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.

Distinctive IgVH Gene Segments Usage and Mutation Status in 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 in Asian countries is lower than that in the Western ones, where CLL is the most common leukemia. To evaluate the frequency and mutation status of immunoglobulin (Ig) variable heavy chain gene (IgVH) expression in Chinese patients with CLL. We investigated IgVH gene segments usage and mutation status by multiplex RT-PCR in 52 CLL patients, and analyzed the relationship between IgVH somatic mutation status and the expression of CD38, ZAP-70 and CLLU1. 38 patients had mutated IgVH, and 14 had unmutated IgVH. The most frequently expressed VH gene family was found to be VH3 (46.2%) followed by VH4 (40.4%), VH1 (5.8%), VH2 (5.8%) and VH7 (1.9%), with no expression of VH5 and VH6 gene families. VH1-69 and VH3-21 which commonly overused in Western CLL weren't detected in our cohort. The frequency of IgVH gene families indicates significant difference in Chinese CLL patients compared with Western patients, suggesting involvement of ethnic and/or environmental factors in CLL disease initiation. IgVH gene mutation status was significantly associated with the expression of CD38 and CLLU1. The expression of them may be simple and reliable surrogates for the identification of IgVH mutations.VH gene family usage and mutation statusVH familynMutated VH geneUnmutated VH geneVH1330VH2321VH324195VH421165VH5000VH6000VH7101

The Prognostic Importance of T and NK-Cells in a Consecutive Series of Newly Diagnosed Patients with Chronic Lymphocytic Leukemia.

Background: Patients with CLL have a variable clinical course. Current novel prognostic indicators (ZAP-70, CD38, IgVH gene mutation status, FISH) are able to identify early-stage CLL patients at high risk of rapid disease progression. These prognostic factors focus on characteristics of the malignant B cell clone and are not currently changeable factors. Identification of modifiable characteristics of the host unrelated to the leukemic B-cell but that relate to CLL-specific survival may provide opportunities for therapeutic intervention. Since the immune system is likely to modulate disease progression in CLL patients, we evaluated critical features of that system in relation to the size of the circulating clonal B-cell population in a CLL cohort.Methods: 186 consecutive patients with CLL who were evaluated at Mayo Clinic within 2 months of diagnosis between 2000 and 2002 were identified. The primary end-point of the study was to assess if host immunity affected time to treatment (TTT). Baseline flow cytometry analysis was available for 166 patients (88%) and was used to calculate the absolute number of T-cell and natural kill (NK)-cells present at diagnosis. The size of the T-cell/NK-cell compartment relative to the size of the malignant monoclonal B-cell (MBC) compartment was then evaluated by calculating the NK:MBC ratio and T:MBC ratio. Relationships with other prognostic parameters and with TTT were evaluated.Results: Patients exhibited substantial variation in the absolute number of T-cells and NK cells as well as T:MBC and NK:MBC ratios at the time of diagnosis. Higher T:MBC and NK:MBC ratios were observed among patients with early Rai stage and mutated IgVH genes (all P≤0.0002). As continuous variables, both the T:MBC ratio (p value=0.03) and NK:MBC ratio (p value=0.02) were associated with TTT. Since they correlated with TTT as continuous variables, thresholds to classify MBC, T:MBC ratio, and NK:MBC were identified using the method of Contal and O'Quigly. On multivariate Cox modeling including stage, CD38, absolute MBC count, T:MB ratio, and NK:MB ratio, the independent predictors of TTT were disease stage, T:MB ratio, and NK:MB ratio (Table 1).Conclusion: Measurable characteristics of the host immune system appear to 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. Additional studies exploring how interactions between the host immune system and the leukemic clone influence clinical outcomes are needed.

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.

BCRs of Mutated and Unmutated CLL Bind Peptides with Distinct Sequence Features: Evidence from Phage Display Libraries.

