Mutated IGHV Genes Research Articles

Autologous Stem Cell Transplantation (ASCT) in CLL. Results of a Phase III Randomized Multicenter Trial.

Abstract 878 Introduction:We investigated, in a prospective randomized study, the place of ASCT in the frontline treatment of CLL. Patients and methods:From March 2001 to December 2007, 241 patients were included in the trial. Eligibility criteria were previously untreated stage B and C CLL patients under 66 years, characterized by Matutes score 4-5, absence of cyclin D1 expression, baseline flow assessment of ZAP 70 and CD38 expression, karyotype and FISH analysis, IgHv mutational status (centralized). Preceding randomization, initial chemotherapy consisted of 3 monthly courses of mini CHOP regimen as previously described, followed by 3 monthly courses of fludarabine, IV or oral. Patients achieving CR (NCI 1996 criteria plus normal CT scan) were randomized between observation and ASCT. Non CR patients were offered cisplatin/cytosine-arabinoside/dexamethasone (DHAP) rescue and randomized whatever the response between ASCT or 3 subsequent monthly IV courses of Fludarabine-Cyclophosphamide (FC). Conditioning regimen for ASCT consisted of cyclophosphamide IV (60 mg/sqm d-5-4) and fractionated total body irradiation (10 Gy). The primary end-point of the study was event-free survival at 3 years. Responses after initial treatment (i.e.before randomization) and after completion of therapy, overall survival, side effects, prognostic significance of clinical and biological characteristics at baseline were other endpoints. Results:Baseline characteristics were: gender (M/F: 3), age (median 56.4 years, range 33.3-66), stage B (185 patients) or C (56 patients). All enrolled cases but five (236 patients) started the treatment. Among them, 206 completed the six planned courses of initial chemotherapy. For the 236 patients, CR rate was 43.6%, and overall response was 89.8%. Forty two patients were not randomized because of treatment failure (19) or patient/physician decisions (23). After an observed median follow-up of 40.2 months (Q1-Q3, 17.9-47.9) at the reference date (1/1/2009), the overall survival for the 241 patients was 87.8% (95% CI, 83.3-92.6) at 3 years and 75.4% (95% CI, 66.2-88.6) at 5 years. For the 199 randomized patients, the overall survival was 90.9% (95% CI, 86.7-95.3) at 3 years. Among them 105 patients were in CR after initial treament and were allocated to ASCT (53 patients) or observation (52 patients) with a 3 years EFS of respectively 78.7% (95% CI, 67.7-91.4) and 31.3% (95% CI, 20.1-48.8) (p<0.00001). The median EFS from the start of the studied treatment was 26.2 months in the observation arm (not reached in the ASCT arm). Randomization (p=0.0001), mutational status (p<0.00001) and 11q deletion (p=0.001) remained independent prognostic factors for EFS in a multivariate analysis. There was no difference between ASCT and observation arms in terms of overall survival with 98% (95% CI, 94.3-100) and 97.5% (95% CI, 92.2-100)respectively. For the 94 patients not achieving CR and rescued with DHAP, the EFS at 3 years was 46.5% (95% CI, 33.6-66.3) in the ASCT arm (46 patients) and 43.2% (95% CI, 29.8-62.5) in the FC arm (48 patients). There was not statistical difference between these 2 arms but for the 34 patients actually autografted the EFS was 57.8% (95% CI, 41.7-80.2). The overall survival was not different for ASCT and FC arms: 81,2% (95% CI, 70.3-93.9) and 85,4% (95% CI, 75.1-97) respectively. In multivariate analysis, the only prognostic factor influencing the EFS (p=0.0001) and the overall survival (p=0.04) was the 17p deletion. The mutational status (non mutated) and the 17p deletion remained adverse prognostic factors in patients actually autografted. For the whole study, 28 patients allocated to ASCT did not receive this treatment mainly because of mobilisation failures (14 pts). Four MDS were recorded within the follow-up frame. Conclusions:ASCT is a safe procedure which significantly improves response duration in patients attaining CR after a first line treatment. For patients not in CR, ASCT or consolidation with 3 FC courses provide similar results on response duration but ASCT could be considered in patients without 17p deletion or with mutated IGHV gene. Disclosures:Van Den Neste:Roche: Research Funding.

Extensive Intraclonal Diversification in a Subgroup of Chronic Lymphocytic Leukemia Patients with Stereotyped IGHV4-34/IGKV2-30 B cell Receptors: Implications for Ongoing Interactions with Antigen.

