: Preventing falls and their complications could lead to major advancements in the quality of home care for patients with PD, which would significantly impact the quality of life of both these patients and their caregivers.

Background: The gene encoding the DNA repair enzyme [O.sup.6]-methylguanine DNA methyltransferase (MGMT) is transcriptionally silenced by promoter hypermethylation in several human cancers, including diffuse large B-cell lymphoma (B-DLCL). MGMT promoter hypermethylation is a favorable prognostic marker in patients with brain tumors treated with alkylating agents. Methods: In a retrospective cohort study, we used methylation-specific polymerase chain reaction to analyze the MGMT promoter methylation status in tumor DNA of B-DLCL patients receiving cyclophosphamide as part of multidrug regimens. Molecular data were compared with patient response with the use of Student's t test. Disease-free survival and overall survival were estimated by the Kaplan--Meier method and compared with the use of the log-rank test. Multivariable survival analyses were performed with the Cox proportional hazards model. All statistical tests were two-sided. Results: Thirty (36%) of 84 B-DLCL patients showed MGMT promoter hypermethylation in their lymphomas. The presence of MGMT methylation was associated with a statistically significant increase in overall survival (hazard ratio for time to death for nonmethylation versus methylation = 2.8; 95% confidence interval (CI) = 1.2 to 7.5; P = .01) and progression-free survival (hazard ratio for time to progression for nonmethylation versus methylation = 2.6; 95% CI = 1.3 to 5.8; P = .02). MGMT promoter hypermethylation was both independent of and stronger than established prognostic factors, such as age, disease stage, serum lactic dehydrogenase level, and performance status. Conclusion: MGMT promoter hypermethylation appears to be a useful marker for predicting survival in patients with B-DLCL treated with multidrug regimens including cyclophosphamide. [J Natl Cancer Inst 2002;94: 26-32]

Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P < .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.

Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P< .001). Multivariate analysis selected NOTCH1mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1mutated patients were devoid of TP53disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1mutations was similar to that of TP53mutated/deleted CLL. NOTCH1mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53abnormalities, and identify cases with a dismal prognosis.

Splenic marginal zone lymphoma (SMZL) is one of the few B-cell lymphoma types that remain orphan of molecular lesions in cancer-related genes. Detection of active NF-κB signaling in 14 (58%) of 24 SMZLs prompted the investigation of NF-κB molecular alterations in 101 SMZLs. Mutations and copy number abnormalities of NF-κB genes occurred in 36 (36%) of 101 SMZLs and targeted both canonical (TNFAIP3and IKBKB) and noncanonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways. Most alterations were mutually exclusive, documenting the existence of multiple independent mechanisms affecting NF-κB in SMZL. BIRC3inactivation in SMZL recurred because of somatic mutations that disrupted the same RING domain that in extranodal marginal zone lymphoma is removed by the t(11;18) translocation, which points to BIRC3disruption as a common mechanism across marginal zone B-cell lymphomagenesis. Genetic lesions of NF-κB provide a molecular basis for the pathogenesis of more than 30% of SMZLs and offer a suitable target for NF-κB therapeutic approaches in this lymphoma.

Splenic marginal zone lymphoma (SMZL) is one of the few B-cell lymphoma types that remain orphan of molecular lesions in cancer-related genes. Detection of active NF-κB signaling in 14 (58%) of 24 SMZLs prompted the investigation of NF-κB molecular alterations in 101 SMZLs. Mutations and copy number abnormalities of NF-κB genes occurred in 36 (36%) of 101 SMZLs and targeted both canonical (TNFAIP3 and IKBKB) and noncanonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways. Most alterations were mutually exclusive, documenting the existence of multiple independent mechanisms affecting NF-κB in SMZL. BIRC3 inactivation in SMZL recurred because of somatic mutations that disrupted the same RING domain that in extranodal marginal zone lymphoma is removed by the t(11;18) translocation, which points to BIRC3 disruption as a common mechanism across marginal zone B-cell lymphomagenesis. Genetic lesions of NF-κB provide a molecular basis for the pathogenesis of more than 30% of SMZLs and offer a suitable target for NF-κB therapeutic approaches in this lymphoma.

Abstract: Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma, originating from naïve B-cells. The blastoid MCL tumors often show complex cytogenetic aberrations. In this review, we summarized the data available on immunoglobulin heavy-chain (IgH) genes rearrangement for their importance in suggesting the MCL normal counterpart B-cell. Some new data suggesting an antigen selection process were also presented in this review.

Several drugs used for diffuse large B-cell lymphoma (DLBCL) treatment rely on DNA damage for tumor cell killing. We verified the prognostic impact of the host DNA repair genotype in 2 independent cohorts of DLBCL treated with R-CHOP21 (training cohort, 163 cases; validation cohort, 145 cases). Among 35 single nucleotide polymorphisms analyzed in the training series, MLH1 rs1799977 was the sole predicting overall survival. DLBCL carrying the MLH1 AG/GG genotype displayed an increased death risk (hazard ratio [HR] = 3.23; P < .001; q =0 .009) compared with patients carrying the AA genotype. Multivariate analysis adjusted for International Prognostic Index identified MLH1 AG/GG as an independent OS predictor (P < .001). The poor prognosis of MLH1 AG/GG was the result of an increased risk of failing both R-CHOP21 (HR = 2.02; P = .007) and platinum-based second-line (HR = 2.26; P = .044) treatment. Survival analysis in the validation series confirmed all outcomes predicted by MLH1 rs1799977. The effect on OS of MLH1, a component of the DNA mismatch repair system, is consistent with its role in regulating the genotoxic effects of doxorubicin and platinum compounds, which are a mainstay of DLBCL first- and second-line treatment.

IGHV3-21–using chronic lymphocytic leukemia (CLL) is a distinct entity with restricted immunoglobulin gene features and poor prognosis and is more frequently encountered in Northern than Southern Europe. To further investigate this subset and its geographic distribution in the context of a country (Italy) with both continental and Mediterranean areas, 37 IGHV3-21 CLLs were collected out of 1076 cases enrolled by different institutions from Northern or Central Southern Italy. Of the 37 cases, 18 were identified as homologous (hom)HCDR3–IGHV3-21 CLLs and were found almost exclusively (16 of 18) in Northern Italy; in contrast, 19 nonhomHCDR3–IGHV3-21 cases were evenly distributed throughout Italy. Clinically, poor survivals were documented for IGHV3-21 CLLs as well as for subgroups of mutated and homHCDR3–IGHV3-21 CLLs. Negative prognosticators CD38, ZAP-70, CD49d, and CD79b were expressed at higher levels in homHCDR3 than nonhomHCDR3–IGHV3-21 cases. Differential gene expression profiling (GEP) of 13 IGHV3-21 versus 52 non–IGHV3-21 CLLs identified, among 122 best-correlated genes, TGFB2 and VIPR1 as down- and up-regulated in IGHV3-21 CLL cases, respectively. Moreover, GEP of 7 homHCDR3 versus 6 nonhomHCDR3–IGHV3-21 CLLs yielded 20 differentially expressed genes, with WNT-16 being that expressed at the highest levels in homHCDR3–IGHV3-21 CLLs. Altogether, IGHV3-21 CLLs, including those with homHCDR3, had a peculiar global phenotype in part explaining their worse clinical outcome.

Posttransplantation lymphoproliferative disorders (PTLDs) are heterogeneous lymphoid proliferations representing a major complication of solid organ transplant. This study details the clinicopathological and molecular features of 17 B-cell PTLDs observed in a single center series of 988 heart and/or lung transplant recipients.

Aberrant somatic hypermutation (SHM) has been identified as a mechanism for genomewide instability in diffuse large B-cell lymphoma (DLBCL). To assess whether aberrant SHM plays a role in the molecular pathogenesis of Hodgkin lymphoma (HL), we investigated microdissected neoplastic cells of nodular lymphocyte-predominant HL (NLPHL; n = 10) and classic HL (cHL; n = 9) for the presence of mutations in the 5′ sequences of 4 previously identified aberrant SHM targets (PIM1, PAX5, RhoH/TTF, c-MYC). Mutations in one or more genes were detected in 80% of NLPHLs and 55% of cHLs, with 50% and 30% of patients carrying mutations in 2 or more genes, respectively. The most frequently involved protooncogene was PAX5, mutated in 7 of 9 patients with NLPHL and 2 of 9 patients with cHL. In total, 34 mutations were detected in NLPHL (frequency, 1.04/1000 bp) and 35 were detected in patients with cHL (frequency, 1.92/1000 bp). Mutations were of somatic origin because they were absent in control T cells and shared most of the features of the immunoglobulin variable (IGV)gene–associated SHM mechanism—ie, single nucleotide substitutions (n = 63) with rare deletions/insertions (n = 6) and a predominance of transitions over transversions with preferential targeting motifs. Our finding that NLPHL and cHL are targeted by aberrant SHM, as is DLBCL, suggests that these lymphomas may share common molecular pathogenetic events.