In CLL, the use of specific IgV genes to code the clone's BCR is non-random and there is an apparent selection for particular genetic and amino acid structures that can be shared by different patients, supporting the hypothesis that antigenic stimulation influences the development and course of CLL. As the binding specificities of the BCR are largely unknown, a vast variety of antigens may affect the BCRs and defined antigens have yet to be identified. Therefore, we used peptide phage display technology to identify ligands for CLL BCRs. BCRs from 2 IgVH unmutated (U-CLL) and 3 mutated (M-CLL) patients were expressed as IgG1 mAbs and used to probe a 12-mer peptide phage display library. In each case, after 3 rounds of selection, we isolated ligands reactive with the CLL mAbs. For the 3 M-CLL mAbs, phage clones carrying peptide inserts with conserved consensus motifs were found. Specificity of the BCR-ligand interactions was demonstrated in direct and indirect ELISA, since selected phage clones and synthetic peptides bound to their respective M-CLL mAb but not to other M-CLL mAbs. Variation of the amino acid sequence of the synthetic peptides significantly altered their reactivity with the corresponding M-CLL mAb. Furthermore, synthetic peptides were bound only by the proper mAb/BCR, but not by mAbs of other M-CLL or U-CLL patients with BCRs comprised of different IgVH genes, supporting the hypothesis that BCRs of M-CLL recognize a defined epitope. In contrast, the mAbs from 2 U-CLL cases did not select phages bearing a consensus motif. Rather these U-CLL mAbs bound multiple phages expressing the same 12-mer peptides, although these differed in sequence between the two U-CLL cases tested. Furthermore, 2 separate selection procedures using 1 U-CLL mAb isolated multiple phage bearing the same 12-mer sequence on each occasion as well as another set of phages with a completely distinct sequence in 1 of 2 selections. ELISAs demonstrated specific binding of all phage clones and of the synthetic peptides by the U-CLL mAbs. Despite this level of specificity, the 2 U-CLL mAbs also reacted with peptides isolated from panning with other CLL mAbs, thereby displaying considerable polyreactivity. Rather than binding only one distinct epitope, mAbs from U-CLL appear capable of interacting with multiple, unrelated structures. Finally, one of the peptides isolated with an U-CLL mAb was bound by all of the CLL mAbs tested, including those from M-CLL cases; therefore this target is antigenically “polyreactive”. Thus, phage display is a feasible approach to identify specific ligands for CLL BCRs. The two classes of BCRs in M-CLL and U-CLL show substantially different binding properties - the former binding shared amino acid motifs and the latter binding multiple ligands of distinct and identical 12-mer amino acid sequences. These peptides can be used to analyze more precisely the binding sites of CLL BCRs as well as the consequences that ensue after BCR crosslinking, and they might help develop BCR-specific therapeutic agents.

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.

Surprising Lack of Correlation between CD38 and Time to First Treatment in Chronic Lympocytic Leukemia.