Abstract 2337Poster Board II-314Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen (Ag) recognition through the clonotypic B cell receptors (BCRs). However, it is still unclear whether Ag involvement is restricted to the malignant transformation phase or whether the putative Ag(s) may continuously trigger the CLL clone. Valuable insight into these issues may be gleaned from the study of intraclonal diversification (ID) within the immunoglobulin (IG) genes through ongoing somatic hypermutation (SHM). Definitive data regarding ID within IG genes in CLL remains limited and conflicting. In the present study we systematically explored the presence of ID within IG genes of CLL, not only at cohort level but also in subgroups defined by BCR stereotypy and IG gene mutational status. We thus conducted a large-scale subcloning study of both IG heavy and light variable genes, in a total of 1496 and 1008 subcloned sequences from 71 and 56 CLL cases, respectively. The analysis was intentionally biased to cases expressing IGHV4-34/IGKV2-30 IGs (subset #4) and IGHV3-21/IGLV3-21 IGs (subset #2) that exhibit distinctive, subset-biased SHM patterns. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and at least 14 colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample (ii) confirmed mutation (CM) - a mutation observed more than once among subcloned sequences from the same sample. Analysis of heavy chain sequences revealed that 40% (28/71) of cases carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, whilst 32% (23/71) of cases carried only UCMs. The remaining 28% (20/71) of cases carried sets of identical IGHV-D-J subcloned sequences. Although most cases showed no or low levels of ID, an intense and, likely, functionally driven ID was evident in selected cases, especially those belonging to subset #4. The distinct ID in subset #4 was statistically significant when compared to all other groups defined by IGHV gene usage and mutation status, BCR stereotypy or heavy chain isotype. Subsequent analysis of the clonotypic light chains revealed that the impact of ID was generally low, with the outstanding exception again relating to subset #4. In fact, of 22 IGKV-J rearrangements exhibiting CMs, 11 (50%) utilized the IGKV2-30 gene and notably 10/11 (91%) of these were expressed by subset #4 cases. In such cases, the expressed IGKV2-30 gene was affected by an active and precisely targeted ID, analogous to their partner IGHV4-34 gene. These findings suggest that the SHM mechanism may continuously operate in certain subsets of CLL patients, particularly those cases expressing stereotyped IGHV4-34/IGKV2-30 BCRs typical of subset #4. In such cases, the observed ID patterns attest to the very precise targeting of the SHM process and may be considered as evidence for a “stereotyped response” to an active, ongoing interaction with Ag(s). Disclosures:No relevant conflicts of interest to declare.

Histopathological EXAMINATION of BONE MARROW Biopsy (BMB) IN Primary Splenic B CELL Lymphomas of Marginal-ZONE ORIGIN (PSMZL). A Reliable Substitute for Spleen Pathology?.

Abstract 1924Poster Board I-947According to the 2008 WHO Classification, PSMZLs mainly comprise splenic marginal-zone lymphoma (SMZL) and splenic diffuse B cell lymphoma/leukemia unclassifiable (SDLU). Until recently, histological examination of splenectomy specimens was considered as a prerequisite for the diagnosis of SMZL though, due to the recent advancement in chemo-immunotherapy therapies, splenectomy is no longer performed in most cases. We evaluated the diagnostic efficiency of BMB examination in the documentation of the diagnosis of PSMZLs and their differential diagnosis from mimickers, including other small B-cell lymphomas, and tested whether BMB may serve as a substitute for spleen microscopic examination in the differential diagnosis of SMZL from SDLU. The study group included 108 BMB samples from patients with a diagnosis of PSMZL based either on examination of splenectomy specimens (n= 46), or clinical presentation, cell morphology and flow cytometry analysis (n= 62). Histological and immunohistochemical results are summarized in Table 1. Molecular analysis of immunoglobulin genes showed that 10/21 analyzed CD27+ cases carried IGHV genes with more than 98% germline identity (unmutated), whereas 5/8 analyzed CD27- cases carried mutated IGHV genes. Furthermore, 7/14 analyzed SIgD+ cases carried mutated IGHV genes. Splenectomy specimens were available in 46/108 cases. Blinded histopathological examination of the spleens revealed 35 SMZL and 11 SDLU. Paired assessment of BMB and spleen specimens did not identify any discriminating pattern of BM infiltration and/or morphology between SMZL and SDLU; notably, the immunophenotypical profile of BMB and spleen specimens was identical when 14 different markers have been utilized (Table 1). In conclusion, our study documents the value of BMB histopathology in the diagnosis of PSMZLs and their differential diagnosis from other small B-cell lymphomas primarily or secondarily involving the spleen. Presently, it is not possible to distinguish SMZL from SDLU based on the only BMB histopathology. Finally, our results confirm and expand the considerable heterogeneity of PSMZL, which could reflect different activation status and/or different histogenesis of the clonogenic cells.Table 1Histology and immunohistochemistry.Pattern of infiltrationPure intrasinusoidal6/108Interstitial19/108Nodular4/108Diffuse8/108Mixed intrasinusoidal-interstitial39/108Nodular combined with other types32/108MorphologySmall lymphocytes105/108plasmacytic differentiation (PC)13/105onocytoid differentiation (MC)20/105Pure MC population3/108ImmunohistochemistryCD20+ CD79á+BCL2+108/108CD43+BCL6+CD10+CyclinD1+0/108CD5+7/98CD23+4//91CD5+CD23+0/91TRAP+31/58DBA44+59/93CD27+44/70SIgD+30/61SIgD+CD27+18/24 Disclosures:No relevant conflicts of interest to declare.

Evidence for Antigen-Driven Development of Molecularly Classified Burkitt Lymphomas.