Posttransplantation lymphoproliferative disorders (PTLDs) represent a serious complication of solid organ transplantation. This study assessed the molecular histogenesis of 52 B-cell monoclonal PTLDs, including 12 polymorphic PTLDs (P-PTLDs), 36 diffuse large B-cell lymphomas (DLBCLs), and 4 Burkitt/Burkitt-like lymphomas (BL/BLLs). Somatic hypermutation (SHM) of immunoglobulin variable (IgV) genes documented that most monoclonal B-cell PTLDs (75% P-PTLDs, 91.3% DLBCLs, 100% BL/BLLs) derive from germinal center (GC)-experienced B cells. B-cell lymphoma 6 (BCL6) mutations occurred in 25% P-PTLDs, 60.6% DLBCLs, and 75.0% BL/BLLs. A first histogenetic category of PTLDs (31.2% DLBCLs) express the BCL6+/multiple myeloma oncogene-1 protein (MUM1-/+)/CD138- profile and mimic B cells experiencing the GC reaction, as also suggested by ongoing SHM in a fraction of these cases. A second subset of PTLDs (66.7% P-PTLDs and 31.2% DLBCLs) display the BCL6-/MUM1+/CD138- phenotype and mimic B cells that have concluded the GC reaction. A third histogenetic category of PTLDs (25.0% P-PTLDs and 31.2% DLBCLs) shows the BCL6-/MUM1+/CD138+ profile, consistent with preterminally differentiated post-GC B cells. Crippling mutations of IgV heavy chain (IgVH) and/or IgV light chain (IgVL) genes, leading to sterile rearrangements and normally preventing cell survival, occur in 4 DLBCLs and 1 BL/BLL that may have been rescued from apoptosis through expression of Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1). Overall, the histogenetic diversity of monoclonal B-cell PTLDs may help define biologically homogeneous categories of the disease. (Blood. 2003;102: 3775-3785)

The pathogenesis of AIDS-related non-Hodgkin lymphomas (AIDS-NHLs) is associated with chromosomal translocations that deregulate the expression of various oncogenes. Recently, a novel mechanism of genetic lesion, termed aberrant hypermutation, has been identified in diffuse large B-cell lymphoma (DLBCL) of immunocompetent hosts. In these tumors, the somatic hypermutation (SHM) process that normally targets immunoglobulin V (IgV) genes in B cells appears to misfire and causes mutations in the 5′ sequences of multiple proto-oncogenes, including PIM-1, PAX-5, RhoH/TTF, and c-MYC. To investigate whether aberrant hypermutation occurs also in AIDS-NHL, we studied the mutation profile of these 4 genes in various histologic subtypes. Mutations in 1 gene or more were detected in 19 of 39 (48.7%) AIDS-NHL cases (10 of 18 AIDS-diffuse large B-cell lymphoma; 4 of 11 AIDS-Burkitt lymphoma; 4 of 6 AIDS-primary effusion lymphoma; 1 of 4 AIDS-primary central nervous system lymphoma), with 9 of 39 (23.1%) cases carrying mutations in 2 or more genes. Overall, PIM-1 was mutated in 5 of 39 (12.8%), PAX-5 in 8 of 39 (20.5%), RhoH/TTF in 9 of 39 (23.1%), and c-MYC in 7 of 27 (25.9%) AIDS-NHL cases. Mutations were represented mainly by single base pair substitutions (n = 63) with rare deletions/insertions (n = 5) and displayed features typical of the IgV-associated SHM process. In addition, a number of mutations in PIM-1 and c-MYC were found to affect coding exons, leading to amino acid substitutions with likely functional consequences. Analysis of intraclonal heterogeneity documented that the aberrant hypermutation activity may be ongoing in at least some cases. These data indicate that aberrant hypermutation is associated with various subtypes of AIDS-NHL and may represent a major contributor to their pathogenesis.

Different biologic features have been associated with a more or less aggressive clinical course in chronic lymphocytic leukemia (CLL). In the present study, 20 patients with highly stable CLL observed at a single institution over a period of 10 to 23 years and who never required treatment were extensively characterized. The aim was to identify a distinct and reproducible biologic profile associated with disease stability that may be used to recognize at presentation CLL patients who are likely to have a very benign clinical course and for whom treatment is not indicated. The results obtained indicate that numerous parameters are closely associated with disease stability: a typical CLL morphology and immunophenotype, the lack of expression of the CD38 antigen, the mutated immunoglobulin (Ig) heavy (H) chain variable (V) pattern, the absence of p53 mutations, a CD4/CD8 ratio more than 1, the lack of 17p and 11q deletions and of complex karyotypic aberrations, and the occurrence of the 13q14 deletion. No case displayed the VH3-21 gene, linked in mutated CLL with a poor outcome. In addition, the VH1-69 gene associated with unmutated CLL cases was never detected. These biologic features were coupled with an indolent clinical course characterized by an unmodified clinical stage over time, and by lack of autoimmune phenomena and of major infections requiring parental antibiotics. At a time when aggressive therapeutic strategies are always more frequently used in the management of CLL, the distinctive features of patients with long-lived stable disease should be prospectively identified at presentation.

Knowledge of the role of cell-cycle regulators in the pathogenesis of acquired immune deficiency syndrome-related non-Hodgkin's lymphomas (AIDS-NHLs) is scarce. Here we analyzed 86 systemic AIDS-NHLs and 20 AIDS-primary central nervous system lymphomas for expression of p27Kip1, a negative regulator of cell-cycle progression belonging to the Kip family of cyclin-dependent kinase inhibitors. In parallel, we investigated the relationship between p27Kip1, the lymphoma proliferation index, Epstein-Barr virus status, expression of cellular cyclin D3 and cyclin D1, and B-cell differentiation stage. We report that AIDS-immunoblastic lymphomas (AIDS-IBLs), either systemic or primarily localized to the central nervous system, consistently express p27Kip1protein (19 of 24 and 10 of 14, respectively) despite the high proliferative rate of the lymphoma clone, suggesting a failure of p27Kip1to inhibit the cell cycle in AIDS-IBL. Conversely, the remaining systemic AIDS-NHLs and AIDS-primary central nervous system lymphomas preferentially fail to express p27Kip1. Expression of p27Kip1in Epstein-Barr virus-positive AIDS-NHLs generally associates with latent membrane protein 1 (LMP1) expression and is related to a late stage of B-cell differentiation, characterized by the BCL-6−/MUM1+/syn-1± phenotypic profile, whereas it seems to be unrelated to the expression of cellular cyclins. In B cells in vitro, induction of LMP-1 expression under the control of inducible promoters up-regulates expression of p27Kip1, thus providing a putative mechanistic explanation for the association between LMP1 and p27Kip1observed in vivo. Overall, these data show that AIDS-IBL pathogenesis is characterized by loss of the inverse relationship between p27Kip1positivity and tumor growth fraction that is otherwise generally observed in normal lymphoid tissues and in most other types of NHLs.

Simian AIDS-related lymphomas (sARL) of cynomolgus monkeys infected with a simian immunodeficiency virus (SIVsm) were studied in relation to growth in severe combined immunodeficient (SCID) mice, karyotype abnormalities, and DNA sequence of the first noncoding region of the Bcl-6 gene. The tumors were diffuse large B cell lymphomas and expressed a simian homolog to Epstein-Barr virus (HVMF-1) in 12 of 13 primary tumors and corresponding cell lines. A tested cell line was tumorigenic in SCID mice. Tumors in the SCID mice showed cell growth features similar to those in the original lymphoma, suggesting that no subpopulation with growth advantage was selected for in the mice. Spectral karyotype analysis of sARL cell lines showed normal cytogenetic features except for a trisomy of monkey chromosome 2 (corresponding to human chromosomes 7 and 21) in two of five sARL lines, which was not recovered in SCID tumors established from the same cell line. Sequence analysis of a Bcl-6 gene fragment showed sequence variations indicative of population polymorphism(s) in 10 of 13 sARLs, and no evidence of Bcl-6 mutations. Thus Bcl-6 mutations in the first noncoding region are irrelevant for sARL development in cynomolgus monkeys and for tumorigenicity of sARL cell lines. We also demonstrate that no cytogenetic alterations are needed for the development of highly aggressive lymphomas in the SIV-immunosuppressed host.

In the present study sequence variations at the C terminus of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1), EBV-encoded latent membrane protein 1 (LMP-1), and EBNA-2 and EBNA-3C genes were investigated in 64 cases of EBV-positive AIDS-related diffuse large cell lymphoma (AIDS-DLCL), both systemic (12) and localized primarily to the central nervous system (52), and in 12 cases of EBV-positive AIDS-related Burkitt's lymphoma (AIDS-BL). Sequence analysis of the EBNA-1 C-terminal region led to the distinction of two major unrelated EBV strains, termed strain P (prototype) and strain V (variant), and their related subtypes, namely P-ala, P-thr, V-leu, V-val, and V-pro. Analysis of the LMP-1 gene was performed to assess the frequency of the C-terminus deletion variant, whereas analysis of EBNA-2 and EBNA-3C genes led to the identification of the distribution of the EBV type 1 and type 2 strains. The frequency of EBNA-1 subtypes was assessed in 49 cases of AIDS-NHL, including 37 cases of AIDS-DLCL and 12 cases of AIDS-BL. The P strain was detected in 45 of 49 cases (91.8%) whereas the V strain was found in 4 of 49 samples (8.1%). A significant difference in the distribution of the P and V strains was found between AIDS-DLCL and AIDS-BL (p < 0.01), because of the exclusive infection by the P strain of the AIDS-DLCL samples analyzed. The frequency of LMP-1 deletion variants and of EBV type 1 and type 2 strains in AIDS-DLCL overlapped with that of the general population, and no correlation was found with the evaluated clinicoepidemiological data of patients, that is, disease site, tumor histology, CD4+ cell counts, and HIV transmission route. In conclusion, we found that the distribution of the EBV genotype in all of the AIDS-NHL samples analyzed is similar to the viral representation found in control individuals of both immunocompetent and immunocompromised populations.

