Unmutated IgVH Genes Research Articles

Germline IgVH, del(17p13.1) and del(11q22.3) Predict Duration of Response in Patients Affected by Chronic Lymphocytic Leukaemia Treated with Oral Fludarabine and Cyclophosphamide as First-Line Therapy.

We test the efficacy and safety of the oral formulation of fludarabine combined with cyclophosphamide as front-line therapy of CLL. We studied the influence of IgVH gene mutation status, interphase cytogenetic abnormalities, and expression of ZAP-70 and CD38 on the overall response rate (OR), progression free survival (PFS), time to re-treatment (TTR) and overall survival (OS). Twenty seven patients were male and 10 were female, with a median age of 68 years (range, 52 to 75 years). Thirty patients were in stage B/II, 1 in stage C/III, and 6 in stage C/IV. The treatment schedule consisted of oral fludarabine (30 mg/m2) and oral cyclophosphamide (250 mg/m2) for 3 consecutive days every 4 weeks, for a maximum of 6 cycles. Eighteen patients had unmutated and 15 had mutated IgVH genes; in 4 patients the mutation status was not determined. Nine patients (24%) had the ‘high risk' cytogenetic abnormality del(11q22.3) or del(17p13.1). Fifteen patients had more than 20% ZAP-70-positive and 8 patients had more than 30% CD38-positive CLL B-cells, respectively. Thirty-five patients were evaluable for response to treatment. Two patients discontinued the treatment protocol because of the development of immune thrombocytopenia after 2 and 4 cycles, respectively. Both of them belonged to the ‘high risk' cytogenetic category. Fourteen patients (40%) obtained a complete response and 13 (37%) a partial response with an ORR of 77%. Four patients showed stable disease and four patients experienced progressive disease after 2, 3, 4 and 6 cycles, respectively. The median PFS was 23 months (range 2–42) and the median TTR was 38 months (range 2–42). The ‘high risk' cytogenetic abnormalities del (17p13.1) and del (11q22.3) were predictive of a lower OR rate (43% vs 85%, p=0.011) as well as a shorter PFS (22 vs 27 months, p= 0.015) and shorter TTR (22 vs 40 months, p=0.031). In addition, unmutated IgVH genes were significantly associated with a reduced TTR (26 vs 41 months, p=0.035), whereas no significant impact was noticed in terms of OR, PFS and OS, even though all four deaths occurred in patients with unmutated IgVH genes. Expression of CD38 and ZAP-70 had no significant impact on clinical outcome. At present, median follow-up is 30 months, with an OS rate of 89%. The median time of OS has not been reached for any of the prognostic categories. In terms of haematological toxicity, ten patients developed grade IV neutropenia during treatment and received G-CSF. Three patients developed grade III and IV anemia that required red blood cell transfusions. Extra-hematological toxicity consisting of nausea was detected in 14 patients (40%) of whom 12 with grade I–II and 2 (6%) with grade III; half of these patients also experienced vomiting (6 patients grade I–II and one patient grade III). One patient developed fever of unknown origin after the first cycle of chemotherapy, which spontaneously resolved without hospitalization. The oral combination of fludarabine and cyclophosphamide is an effective, safe and well tolerated regimen that appears especially appropriate for a subset of previously untreated CLL patients characterized by mutated IgVH genes and the absence of “high risk” cytogenetic abnormalities.

TCL1 Expression in Chronic Lymphocytic Leukemia Correlates with the Intensity of 11q Deletions and ZAP-70.

The TCL1 (T-cell leukemia/lymphoma1) oncogene is a coactivator of the AKT oncoprotein, an essential molecule in the transduction of antiapoptotic signals in T and B cells. Eμ-TCL transgenic mice with B cells with high TCL1 expression develop the aggressive phenotype of chronic lymphocytic leukemia (CLL). Studies in human CLL have found that expression of TCL1 correlates with high expression of ZAP-70 and use of unmutated IgVH genes. The expression of TCL1 may be regulated in part by microRNA, miR-29 and miR-181, which map to chromosome 11(11q). Because aberrations at 11q have been associated with poor prognosis in CLL, we interrogated the relationship between deletions at 11q and expression of TCL1 and ZAP-70. We used a direct immunophenotyping method to investigate the relative co-expression levels of TCL1 and ZAP-70 within CLL cells and examined the relationship between such levels and the proportion of leukemia cells within the CLL population bearing 11q deletions, as detected by FISH analysis. Direct staining of intracellular TCL1 protein was performed by using the monoclonal anti-TCL1 antibody (clone 1–21) labeled with Alexa647 in combination with the established ZAP-70 protocol (NEJM 2004; 351:893) together with mAb directed against CD5 and B cell surface antigens. Negative staining levels were set using isotype control antibodies. FISH was performed on interphase nuclei by using uniform and cross-validated procedures at all CRC sites using the CLL-panel from Vysis. Chromosomal abnormalities were detected in 76% (520) of the 680 CLL samples analyzed. Sixteen percent of the patients had leukemia cells with monoalleleic deletions at 11q. We performed flow cytometry for intracellular co-expression TCL1 and ZAP-70 on cryopreserved samples obtained from 25 CLL patient samples with varying proportions of cells with the 11q deletion (10% to 98% abnormal cells with 11q deletion, mean 70%) and 30 CLL samples lacking any chromosomal abnormalities. We detected significantly higher levels of TCL1 in CLL cells that expressed ZAP-70 and/or unmutated IgVH genes. The ZAP-70pos cases (39/55) had a median percent of TCL1pos cells of 34% compared to 15% for the ZAP-70neg cases. The cases using unmutated IgVH genes (43/55) had a median percent of TCL1pos cells of 31%, which was greater than the 19% median observed for cases that used mutated IgVH genes. Multiparameter analyses revealed that the ZAP-70 positive fraction of each CLL clone had significantly higher levels of TCL1 than did the ZAP-70 negative cells (mean=45% versus 29%, respectively, p=0.002). We observed a significant difference between the expression levels of TCL1 for CLL cells that had deletions in 11q relative to that of CLL cells lacking any chromosomal abnormalities (mean=41% versus 18%, respectively p=0.0002). In addition, we observed a relationship between the levels of TCL1 expressed in leukemia cell populations and the relative proportion of leukemia cells with deletions at 11q. This study reveals a relationship between the levels of TCL1 expression in CLL leukemia-cell expression of ZAP-70, and the relative proportions of leukemia cells having deletions at 11q. Studies are in progress to define whether these relationships can be explained by altered expression of microRNA that map to 11q, which might also account for the noted adverse prognosis of CLL that has deletions at 11q.

Unfavourable Outcome and Heterogeneity of Partner Chromosomes in Chronic Lymphocytic Leukemia with 14q32/IGH Translocations.