Abstract 317 Evidence for Antigen-driven Development of Molecularly Classified Burkitt LymphomasThe presence of somatic mutations and intraclonal diversity (ID) in the variable region of rearranged immunoglobulin (Ig) genes is commonly used as a marker of the cellular passage of B cells through the germinal center (GC) reaction in secondary lymphoid tissues following an in vivo antigen encounter. Such features have also been documented in different subgroups of aggressive B-cell lymphomas, suggesting that antigen selection pressure has taken place during lymphoma development. We undertook a detailed analysis of the Ig repertoire and somatic hypermutation (SHM) status of 54 molecularly classified Burkitt lymphomas (BL) (Hummel et al. 2006). The IGHV gene repertoire was remarkably biased, with only three genes accounting for 40.8% of all cases (IGHV4-34, 18.5%; IGHV4-39, 11.1%; IGHV3-30, 11.1%). Of note, this bias was even more evident when cases were grouped according to age < or > 14 years, with the IGVH4-34 and IGVH4-39 rearrangements occurring almost exclusively in pediatric BL (7/11 and 5/6 cases respectively). Only 2/54 cases (3.7%) carried unmutated IGHV genes (100% germline identity, GI); 12/54 cases (22.2%) carried IGHV genes of borderline/minimally mutated status (98–99.9% GI); finally, the remaining 40/54 cases (74.1%) carried mutated IGHV genes (<98% GI). The median GI of the cohort was 95.2% (range: 86.4–100%) with a significantly different mutation load according to age below or above 14 years (GI 95.8 vs. 92.9 respectively, p=0.0168). Shared replacement mutations (“stereotyped” amino acid (AA) changes) at certain codon positions were identified in 12/54 (22.2%) BL cases. Stereotyped AA changes were found only amongst rearrangements utilizing the IGHV4-34 and IGHV4-39 genes, strongly indicating evidence for an antigen driven SHM process in BL. The majority of stereotyped AA changes identified here were distinct from those previously reported in other entities, in particular CLL, and thus can be considered as BL-biased. Also, of note, the IGVH4-34 specific motif responsible for binding the N-acetyl-lactosamine antigenic determinant was found intact in 11 IGVH4-34 BL sequences, implying that these clones retained the ability to bind to and be activated by superantigens. Furthermore, the distribution of mutations was different in IGHV4-34 vs. non-IGHV4-34 cases, as evidenced by low R-to-S ratios in the heavy complementarity-determining regions (especially HCDR1), indicating a different selection pattern of VH-genes according to varying antigens.To assess the presence of ID through ongoing SHM, we evaluated 411 subcloned sequences from 11 IGVH4-34 and 6 IGVH4-39 cases. Only one case carried identical sets of subcloned sequences; seven cases carried mutations in single subcloned sequences that were considered as unconfirmed; finally, 9 cases carried at least one confirmed mutation among two or more subcloned sequences and, thus, were considered as exhibiting confirmed ID. However, analysis of the distribution of the so called unconfirmed changes provided further evidence for the very precise targeting of mutations introduced as part of the ID process. Thus, certain changes identified in single subcloned sequences from one case were shared by most or all subcloned sequences of another case utilizing the same IGHV gene (“confirmed by another case”). In conclusion, our data demonstrate that BL is characterized by a highly distinctive IG gene repertoire with a precisely targeted SHM process. In addition, they provide strong evidence for a functionally driven SHM, supporting a role for antigen-driven selection of the clonogenic progenitors. Disclosures:No relevant conflicts of interest to declare.

The Green Tea Extract EGCG Demonstrates Synergist Activity against CLL B-Cells When Combined with Fludarabine and Chlorambucil.