This study was aimed at defining the histogenesis of the pathologic spectrum of lymphoma arising in the context of human immunodeficiency virus (HIV) infection. Toward this aim, 87 AIDS-related non-Hodgkin lymphomas (AIDS-NHL) and 16 Hodgkin lymphomas arising in HIV+ patients (HIV-HL) were comparatively analyzed for the expression pattern of several B-cell histogenetic markers, including BCL-6 (expressed by centroblasts and centrocytes), MUM1/IRF4 (expressed by late centrocytes and post–germinal center [GC] B cells), and CD138/syn-1 (expressed by post-GC B cells). Expression of MUM1, BCL-6, and syn-1 segregated 3 major phenotypic patterns among AIDS-NHL and HIV-HL: (1) the BCL-6+/MUM1−/syn-1− pattern, selectively clustering with a large fraction of AIDS-Burkitt lymphoma (17 of 19) and of systemic AIDS–diffuse large cell lymphoma (12 of 16); (2) the BCL-6−/MUM1+/syn-1−pattern, associated with a fraction of AIDS-immunoblastic lymphoma (8 of 24); and (3) the BCL-6−/MUM1+/syn-1+ pattern, associated with systemic and primary central nervous system immunoblastic lymphoma (14 of 24) and with primary effusion lymphoma (10 of 10), plasmablastic lymphoma of the oral cavity (7 of 7), and HIV-HL (15 of 16). Analysis of nonneoplastic lymph nodes showed that the 3 phenotypic patterns detected in AIDS-NHL and HIV-HL correspond to distinct stages of physiologic B-cell development—centroblasts (BCL-6+/MUM1−/syn-1−), late GC/early post-GC B cells (BCL-6−/MUM1+/syn-1−), and post-GC B cells (BCL-6−/MUM1+/syn-1+). Expression of the Epstein-Barr virus-encoded latent membrane protein-1 clustered with the BCL-6−/MUM1+/syn-1+profile throughout the clinicopathologic spectrum of AIDS-NHL and HIV-HL. Overall, these results define novel histogenetic subsets of AIDS-NHL and HIV-HL and may provide novel tools for refining the diagnosis of these disorders.

BCL-6 mutations are accumulated during B-cell transit through the germinal center (GC) and provide a histogenetic marker for B-cell tumors. On the basis of a comprehensive analysis of 308 B-cell neoplasms, we (1) expand the spectrum of tumors associated withBCL-6 mutations; (2) corroborate the notion that mutations cluster with GC and post-GC B-cell neoplasms; and (3) identify heterogeneous mutation frequency among B-lineage diffuse large cell lymphoma (B-DLCL) subsets. Mutations are virtually absent in acute lymphoblastic leukemia (P < .001) and mantle cell lymphoma (P < .05), whereas they occur frequently in GC or post-GC neoplasms, including lymphoplasmacytoid lymphoma, follicular lymphoma, MALT lymphomas, B-DLCL and Burkitt lymphoma. Among B-DLCL, mutations occur frequently in systemic nodal B-DLCL, primary extranodal B-DLCL, CD5+ B-DLCL, CD30+ B-DLCL, and primary splenic B-DLCL, suggesting a similar histogenesis of these B-DLCL subsets. Conversely, mutations are rare in primary mediastinal B-DLCL with sclerosis (10.0%; P < .01), supporting a distinct histogenesis for this lymphoma. Longitudinal follow-up of B-DLCL transformed from follicular lymphoma shows that theyBCL-6 mutations may accumulate during histologic progression. Mutations also occur in some B-cell chronic lymphocytic leukemias, small lymphocytic lymphomas, and hairy cell leukemias, consistent with the hypothesis that a fraction of these lymphoproliferations are related to GC-like cells. Finally, the molecular pattern of 193 mutational events reinforces the hypothesis that mutations ofBCL-6 and immunoglobulin genes are caused by similar mechanisms.

Primary central nervous system lymphoma (PCNSL) is a major complication of the late stages of human immunodeficiency virus (HIV) disease. Epstein Barr virus (EBV) infection is the only genetic lesion consistently associated with this neoplasia. Recently, it has been proposed that the pathogenesis of AIDS‐related PCNSL (AIDS‐PCNSL) may be associated with infection by human herpesvirus‐8/Kaposi's sarcoma associated herpesvirus (HHV‐8/KSHV), although at present such association remains controversial. In order to conclusively assess the link between HHV‐8/KSHV infection and AIDS‐PCNSL, we performed a comprehensive study based on multiple molecular assays on cerebral tissues and cerebrospinal fluid (CSF) as well as specific immunologic assays on patients’ sera. A well characterized panel of 33 Italian patients with AIDS‐PCNSL and 13 controls with other HIV‐related brain focal diseases from the same geographical area was analyzed. No signs of HHV‐8/KSHV infection were detected in cerebral tissues by single‐step PCR. Cerebral tissues of all AIDS‐PCNSL scored negative for HHV‐8/KSHV DNA sequences also by nested PCR, with the exception of one single patient who was simultaneously affected by Kaposi's sarcoma. All CSF samples analyzed were consistently devoid of HHV‐8/KSHV sequences by molecular assays. By serologic assays, detecting both latent and lytic HHV‐8/KSHV antigens, a specific immunoreactivity was observed in 16/33 (48%) AIDS‐PCNSL and in 6/13 (46%) controls (P=0.88). A significant correlation with HHV‐8/KSHV serum reactivity was seen with a homosexual route of HIV transmission (P=0.018), but not with the presence of AIDS‐PCNSL. The results of our analysis conclusively assess that HHV‐8/KSHV infection is not a feature of AIDS‐PCNSL.

This study was aimed at defining the histogenesis of the pathologic spectrum of acquired immunodeficiency syndrome–related non-Hodgkin's lymphomas (AIDS-NHL), including AIDS-related small noncleaved cell lymphoma (AIDS-SNCCL), AIDS-related large noncleaved cell lymphoma (AIDS-LNCCL), AIDS-related large cell immunoblastic lymphoma plasmacytoid (AIDS-IBLP), and AIDS-related primary effusion lymphoma (AIDS-PEL). Forty-six cases of AIDS-NHL were investigated for the expression pattern of BCL-6, a protein specifically expressed by germinal center (GC) B-cells, and CD138/syndecan-1 (syn-1), a marker of post-GC B-cell differentiation. Expression of BCL-6 and syn-1 segregated two major phenotypic patterns among AIDS-NHL: (1) the BCL-6+/syn-1- pattern associated with AIDS-SNCCL and AIDS-LNCCL; (2) the BCL-6-/syn-1+ pattern associated with AIDS-IBLP and AIDS-PEL. Among systemic AIDS-NHL infected by Epstein-Barr virus (EBV), expression of the EBV-encoded latent membrane protein-1 (LMP-1) preferentially associated with the BCL-6-/syn-1 + profile. Analysis of nonneoplastic lymph nodes showed that the two phenotypic patterns detected in AIDS-NHL correspond to physiologic stages of B-cell development, ie, GC B-cells (BCL-6+/syn-1-) and preterminally differentiated post-GC B-cells (BCL-6-/syn-1+). Thus, BCL-6+/syn-1- AIDS-NHL reflects a GC stage of differentiation, whereas AIDS-NHL which are BCL-6-/syn-1+, and LMP-1+when infected by EBV, derive from B cells that have entered post-GC plasmacell differentiation. These findings are relevant for the pathogenesis and differential diagnosis of AIDS-NHL.

The tumor cells in most cases of Hodgkin's disease (HD) have been recently recognized to originate from the B-cell lineage, but their precise differentiation stage is not fully clarified. Recently, we have reported that the histogenesis of B-cell lymphomas may be assessed by monitoring the expression pattern of BCL-6, a transcription factor expressed in germinal center (GC) B cells, and CD138/syndecan-1 (syn-1), a proteoglycan associated with post-GC, terminal B-cell differentiation. In this study, we have applied these two markers to the study of HD histogenesis. We have found that in nodular lymphocyte predominance HD (NLPHD) tumor cells consistently display the BCL-6+/syn-1- phenotype, indicating their derivation from GC B cells. Conversely, classic HD (CHD) is heterogeneous because the tumor cells of a fraction of CHD display the BCL-6-/syn-1+ phenotype of post-GC B-cells, whereas another fraction of CHD is constituted by a mixture of tumor cells reflecting the GC (BCL-6+/syn-1-) or post-GC (BCL-6-/syn-1+) phenotypes. BCL-6-/syn-1+ tumor cells of CHD are mostly found surrounded by T cells expressing CD40L, consistent with the observation that CD40 signaling downregulates BCL-6 expression. These data indicate that tumor cells of NLPHD uniformly display a GC B-cell phenotype, whereas the phenotype of tumor cells of CHD appears to be modulated by the surrounding cellular background, particularly CD40L+ reactive T cells.

Acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (AIDS-NHL), a major source of morbidity and mortality among human immunodeficiency virus (HIV)-infected individuals, are derived from B cells and are classified into two major categories, Burkitt's lymphoma (BL) and diffuse large cell lymphoma (DLCL). Anaplastic large cell lymphoma (ALCL) and body-cavity-based lymphoma (BCBL) represent less frequent AIDS-NHL types. The molecular pathogenesis of AIDS-NHL is characterized by distinct genetic pathways, including chromosomal rearrangements of c-MYC and BCL-6 in AIDS-BL and AIDS-DLCL, respectively. In addition to gross rearrangements, recent evidence has suggested that BCL-6 may also be affected by mutations of the gene 5' noncoding regions. Here we have investigated the distribution of BCL-6 mutations in a panel representative of all the AIDS-NHL subtypes. Forty-three AIDS-NHL were analyzed for mutations in the first exon-first intron boundary region of BCL-6. Mutations were detected in all categories of AIDS-NHL (25 of 43 cases; 58%), including 12 of 20 AIDS-BL, 10 of 15 AIDS-DLCL, two of three AIDS-ALCL, and one of five of AIDS-BCBL. BCL-6 mutations occurred independent of BCL-6 rearrangements and presence of other genetic lesions frequently associated with AIDS-NHL. These results indicate that mutations of BCL-6 5' noncoding regions represent the most common genetic alteration presently detectable in AIDS-NHL. The frequency of these mutations, as well as their location in the proximity of BCL-6 regulatory sequences, suggest that they may play a role in AIDS-related lymphomagenesis.