BACKGROUND: Although IgVH gene mutation status and expression of CD38 are accepted prognostic markers of patient survival in chronic lymphocytic leukemia(CLL), the relative value of these two markers continues to vex even experts in the field.METHODS: To address this issue we evaluated mutation status and CD38 expression in 159 patients and examined time to first treatment(TTT), as a surrogate endpoint for survival, in various combinations of factors. IgVH gene and CD38 analyses were performed according to standard practice. TTT was analyzed using the product limit method and compared using the log rank test. For these analyses, subjects who had not yet started treatment at the time of cut-off (July 2007) or were lost-to-follow-up were considered censored. A Cox proportional hazards model was used to examine the joint effects of CD38 and mutation status on starting treatment.RESULTS: The two tables show TTT for each CD38 and mutation status independently and in combination. Median TTT for CD38- patients(n=88) was 79 months(95% CI: 60–144) vs 60 months(95% CI: 45–153) for CD38+ patients(n=71). This did not represent a statistical difference(p=0.1891). On the other hand, there was a statistical difference in TTT based on mutation(p<0.0001). Median TTT for mutated patients(n=73) was 192 months(95% CI: 96-not estimable(NE)) vs 48 months(95% CI: 45–60) for unmutated patients(n=86). Cox regression showed that unmutated patients were 2.7 times more likely to start treatment than patients who were mutated(p<0.006). There was a significant difference in TTT based on CD38 status and mutation status combined(p<0.0009). Pair-wise comparisons indicate that: TTT was significantly longer for patients who were CD38-/mutated than for patients who were CD38+/unmutated(p=0.0013); TTT was significantly longer for patients who were CD38-/mutated than for patients who were CD38-/unmutated(p=0.0137). Although the median TTT is longer for CD38+/mutated patients(192 mo) than for the CD38-/mutated patients(120 mo), it is not statistically significant(p=0.7725) and may be effected by the relatively small sample size of the former(n=18).CONCLUSIONS: These data confirm the significant prognostic value of mutation status and time to first treatment in CLL. Surprisingly, and in contrast to a recent report(Rassenti et al, Blood, in press), our results with CD38 expression reveal a lesser level of influence. These findings must be cautiously interpreted to avoid potentially confounding factors. First, a selection bias in patients referred to a tertiary care institution in terms of disease severity or aberrant clinical course may exist. Second, TTT decisions are, to some extent, subjective and our threshold for initiating treatment may be higher than of other hematologists-oncologists. Our data confirm that newly identified prognostic markers need further evaluation.Univariate AnalysisnMedian Time to Treat(95% CI)Total unmutated8648 mo (45–60)Total mutated73192 mo (96-not estimable)Total CD38+7160 mo (45–153)Total CD38–8879 mo (60–144)Multivariate AnalysisnMedian TTT(95% CI)unmutatedCD38+5457 mo (36–72)mutatedCD38+17192 mo (72-not estimable)unmutatedCD38–3248 mo (42–66)mutatedCD38–56120 mo (79-not estimable)

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]

CD38 and ZAP-70 Mark Chronic Lymphocytic Leukemia (CLL) Cells with High Migratory Potential and Regulate Chemotaxis.

The absence of mutations in the IgVH genes along with the expression of the cell surface receptor CD38 and the cytoplasmic kinase ZAP-70 are independent molecular markers of unfavourable outcome in CLL patients. Our group has been interested in unravelling the role of CD38 in CLL not only as disease marker but also as pathogenetic agent. We have shown that CD38 can positively affect proliferation and survival of CLL cells through binding CD31, the ligand expressed by stromal, endothelial and nurse-like cells (NLC). Starting points of our current investigation are thati.NLC produce high levels of the attractant stromal cell-derived factor-1 (SDF-1α/CXCL12),ii.CD38 is an essential mediator of migration and homing in murine neutrophils, andiii.ZAP-70 is a key element in CXCR4 (SDF-1α receptor) pathway in T cells.The consequent working hypothesis is that CD38 and ZAP-70 intertwine to influence CLL cell migration. The analysis of a cohort of 56 clinically and molecularly characterized CLL patients demonstrated that CD38+/ZAP-70+ samples migrate significantly better towards CXCL12 than all the other CLL subgroups, including the CD38+/ZAP-70− one, suggesting a functional association between CD38 and ZAP-70. CD38 engagement by agonistic monoclonal antibodies (mAbs) leads to phosphorylation of the activatory tyrosines present in ZAP-70 in 26 CLL cell cases and in an ad hoc modified B line expressing a ZAP-70-myc construct. More importantly, ZAP-70 is required for CD38 signalling, as the CD38 pathway is impaired in CD38+/ZAP-70− CLL cells. Microarray analyses performed on a subgroup of 20 CLL patients indicate the existence of a genetic signature marking CLL samples endowed with a high migratory potential. These studies resulted in the identification of a panel of 24 differently expressed genes, many of which are known to be involved in adhesion and migration. Taken together, the above data point to a direct role for the CD38/ZAP-70 pathway in CLL migration towards CXCL12. Indeed, CXCL12-mediated chemo taxis of CLL cells and of the NALM-6 pre-B cell line can be efficiently blocked by anti-CD38 mAbs in a dose-dependent fashion. Out of the 9 different anti-CD38 mAbs tested, only the ones binding epitopes close to the C terminus of the molecule interfere with migration, while the ones binding closer to the plasma membrane do not. Pre-treatment of CLL and NALM-6 cells results in the almost complete block of the CXCR4 signalling pathway. This was shown by monitoring Ca2+ mobilization, actin polymerization and ligand-induced internalization. Further, upon exposure to CXCL12, CD38 and CXCR4 become physically associated, as shown using confocal microscopy and co-immunoprecipitation approaches. CD38 however, does not internalize upon CXCL12 biding to CXCR4. Collectively, these data suggest that the blocking effects of the anti-CD38 mAbs are due to steric hindrance. In conclusion, we believe that these results offer biological evidence explaining why the combination of CD38 and ZAP-70 results in a more efficient identification of CLL patients with aggressive disease. Further, it may provide a rationale for using anti-CD38 ligands or inhibitors as an adjunct tool for the therapy of aggressive CLL.