Abstract 3452Poster Board III-340 PURPOSEChronic Lymphocytic Leukemia (CLL) is incurable with current chemotherapy treatments. Epigallocatechin 3 gallate (EGCG), the major catechin in green tea, has been shown to induced caspase-dependent death CLL B-cells and down regulate anti-apoptotic proteins (Mcl-1; XIAP) known to increase the resistance of CLL B-cells to apoptosis (Blood 104:788-94). In Phase I testing for patients with early stage CLL (Rai 0-II), EGCG treatment was well tolerated and induced a decline in absolute lymphocyte count (ALC) and/or lymphadenopathy in the majority of treated patients without myelosuppression (JCO 27:3808-14). Although peak plasma EGCG levels were not measured in this study, trough plasma EGCG levels as high as 4 μg/ML were achieved. To further our understanding of the potential clinical applications of EGCG for patients with CLL, we evaluated the effects of EGCG on the viability of CLL B-cells when combined with fludarabine (F), chlorambucil (C), or fludarabine and chlorambucil in combination (FC). METHODSPrimary CLL B-cells were treated with various doses of F (0.25-1 μM), C (10-30 μM), or FC either alone or in combination EGCG (50 to 100 μM). In other experiments, CLL B-cells were treated with various doses of F (0.25-1 μM), C (10-30 μM), or FC with or without a fixed dose of EGCG known to be physiologically achievable in vivo (4 μM). After 48 hours (experiments with C +/− EGCG) or 72 hours (experiments with F +/− EGCG or FC +/− EGCG), cells were harvested, stained with annexin/PI and viability analyzed by flow cytometry. After concentration-effect curves were generated for each agent, data were also analyzed using the CalcuSyn software program (Biosoft, Cambridge, UK) which uses the method of Chou and Talalay to determine whether combination treatment yielded greater effects than expected from summation alone. A combination index (CI) of 0.8 – 1.2 indicates an additive effect, a CI >1.2 indicates an antagonistic effect and a CI <0.8 indicates a synergistic effect. RESULTSPrimary leukemic B-cells from 56 CLL patients were cultured in vitro with various doses of EGCG alone or in combination with F, C, or FC. The median LD50 was approximately 100 μM. Although % apoptotic cells at the 100 μM EGCG dose did not vary based on Rai stage, ZAP-70 status, IGHV gene mutation status, or cytogenetic abnormalities by FISH, CD38 negative patients had greater cell death than CD38 positive patients (67% vs. 49%; p=0.05). In co-titration experiments, EGCG had an additive (CI 0.8 – 1.2) or synergistic (CI <0.8) effect on apoptosis when combined with C in the majority of patients (13/15; additive 11, synergism 2) with rare individuals demonstrating antagonism (CI >1.2; 2/15). The effects of EGCG when combined with F in co-titration experiments were more variable with a relatively even distribution between antagonism (8/18), additive effects (4/18), and synergy (6/18). However, co-titration experiments of FC with or without EGCG demonstrated additive or synergistic effect in most patients (8/10; additive 7, synergism 1). Next we evaluated the effect of F (0.25 - 1 μM), C (10 - 30 μM), or FC with or without a fixed dose of EGCG known to be physiologically achievable in vivo (4 μM). This dose of EGCG had an additive or synergistic effect in the majority of samples across the spectrum of dose levels under all 3 conditions (F, C, and FC). For example, at the 10 μM dose of C the addition of 4 μM EGCG had an additive or synergistic effect in 11/14 patients (synergistic 6; additive 5). With F at the 2.5 μM dose level the addition of 4 μM EGCG had an additive or synergistic effect in 13/16 patients (synergistic 7, additive 6). The addition of 4 μM EGCG appeared particularly beneficial when given in combination with FC (Fig.). For example, at F 2.5 μM in combination with C 5 μM the addition of 4 μM EGCG had an additive or synergistic effect in 10/10 patients (synergism 7; additive 3). CONCLUSIONSPhysiologically achievable doses of EGCG appear to enhance the efficacy of alkylating agents, purine nucleoside analogues, and alkylating agent/purine analogue combination therapy for the majority of CLL patients on in vitro testing. The favorable toxicity profile of EGCG and lack of myelosuppression with this agent in the phase I trial (JCO 27:3808-14) make it an attractive agent to test in combination with purine analogue and alkylator based chemo-immunotherapy for patients with CLL. [Display omitted] DisclosuresKay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon Pharma : Patents & Royalties, Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding.

Influence of Age at Diagnosis On Utility of Prognostic Testing in Patients with CLL.

Abstract 2342Poster Board II-319 PURPOSE:Numerous clinical and biologic parameters are able to predict time to first treatment (TFT) and overall survival (OS) for patients with CLL. The utility of prognostic testing in CLL patients may vary based on age at diagnosis given the higher mortality from other causes among older individuals. The goals of the present study were to i) evaluate differences in natural history of CLL patients based on age at diagnosis, ii) compare survival to age-matched individuals in the general population, and iii) determine the age-stratified utility of prognostic testing. METHODS:All 2487 CLL/SLL patients diagnosed between January 1995 and June 2008 and cared for in the Mayo Clinic Division of Hematology were categorized by age at diagnosis (<55 years, 55-64 years, 65-74 years, or ≥75 years) and evaluated for differences in clinical characteristics, TFT, and OS using chi-square tests. IGHV gene mutation analysis, ZAP-70 status, CD38 status, and cytogenetics abnormalities by interphase FISH testing were performed as part of clinical and/or research studies using methods previously described by our group. Cox regression was used to evaluate the ability of prognostic parameters to predict clinical outcome stratified by age at diagnosis. Patient survival was compared to that of the age matched Minnesota population. RESULTS:When grouped by age, 593 patients were age <55, 713 age 55-64, 748 age 65-74, and 433 age ≥75 at diagnosis. As of last follow-up, 268, 282, 296, and 108 patients in these age groups have been treated, respectively and 112, 171, 254, and 199 patients have died. Survival decreased as the age at diagnosis increased (p<0.001). OS of CLL patients was significantly shorter than that of the age-matched general population for patients age <55 years (p<0.001), 55-64 years (p<0.001), and 65-74 years (p<0.001) at diagnosis but not those ≥75 years (p=0.136). Among Rai stage 0 patients, survival was also shorter than that of the age-matched general population for CLL patients age <55 years (p<0.001), 55-64 years (p<0.001), and 65-74 years (p<0.001) but not those ≥ age 75 at diagnosis (p=0.07).No statistically significant differences were observed in CD38 status, IGHV mutation status, ZAP-70 status or the frequency of common cytogenetic abnormalities on FISH based on age at diagnosis category. Consistent with prior reports, all of these parameters were powerful predictors of TFT and OS for the overall cohort on univariate analysis (all p<0.001 for both TFT and OS). Given the variation in TFT and OS based on age at diagnosis, we next evaluated the utility of these prognostic parameters for predicting TFT and OS in each age category after adjusting for stage. CD38, IGHV mutation, and ZAP-70 each predicted TFT independent of stage for all age groups (all p <0.05). FISH results predicted TFT independent of stage for patients age 55-64 and 65-74 however did not reach statistical significance for those age <55 (p=0.18) or ≥75 (p=0.07). The utility of these parameters for predicting OS independent of stage was less consistent and varied by age. IGHV predicted OS independent of stage for patients <age 55 (p<0.001), 55-64 (p=0.007), and 65-74 (p'0.001) but was not a significant predictor of OS among those ≥75 (p=0.15). CD38, ZAP-70, and FISH each predicted survival independent of stage for only 2 of the 4 age categories (ZAP-70 for age <55 and 55-64; FISH for age <55 and 65-74; CD38 for 55-64 and ≥75). CONCLUSIONS:Survival of CLL patients is shorter than that of the age-matched general population for individuals age <75 at diagnosis regardless of disease stage. In contrast, survival does not differ from the age-matched general population among CLL patients ≥age 75 at diagnosis. Prognostic testing using CD38, IGHV mutation, and ZAP-70 is useful for predicting TFT independent of stage for CLL patients of all ages (including those ≥age 75) but has less value for predicting OS particularly as the age at diagnosis increases. IGHV appeared to be the best predictor of OS independent of stage among all patients age <75. Unexpectedly, prognostic testing had little utility for predicting OS independent of stage among patients age ≥75. These findings have important implications regarding the use of prognostic tests in patients with CLL as well as the use of test results to select patients for clinical trials testing early treatment for asymptomatic individuals. Disclosures:Shanafelt:Cephalon: Research Funding; Celgene: Research Funding; Polyphenon Pharma : Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding. Zent:Genentech, Bayer, Genzyme, Novartis: Research Funding.