Translocations of 14q32/IgH have a low incidence in CLL. Partner chromosomes and the prognostic significance are poorly defined. Four hundred thirty cases of CLL seen at the Hospitals of Ferrara, Salamanca and Siena between 1992 and 2006 were studied. Inclusion criteria were:a.diagnosis of CD5/CD19+ CLL with k/λ restriction,b.cytogenetic/FISH data,c.immunophenotypic data,d.complete hematological and clinical data. Lymphomas in leukemic phase were excluded.FISH was performed for 17p13/TP53, 11q22.3/ATM, 6q21, chr 12 centromere, 13q14 and 14q32/IGH. Patients with no detectable aberration or isolated 13q− were included into a favourable cytogenetic group (group 1), those with +12, 6q− or 1–2 aberrations into an intermediate risk group (group 2) and those with 17p−, 11q−, ≥ 3 aberrations into an unfavourable group (group 3). Cases with 14q32/IGH translocation as primary chromosome change represented a specific category (group 4) and were studied with FISH probes for the detection of partners (BCL1, BCL2, BCL3, BCL6, c-MYC, BCL11A, PAX5, CCND3, CDK6). One hundred eighty-six cases were allocated into group 1; 158 into group 2; 64 into group 3, and 22 into group 4. Additional aberrations were found in a minority of cells in 8 patients in group 4. Being the aim of the study to assess whether the 14q32/IGH translocation represented an unfavourable parameter as compared with cases in the favourable or intermediate cytogenetic risk groups, cases within group 3 were excluded from the analysis and the data presented here will refer to 366 patients belonging to groups 1,2 and 4. Translocation partners of 14q32/IGH were identified in 9/22 cases: 2p13/BCL11A, (n 1); 6p21/CCND3 (n 1); 7q21/CDK6 (n 1); 18q21/BCL2 (n 6). Thirteen cases did not show involvement of the loci studied. Cases with 14q32/IGH translocations were characterized by typical morphology and classical immunophenotype (score 4–5 in 92% of the cases). Unmutated IGVH genes were found in 11/18 cases tested (61%); ZAP-70 was positive in 3/5 cases tested. Median age was 63.5 years (range 18–97), male:female ratio 240/126; 345 patients were in Rai stage 0-II, 21 were in stage 3–4; CD38 was positive in 137/366 cases. There was no difference between groups 1,2 and 4 for age, stage, male:female ratio, hematological parameters at diagnosis, IGVH mutations and ZAP70. CD38 was more positive in group 4 than in group 1 (p=0.028). There was no difference in survival and treatment free interval (TFI) when comparing cases in group 4 with and without additional aberrations. Cases in group 4 had a shorter TFI and a shorter survival when compared with group 1 (p=0.02) and group 2 (p=0.02). The difference maintained its statistical significance at multivariate analysis for TFI (p=0.02) along with stage (p<0.0001) and CD38 positivity (p<0.0001) and for survival (p=0.02) along with sex (p=0.006) and stage (p=0.0001). In conclusion, the 14q32/IGH translocation in CLL shows heterogeneity of partner chromosomes and it represents a cytogenetic marker predicting for an evolutive form of CLL.

Cytotoxicity of the Type 4 Phosphodiesterase Inhibitor CD160130 for Freshly Isolated Human CLL Cells In Vitro.

B cell chronic lymphocytic leukemia (CLL), the most common subtype of leukemia in the United States of America and in Europe, is treatable but incurable. New drugs are needed for its management. Phosphodiesterase inhibitors (PDEi) block catabolism of cyclic nucleotides resulting in accumulation of cellular cAMP and cGMP. These PDEi also decrease production of inflammatory cytokines such as tumor necrosis factor. Furthermore, they inhibit expression of inducible nitric oxide synthase (NOS2) mRNA and NO production. Researchers have previously noted that nonspecific PDE inhibitors such as theophylline as well as a relatively specific PDE4 inhibitor (rolipram) cause CLL cell death in vitro, while relatively sparing normal peripheral blood mononuclear cells (PBMC). The purpose of this study was to evaluate the effectiveness of CD160130 in the killing of freshly isolated CLL cells in vitro. CD160130 is a novel, orally available heterocyclic pyrimido-indole derivative that is relatively PDE4-specific. Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CLL cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. Samples from 10 CLL patients (6 male and 4 female) and 10 normal controls were examined. Nine of 10 patients were stage 0 at presentation, and 1 was stage 2. They had been followed 6.1 yr (median; range 0.7–19.1 yr). One of 10 was CD38 positive, and 6 of 10 were Zap-70 positive. Of nine analyzed, one had unmutated IgVH gene, and 9 were mutated. Six patients had not been treated, and 4 had been treated with chlorambucil, fludarabine, and/or rituximab. Four had normal cytogenetics by FISH analysis; one had trisomy 12; three had 13q14 del; and one had 17p del. Of seven determined, two had elevated CLL cell lipoprotein lipase mRNA elevated. CD160130 induced cell death in a dose-dependent fashion in all patients' samples, with a mean cytotoxicity of 96% at 12.5 uM. The mean ED50 for killing was 233 nM in media with FBS and 314 nM in serum-free medium, while that for PBMC was higher at 7500 nM with FBS and 5210 nM with serum-free medium. The agent was 32.2 fold more potent for killing of CLL cells compared to PBMC in medium with FBS and 16.6 fold more potent for CLL cells in serum-free medium. In summary, the PDE4 inhibitor CD160130 potently kills freshly isolated CLL cells in vitro in the presence or absence of serum. The killing is relatively selective for CLL cells compared to normal PBMC. In vivo trials of CD160130 in patients with CLL should help determine the toxicity and efficacy in patients.

Immunoglobulin Gene Translocations Are Frequent in Unclassifiable CD5+ Small B-Cell Leukemias and in Particular, a Novel Subtype Characterised by t(14;19)(q32;q13) and Deregulation of BCL3 Is Described.