Primary central nervous system lymphoma (PCNSL) is a major cause of morbidity and mortality among human immunodeficiency virus (HIV)-infected individuals. The precise histogenetic derivation and the molecular pathogenesis of PCNSL is poorly understood. In an attempt to clarify the histogenesis and pathogenesis of these lymphomas, 49 PCNSL (26 acquired immunodeficiency syndrome [AIDS]-related and 23 AIDS-unrelated) were analyzed for multiple biologic markers, which are known to bear histogenetic and pathogenetic significance for mature B-cell neoplasms. PCNSL associated frequently (50.0%) with mutations of BCL-6 5' noncoding regions, which are regarded as a marker of B-cell transition through the germinal center (GC). Expression of BCL-6 protein, which is restricted to GC B cells throughout physiologic B-cell maturation, was detected in 100% AIDS-unrelated PCNSL and in 56.2% AIDS-related cases. Notably, among AIDS-related PCNSL, expression of BCL-6 was mutually exclusive with expression of Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP)-1 and, with few exceptions, also of BCL-2. All but one PCNSL expressed hMSH2, which among mature B cells selectively stains GC B cells. These data suggest that PCNSL may be frequently related to GC B cells and may be segregated into two major biologic categories based on the expression pattern of BCL-6, LMP-1, and BCL-2. BCL-6+/LMP-1-/BCL-2- PCNSL occur both in the presence and in the absence of HIV infection and consistently display a large noncleaved cell morphology. Conversely, BCL-6-/LMP-1+/BCL-2+ PCNSL are restricted to HIV-infected hosts and are represented by lymphomas with immunoblastic features. These data are relevant for the pathogenesis and histogenesis of PCNSL and may be helpful to segregate distinct biologic and prognostic categories of these lymphomas. © 1998 by The American Society of Hematology.

Low grade B-cell non-Hodgkin's lymphomas (B-NHL) represent a markedly heterogeneous group of lymphoproliferative disorders, including B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CCL/SLL), lymphoplasmacytoid lymphoma (LPL), follicular lymphoma (FL), mucosa-associated lymphoid tissue lymphoma (MALTL), and splenic lymphoma with villous lymphocytes (SLVL). The molecular pathogenesis of low grade B-NHL is characterized by distinct genetic pathways which selectively associate with each clinico-pathologic category. At diagnosis, B-CLL/SLL frequently display deletions of 13q14 and tri-somy 12, whereas evolution to Richter's syndrome associates with disruption of p.53. LPL carries t(9;14)(p13;q32) in 40-50% of the cases, leading to the deregulated expression of the PAX-5 gene. FL consistently harbors rearrangements of BCL-2 independent of the cytologic variant. With time, a fraction of FL cases accumulates mutations of p.53 and evolves into a high grade B-NHL. Low grade MALTL are characterized by the frequent occurrence of tri-somy 3 and, occasionally, by p53 mutations. SLVL cames p.53 mutations in a fraction of cases. The identification of distinct genetic categories among low grade B-NHL may help in the therapeutic stratification of these disorders. In addition, genetic lesions of low grade B-NHL have proved to be a useful molecular marker for monitoring minimal residual disease.

Human herpesvirus type-8 (HHV-8) is a lymphotropic herpesvirus originally identified in Kaposi's sarcoma. Among lymphoproliferative disorders, HHV-8 infection is restricted to body-cavity-based lymphoma (BCBL) and multicentric Castleman's disease (MCD). BCBL are B-cell lymphomas growing in liquid phase in the body cavities and most frequently associated with AIDS. BCBL express indeterminate phenotypes, in all cases are associated with HHV-8 infection, and frequently carry Epstein-Ban virus genomes in the absence of c-MYC rearrangements or other genetic lesions characteristic of B-cell lymphomas. The clinical outcome of BCBL is poor with a median survival of only few months. MCD is an atypical lymphoproliferative disorder which displays marked vascular hyperplasia and is commonly associated with Kaposi's sarcoma. HHV-8 infection occurs in 100% of AIDS-related MCD and in approximately 40% of AIDS-unrelated cases. Overall, the consistency of HHV-8 infection in BCBL and MCD, its selectivity throughout the spectrum of lymphoproliferative disorders and the high copy number of HHV-8 DNA sequences in infected cells suggest that the virus plays a pathogenetic role in these disorders.

Common-variable immunodeficiency (CVI) patients develop non-Hodgkin's lymphomas (NHL), mainly B-lineage diffuse large-cell lymphomas (DLCL), with a high relative risk. The molecular pathogenesis of CVI-related NHL (CVI-NHL) is unknown. Here we aimed at providing a detailed molecular characterization of CVI-NHL. Rearrangements of BCL-6were detected in two thirds of CVI-NHL cases examined. All 3 CVI-NHL also harbored point mutations of the BCL-65` noncoding regions, which constitute a marker of B-cell transit through the germinal center (GC). The number and molecular pattern of BCL-6mutations in CVI-NHL were similar to that detected in DLCL of immunocompetent hosts and in DLCL arising in other immunodeficiency settings. Microsatellite instability occured in one CVI-NHL devoid of a BCL-6rearrangement. All CVI-NHL scored negative for genetic lesions of BCL-2, p53, c-MYC, RELas well as for viral infection by EBV and HHV-8. Overall, these data indicate that: similarly to other immunodeficiency-related NHL, involvement of BCL-6occurs frequently also in CVI-NHL; and because BCL-6mutations are acquired by B cells during GC transit, their occurrence in CVI-NHL suggest that these lymphomas are histogenetically related to GC B cells. HUMPATHOL31:871-873.

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Abstract 283

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The identification of stereotyped immunoglobulin (IG) receptors has improved our knowledge on the pathogenesis of several B-cell malignancies, suggesting the role of antigen-driven stimulation in chronic lymphocitic leukemia (CLL), marginal-zone lymphoma (MZL) and mantle-cell lymphoma (MCL). Multiple myeloma (MM) is a terminally-differentiated neoplasm no longer expressing surface IG; however some reports suggest the existence of early B-lymphocyte precursors which could be susceptible to antigen-driven stimulation. IG heavy chain (IGH) repertoire has not been extensively investigated in MM, with the largest available reports containing less than 80 complete sequences.To address this issue we created a database of MM IGH sequences including our institutional records (mostly derived from minimal residual disease studies) and sequences available from the literature. We planned a two-step analysis: a) first we characterized the MM repertoire and performed intra-MM clustering analysis; b) then we compared our MM series to a large public database of IGH sequences from neoplastic and non-neoplastic B-cells in search of similarities between MM sequences and other normal or neoplastic IGH repertoires.131 MM IGH genes were amplified and sequenced at our Institutions and belonged to Italian patients, while 214 MM IGH sequences from non-Italian patients were derived from published databases (NCBI-EMBL-IMGT/LIGM-DB) for a total of 345 fully interpretable MM sequences (out of 396). 28590 IGH sequences from other malignant and non-malignant B-cells were retrieved from the same public databases, including approximately 4500 CLL/Non-Hodgkin lymphoma (NHL) sequences and comprising 500 sequences from Italian patients. All sequences were analyzed using the IMGT database and tools (Lefranc et al., Nucleic Acid Res. 2005; http://imgt.cines.fr/) to identify IGHV-D-J gene usage, to assess the somatic hypermutation (SHM) rate and to identify HCDR3. HCDR3 aminoacidic sequences were aligned together using the ClustalX 2.0 software (Larkin et al., Bioinformatics, 2007; http://www.clustal.org/). Subsets of stereotyped IGH receptors were defined according to Stamatopoulos et al. (Blood, 2007).IGHV analysis in MM was almost in keeping with the normal B-cell repertoire, showing a less remarkably biased IGH usage compared to CLL, MCL and MZL (with seven genes accounting for 40% of cases, compared to respectively five, three and two genes). However, a modest but significant over-representation of IGHV1-69, 2–5, 2–70, 3–21, 3–30-3, 3–43, 5–51 and 6-1 genes and under-representation of the IGHV1-18, 1–8, 3–30, 3–53 and 4–34 was noticed. The rate of somatic hypermutation in MM followed a Gaussian distribution with a median value of 7.8%. Intra-MM search for HCDR3 similarities never met minimal requirements for stereotyped receptors. When MM sequences were compared to non-MM database, only a minority of MM sequences (2.6%, n=9) clustered with sequences from lymphoid tumors and normal B-cells (figure 1A). In particular two non-Italian MM sequences clustered with previously characterized, uncommon CLL subsets (n.37 and n.71 according to Murray et al., Blood 2008). Moreover, novel provisional clusters were observed including three MM-CLL subsets, one MM-NHL subset, and three MM-normal B-cell subsets. While the MM-normal B-cell clusters involved non-Italian patients, we unexpectedly noticed that the four MM-CLL/MM-NHL clusters were composed exclusively of Italian patients, as shown in figure 1B, although Italian subjects represented less than 12% of the entire CLL-NHL database.The analysis of the largest currently available database of MM IGH sequences indicates the following: 1) MM IGH repertoire is closer to physiological distribution than that of CLL, MCL and MZL; 2) MM specific clusters do not occur to a frequency detectable with currently available databases; 3) 98% of MM sequences are not related to other “highly-clustered” lymphoproliferative disorders; 4) Uncommon clustering phenomena may follow a geographical rather than a disease-related pattern.No relevant conflicts of interest to declare.