Defining origins of malignant B cells: a new circulating normal human IgM+D+ B-cell subset lacking CD27 expression and displaying somatically mutated IGHV genes as a relevant memory population

In probing the cell of origin in malignant B cells, an imprint of somatic hypermutation (SHM) in immunoglobulin (Ig) variable (V) region genes delineates antigen encounter, and identifying the precise pathway generating SHM in the normal B-cell counterpart becomes relevant. SHM remains the definitive memory imprint in normal human B cells, but CD27 expression also delineates memory. Recently, dye extrusion adenosine triphosphate-binding transporter assays identified circulating isotype-switched memory B cells that lacked CD27, yet exhibited low levels of SHM. To extend findings, we report a pre-switched CD27(-ve) circulating memory B-cell population in normal blood using comparable assays, and isolated CD19(+)IgM(+)D(+)CD27(-ve) cells (>99% purity) for the analysis of IGHV5/IGHV3-IGHM transcripts. Of these (n=334), approximately 78% were germ line and naive B cell derived. Strikingly, 21.9% of the transcripts were mutated. They showed 3-5 mutations (13.5% of sequences) and >5 mutations (8.4% of sequences) per transcript. Accrual of mutations in a subset of CD19(+)IgM(+)D(+)CD27(-ve) cells define a new circulating pre-switched memory B-cell pool, present in substantial numbers in the population harboring naive B cells. These CD19(+)IgM(+)D(+)CD27(-ve) memory B cells may have a distinct lineage and function, and seem relevant to understanding origins of malignant B cells, in particular those of hairy cell leukemia cells, which display mutated V genes yet lack CD27 expression.

Clinical features and outcome of Chinese patients with monoclonal B-cell lymphocytosis

B-cell chronic lymphocytic leukemia (CLL) is the most common type of adult leukemias in the Western countries, however, infrequent in the Eastern. A diagnosis of CLL requires a count of B-lymphocytes ≥5.0×109/L. Asymptomatic person with <5.0×109/L B-lymphocytes is defined as monoclonal B-cell lymphocytosis (MBL). To compare the clinical characteristics, prognostic factors, and outcome of Chinese patients with MBL and CLL, we present a study from our single centre of 20 patients with MBL and 136 patients with CLL. The factors included: age at diagnosis, gender, direct antiglobulin test (DAT), immunoglobulin heavy chain variable gene (IgHV) mutational status, ZAP-70 protein, CD38 expression level, and molecular cytogenetic aberrations were analyzed in MBL and CLL subgroups. The Kaplan–Meier method was used to construct survival curves, and results were compared using the log-rank test. Patients in the MBL category were slightly older than in the CLL category. There was no significant difference of these clinical and biological characteristics between patients in MBL subgroup and early stage CLL (Binet A). The incidence of positive DAT was significantly increased in CLL patients at Binet B and C, compared with MBL (P=0.036). IgHV gene mutation in MBL is skewed, with more than 92.3% of subjects harbored mutated IgVH genes (P=0.025). The proportion of MBL patients with a 13q14 deletion or trisomy 12 was similar to that of CLL patients. Moreover, markers associated with poor prognosis (deletion of 11q22 or 17p13) in these MBL populations were less than those in Binet B and C CLL patients (P=0.025). No statistically significant differences in ZAP-70 and CD38 status were observed between the MBL and CLL subgroups. During a median follow-up period of 45.5 months, MBL patients had a low probability of progression, with no patients transformed to aggressive non-Hodgkin's lymphoma or dying of CLL-related causes. The overall survival of MBL was very similar to Binet A CLL, but longer than that of CLL patients at advanced stages (Binet B and C) (P=0.024). Our study demonstrated that a more indolent clinical course and superior clinical outcome for patients with MBL compared to CLL.