We describe the features of 24 cases of atypical CD5+ small B-cell leukaemia unclassifiable by current methods. The male/female ratio was 3 (18M/6F), with a median age at diagnosis of 58 years (range 39–84 years). All cases were CD5 positive, but with an immunophenotype and morphology atypical for CLL. All cases were negative for t(11;14) and other B cell malignancies were excluded. FISH analysis was carried out using probes for IGH break apart, t(14;19) involving BCL3, trisomy 12, 13q14, 11q22–23 (ATM) and 17p13 (p53 locus). IgVH somatic mutations were analysed using direct sequencing (2% cut off). FISH using IgH break apart probe showed 8 (32%) cases with a split at 14q32. These 8 cases were further investigated using a FISH probe for t(14;19). Four cases (16%) exhibited t(14;19) ie. showed the IgH/BCL3 rearrangement in >70% of cells. Further characterisation of the 24 cases, at additional loci showed that 4 (16%) patients had 17p deletion, 3 (12%) 11q deletion, 3 (12%) 13q deletion, and 14 cases (56%) trisomy 12. The 8 cases showing a split IgH signal did not show deletions of 17p, 11q, or 13q but trisomy 12 was present in 7/8 (88%) patients. Sixteen out of 24 (67%) cases had unmutated IgVH genes with VH3 and VH4 families being used in 91% (22/24). Interestingly, unlike in chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL), the VH1 family was not used in this group of patients. All 4 cases with t(14;19) showed unmutated IgVH genes. Of the sixteen patients without involvement of 14q32, only 2 patients have died with a median follow up of 5.5 years. Of the 4 cases with the t(14;19) (IgH/BCL3 rearrangement), 3 required treatment within 2.5 years of diagnosis and two out of four patients died 5 years after diagnosis, following Richter's transformation suggesting that this rearrangement is a poor prognostic factor and defines a group with a distinct clinical outcome. Our findings show a high frequency of 14q abnormalities in atypical CD5+ small B-cell leukaemia. BCL3 rearrangement is seen in a significant subset of these cases. The BCL3 gene encodes a nuclear protein that belongs to the IkB family of inhibitors of NFkB. It is involved in the regulation of a variety of transcription factors and it is able to stimulate the expression of cyclin D1. The dysregulation of BCL3 via its juxtaposition to the regulatory element of the IgH locus in our cases suggests a pathogenic role for BCL3 in these B cell disorders and may define a group of patients with a more aggressive clinical course. Further, we have examined these cases for signatures, defined in myeloma, characteristic of NFkB deregulation. We suggest that in cases of CD5 positive B-cell disorders with an immunophenotype and morphology atypical for CLL, and negative for t(11;14), rearrangements involving the IgH locus are frequent and have allowed us to identify cases with poor prognosis and with a pathogenesis that involves deregulation of NFkB.

The Prognostic Significance of 11q Deletion Detected by Fluorescence In Situ Hybridization (FISH) in Untreated Chronic Lymphocytic Leukemia (CLL): The MDACC Experience.

Introduction: The identification of an 11q deletion (del) by FISH analysis is reportedly an independent predictor of poor survival in CLL (Döhner et al, N Engl J Med 343: 1910). The aim of this study was to assess the presenting features and clinical outcomes of a more recent cohort of untreated CLL patients carrying the 11q del.Methods: We searched our database for untreated CLL patients who presented to MDACC from 10/03 to 4/07 with an 11q del and reviewed their medical records. Patient bone marrow samples were assessed at presentation by FISH analysis using probes for trisomy 12, ATM, LAMP1, D13S319, and P53 genes.Results: Overall, 69 of 708 (9.7%) patients analyzed using FISH, had an 11q del without a 17p del. In addition to 11q del, 62% had 13q del, and 3% had trisomy 12. Their median age was 59 yrs; and 80% were males. Zubrod performance status was > 0 in 53%; and 13% had Rai stage 3–4. Lymphadenopathy was present in 96% (> 5 cm, 11%), splenomegaly in 19%, anemia in 9%, and thrombocytopenia in 4%. ZAP-70 was expressed in 74% (34/46), CD38 in 61% (33/54), and 89% (39/44) had an unmutated IgVH gene. The median absolute lymphocyte count (ALC) was 24 × 109L (range, 2–513 × 109L), and the median proportion of cells that exhibited an 11q del was 53% (range, 9–98%). Cytogenetics were as follows: del(11)(q13q23) in 17/63 (27%) patients, normal in 37 (59%), del6(q13q25) in 1, complex in 2, and other in 6 patients. The median time from presentation at MDACC to treatment was 1 month (range, 0–28) and from diagnosis to treatment was 15 months (range, 1–97). The median ALC doubling time was 237 days. Forty patients required therapy for progressive disease (lymphadenopathy 17, ALC doubling time <1 yr 14, B-symptoms 6, splenomegaly 2, and anemia 1); and 38 were evaluable for response: 29 treated with FCR (fludarabine, cyclophosphamide, and rituximab), 4 with CFAR (FCR+alemtuzumab), 3 with R-GMCSF (rituximab+GMCSF), and 2 with other therapies (FC 1, CVP+R 1). The overall response rates for FCR+CFAR, R-GMCSF, and other therapies were 100%, 33%, and 100%, respectively; and the CR rates were 88%, 0%, and 100%, respectively. Among 23 patients who were monitored by FISH analysis after treatment, 21 had disappearance of 11q del. One patient who developed Richter's syndrome (RS) and another patient with relapsed CLL had recurrence of the 11q del clone (both ZAP-70 positive, IgVH gene unmutated). The median follow-up of surviving patients was 13 months. Only 3 patients have died, including the RS patient, who died from sepsis after therapy, and 2 patients who died from pancreatic cancer and glioblastoma. At 1 and 3 yrs, the survival rates were 97% and 91%, respectively. Three patients developed relapsed disease at 16, 17, and 18 months. Time to treatment, survival, and relapse-free survival (RFS) are shown in Fig.Conclusions: CLL with an 11q del was associated with high rates of response, survival, and RFS when treated with FCR-containing therapy. This cohort of patients will be followed over the long term to establish the natural history of 11q del in the chemoimmunotherapy era. [Display omitted]

Mutated IGHV 1-69 B-CLL Patients Have Indolent Disease and Short CDR3 Sequences.

Background B-CLL is a heterogenous disorder with a highly variable clinical course. Although certain acquired cytogenetic aberrations, unmutated IGVH genes, ZAP-70 positivity and an atypical phenotype have been linked with more aggressive disease and a poorer outcome, the search for further markers of poor prognosis is ongoing is ongoing. IGHV 3–48, 3–53 and 3–21 gene usages have previously been associated with more aggressive disease and shorter survival times. Furthermore, IGHV 1–69 is expressed at a high frequency in B-CLL suggesting a role for antigenic selection in disease pathogenesis. It has been suggested that comparison to germline immunoglobulin sequence using the NCBI/Ig blast only may result in over estimation of mutated IGHV frequency associate with IGHV 1–69 rearrangements in B-CLL. Thus the clinical significance of IGHV 1–69 gene usage remains to be clarified. Aims The aims of this study were to determine the frequency of IGVH1–69 gene usage in B-CLL, to assess the structural features of the CDR3 region, mutational status of the IGHV 1–69 gene using the up-dated IgBLAST and V-QUEST databases and to and to determine if patients with IGHV1–69 rearrangements differ in clinical course to those patients with alternative IGHV gene usage.Methods Two hundred and seventy patients were recruited for this study. IGHV gene usage was determined using multiplex BIOMED-2 primers (InVivoScribe Technologies) and protocol. IGHV mutational status was determined by sequence analysis using BigDye chemistry and homology comparison with the up-dated NCBI/IgBLAST and IMGT/V-QUEST databases. Interphase FISH analysis was performed to screen for common cytogenetic aberrations and serum thymidine kinase (TK) levels were measured using a radioenzyme assay.Results We identified IGHV1–69 gene usage in 31/270 CLL patients (11.5%), 30 of which were characterised further. We found that it was associated with unmutated IGHV status (80% of cases) and a preponderance of males (73%). Poor/intermediate prognosis cytogenetic abnormalities were detected in 10 of 15 (67%) cases analysed. Although 21 patients (70%) were found to have Binet stage A disease upon presentation, the majority have since progressed to stage B or C. The median TK level was 16.1 U/L (>8.5 U/L is associated with progressive disease). IGHV somatic hypermutation was found in 6 cases (20%) and was associated with shorter CDR3 regions, IGHJ 4 gene usage and lower TK levels (median of 6.8 U/L). These latter patients have early stage, apparently indolent disease, with no evidence so far of progression.Summary/conclusions We have found that IGHV1–69 is over-expressed in B-CLL, is associated with advanced disease and may have potential as an independent prognostic marker of progressive disease. However, this remains to be determined as its strong association with IGHV status may be the determining factor. We present evidence that IGHV1–69 B-CLL cases can be divided into mutated (n=6) and unmutated (n=24) sub-groups and that mutated cases are defined by short CDR3 regions, biased IGHJ 4 gene usage and indolent disease. We are currently investigating the possible common antigenic stimuli which may be involved in leukaemogenesis in this sub-group.