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In the last years, the B cell receptor (BCR) has become a key molecule in chronic lymphocytic leukemia (CLL), given the correlation between mutational status of immunoglobulin heavy chain variable (IGHV) genes and disease prognosis. Recently, a fraction of CLL has been shown to preferentially express specific IGHV genes, often in a non-random combination with homologous heavy chain complementarity-determining region-3 (HCDR3) and peculiar light chains. Some of these stereotyped BCR mark CLL subsets with peculiar clinical behavior regardless of IGHV mutations. These data suggest a role for BCR in defining the clinical and biological features of CLL, also beyond the mutational status of IGHV genes.A HCDR3-driven clustering of 1,426 IG sequences (1,398 patients) was performed using ClustalX(1.83). Time to treatment (TTT) intervals, Rai staging, IGHV mutational status, CD38, ZAP-70, and karyotype abnormalities evaluated by FISH were available for 617 patients. Gene expression profiling (GEP) and quantitative real-time PCR experiments (QRT-PCR) were performed on purified CLL cells.IGHV3-23 was totally absent in 71 identified stereotyped clusters despite being the second most frequently used IGHV gene, such distribution was significantly skewed (p<0.0001), compared with the distribution of IGHV genes belonging to stereotyped BCR clusters observed in our series. Although 109/134 IGHV3-23 were mutated (M), alignment of IGHV sequences revealed a high degree of conservation in the context of the 13 AA positions involved in superantigen binding by IGHV3 subgroup genes, suggesting that the majority of M IGHV3-23 cases maintained the capacity to mediate superantigen recognition and binding. Median TTT (73 months) of 43 M IGHV3-23 CLL was significantly shorter than median TTT (253 months, p=0.0153) of 333 M CLL, as well as of 326 M CLL in which 7 cases belonging to the bad prognosis IGHV3-21/IGLV3-21 cluster were excluded (253 months, p=0.0082). Multivariate Cox proportional hazard analyses selected IGHV3-23 usage (p=0.029), Rai stage (p<0.0001) and FISH group (p<0.0001) as independent markers of disease progression for 376 M CLL, and for the cohort in which 7 M CLL from the IGHV3-21/IGLV3-21 cluster were excluded. Comparing 5 M IGHV3-23 and 22 M non-IGHV3-23 CLL for their differential GEP, 212 genes were selected, 108 up-regulated and 104 down-regulated in M IGHV3-23 CLL. Using the “Gene-Ontology Tree Machine” platform, a set of growth/tumor suppressor genes (PDCD4, TIA1, RASSF5), all down-regulated in M IGHV3-23 CLL, was constantly found in several gene-ontology categories related to apoptosis. QRT-PCR confirmed a significant down-regulation of these genes in 15 M IGHV3-23 compared to 35 M non-IGHV3-23 CLL. Given the notion that PDCD4 and TIA1 are among the genes under control of miR-15a and miR-16-1 a “Gene Set Enrichment Analysis” carried out on the 212 differentially expressed genes, confirmed that M IGHV3-23 samples were significantly deprived in genes whose expression is under control of miR-15a and miR-16-1. Accordingly, QRT-PCR experiments performed on 15 M IGHV3-23 and 35 M non-IGHV3-23 CLL revealed significant higher levels of both miR-15a (p=0.0007) and miR-16-1 (p=0.0031) in M IGHV3-23 cases. No difference was found in the distribution of patients with 13q14 deletion between M IGHV3-23 CLL and M non-IGHV3-23 CLL (p=0.19). Considering the cases used for microRNA expression experiments (data available in 47/50 cases), 8/15 M IGHV3-23 CLL bore the 13q14 deletion in more than 20% of nuclei, against 19/32 cases in the group of M non-IGHV3-23 CLL (p=0.94).Expression of IGHV3-23 marks a subset of M CLL with a worse prognosis; such a peculiar clinical behavior may be related to superantigen stimulation combined with down-regulation of specific growth/tumor suppressor genes and up-regulation of miR-15a and miR-16-1.No relevant conflicts of interest to declare.

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Characterization of the IgH receptor provides useful insights to understand the pathogenesis and natural history of lymphoid tumors. For example the recognition of stereotyped clusters of immunoglobulin receptors has been a major step forward to understand the pathogenesis of chronic lymphocytic leukemia (CLL). IgH rearrangements have been less extensively investigated in MM, mostly because of lack of large databases of IgH sequences. At our Institution a large number of MM patients has undergone IgH sequencing for minimal residual disease (MRD) evaluation. This database has been merged with MM IgH sequences available in the literature, resulting in 308 MM sequences which have been employed to comprehensively investigate the characteristics of the IgH rearrangement in this tumor.131 IgH genes from MM patients were amplified and direct sequenced at our Institution (mostly for MRD detection purposes) from specific cDNA obtained at diagnosis, as already described (Voena et al, Leukemia 1997). 177 MM IgH sequences were derived from published databases (NCBI and EMBL). To further characterize the IgH repertoire, we have then compared the MM complementarity-determining region 3 (HCDR3) amino acid (AA) sequences to a panel of productive, non redundant 27413 HCDR3 AA sequences retrieved from public databases (EMBL, NCBI, IMGT/LIGM-DB: 25655 sequences) and from our unpublished laboratory database (1758 sequences), including sequences from malignant B-cell clones (3197 CLL, 1217 lymphomas) and from non malignant repertoire (2685 autoreactive, 4408 immunederegulation/immunodeficiency, 15429 normal and 477 phage display libraries). All the sequences have been analyzed using the IMGT database and tools (Lefranc et al., Nucleic Acid Res. 2005; http://imgt.cines.fr/) to identify IGHV, IGHD and IGHJ gene usage, to assess the somatic hypermutation (SHM) rate and to identify HCDR3 AA sequences. HCDR3 AA sequences were aligned together, in search of subsets of stereotyped receptors, using the ClustalX 2.0 software (http://www.clustal.org/). To define subsets of stereotyped IgH receptors, we followed the criteria proposed by Messmer et al. (J Exp Med. 2004) and Stamatopoulos et al. (Blood, 2007).No significant differences were noted between our Institutional and published MM databases. Overall IGHV usage in MM appeared in keeping with the normal B cell repertoire with predominance of IGHV3 family (53.9%) followed by IGHV4 (slightly under-represented in MM, 17.2% vs 23.2%, p=0.02) and IGHV1 (12%); besides an over-representation of IGHV2 (7.8% vs 2.3%, p<0.001) was noticed. A modest but significant (p<0.05) over-representation of the IGHV3-9 (5.2% vs 2.6%), IGHV3-21 (4.5% vs 2%), IGHV5-51 (4.5% vs 2.2%) genes and under-representation of the IGHV3-23 (8.1% vs 12.2%) and IGHV4-34 (1% vs 6.5%, p<0.001) were observed. IGHD and IGHJ followed a distribution similar to that of normal IgH repertoire. The median SHM rate was 7.5% (range 0-28%): interestingly we found one single patient with 100% identity to the germline sequence and only three patients with >98% identity. The median length of the HCDR3 was 15 AA (range 7-29), again in line with normal IgH repertoire. Intra MM search for HCDR3 similarity showed no association which met minimal requirements to define stereotyped receptors. The comparison of MM sequences with non MM database showed that 98% of MM sequences are unrelated to known CLL subsets. Among the remaining, 3 MM sequences could be assigned to previously identified CLL subsets (namely n. 25, n. 37 and n. 71 according to Murray et al., Blood 2008) whereas 2 MM formed 2 novel provisional subsets with CLL. When HCDR3 MM were compared to the database of non MM/CLL HCDR3, similarities were found with clones from normal B cells, while similarities with autoreactive clones and other B cell malignancies were sporadic.The analysis of the largest database of MM IgH sequences so far reported indicate the following: 1) Family usage in the MM IgH repertoire follows a nearly physiological distribution apart for modest skewing of a number of IGHV genes; 2) MM specific HCDR3 clusters do not occur to a frequency detectable with currently available databases; 3) The vast majority of MM sequences are not related to known CLL clusters; 4) MM IgH sequences share more similarities with normal IgH sequences compared to those derived from pathological B lymphoid cells. Thus to state of current knowledge there is little evidence in favor of an antigen driven pathogenesis for this neoplasm.No relevant conflicts of interest to declare.