Abnormal serum free light chain ratios are associated with poor survival and may reflect biological subgroups in patients with chronic lymphocytic leukaemia

The measurement of immunoglobulin serum free light chains (sFLC) has prognostic significance in plasma cell dyscrasias but its role in chronic lymphocytic leukaemia (CLL) is unknown. This retrospective study from three UK hospitals analysed sFLC in 181 untreated/pre-treatment CLL patients and 78 treated CLL patients, with samples taken later in their disease. An abnormal sFLC ratio was significantly associated with poor overall survival for the 181 untreated/pre-treatment patients (P = 0.0001) and for all patients (P = 0.002), irrespective of cause of death. Using multivariate analysis (n = 194), four independent prognostic variables for overall survival were identified namely Zap-70 (P = 0.0001), beta2M (P = 0.01), IGHV mutation status (P = 0.017) and an abnormal sFLC ratio (P = 0.024). For CLL patients with unmutated IGHV genes, elevated kappa/lambda ratios were adversely prognostic. For patients with mutated IGHV genes, reduced kappa/lambda ratios were adversely prognostic and associated with the poor prognostic IGHV3-21, IGHV3-48 and IGHV3-53 subgroups, suggesting an abnormal sFLC ratio may reflect biological subgroups within CLL. Abnormal sFLC ratios need to be studied prospectively in CLL patients and the biological rationale for their abnormality investigated.

High Resolution Screening of Copy-Number Alterations in Chronic Lymphocytic Leukemia Using Affymetrix 250K SNP-Arrays Reveals a Higher Complexity of Genomic Alterations in Patients with Unmutated IGHV Genes

Chronic lymphocytic leukemia (CLL) is a biologically heterogeneous disease where no common genetic aberration so far has been described. Although recurrent genomic aberrations of prognostic significance are well established (e.g. deletions of 11q, 13q, 17p and trisomy 12), less is known about the overall genetic complexity. Recent development of high-resolution single nucleotide polymorphism (SNP)-arrays will allow screening of genetic complexity, which includes detection of smaller copy-number alterations (CNAs) in addition to the known, recurrent aberrations. We here applied the Affymetrix GeneChip® Mapping 250K Nsp arrays and screened peripheral blood samples from 203 newly diagnosed CLL patients (≥70% tumor cells) from a population-based Scandinavian cohort. The male:female ratio was 2:1, the majority of patients was in stage A (70%) and the median age at diagnosis 61 years. Sixty percentage of cases displayed mutated IGHV genes whereas 35% showed unmutated IGHV genes. The Nexus copy-number software (Biodiscovery, Inc.) was applied and CNAs were identified in all CLL samples investigated, where deletions were more commonly detected than gains (in average, 3.5 vs. 2.2 per sample, respectively). The average length of deletions was 3.5 Mb while gains had an average length of 9.5 Mb (3.6 Mb when excluding trisomy 12). More than 50% of deletions and gains had a size ranging between 0.1–1 Mb, whereas 22% of CNAs were less than 0.1 Mb and 25% larger than 1 Mb. When investigating known, recurrent alterations, 105 samples (52%) carried del(13)(q14); 82 samples displayed a mono-allelic deletion, 15 samples a bi-allelic deletion and 8 samples carried 2 mono-allelic deletions of different sizes. The minimal overlapping region was 0.44 Mb and most of the 13q14 deletions covered the genomic region of miR-15, miR-16 and/or DLEU7. Twenty-one samples (10%) showed trisomy 12 and del(11q) was detected in 27 samples (13%), all covering the ATM gene. del(17p) was detected in 7 patients (3.4%) with one patient also carrying several gains and losses of 17q. Moreover, large alterations involving chromosome arms or entire chromosomes were recurrently observed among the samples, for instance, 3 large 8p losses and 4 large 8q gains. Interestingly, 5 samples, which all carried del(11q), displayed a gain covering whole or large parts of the p-arm of chromosome 2, a region which covers MYCN, REL and BCL11A. Furthermore, comparison of samples with different IGHV mutation status (M vs. UM) illustrated a significantly higher frequency of del(11q) (32% of UM vs. 4% of M), trisomy 12 (22% of UM vs. 5% of M) and gain of 2p (12% vs. 0% in M) in samples with unmutated IGHV genes. As expected, patients with mutated IGHV genes showed a higher frequency of del(13q) (61% of M vs. 31% of UM). In addition, large aberrations (>1Mb) were more common in UM samples which in average displayed 0.57 gains and 1.22 deletions compared to 0.26 gains and 0.67 deletions in M samples. In conclusion, with whole-genome screening using high resolution SNP-arrays, higher complexity was revealed in CLL, including a higher number of gains and losses and a higher frequency of larger aberrations in UM compared to M samples. A novel, recurrent combination of del(11q) and gain of 2p was demonstrated which deserves further investigation.