Binding of CLL B-Cell Receptors to Viable and Apoptotic Human Cells Offers Insight into the Role of Autoantigens in Leukemic Transformation.

IgVH gene mutation status correlates with clinical course in chronic lymphocytic leukemia (CLL), with the unmutated group usually having a worse outcome. Because the binding domains of B-cell receptors (BCRs) among patients exhibit considerable genetic and structural similarity, it has been suggested that a limited set of antigens drive clonal expansion in CLL. We reasoned that characterizing antigenic reactivities of CLL BCRs would provide insight into the development of the disease. Hence we cloned and expressed BCRs from 28 CLL patients (18 unmutated and 10 mutated) as monoclonal antibodies (mAbs). We tested these by immunofluorescence for reactivity with the human epithelial cell line HEp-2, often used to identify nuclear autoantigens in autoimmune disorders such as systemic lupus erythematosus (SLE). Furthermore since apoptotic cells are a source of autoantigens in autoimmune conditions, we also tested the mAbs for reactivity with apoptotic and viable B and T cells, from RAMOS (B) and Jurkat (T) cell lines and from normal subjects and CLL patients. All 28 mAbs showed binding to intracellular structures of HEp-2 cells, reminiscent of SLE. Unlike SLE, the binding was predominantly cytoplasmic. Only one mAb, expressing VH4-59, bound HEp-2 cell nuclei. When tested for reactivity with B and T cell surfaces by indirect immunofluorescence and flow cytometry, only 13 (10 unmutated and 3 mutated) of the 28 mAbs bound at least one lymphoid cell type. Among the cell-reactive mAbs, 8 bound apoptotic cells without discriminating between B and T cells. All these expressed unmutated IgVH genes; 5 were VH1 family members (3 exhibited VH1-69-D3-3-JH6 with different HCDR3s and L chain rearrangements, 1 expressed VH1-03, and 1 expressed VH1-02) and the last 3 belonged to a stereotypic set comprising VH4-39-D6-13-JH5 + Vk1D-39-Jk1. Notably, 5 mAbs, expressing VH4-34, reacted only with viable cells. Two of these used unmutated 4-34 genes and bound both B and T cells; altering their companion VLJL rearrangements eliminated virtually all cell binding. The other 3 mAbs, members of the stereotypic set with a mutated VH4-34-D5-18-JH6 rearrangement, bound only viable B cells, predominantly of the naïve subset. Substitution of their L chains led to a decrease in B cell binding, although to a lesser degree than for the unmutated VH4-34 mAbs. The one mAb reacting with HEp-2 nuclei failed to react with apoptotic or viable B and T cells. Of note, no VH3-expressing mAbs reacted with cell surface targets. In sum, even though all 28 CLL mAbs reacted with intracellular targets, only ~45% (n=13) reacted with lymphoid cell surfaces. Of those, 8 bound only apoptotic cells and 5 bound viable cells. All mAbs binding viable cells expressed VH4-34, with mutated mAbs binding solely B cells, and unmutated binding both. Interestingly, although the 8 mAbs binding apoptotic B or T cells were all unmutated, another 8 unmutated, but HEp-2-reactive, mAbs failed to bind any surface targets. Thus unmutated polyreactive BCRs exhibit a degree of specificity in antigen binding conferred by VH family and gene use and their companion VHDJH and VLJL. Finally, these data highlight a role for apoptosis as a source of already existing or newly created autoantigens in the promotion and evolution of CLL.

CLL-Cell Proliferation Measured by In Vivo Deuterium Labeling in Early Stage Patients and Correlation with Other Prognostic Markers.

Previous work has suggested a relationship between disease activity and in vivo birth rates of chronic lynphocytic leukemia (CLL) cells, as measured by incorporation of deuterium into DNA during heavy water (2H2O) consumption. By extension, CLL-cell kinetics may be a useful prognostic marker in early stage disease, with an increased birth rate predicting shorter time to disease progression and poor prognosis. We are conducting a multi-center study to evaluate the relevance of proliferation rates to both known prognostic markers and to disease progression during 2.5 years of clinical follow up in previously untreated patients with early stage disease. Patients are given a daily dose of 2H2O for 6 weeks and CLL-cell birth rates are measured. We examined the leukemia cells of 36 patients for known prognostic markers (ZAP 70 expression, CD38 expression, IgVH mutation status) and CLL birth rates. Subjects were Rai Stage 0 (n=15), 1 (n=17), or 2 (n=4); mean age 57 +/− 9 (range 41–78); mean time from diagnosis to enrollment 2.1 +/− 2.2 y (range 0.1–10 y). ZAP-70 expression (20% cutoff) was positive in 44%, CD38 expression (30% cutoff) was positive in 35%, and use of unmutated IgVH genes was found in 45% of the subjects. CLL-cell birth rates ranged from 0.09 to 0.97% per day. Utilizing the previously published cut-off value of 0.35% newly divided cells per day, 33% of subjects had a high proliferation rate. Data on all biomarkers were available for 33 subjects. Nine subjects had 4/4 favorable prognostic indicators and 5 subjects had 4/4 unfavorable prognostic indicators. Agreement between prognostic markers, as assessed by kappa coefficient, was strongest for the ZAP-70 v. IgVH mutational status comparison. Comparisons were also significant for ZAP-70 v. CD38 and IgVH mutational status v. CD38. Birth rate was not significantly correlated with any of the other prognostic markers, suggesting it may be a useful independent predictor of disease progression. Isolation of CLL-cells from the bone marrow at the end of the 6 wk labeling period in 5 subjects revealed matching proliferation rates in the marrow and blood in 3 subjects. In 2 subjects, marrow enrichment was lower, suggesting a more slowly proliferating pool in the marrow, the pathophysiologic significance of which is unclear. In 5 subjects, kinetic analysis revealed a very delayed entry of newly divided cells into the blood compartment, presumably from tissue sites of proliferation. This parameter may reflect pool size of CLL-cells in tissues and correlation with lymph node burden is being evaluated. In summary, analysis of CLL-cell birth rates in vivo in these early stage patients provides unique information about the biology of CLL. Correlations with disease progression during the follow-up period will help determine the utility of this test as a prognostic biomarker.