All somatic cell types, including all B lineage cells, constitutively express a number of DNA repair proteins to maintain genomic stability and thwart tumorigenesis in the face of ongoing constitutive levels of DNA damage. Compromises in expression of these essential DNA repair factors have been shown to readily induce the development of various cancers including B cell lymphomas, suggesting expression levels are tightly regulated and limited in quantity. In germinal center (GC) B cells, however, additional DNA repair capacity is likely required to counterbalance the heightened level of mutagenic activity owing to the induced expression of activation-induced cytidine deaminase (AID) during somatic hypermutation (SHM). AID is a DNA editing enzyme which introduces somatic mutations into immunoglobulin (Ig) V regions at an estimated rate which is almost a million-fold higher than the spontaneous rate in somatic cells.We hypothesize that to maintain the genomic wellness of GC B cells which are undergoing SHM, there is a need for induction of an accompanying robust DNA repair system. We further hypothesize that inefficient induction of these repair genes may predispose to malignant transformation.Using tonsillar tissue sections and purified tonsillar B cell subpopulations, we compared expression levels of various DNA repair genes and related these levels to AID expression across the subsets using immunohistochemistry, real-time RT-PCR, and Western blot analysis. To characterize the nature of signals capable of inducing expression levels of AID and/or DNA repair proteins, peripheral blood B cells were activated in vitro using a panel of stimuli, including coculture with activated CD4 T cells. As a surrogate measure of mismatch repair (MMR) activity in the relative absence of T cell help, we quantitated the number of somatic hypermutations at A/T sites in the Ig heavy chain variable (IGHV) region genes in a collection of IGHV sequences obtained from normal B cells and HIV-related lymphoma cells.Using immunohistochemistry, we observed that, similar to the expression of AID, DNA MMR genes are significantly induced in tonsillar GC B cells. These results were further validated using a more sensitive real-time RT-PCR assay and analysis by Western blotting. By expanding our DNA repair gene panel, we observed that proteins of homologous recombination, base excision repair and DNA single strand break signalling pathway are also similarly induced in GC B cells at RNA, protein, and functional levels compared to their expression in naïve and memory B cells. By contrast, expression of non-homologous end joining and DNA double strand break signalling molecules are unchanged. We have termed this selective induction of repair mechanisms in GC B cells as somatic hyperrepair (SHR). To identify pathways that lead to the activation of AID and SHR, we used an in vitro system and a variety of stimuli and we discovered that multiple B cell stimuli including CpG, CD40L, and anti-BCR could each independently induce the expression of AID while SHR induction strictly required the engagement of CD4+ T cells. This provocative observation suggests a novel role for CD4+ T cells in mitigating tumorigenesis of post-GC B cells through their ability to induce the SHR pathway in cells that have been induced to undergo SHM. To demonstrate the possible role of SHR in lymphomagenesis, we analysed the mutation pattern of IGHV genes from a panel of B cell lymphomas obtained from HIV infected (CD4+ T cell suppressed) patients. We found that HIV-related lymphoma cells displayed a significantly lower frequency of SHM at A/T positions relative to normal memory B cells, indicative of compromised MMR of their precursor cells during GC transit. Our findings resolve the lingering paradox that B cell malignancies are overwhelmingly prevalent under T cell suppression conditions such as HIV infection, post-organ transplant, and aging. Finally, our results also suggest for the first time that mounting efficient tumor suppression for some cells may depend on signals transmitted by neighboring cells and the specific microenvironment.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Scant information is available on the impact of the host genetic background as a predictor of outcome and toxicity in diffuse large B cell lymphoma (DLBCL) treated with R-CHOP. We verified whether relevant single nucleotide polymorphisms (SNPs) of the host may further refine prognostic stratification and toxicity prediction in DLBCL treated with R-CHOP21. The study was based on 106 consecutive DLBCL provided with a prospectively collected dataset. Candidate SNPs belonged to genes known to be involved in the pharmacogenetics of cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab, and included SNPs affecting: drug metabolism (cytochrome-P450: CYP2B6- 25504C>T, CYP2B6-15630G>T, CYP2C19-19153G>A, CYP3A4--392A>G, CYP3A4- 14365T>G, CYP3A5-6980G>A); drug detoxification: (gluthatione-S-transferase: GSTA1--4621T>C, GSTM1-null, GSTP1-1374A>G, GSTP1-2264C>T); drug transport (multidrug-resistance-related proteins: ABCC1-129623G>T, ABCC2-53394T>A, ABCC2-68692G>A, ABCB1-90855C>T, ABCB1-49691G>A, ABCG2-8824C>A, ABCG2-33G>A; and drug pharmacodynamics (NADPH-subunits: NCF4--368A>G, RAC2-7418T>A, CYBA-4185C>T; TP53: p53-440G>C; Fcγ receptor: FCGR2A- 4487A>G, FCGR3A-5092T>G, FCGR3A-1301T>G/A; glucocorticoid receptor: GR- 1087A>G, GR-67G>A, GR-1829C>G). Genotyping of candidate SNPs was performed on PBMC collected at DLBCL diagnosis by SNP-minisequencing. Primary end points were event free survival (EFS) and toxicity. Associations of SNPs with primary endpoints were controlled for multiple testing by false discovery rate (FDR) analysis. Univariate log-rank analysis controlled for multiple comparisons by FDR testing identified GSTA1-- 4621C>T, CYBA-4185C>T, and ABCC2-53394T>A as predictors of EFS. GSTA1 encodes a glutathione S-transferase facilititating solubility and excretion of cyclophosphamide and its derivatives. Patients carrying the GSTA1--4621 CT/TT genotypes displayed a better EFS (3-year EFS: 68.4%; SE: 5.9%) compared to GSTA1--4621 CC carriers (3-year EFS: 39.2%; SE: 9.2%) (p=.025; q=.200). CYBA encodes a NAD(P)H oxidase subunit. Patients carrying the CYBA-4185 TT genotype displayed a poorer EFS (3-year EFS: 41.7%; SE: 12.4%) compared to CYBA-4185 CT/CC carriers (3-year EFS: 61.2%; SE: 5.8%) (p=.008; q=.156). ABCC2 encodes an ATP-binding cassette transporter (MRP2) acting as an efflux system for doxorubicin and vincristine. Patients carrying the ABCC2-53394 AT/AA genotypes displayed a poorer EFS (3-year EFS: 34.1%; SE: 11.5%) compared to ABCC2- 53394 TT carriers (3-year EFS: 64.0%; SE: 5.8%) (p=.013; q=.156). Multivariate analysis identified GSTA1--4621 CT/TT (HR: 0.39; 95% CI 0.21–0.73; p=.004) and CYBA-4185 TT (HR: 2.01; 95% CI 1.01–3.97; p=.045) as independent predictors of EFS in DLBCL treated with R-CHOP21, along with IPI 3–5 (HR: 4.88; 95% CI 2.24–8.55; p= 1.5 × 10−4). CYBA- 4185C>T and GSTA1--4621C>T also predicted outcome in DLBCL subgroups by IPI. The impact of SNPs was also evaluated for toxicity in 658 R-CHOP21 courses by logistic regression analysis adjusted for age, sex, ECOG PS, IPI, comorbidities, organ function, and doses of cyclophosphamide, doxorubicin and vincristine. Generalized estimating equations (GEE) were used to build a multivariate model for toxicity, which adjusts for the clustering of treatment courses within a patient. NCF4--368AG/GG was an independent predictor of low risk of hematologic toxicity G3-4 (HR:0.45; p=.018), infection (HR:0.46; p=.003), and cardiac toxicity (HR:0.37; p=.023). The implications of these results are twofold. First, host SNPs affecting doxorubicin and/or cyclophosphamide pharmacodynamics (CYBA-4185C>T) and detoxification (GSTA1--4621C>T) are independent predictors of outcome in DLBCL treated with R-CHOP21. Poor EFS heralded by CYBA-4185C>T, a nonsynonimous SNP of NADPH-oxidase p22phox, may be due to reduced production of reactive oxygen species (ROS) that mediate doxorubicin cytotoxicity. Favorable EFS associated with GSTA1--4621C>T may be related to reduced expression of glutathione- S-transferase A1, a phase II enzyme involved in cyclophosphamide and doxorubicin detoxification. Second, NCF4--368AG/GG, a SNP belonging to NADPH-oxidase p40phox and regulating ROS generation, has an independent protective role against both hematologic and non-hematologic toxicity.

CLL harboring del17p13 displays a dismal prognosis and a high risk of chemorefractoriness. We tested whether TP53 inactivation through mutation harbors the same prognostic role as del17p13 on 308 consecutive CLL. TP53 mutation status was assessed at diagnosis by direct sequencing of TP53 exons 2–10. Thirty-two TP53 mutations were observed in 31/308 (10.0%) CLL. Among 26 missense mutations, the median residual transactivation activity compared to germline TP53 was 8.9% (25th–75th percentile: 0.6–13.0%), and was ≤20% in 22/26 (84.6%) mutations. All microdeletions and microinsertions (4/32; 12.5%) led to frameshift. One non-sense mutation introduced a stop codon at position 213, and one mutation affected the 3′ splicing site of exon 8. Despite the statistical association between TP53 mutations and del17p13 in the whole cohort, the overlap between the two genetic lesions was restricted to a fraction of patients. By combining results of FISH and TP53 mutation analysis, 44/297 (14.8%) CLL carried TP53 inactivation: 18/44 (40.9%) showed TP53 mutation paired to del17p13; 10/44 (22.7%) showed TP53 mutations in the absence of del17p13; and 16/44 (36.3%) showed del17p13 in the absence of TP53 mutations. Univariate log-rank analysis identified both TP53 mutations and del17p13 as risk factors for short overall survival (OS). Median OS for patients with TP53 mutations was 79.6 months (5-year OS 66.2%), whereas median OS for patients without TP53 mutations was not reached and the 5-year OS was 85.2% (p<.001). Multivariate analysis selected TP53 mutations (HR 3.20; p=.002), along with age >65 years (HR 4.98; p<.001) and advanced Binet stage (HR 3.01; p<.001), as independent predictors of OS after adjustment for del17p13 and other potentially confounding biological and clinical covariates. Univariate log-rank analysis identified both TP53 mutations and del17p13 as risk factors of short time to chemorefractoriness. Median time to chemorefractoriness for patients with TP53 mutations was 6.3 months versus 72.7 months for patients without TP53 mutations (p<.001). Multivariate analysis selected TP53 mutations (HR 3.97; p<.001) as independent predictor of short time to chemorefractoriness after adjustment for del17p13 and other potentially confounding covariates. OS and time to chemorefractoriness did not differ between CLL harboring TP53 mutations in the absence of del17p13 and CLL harboring del17p13. Patients harboring TP53 mutations only and patients harboring both TP53 mutation and del17p13 were characterized by short time to chemorefractoriness compared to patients without any TP53 alteration or to patients harboring del17p13 only (p<.05). CLL that at diagnosis harbored del17p13 without TP53 mutations showed a significantly longer time to chemorefractoriness compared to CLL who at diagnosis carried both del17p13 and TP53 mutations (median: 55.2 months vs 5.5 months, respectively; p=.003). Notably, at diagnosis, the percentage of deleted nuclei was significantly lower in CLL harboring del17p13 only (median: 45.0%, 25th–75th percentiles: 7.3–67.4%) compared to CLL with both del17p13 and TP53 mutations (median: 66.5%, 25th–75th percentiles: 52.1–77.9%, respectively) (p=.020). The value of TP53 mutations in predicting chemorefractoriness was also assessed in patients who had been exposed to fludarabine (n=88). Multivariate analysis selected TP53 mutations as the sole independent predictor of short time to fludarabine refractoriness (HR 6.72; p<.001). Sequential samples were available for 14 CLL who had no TP53 abnormalities at diagnosis and who later developed chemorefractoriness. At the time of chemorefractoriness, TP53 abnormalities were acquired in 5/14 (35.7%) cases. The implications of this study are threefold: mutation in the absence of del17p13 is the sole mechanism of TP53 inactivation in 20% CLL harboring TP53 disruption; TP53 mutations carry the same prognostic relevance as del17p13 in terms of CLL progression, survival and risk of chemorefractoriness; because of the practical implications for choice of therapy, screening for TP53 mutations, in addition to del17p13 assessment, should be included in the initial prognostic assessment of CLL.