A subset of Binet stage A CLL patients with TP53 abnormalities and mutated IGHV genes have stable disease

A subset of Binet stage A CLL patients with TP53 abnormalities and mutated IGHV genes have stable disease

FCRL2 expression predicts IGHV mutation status and clinical progression in chronic lymphocytic leukemia

AbstractCD38 and ZAP-70 are both useful prognostic markers for B-cell chronic lymphocytic leukemia (CLL), but are variably discordant with IGHV mutation status. A total of 5 human Fc receptor–like molecules (FCRL1-5) have tyrosine-based immunoregulatory potential and are expressed by B-lineage subpopulations. To determine their prognostic potential in CLL, FCRL expression was compared with IGHV mutation status, CD38 and ZAP-70 expression, and clinical features from 107 patients. FCRL1, FCRL2, FCRL3, and FCRL5 were found at markedly higher levels on CLL cells bearing mutated IGHV genes than on unmutated CLL cells or CD19+ polyclonal B lymphocytes. Univariate comparisons found that similar to CD38 and ZAP-70, FCRL expression was strongly associated with IGHV mutation status; however, only FCRL2 maintained independent predictive value by multivariate logistic analysis. Strikingly, FCRL2 demonstrated 94.4% concordance with IGHV mutation compared with 76.6% for CD38 and 80.4% for ZAP-70. Compared with other indicators, FCRL2 was also superior at predicting the time to first therapy; the median treatment-free interval was 15.5 years for patients with high FCRL2 expression compared with 3.75 years for FCRL2-low patients. Our studies indicate that FCRL2 has robust predictive value for determining IGHV gene mutation status and clinical progression and thus may further improve prognostic definition in CLL.

Prognostic importance of T and NK‐cells in a consecutive series of newly diagnosed patients with chronic lymphocytic leukaemia

Patients with chronic lymphocytic leukaemia (CLL) have a variable clinical course. The identification of modifiable characteristics related to CLL-specific survival may provide opportunities for therapeutic intervention. The absolute number of T-cell and natural killer (NK)-cells was calculated for 166 consecutive patients with CLL evaluated by flow cytometry at Mayo Clinic < or = 2 months of diagnosis. The size of the T-cell/NK-cell compartment relative to the size of the malignant monoclonal B-cell (MBC) compartment was evaluated by calculating NK:MBC and T:MBC ratios. Patients exhibited substantial variation in the absolute number of T- and NK-cells as well as T:MBC and NK:MBC ratios at diagnosis. Higher T:MBC and NK:MBC ratios were observed among patients with early stage and mutated IGHV genes (all P < or = 0.0003). As continuous variables, both T:MBC ratio (P-value = 0.03) and NK:MBC ratio (P-value = 0.02) were associated with time to treatment (TTT). On multivariate Cox modelling including stage, CD38, absolute MBC count, NK:MBC ratio and T:MBC ratio, the independent predictors of TTT were stage, T:MBC ratio and NK:MBC ratio. These findings suggest that measurable characteristics of the host immune system relate to the rate of disease progression in patients with newly diagnosed CLL. These characteristics can be modified and continued evaluation of immunomodulatory drugs, vaccination strategies and cellular therapies to delay/prevent disease progression are warranted.

TCR Gene Rearrangement in B-Cell Chronic Lymphocytic Leukaemia.

Background: Chronic lymphocytic leukaemia (B-CLL) is a heterogenous B cell disorder. However, clonal/oligoclonal T cell expansion has also been detected at a low frequency in B-CLL using a range of methodologies including Southern blotting and flow cytometric analysis. The underlying cause of this phenomenon is unclear and it has even been suggested that this clonal T cell receptor (TCR) gene rearrangement occurred in the leukaemic cells, leading to the concept of lineage infidelity. Other suggestions include that the observed T cell clonality may simply be related to the normal aging process or that it may result from the immune response to malignant cells or viral infection. During normal T cell maturation in the thymus, the TCR genes undergo a complex process of V(D)J segment rearrangement to produce a wide repertoire of antigen specificity. As it is an early event in T cell development, TCRγ gene rearrangement is commonly used as a marker of clonality. The TCRβ gene is also routinely investigated due to its greater combinatorial diversity. Lack of sufficiently sensitive detection methodology is often a limiting factor in the discrimination between clonal and polyclonal T-cell populations.Aims: The aim of this study was to use high resolution heteroduplex gel and fluorescence GeneScan analysis to screen for TCRγ and TCRβ gene rearrangements respectively in a cohort of B-CLL patients and to ascertain if the clonal rearrangement occurred in B cells or T cells.Methods: B and T cells were purified from peripheral blood from 34 B-CLL patients using a CD19+ or CD3+ magnetic bead system (autoMACS). DNA was extracted from purified B or T cells and the TCRγ and TCRβ genes were amplified by BIOMED-2 multiplex PCR assays (InVivoScribe Technologies). Heteroduplex and GeneScan analysis were then performed to detect clonal TCR gene rearrangments.Results: Clonal TCR gene rearrangements were detected in11/34 (32%) of cases in purified T cell fractions. No clonal TCR gene rearrangements were found in purified B cell fractions. Clonal TCRβ rearrangements were found in 10/34 (29%) and clonal TCRγ in 8/34 (23%). In most instances a weak clonal pattern was observed. Clonal TCR rearrangements were detected in 7 males and 4 females. Five patients had mutated IGHV genes, while the remaining 6 possessed unmutated IGHV genes. Seven patients presented with more advanced clinical stage (Binet B or C), and 8 patients received treatment.Summary/conclusions: These results are in agreement with previous work demonstrating that T-cell clonal/oligoclonal expansions occur frequently in B-CLL patients. However this is the first report to demonstrate that the clonal expansions occur in T cells and not in B cells. In most instances a weak clonal pattern was observed which was probably due to minor clonal T-cell populations. It is possible that T-cell clonality may be associated with a poorer prognosis in this disease category. Of interest, we found that 8/11(73%) patients in our B-CLL sub-group with T-cell expansion also had advanced stage disease and/or unfavourable molecular (IGHV gene usage/mutational status) markers. We are currently investigating the possibility of a specific antigenic stimulus resulting in this clonal T-cell expansion.