The Expression of the GTPase-Deficient, Hematopoietic-Specific RhoH GTPase Is Implicated in Development of Chronic Lymphocytic Leukemia (CLL).

RhoH is a hematopoietic-specific small GTPase which is constitutively active due to lack of intrinsic GTPase activity, suggesting that expression levels of RhoH regulate cellular function. Our lab has previously shown that RhoH in T-cells directly interacts with ZAP-70, a critical protein tyrosine kinase in TCR cell signaling and function (Gu et al., Nature Immunol., 2006). As in human B-CLL ZAP-70 expression has been correlated with an unmutated IgVH gene status and poor prognosis, we have investigated RhoH expression and function in B-CLL. PBMC samples from 29 B-CLL patients, all containing >90% CD19+/CD5+ human B-CLL cells, were compared to CD19+ B-cells from normal donors. Significantly higher RhoH mRNA levels were detected in CLL samples by quantitative PCR (p<0.001) with a 2–4 fold increase in the majority of samples. In addition, RhoH levels in these CLL samples were directly correlated with ZAP-70 expression (p<0.01). To further delineate the role of RhoH in the development and progression of B-CLL, we utilized a genetic mouse model of B-CLL, the Eμ-TCL1 transgenic mouse (Bichi et al. PNAS, 2002), and interbred these with Rhoh−/− mice. Starting at 2 months of age peripheral blood of these mice was analyzed every month for different blood cell lineages and in particular for the number of IGM+/CD5+/B220low cells as a readout for the progression of the CLL-like disease. In TCL1 transgenic mice with normal RhoH expression (Rhohwt; TCL1Tg) and with one Rhoh allele knocked out (Rhoh+/−; TCL1Tg) an accumulation of IgM+/CD5+ B-cells was observed which started as early as an age of 3 and 4 months, respectively, and increased dramatically thereafter. In contrast, Rhoh−/−; TCL1Tg showed no significant increase of IgM+/CD5+ B-cells until month 5 with a modest increase thereafter and values were significantly reduced in comparison to Rhohwt; TCL1Tg from month 3–5. As expected, no increase of IgM+/CD5+ B-cells was observed in the nontransgenic controls Rhoh−/+; TCL1NTg, Rhoh−/−; TCL1NTg (data not shown), and Rhohwt; TCL1NTg (Table 1).Table 1:IGM+/CD5+ cells in the peripheral blood.Age [months]Rhohwt; TCL1TgRhoh+/−;TCL1TgRhoh−/−;TCL1TgRhohwt;TCL1NTg20.12 ± 0.02 (10)0.28 ± 0.07 (8)0.15 ± 0.04 (9)0.08 ± 0.02 (9)30.33 ± 0.09 (11)0.22 ± 0.06 (8)0.07 ± 0.03 (8)*0.07 ± 0.01 (10)40.83 ± 0.38 (11)0.57 ± 0.20 (18)0.11 ± 0.05 (16)*0.08 ± 0.01 (6)53.32 ± 2.27 (6)1.35 ± 0.46 (17)0.26 ± 0.06 (15)*0.11 ± 0.03 (5)64.38 ± 2.41 (7)5.01 ± 3.01 (9)0.66 ± 0.23 (8)0.07 ± 0.02 (5)Mean ± SEM (x1000/ul); () animals analyzed; * p<0.05 in comparison to Rhohwt; TCL1NTG (data collection still in progress)When mice from each genotype were sacrificed at 6 months of age the number of IgM+/CD5+ B-cells detected in Rhoh−/− TCL1Tg vs RhohwtTCL1Tg mice was 6-fold reduced in the peritoneal cavity, 3-fold reduced in the bone marrow and a 2-fold reduced in the spleen. In summary our data strongly suggest that RhoH plays a role in the progression of the B-CLL-like disease in the transgenic Eμ-TCL1 mouse model. This will allow detailed molecular studies of the role of RhoH in pathways altered in TCL1-induced expansion of B-cell precursor populations in this model.

IgVH Mutational Status and Clonality Analysis of Richter's Transformation

Approximately 5% of B-cell chronic lymphocytic leukemia (B-CLL) patients develop a secondary aggressive lymphoma, usually of diffuse large B-cell type (DLBCL), termed Richter's transformation (RT). Rarely, classic Hodgkin lymphoma (HL) is observed. Published small series suggest that tumor cells in DLBCL and HL can be clonally identical to the B-CLL clone or arise as an independent, secondary lymphoma. We describe the morphology, immunophenotype, and clinical features of 34 classic RT patients with DLBCL, 6 cases of B-CLL with HL, and 8 cases with scattered CD30-positive Hodgkin and Reed-Sternberg (HRS)-like cells. The clonal relationship of the 2 components was analyzed using sequencing analysis of immunoglobulin heavy chain variable region (IgVH) genes. In classic RT, 18/23 B-CLL cases (78%) showed clonal progression to DLBCL with identical IgVH sequences in both lymphoma components, whereas in 5 cases (22%) the DLBCL was clonally unrelated. Among clonally related RT samples, 73% carried unmutated IgVH genes, whereas 4/5 unrelated cases were mutated. Immunophenotypically, most cases of DLBCL irrespective of clonal relatedness showed significant differences in phenotype compared with the B-CLL, with common loss of CD5 and CD23. Using immuno-laser capture microdissection, sequencing of the IgVH CDR3 region of isolated HRS cells showed that 2/2 cases with HL were clonally unrelated, whereas they were clonally identical in 1/2 cases of B-CLL with scattered HRS-like cells. HRS or HRS-like cells in all 3 unrelated cases showed evidence of Epstein-Barr virus infection. Of interest, 5/6 cases of B-CLL with HL, and 5/6 cases of B-CLL with HRS cells showed mutated IgVH genes.