Background and aims. In CLL, TL has been associated to outcome. However a larger patient sample and an analysis on a blinded validation series are required to fully establish the independent prognostic value of TL and to define its impact on prognostic subgroups defined according to established predictors. These issues have been addressed on a CLL learning cohort and validated on an independent blinded cohort, overall accounting for 401 CLL patients. Also, we have tested TL as a risk factor of Richter’s Syndrome (RS), an extremely severe event that most of the currently available biomarkers fail to predict. Methods. The learning series (LS) included 191 patients from the university of Torino (UT), while the blinded validation series (BVS) included 210 patients from University of Eastern Piedmont (UEP). TL was assessed on PBMC collected at diagnosis by Southern blotting and no biological or clinical feature of BVS patients was available to the laboratory performing the analysis. Detailed clinical history, clinical parameters at diagnosis as well as VH-mutational status, cytogenetics, CD38 expression and Zap-70 were available for the vast majority of patients and employed together with TL as covariates for multivariate survival analysis. Results. The two series showed no differences for any clinical and biological features except age that was slightly higher in the BVS series. Also TL distribution was similar in the two series (median TL 6024 and 5959 bp respectively). By applying ROC analysis and Youden’s index to the LS we identified a cut-off point of 5000bp segregating 26% (104/401) of patients in the high-risk subgroup. TL was a powerful and independent outcome predictor for both TFS (24.6 vs 73 months p<.001, Fig.1A) and OS (105.5 vs 281 months p<0.001, Fig. 1B). The same cut-off was then applied to the BVS and again TL emerged as strong predictor for TFS (15.2 vs 130.8 months; p<.001) and OS (79.8 vs not reached; p<.001). Multivariate analysis in the BVS selected TL as an independent predictor for both TFS (p=.002) along with peripheral blood lymphocytes (p<.001), CD38 expression (p<.001), beta-2-microglobulin levels (p=.001), and unfavorable FISH karyotype (p=.046), and OS (p.035) (along with age >65 years, p<.001 and advanced Binet stage p=.015). The strict biopsy policy adopted at UEP in case of suspected RS allowed to demonstrate that TL≤5000 acts as an independent predictor also for this event (five-year risk: 18.9% vs 6.4%; p=.016 HR 2,70) (Fig 1C). Finally we analyzed how TL performed in conjunction to established prognostic indicators: TL segregated a CLL group displaying short TFS and OS despite being characterized by Binet A (p<.001), IGHV-homology< 98% (p<.001), CD38<30% (p<.001) and ZAP70<20% (p<.001) and favorable FISH (p<.001). Also, among CLL carrying unfavorable prognosticators, TL<5000 bp identified a CLL subgroup with longer OS despite being characterized by age >65 years (p=.004), Binet stage B-C (p<.001), or unfavorable cytogenetics (p=.001). Conclusions. These results demonstrate that TL is a powerful independent predictor of multiple outcome events in CLL and contributes to refine the prognostic assessment of CLL when utilized in combination with other prognostic markers. We thus recommend a more widespread use of this biomarker in CLL. Figure Figure

Non-Hodgkin’s lymphomas (NHL) represent a frequent complication of HIV infection and a major source of morbidity and mortality amongst patients affected by acquired immunodeficiency syndrome. HIV-related NHL (HIV-NHL) are generally of B-cell origin and are phenotypically and histogenetically related to germinal center (GC) or post-GC B-cells. The pathogenesis of HIV-NHL is a multistep process involving factors provided by the host as well as alterations intrinsic to the tumor clone. Data on the underlying genetics of HIV-NHL are still scarce and mainly based on screening of genes known to be involved in the pathogenesis of lymphomas in an immunocompetent (IC) host. In order to elucidate the molecular pathogenesis of HIV-NHL, we performed a genome-wide DNA profiling based on microarray comparative genomic hybridization in 48 systemic HIV-NHL, including 28 HIV-diffuse large B-cell lymphomas (HIV-DLBCL), 13 HIV-Burkitt lymphomas (HIV-BL), and 7 HIV-Burkitt-like lymphomas (HIV-BLL), as well as in 4 HIV-primary central nervous system lymphoma (HIV-PCNSL) and in 2 HIV-primary effusion lymphomas (HIV-PEL). DNA samples, extracted from frozen biopsies, were analyzed using the Affymetrix Human Mapping 250K Nsp arrays. Aberrations occurring in 20% or more of the cases were defined as recurrent. HIV-DLBCL cases had recurrent gains at 2p15, 3q29, 4q13.2, 8p23.1, 10p12.31, 11q22.1–q24.1, 14q11.2, 14q32.33, 12p13.31-pter, 15q11.2, and recurrent losses at 1p36.32–p36.33, 2p11.2, 5p12, 3p14.2, 7q22.1, 8p23.1, 9p23, 9p24.1 12p11.1, 14q11.2, 15q11.2, 17p13.1–p13.3 and 22q11.1. In less than 20% of cases, gains of 1q, 8q, 21q and loss of 3q, 6q, 8p and 18q were also observed. Remarkably, there were no gains of 18q. HIV-BL had recurrent gains in 1q25.2, 3q29, 8p23.1, 15q11.2, 12p12.1-pter, 20q13.12 and recurrent losses at 15q11.2 and 17p11.2–p13.2. Regions of uniparental disomy longer than 5 Mb, were detected in 45% of the cases, including 7 (58%) HIV-BL, 10 (37%) HIV-DLBCL, 3/7 HIV-BLL and 2/2 HIV-PCNSL. One HIV-DLBCL and a cell line showed intragenic deletions of both the fragile histidine triad gene (FHIT), in the recurrently lost 3p14 region, and of the WW domain-containing oxidoreductase gene (WWOX), at 16q23.1, suggesting a synergistic effect of the loss of the two genes. Inactivation of FHIT gene by aberrant methylation of regulatory regions was investigated by methylation specific-PCR and was observed in 12 (25%) samples (8 HIV-DLBCL, 2 HIV-BL and 1 HIV-PCNSL), all with no 3p deletion. In conclusion, our data show a series of previously unreported recurrent aberrations in HIV-NHL. Also, HIV-BL show less copy number aberrations than HIV-DLBCL, and the pattern of gains and losses in HIV-DLBCL is more similar to the genetic profile observed in IC DLBCL derived from germinal center B-cells than that from activated B-cells (ABC). Finally, FHIT inactivation by DNA loss or promoter methylation, might have a pathogenetic role in at least 50% of HIV-NHL.

Hairy cell leukemia (HCL) is a rare B-cell neoplasm with largely unravelled features of lymphomagenesis. Molecular mechanisms of disease are largely unknown and genomic aberrations have infrequently been investigated in HCL. In order to elucidate the genetic mechanisms of HCL pathogenesis, we analyzed copy number changes and loss of heterozigosity (LOH) by genome wide DNA profiling with the Affymetrix Human Mapping 250k Nsp arrays in 16 HCL. All investigated cases expressed multiple pre- (IgM+/−D) and post-switch (IgG+/−A) isotypes. Tumor IGHV gene transcript homology to germline ranged from 88.4 to 98.6%, with 4/16 HCL having >98% homology. Four of 16 patients were refractory or had progressive disease after first line purine-analogue-based treatments. CD103+ve hairy cells were >60% in all test samples. Genome wide DNA profiling demonstrated gross non recurrent copy number deletions in only 4/16 cases (25%), affecting 14q24-q32, 3q23, 3q27.3-q29, 8p12-pter 10q21-q23, 10q25-q26, 17p11-pter, 17q11-q21, 17q22–23, Xp21.2-p22.11, Xq13.3, Xq21.1-q21.33, Xq22.2–q28, the whole chromosomes 21 and X. Five of 16 patients (31%) presented LOH. Most interestingly, the 5 patients showed LOH regions, with a region of overlap in two patients at 6q25.1, without any copy number changes, indicative of uniparental disomy. Genes involved in bone marrow fibrosis, multiple isotype expression and response to treatment were specifically investigated. Interestingly, among genes critical for fibrosis, FGF12, a member of the fibroblast growth factors family, was specifically targeted by a complex chromosomal rearrangement at 3q27.3-q29, and reverse transcript-polymerase chain reaction confirmed FGF12 transcript expression. Among genes associated with poor response, p53 deletion was documented in a non-responder HCL. Conversely, no abnormalities at 14q32 locus (duplications or gains) were observed at IgH 14q32 region in these multiple IgH isotype expressing HCL. Overall, our analysis showed a highly stable genome with lack of gross recurrent abnormalities, which is an additional unique feature of HCL among B-cell neoplasms. However, our genomic analyses were able to identify specific chromosomal rearrangements targeting genes involved in fibrosis. Lack of gains of 14q32 locus was consistent with our hypothesis that multiple IgH isotypes are generated at RNA level in multiple IgH expressing HCL. The present analysis provides the basis for further molecular and epigenetic investigations.