IgM+ B-Cells Lacking CD27 Expression Display Somatically Mutated Ig VH Genes and Define a New Memory Population.

Somatic hypermutation (SHM) and isotype class switch recombination are critical mechanisms that diversify normal B-cells in response to antigen, and are generally confined to the germinal center (GC). Post-GC B-cells differentiate either into memory B-cells or antibody forming cells maturing as plasma cells. Memory B-cells which exit the GC have been regarded as invariably expressing CD27 and this population comprises both IgM+ and switched B-cells. It is, however, the presence of SHM that remains the defining feature of memory status in B-cells. Recently, it has been reported that dye extruding membrane ATP-binding transporters expressed by naive B-cells can distinguish them from memory B-cells which lack transporters and this, notably, led to the identification of IgG+ memory B-cells that did not express CD27. However, these observations were not correlated with SHM status. Here, we have examined these findings further and focused specifically on IgM+D+ B-cells in relation to our interest in mapping origins of B-cell tumors. Using dye-based transporter assays, we identified a substantial IgM+CD27− population in PBMNC from healthy individuals which lacked transporter activity. These cells were next isolated using a different strategy, employing IgD and CD27 expression, and a highly purified (>99%) IgD+CD27− fraction obtained by FACS sorting. There was virtually nil contamination by CD27+ B-cells. IgM transcripts were then specifically analyzed in this CD27− population for VH gene mutations by using Cmu downstream primers. We examined VH3 and VH5 gene transcripts from 2 separate sorts from a healthy donor following amplification by primary and nested RT-PCR and cloning products for sequence analysis. Of 104 VH3 clones from this CD27− fraction, 38 (37%) were germline and the remainder mutated (55% with 98–99.9% homology to germline, 9% <97.9%). Of these, 27% displayed 3–5 or more nucleotide mutations. Analysis of 124 VH5 clones from the same fraction showed 50% to be germline and the remainder mutated (45% with 98–99.9% homology, 5% <97.9%), of which 16% exhibited 3–5 or more mutations. Only 1 VH5 donor germline gene exists in the IgH repertoire, but 4 allelic variants which differ by 1 nucleotide are known. We mapped the VH5 germline gene in our donor using recombination sequence primers and only 1 allele was identified, identical to the donor gene mapped by database alignment, eliminating polymorphisms as a source of apparent base changes. Mutational load was also over and above a low level of PCR error which was quantified in the analysis. This confirmed that the levels of mutation detected in the rearranged VH5 transcripts in the IgM+D+CD27− population were generated by SHM. In contrast, between 74–90% of VH3 and VH5 transcripts from the IgM+D−CD27+ population were mutated, with a higher frequency of sequences (65–87%) displaying 3–5 or more mutations. Our data reveal the existence of a novel IgM+D+ population of normal B-cells which are somatically mutated but lack CD27. Unmutated IgM+D+ B-cells in this population point to heterogeneity in the CD27− B-cell pool. As yet, the clonal derivation of these mutated CD27− cells is undefined. Importantly, they have relevance for understanding the cell of origin in Hairy Cell leukaemia and Waldenstrom's macroglobulinemia, where tumor cells are mutated yet lack CD27 expression.

Molecular Prognostic Factors and Outcome among a Cohort of Patients with Monoclonal B-Cell Lymphocytosis (MBL).