CD5+ B cells with the features of subepithelial B cells found in human tonsils

This study describes a CD5+ B cell that differs from the majority of the CD5+ B cells from human tonsils. This cell, isolated from in vivo activated B cells, expressed activation markers and featured a CD23-, IgMhigh, IgDlow surface phenotype, responded to T cell-independent type-2 antigens in vitro, and was detected in the subepithelial (SE) areas, the tonsil equivalent of the splenic marginal zone (MZ). Most of the cells utilized unmutated Ig VH genes, although cells with mutated genes also were found, a finding confirmed by single-cell studies. Mutated sequences were more frequent in suspensions enriched for CD27+ cells. Repeated VDJ gene sequences were observed in different molecular clones from the same cell suspension, suggesting in situ expansion. These CD5+ B cells seem to share features with previously characterized tonsil CD5- SE B cells and differ from the majority of tonsil CD5+ B cells, which have the surface phenotype of follicular mantle B cells, lack activation markers, do not respond to T cell-independent antigens, and utilize unmutated VH genes. These data are discussed considering the present views on the origin of B cell subset populations and the relationships between MZ and B1 cells.

Improved risk assessment in B-cell chronic lymphocytic leukemia based on sex-specific molecular profiling

7090 Background: B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with some patients having an indolent course and others a rapidly progressing one. As underlying molecular mechanisms of differential disease activity, genomic aberrations, the mutational status of the variable region of the immunoglobulin heavy chain (Ig VH) and distinct expression levels of Zap-70 and CD38 have recently been identified, and their capacity to interact with survival and proliferation signals of B-CLL cells has directly been linked to progression and response to therapy. Although male sex has also been identified as risk factor for a more aggressive disease in B-CLL, the molecular basis for this interaction remains elusive. Methods: We compared the prognostic value of the above-mentioned molecular risk factors between female (n=88) and male patients with B-CLL (n=116). Differences in their frequency was evaluated using the Chi-square test. Their impact on treatment-free and overall survival of patients was analyzed using the Kaplan-Meier method and the log-rank test. The prognostic value of each molecular risk parameter in female and male patients was determined using the Cox proportional hazards regression model. Results: High CD38 expression (=30%), unmutated Ig VH gene (=98%) and unfavorable genetic aberrations (i.e. del17p, del11q or tri12) detected in the neoplastic clone identified patients with significantly shortened treatment-free survival, irrespective of the sex of patients. In contrast, Zap70 expression in more than 20% of B-CLL cells was negatively associated with treatment-free survival only in female (RR: 3.5, 95% CI 1.5 –7.9, P=.003) but not in male patients (RR: 1.6, 95% CI 0.9 –2.9, P=.089). In addition, we identified high CD38 expression in T-cells as independent negative prognostic marker in male (RR: 2.8, 95% CI 1.5 –5.0, P=.0006) but not in female patients (RR: 1.9, 95% CI 0.8 –4.6, P=.17). Based on these results we developed a novel sex-adapted algorithm for improved risk assessment in B-CLL. Conclusions: We identified sex-specific differences in the prognostic value of Zap-70 and CD38 in B-CLL. Whether this can be explained by modulation of their specific signaling pathways by sex hormones will have to be addressed in future studies. No significant financial relationships to disclose.

A comprehensive genetic and histopathologic analysis identifies two subgroups of B-cell malignancies carrying a t(14;19)(q32;q13) or variant BCL3-translocation

The biologic and pathologic features of B-cell malignancies bearing a translocation t(14;19)(q32;q13) leading to a fusion of IGH and BCL3 are still poorly described. Herein we report the results of a comprehensive cytogenetic, fluorescence in situ hybridization (FISH), molecular and histopathological survey of a large series of B-cell malignancies with t(14;19) or variant translocations. A total of 56 B-cell malignancies with a FISH-proven BCL3 involvement were identified with the translocation partners being IGH (n=51), IGL (n=2), IGK (n=2) and a non-IG locus (n=1). Hierarchical clustering of chromosomal changes associated with the t(14;19) indicated the presence of two different groups of IG/BCL3-positive lymphatic neoplasias. The first group included 26 B-cell malignancies of various histologic subtypes containing a relatively high number of chromosomal changes and mostly mutated IgVH genes. This cluster displayed three cytogenetic branches, one with rearrangements in 7q, another with deletions in 17p and a third one with rearrangements in 1q and deletions in 6q and 13q. The second group included 19 cases, mostly diagnosed as B-cell chronic lymphocytic leukemia (B-CLL), and characterized by few additional chromosomal changes (e.g. trisomy 12) and unmutated IgVH genes. In conclusion, our study indicates that BCL3 translocations are not restricted to B-CLL but present in a heterogeneous group of B-cell malignancies.

The prognostic role of CXCR3 expression by chronic lymphocytic leukemia B cells

Chemokine receptors are involved in tumor progression and several of these receptors, including CXCR3, are expressed by chronic lymphocytic leukemia (CLL) B cells. This study was aimed to examine a possible relationship between CXCR3 expression in CLL and the clinical evolution of the disease. Using flow activated cell sorting (FACS), we analyzed the level of expression of CXCR3 on blood CLL B cells from 76 consecutive patients. The results were correlated with CD38 expression, IgVH gene status and clinical outcome. CXCR3, measured as mean fluorescence intensity (MFI), was unimodally expressed by blood tumor cells at various levels (range, 3.5 to 232.3) but levels within individual patients were remarkably stable over time. Low CXCR3 expression by CLL B cells was strongly associated with Rai disease stages III and IV (p<0.0001) and a pattern of diffuse tumor infiltration of the bone marrow (p<0.0001). In the 28 cases available for genetic studies, low CXCR3 expression also showed good concordance with tumor unmutated IgVH gene status (p<0.04), and tended to correlate with high CD38 expression (p<0.06). Patients with low CXCR3 expression (MFI < or =15) had a shorter survival (p<0.0001) and, in multivariate analysis, low CXCR3 expression (MFI pound15) was an independent predictor of poor outcome (hazard ratio 24.5; p<0.01). INTERPRETATION AND CONCLUSIONSL: CXCR3 expression by CLL B cells appears to be stable within individual patients. Tests to assay this chemokine receptor are cheap and easy to perform and the results could be of prognostic value in CLL.