BCL2 expression is increased in CLL cells and associates with decreased survival of CLL patients. BCL2C938A polymorphism is localized in the inhibitory P2 promoter of BCL2and modulates BCL2expression. Accordingly, BCL2expression is significantly increased in CLL carrying BCL2938 AA genotype, compared to CLL carrying BCL2938 CC genotype. We aimed at testing the prognostic impact of BCL2C938A in a series of 182 consecutive CLL. Genotyping of BCL2C938A was performed by SNP minisequencing. Biological covariates were CD38 expression, IGHV mutation, FISH karyotype, and p53 mutations. Clinical covariates were age, sex, Rai and Binet stages, number of nodal areas, largest lymph node size, splenomegaly, lymphocyte count, Hb, platelet count, bone marrow lymphocytes, pattern of bone marrow involvement, lymphocyte doubling time (LDT), beta-2-microglobulin (B2M), LDH, alkaline phosphatase, albumin. Genotype distribution of BCL2C938A in CLL was 47/182 (25.8%) AA, 96/182 (52.7%) AC, and 39/182 (21.5%) CC. Allele frequencies were in accordance with Hardy-Weinberg equilibrium (A: 0.522; C: 0.478; p=0.749, χ2). Biological and clinical variables at diagnosis distributed without significant differences among AA, AC and CC genotypes. Treatment free survival (TFS) did not differ in AA, AC and CC genotypes when analyzed separately (5-year TFS AA=58.1%; AC=46.4%; CC=50.0%; p=.228), after pooling C-allele carriers (5-year TFS AA=58.1%; AC/CC=47.6%; p=.414), or after pooling A-allele carriers (5-year TFS AA/AC= 50.1%; CC=50.0%; p=.247). Overall survival (OS) did not differ in AA, AC and CC genotypes when analyzed separately (5-year OS AA=80.1%; AC=76.8%; CC=76.1%; p=.900), after pooling C-allele carriers (5-year OS AA=80.1%; AC/CC=76.5%; p=.837), or after pooling A-allele carriers (5-year OS AA/AC= 77.9%; CC=76.1%; p=.649). The independent prognostic value of BCL2C938A was assessed separately for biological and clinical variables by Cox multivariate analysis. When tested along with biological covariates, BCL2C938A was not an independent predictor of TFS or OS. Biological covariates independently predicting TFS were del11q22–23 (HR 5.52; 95%CI 2.26–11.87; p<.001), p53 inactivation by deletion and/or mutation (HR 4.02; 95%CI 2.14–7.52; p<.001), +12 (HR 3.08; 95%CI 1.67–5.68; p<.001), IGHV unmutated (HR 2.26; 95%CI 1.33–3.84; p=.002), CD38 expression (HR 1.87; 95%CI 1.12–3.13; p=.016), and normal FISH karyotype (HR 0.37; 95%CI 0.12–0.73; p=.004). Also, when tested along with clinical covariates, BCL2A938C was not an independent predictor of TFS or OS. Clinical covariates independently predicting TFS were LDT <12 months (HR 10.83; 95%CI 5.37–21.8; p<.001), Rai III/IV (HR 4.32; 95%CI 2.09–8.91; p<.001), lymphocyte count >20 × 109/l (HR 2.85; 95%CI 1.75–4.64; p<.001), involvement of ≥3 nodal areas (HR 2.42; 95%CI 1.42–4.14; p=.001), and B2M (HR 2.08; 95%CI 1.21–5.55; p=.007). Clinical covariates independently predicting OS were age >65 years (HR 8.93; 95%CI 3.26–24.4; p<.001), albumin < 35 g/l (HR 4.23; 95%CI 1.84–9.71; p=.001), Binet B/C (HR 3.76; 95%CI 1.78–7.92; p<.001), and male sex (HR 2.88; 95%CI 1.33–6.23; p=.007). At variance with previous studies (Nückel et al., Blood 2006; 109: 290), BCL2C938A does not display prognostic relevance in our CLL series. This discrepancy may be ascribed to the more balanced distribution of BCL2C938A genotypes throughout CLL stages in our series. Alternatively, differences in the prognostic role of BCL2C938A may be the consequence of the different genetic background of the populations investigated.

CLL subsets with stereotyped HCDR3 and IGH/IGK-L gene combinations have been reported among cases with both mutated (M) and unmutated (UM) IGHV gene status. Here we evaluated the characteristics of stereotyped combinations in 1428 IG sequences (848 M and 580 UM) from 1399 CLL patients ordered according to a HCDR3-driven clustering, performed aligning HCDR3 amino acid sequences (codons 107–117 IMGT) by the multiple sequence alignment software ClustalX (1.83). Clusters with stereotyped HCDR3 were selected for having a mean alignment score>60/100. We identified 73 different clusters with stereotyped HCDR3, 32/73 with >3 cases (range 3–32) for a total of 247 cases (17.4%), 81 with M and 166 with UM IGHV genes; 17/32 clusters (200 cases) were previous described, while 15 (47 cases) were novel. Seventeen/32 clusters (156 cases) were composed by UM cases (UM/M = 153/3), 14/32 clusters (59 cases) were of M cases (UM/M = 3/53), and a single cluster (32 IGHV3-21 cases) was a mixed one (UM/M=10/22). In our series, 64% of cases (158/247) utilized four specific IGHV genes, i.e. 1–69 (60/169 cases, all UM, split into 6 clusters), 3–21 (32/55 cases, all in a single cluster), 4–34 (19/129 cases, 17 M and 2 UM, split into 2 clusters), 4–39 (17/55 cases, 14 M and 3 UM, split into 3 clusters) or mixed IGHV genes of the IGHV1 family/clan (IGHV1-2/1-3/1-18/1-46/7-4), all in a single cluster (30/153 cases, all UM). Clinical data (time-to-treatment, TTT) were available for 637/1399 patients. UM cases (251) had significantly shorter TTT (p<0.00001), as compared to M (386) cases. Similarly, TTT were significantly shorter (p<0.0005) in 113 CLL patients belonging to clusters (Hom) as compared to 492 CLL patients not belonging to clusters (Het), although these differences were no longer observed when TTT of Hom vs. Het cases were compared in the context of M (Hom/Het = 34/336) and UM (Hom/Het = 79/156) CLL. Patients belonging to the IGHV3-21 cluster had TTT similar to 241 UM cases and significantly shorter (p<0.0001) than 370 M CLL. Shorter TTT were also documented in 15 cases belonging to the IGHV3-21 cluster when compared to 13 IGHV3-21-expressing Het cases either considered altogether or split into M and UM cases (p=0.03). Finally, patients belonging to the IGHV1-2/1-3/1-18/1-46/7-4 mixed cluster had shorter TTT in comparison either with all the other M and UM cases, or with the cases expressing the same IGHV genes but with Het HCDR3 gene, even if this latter group was split in M and UM cases (p = 0.002). The identification of stereotyped HCDR3 CLL suggests a role of specific antigen(s) in defining the biological and clinical features of this disease.

RS occurs in 5–15% CLL, depending on follow-up length and re-biopsy policy. The value of biological and clinical risk factors in predicting RS is unknown and represents the aim of this study. The analysis was based on a consecutive series of 185 CLL. Diagnosis of RS was based on lymph node or extranodal tissue biopsy. RS was diagnosed in 17 cases all represented by diffuse large B-cell lymphoma (DLBCL). Cumulative incidence of RS at 5 and 10 years was 13.6% (95%CI: 7.0–20.1%) and 16.2% (95%CI: 8.0–24.4%), respectively. Transformation plateaued at 82 months of follow-up. Median time to transformation was 23.0 months (95%CI: 15.9–30.1 months) from CLL diagnosis. IGHV studies documented clonal relationship between CLL and DLBCL in 15/17 (88.2%) cases. At the time of CLL diagnosis, molecular variables predicting RS were: IGHV4–39 usage (5-year risk IGHV4-39: 56.2% vs IGHVnon-VH4-39: 11.2%; p<0.001), IGHV homology ≥98% (5-year risk IGHV homology ≥98%: 28.3% vs IGHV homology <98%: 7.0%; p=0.006), absence of del13q14 (5-year risk non-del13q14: 23.1% vs del13q14: 3.8%; p=0.006), and presence of +12 (5-year risk +12: 19.5% vs non +12 10.0%; p=0.027). CLL with p53 inactivation by p53 mutation and/or del17p13 had a trend toward an increased risk of transformation (5-year risk del17p13/p53 mutation: 24.6% vs non-del17p13/p53 mutation: 12.1%; p=0.061). Cluster analysis revealed that 6/17 (35.2%) CLL evolving to RS carried stereotyped CDR3s. Utilization of stereotyped IGHV4-39 or stereotyped IGHV1-2/1-3 predicted transformation (5-year risk stereotyped CDR3: 63.0%; p<0.001). Del11q22-q23, normal FISH katyotype, BCL2 C938A polymorphism, and telomere length did not predict RS. Among phenotypic markers at CLL diagnosis, expression of both CD38 (5-year risk CD38+: 25.4% vs CD38−: 4.7%; p<0.001) and ZAP70 (5-year risk ZAP70+ 27.9% vs ZAP70− 6.7%; p=0.002) predicted RS. At the time of CLL diagnosis, clinical variables predicting RS included: lymph node size ≥3 cm (5-year risk lymph node ≥3 cm: 49.1% vs lymph node <3 cm: 6.1%; p<0.001), involvement of 3 nodal areas (5-year risk 3 nodal areas: 31.8% vs nodal areas <2: 8.3%; p<0.001), Binet B stage (5-year risk Binet B: 40.8% vs Binet C 20% vs Binet A: 7.1%; p=0.003), and LDH ≥1.2 × ULN (5-year risk LDH >1.2 × ULN: 37.5% vs LDH <1.2 × ULN: 8.6%; p=0.001). Age, sex, Rai stage, splenomegaly, Hb, platelet count, percentage of BM lymphocytes, B2M, ALP, albumin and lymphocyte doubling time did not predict RS. Landmark analysis at 12, 24, 36, 48 and 60 months documented: a significantly increased risk of subsequent RS in progressive CLL vs stable CLL starting from 24 months; and no RS in stable CLL after 48 months of follow up. Multivariate analysis identified CD38 expression (HR 4.22, 95%CI 1.26–14.05; p= 0.019) and IGHV4-39 usage (HR 4.29, 95%CI 1.28–14.29; p=0.018) as biological predictors of RS, and lymph node ≥3 cm as clinical predictor of RS (HR 8.73, 95%CI 2.45–31.05; p=0.001). Our data suggest that RS is predicted by: predominant nodal disease at diagnosis; CD38 expression; and usage of specific IGHV genes, namely IGHV4-39. These results suggest the need for a close monitoring and a careful biopsy policy in patients with molecular, phenotypic and clinical predictors of RS identified at the time of CLL diagnosis.