Trisomy 19 is associated with trisomy 12 and mutated IGHV genes in B‐chronic lymphocytic leukaemia

The occurrence of trisomy 19 was investigated in 705 cases of B-chronic lymphocytic leukaemia (CLL) by metaphase cytogenetic and/or fluorescence in situ hybridisation analyses. Trisomy 19 was detected in 11 cases (1.6%), all of which also carried a trisomy 12; nine of 10 had mutated IGHV genes. In contrast, B-CLL cases with trisomy 12 lacking trisomy 19 mostly had unmutated IGHV genes. Karyotypes of the present study and the literature identified a strong correlation to trisomy 18 in addition to trisomy 12. Trisomy 19 seems to be a secondary event in B-CLL with trisomy 12, mostly originating from mutated B cells.

Splenic Marginal-Zone Lymphoma: One or More Entities? A Histological, Immunohistochemical and Molecular Study of 41 Cases.

The considerable heterogeneity of splenic marginal-zone lymphoma (SMZL) hinders firm conclusions concerning histogenetic origin and differentiation stage of the neoplastic cells. We analyzed a series of 41 SMZL cases, diagnosed on splenectomy specimens after established WHO criteria, and correlated histological, immunohistochemical and molecular findings in an attempt to gain insight into the origin(-s) of the currently elusive SMZL clonogenic cell(-s). A predominantly nodular growth pattern was observed in 23 cases; the remainder showed predominantly (11 cases) or exclusively (7 cases) diffuse infiltration. Twenty cases showed the “classical” biphasic appearance; the remainder were monophasic, mainly with MZ morphology; 8/41 cases were composed predominantly of small cells. CD5 was expressed in 2/37 analyzed cases; all cases were negative for CD43. CD21 and CD35 were expressed by a minority of cases (11/40 and 16/37 cases, respectively), generally in a concordant fashion (double positive or negative cases). Twenty-two out of 40 analyzed cases were DBA.44-positive; 4/39 evaluable cases co-expressed CD21/DBA.44, while 10/39 cases were double negative. Similar results were obtained for CD35/DBA.44 co-expression. Seventeen out of 37 analyzed cases were SIgD+; 12/22 analyzed cases were SIgM+SIgD+, 7/22 were SIgM+SIgD-, while one case was SIgM-SIgD+; 5/36 analyzed cases were SIgG+. Plasmacytic differentiation was observed in 4/41 cases. Fifteen out of 36 evaluable cases were CD21/SIgD double negative, while 5/36 co-expressed CD21/SIgD. Strong/weak CD27 staining was observed in 22/27 analyzed cases; 8/19 CD27+ cases were SIgD+. Among five CD27-negative cases, 3 were SIgD-positive. Forty IGHV-D-J rearrangements were amplified in 35/41 analyzed cases; double in-frame rearrangements were amplified in three cases. IGHV1/3/4 subgroup genes were used in 8/11/17 sequences. Twenty-five IGHV genes (all IGHV3 and 10/17 IGHV4) were mutated (<98% homology to germline); 5/15 and 7/15 unmutated sequences utilized, respectively, IGHV1 and IGHV4 genes. Two IGHV1-69/IGHD3-16/IGHJ3 rearrangements (one mutated, one unmutated) had homologous HCDR3 regions, after a pattern previously described by several groups in CLL patients (Blood . 2004; 104:2499–2504; J Exp Med . 2004; 200:519–525; Blood . 2004; 104:2879–2885). Clonal IGKV-J and IGLV-J gene rearrangements were amplified in 29/41 cases; 13/29 IGKV/IGLV sequences were mutated. Five out of 25 mutated IGHV genes and 3/13 mutated IGKV genes had high (>3.0) replacement to silent (R:S) mutation ratios in the CDRs along with low (<2.0) R:S ratios in the FRs. Five out of 17 CD27+ cases with available IG data carried unmutated IGHV genes, while 2/5 CD27- cases were IGHV-mutated (both SIgD+DBA.44+). Six SIgD+ cases carried mutated IGHV genes. The results of the present study:confirm the considerable histological, immunohistochemical and molecular heterogeneity of SMZL lymphoma;indicate an origin from the diverse resident B cell populations of the normal splenic marginal zone and do not support a mantle-zone cell derivation;allude to the role of selective antigenic pressures in the pathogenesis of at least a subset of SMZL cases.