The prognostic evaluation of CLLU1 expression levels in 50 Chinese patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the relentless accumulation of monoclonal B cells with the appearance of small mature lymphocytes and a characteristic CD5 and CD19 co-expression immunophenotype. The incidence of CLL is lower in Asian countries than in Western countries, where CLL is the most common leukemia. To investigate CLLU1 expression in CLL and explore the relationship between CLLU1 expression and alternative prognostic markers, we measured CLLU1 expression levels by semiquantitative RT-PCR in a cohort of 50 Chinese patients with CLL. Analyses of IgVH somatic mutational status, ZAP-70 expression, CD38 expression, and chromosomal aberrations were also performed. The expression of CLLU1 mRNA was determined in 26 of the 50 cases (52%), among which 7 at Binet A (7/21, 33.33%) and 19 at Binet B + C (19/29, 65.52%). The expression levels of CLLU1 were significantly increased in B + C CLL patients at Binet stage compared with those at Binet stage A (P = 0.005). Data for the IgVH somatic mutational status were available for 20 patients with known CLLU1 expression. Five (25%) patients, all expressed CLLU1 mRNA, displayed unmutated IgVH gene usage. While in 15 patients (15/20, 75%) with mutated IgVH gene, only 6 were CLLU1 positive. Patients with unmutated IgVH genes expressed higher levels of CLLU1 than did those with IgVH mutations (P < 0.05). Among 24 CD38+-CLL cases, 17 (70.83%) were CLLU1 positive, whereas only 9 (34.62%) positive cases were identified in 26 CD38−-CLL patients. Thus, the expression of CLLU1 in CD38+-CLL was significantly higher than that in CD38−-CLL. However, no significant difference of CLLU1 expression was found between ZAP-70+ (14/22, 63.64%) and ZAP-70− (12/28, 42.86%) patients (P > 0.05). We conclude that CLLU1 expression was significantly associated with clinical stages, IgVH somatic mutational status and CD38 expression, and might be an important prognostic factor in CLL patients.

Targeting DNA-PK and ATM Increases Sensitivity to Chlorambucil and Fludarabine in Chemoresistant and p53-Defective B-CLL Cells.

A subgroup of B-cell chronic lymphocytic leukemia (CLL) patients develop a more aggressive form of the disease characterised by ZAP70 expression and unmutated IgVH genes. Mutational inactivation of p53 and ataxia telangiectasia-mutated kinase (ATM) are frequent in these patients and confer chemo-resistance. Overexpression of DNA-dependent protein kinase (DNA-PK) correlates with clinical resistance to Chlorambucil (CLB). Thus, alterations in DNA damage signaling pathways are associated with drug resistance. We previously demonstrated that inhibition of DNA-PK sensitizes leukemia cell lines to DNA topoisomerase II poisons and ionising radiation (IR) by inhibition of non-homologous end joining of DNA double strand breaks (DSB). Loss of ATM function compromises the complementary DSB repair pathway, homologous recombination. We hypothesised that repair of DNA damage induced by CLB and Fludarabine may be mediated in part via DNA-PK and ATM and that inhibiting these enzymes would sensitize CLL cells to these agents.NU7441 and KU55933 are potent small molecule inhibitors of DNA-PK and ATM respectively, developed in collaboration with KuDOS Pharmaceuticals (Cambridge, UK). We used these inhibitors in ex-vivo drug combination experiments on CLL lymphocytes derived from a cohort of 60 patients. Lymphocytes were treated with CLB, Fludarabine, Cyclophosphamide, Mitoxantrone or Etoposide in the presence or absence of NU7441 (1μM) or KU55933 (10μM). We observed a concentration-dependent decrease in viability in response to these drugs (XTT assay), and in 70% of cases this decrease was potentiated in the presence of NU7441 (e.g. CLB 2–20 fold potentiation, Fludarabine 2–14 fold). Drug induced caspase 3/7 activity was further increased by NU7441, indicating that DNA-PK inhibition increased cell death via apoptosis. Early data shows that KU55933 also sensitizes tumors to these agents. Increased drug-sensitivity by NU7441 or KU-55933 was independent of disease stage, progression or CD38 status.We quantified γH2AX foci formation (a surrogate marker for DSB) after drug treatment. Fludarabine induced γH2AX foci formation (7 tumors), and this novel observation suggests that Fludarabine induces DSB in quiescent CLL cells. NU7441 increased the number of fludarabine-induced γH2AX foci, implicating DNA-PK as a mediator of DSB repair following Fludarabine treatment. To investigate the significance of p53 functionality on sensitization by inhibitors, we stratified 14 samples from the cohort according to ability to upregulate p21 after IR treatment (FACS analysis). 6/10 cases that displayed impaired p21 upregulation (associated with defects in p53 or ATM function) were sensitized to either CLB or fludarabine by NU7441. 4/4 cases with normal p21 response were sensitized by NU7441. These data are key, since they suggest that NU7441 may have efficacy in cases with p53 dysfunction, a group of tumors representing poor prognosis, drug-resistant patients. Current studies are characterising the cohort (11q,17p deletions) and measuring kinetics of DNA DSB formation and repair. Future studies will further investigate effects of targeting ATM in CLL, and targeting DNA-PK in ATM-defective tumors. Use of DNA-PK and ATM inhibitors represents a targeted and novel therapeutic approach for poor prognosis CLL.

Increased Expression of CD200 Antigen, a Potential Immunotherapeutic Target, Is Consistently Found in CLL Cells Irrespective of Biological Features.

Recently published data has identified the type-1 membrane glycoprotein CD200 as a potential therapeutic target in the treatment of CLL. CD200 has been reported to be expressed at significantly higher levels in CLL compared to normal mature B cells. CD200 has an immunosuppressive function and it's expression by CLL cells may be involved in disease progression by exerting an immunomodulatory effect facilitating escape from host immune responses. In order to further evaluate the clinical and laboratory utility of this antibody the expression of CD200 has been assessed in a range of specimens. We have assessed CD200 expression in 41 CLL patients (36 peripheral blood and 5 bone marrow) and 12 normal samples (5 peripheral blood, 5 bone marrow, 2 lymph node biopsies). Biological features were assessed in 24 known CLL patients (male to female ration 3:1, median age 70 years), 15 untreated and 9 post treatment. Patients were defined as being poor risk if they were ZAP-70+, had deletions of p53, ATM and/or unmutated IgVH genes. Using these criteria 50% of cases were defined as good risk, and 50% as poor risk. Leucocytes were isolated from the specimens by ammonium chloride lysis and an aliquot of 1x106 cells were analysed by 6 colour flow cytometry using the FACS Canto and analysed using FACS Diva software (Becton Dickinson, UK). The level of expression of CD200 above an isotype control was reported for Lymphocytes, Monocytes, Granulocytes, Mature B cells, Germinal centre B cells, Plasma cells and CLL cells. Mean fluorescence intensities were also recorded for each of these groups (table 1). This data shows that CD200 is expressed above control levels by the majority of CLL cells, expression levels were not significantly different between patients with poor and good risk disease (median mfi 1921 and 1677 respectively p=0.42). CD200 was not increased in normal CD5+ B-cells. The very weak expression seen in non-CLL leucocytes may be an artefact of the lower background fluorescence of the control antibody relative to the CD200. Expression levels of CD200 relative to control were to the same magnitude as CD52 and several fold higher than CD20 expression levels, suggesting that CD200 is potentially an excellent target for immunotherapy. In conclusion our data shows that CD200 expression is uniformly higher in CLL than in any other peripheral blood or bone marrow leucocytes. The level of expression appears to be unaffected by prognostic risk factors, suggesting that treatments which target CD200 would be applicable in all patients with CLL. In addition, the lack of CD200 expression on normal leucocytes, particularly on T-cells, is potentially important. The uniformly increased expression of this molecule in CLL also makes this a potential marker for diagnostic and minimal residual disease assays.Table 1Cell TypeMedian %CD200+ (range)Mean MFICLL96.60 (65.2–100)2773.23Mature B cells23.89 (6.06–36.75)69.29Germinal Centre B cells35.60 (2.7–48)871.86Plasma Cells42.50 (2.8–80.9)1939.5T cells0.25 (0.11–0.48)7.73Monocytes3.65 (0.23–6.31)31.86Granulocytes4.47 (0.13–13.25)34.63CD200 Expression in CLL is consistently higher than in normal leucocytes

Prognostic Factors in the UK LRF CLL4 Trial.

The LRF CLL4 trial randomised 777 previously untreated patients with Binet stage progressive A, B or C disease between January 1999 and October 2004 to receive either Chlorambucil, Fludarabine or Fludarabine and Cyclophosphamide. Interphase FISH for deletions of chromosome 6q, 11q, 13q, 17p and trisomy 12, IgVH gene mutational status (98% cut off), CD38 (7% cut off) and ZAP70 (10% cut off) expression were measured at randomisation on 579, 523, 535 and 478 patients respectively. Leukemic cells from 39 patients utilised the VH3-21 gene of whom 33 had homologous CDR3's. Among the biological markers, log rank analysis showed that >20% p53 loss, del 11q, unmutated VH genes, high CD38 and high ZAP 70 correlated with disease progression or death (Table 1) but not deletion of chromosome 6q, 13q and trisomy 12 (p=0.7, 0.3 and 0.2 respectively). There was no difference in PFS or response duration between the 52 patients with 5–20% p53 loss and the 494 patients with no p53 loss. Multivariate Cox regression analysis showed that >20% p53 loss (p<0.0001), unmutated IgVH genes (p=0.0001), deletion of 11q (p=0.02) and male gender (p=0.03) were independent risk factors for short PFS. The effects of stage and age were overridden by FISH abnormalities. High ZAP70 expression was only significant when VH gene mutation status was not included in the model. CD38 expression was only significant in univariate analysis.Table 1VariableProgression or Death/NUnivariate p-value(log rank test)GenderMale384/5730.002Female111/20417q(p53)No131/546<0.00005Yes21/33IgVHUnmutated105/203<0.00005Mutated225/320del 11qNo288/463<0.00005Yes87/116ZAP70Negative140/2420.003Positive158/236CD38Negative110/2010.0001Positive227/334Among the 320 unmutated cases there was no significant difference in PFS between those with 100% homology (227 cases) and those with 99% or 98% homology to the germline sequence (93 cases). Mutated VH3-21 cases were more likely to express ZAP70 than other mutated cases, p=0.004. Excluding patients with >20% p53 loss, patients using the VH3-21 gene had similar progression free survival (PFS) to those remaining patients with unmutated VH genes and an inferior PFS to those with mutated VH genes (2p=0.0001). The adverse prognostic significance of 11q deletions was not clearly evident in an interim analysis presented at ASH ‘05. Patients can now be divided into 3 risk groups (Table 2). This risk stratification provides the basis of evaluating differing treatment modalities for each risk group in subsequent clinical trials.Table 2Risk GroupDefinitionProgression or Death/NUnivariate p-value (log-rank test)3 yr PFSPoor>20%p53 loss28/330%StandardUnmutated VH or 11q deletion or VH3-21208/292<0.0000124.7%GoodMutated VH(excl VH3–21)79/16155.0%

Angiopoietin-2 Expression in B-Cell Chronic Lymphocytic Leukemia: Association with Clinical Outcome and Immunoglobulin Heavy-Chain Mutational Status.

B-cell Chronic Lymphocytic Leukemia (B-CLL) follows an extremely variable clinical course. For some patients CLL is an indolent disease that never progresses to the point of requiring therapy and these patients have a survival time similar to age-matched controls. On the contrary, other patients experience rapidly deteriorating blood count and organomegaly which requires prompt treatment. The overall survival (OS) times range from months to decades. B-CLL patients can be divided into two subgroups on the basis of the presence or absence of somatic mutations in the specific immunoglobulin heavy-chain variable region (IgVH) genes used by leukemic cells. Patients with unmutated IgVH genes usually have an advanced stage and an unfavourable cytogenetic features, require therapy and have a short survival. The biological reasons of the different behaviour of Ig-mutated and Ig-unmutated leukemic clones have not been fully elucidated yet. Angiogenesis is a very complex network which is closely regulated by the orchestrating functions of many angiogenic factors. The balance of cellular expression of all these angiogenic factors determines vascular stabilization or angiogenic remodelling and sprouting or vessel regression. Increasing evidence shows that neovascularization plays a role in the biology of chronic lymphocytic leukemia. The goal of this study was to evaluate the angiogenic status of Ig-mutated and Ig-unmutated CLL in the attempt to identify a possible role of angiogenesis in the adverse clinical outcome of Ig-unmutated CLL patients. So, we first performed a large scale gene-expression analysis on 29 B-CLL patients using microarrays comprising about 20,000 probes and 208 angiogenesis-related genes. We identified 64 up-regulated genes in Ig-unmutated CLL relative to Ig-mutated CLL. Among them, we found angiopoietin-2 (Ang-2) as one of the highest differentially expressed gene (p=3.02x10−6). Then, we evaluated the Ang-2 expression both at transcript and protein level in a wide cohort of CLL, in normal controls and in other haematological malignancies. The data showed an extremely wide range of Ang-2 expression in B-CLL: Ang-2 high-expressing cases were characterized by advanced Binet stage (p=0.032) and significantly shorter progression-free survival than Ang-2 low-expressing subset (median, 16 vs. 146 months) (p=0.006). Moreover, there was a strong correlation between the IgVH mutational status and the Ang-2 gene expression level (p<0.0001). Ig-mutated CLL exhibited very low Ang-2 expression, absolutely similar to the levels measured in healthy controls (median, 0.27 vs. 0.32, p=0.959). On the contrary, Ig-unmutated CLL expressed up to one hundred-fold higher levels of Ang-2 than normal controls(median, 38.61 vs. 0.32, p=0.002). Moreover, Ang-2 was up-regulated in all investigated CML, ALL and AML samples. We observed extremely high levels of expression in CML and ALL (median Ang-2 mRNA, 3948.96 and 190.00, respectively), whereas moderately high levels of Ang-2 were found in AML patients (median, 16.21). These data suggest that increased angiogenesis due to high Ang-2 expression may be involved in adverse clinical outcome of Ig-unmutated CLL patients.