Background: Monoclonal B-cell lymphocytosis (MBL) is characterized by the presence of a circulating population of clonal B-cells where the total B-cell count is <5 × 109/L and criteria for the diagnosis of other lymphoproliferative disorders are not present. In most patients the immunophenotype is similar to that of chronic lymphocytic leukemia with co-expression of CD19 and CD5. The natural history of MBL is unclear. We used the Mayo Clinic hematopathology database to identify patients with MBL and abstracted clinical information on natural history and prognostic parameters from clinical and research records.Methods: After IRB review and approval, we reviewed the hematopathology records of 2292 patients seen at Mayo Clinic between August 1998 and May 2006 who had a clonal B-cell population on flow cytometry phenotypically consistent with CLL and fewer than <5 × 109/L B-cells. Clinical records were then reviewed to identify patients who fulfilled the criteria for MBL (no evidence of other lymphoproliferative disorders, no adenopathy on exam). Patient characteristics, prognostic parameters, and clinical outcomes were abstracted from the clinical and research records of these patients. Cox regression analysis was used to evaluate association between prognostic markers and time to treatment.Results: A total of 118 patients meeting the criteria for MBL were identified. Median age at the time of MBL diagnosis was 69.4 years (range 39.0 – 91.4). A slight male preponderance was observed (54% male). CD38 status was available for 106 (90%) patients of whom 20% were CD38 positive using the 30% threshold to classify CD38. Cytogenetic analysis by fluorescent in situ hybridization (FISH) was available in 58 (49%) patients. Using the hierarchical classification system of Dohner and colleagues, 26 (44.8%) were 13q-, 10 (17.2%) were trisomy 12, 2 (3.4%) were 11q-, 1 (1.7%) was 17p-, and 19 were (32.8%) no defect was detected. ZAP-70 status was available for 32 patients of whom 22% were ZAP-70+ positive. IgVH gene mutation status was only available for 15 (12.7%) patients of whom 40% were unmutated. Time from MBL diagnosis to treatment is shown in Figure 1. Survival free of treatment was 92.7% (95%CI: 86.8–99.1%) at 1 year, 90.9% (95%CI: 84.2–98.2%) at 2 years, and 87.5% (95% CI: 78.7–97.4%) at 5 years. While age (p=0.68) and gender (p=0.14) were not found to be associated with time to treatment (TTT), CD38 status showed a strong relationship with TTT (p<0.001, HR: 22.7, 95% CI: 3.4–239.6).Conclusion: The distribution of prognostic parameters (e.g. CD38, FISH) in patients with MBL appears similar to that of patients with early stage CLL. We observed a low rate of progression, with 5 year survival free of treatment greater than 87%. TTT among newly diagnosed MBL patients was associated with the CD38 status of clonal B-cells. [Display omitted]

CD26 (Dipedtidyl Peptidase IV) Expression in B-CLL.

Background CD26 (dipeptidyl peptidase IV) is a type II transmembrane glycoprotein with prolyl oligopeptidase activity and diverse biological functions including T-cell activation and chemokine regulation. The role of CD26 in tumour cell transformation and development is incompletely understood. Loss of CD26 expression has previously been associated with malignant transformation in melanoma. CD26 appears to be differentially expressed in T-cell malignancies and has been associated with reduced survival and shorter complete remissions in T-cell NHL. CD26 is also expressed on normal B-cells. However, the significance of its expression in malignant B-cell disorders remains unclear. Aims The aim of this study was to investigate the clinical and biological significance of membrane-associated CD26 expression in B-CLL.Methods Two hundred and thirty-two B-CLL patients were recruited for this study. Information on age at diagnosis, Binet clinical stage and time to treatment was available on all patients. Membrane expression of CD26 was assessed by 2-colour flow cytometric analysis of CD19 positive cells using anti CD26 PE. A value of ≥15% positivity was assigned to define a sub-group of patients (high CD26) with a relative increase in surface CD26 expression. IGHV mutational status and gene usage were determined using multiplex BIOMED-2 primers (InVivoScribe Technologies) and protocol and by sequence analysis. Interphase FISH analysis was performed to screen for common cytogenetic aberrations.Results A statistically significant increase in the proportion of males was detected in the high CD26 sub-group (80% versus 58.3% in the low CD26 sub-group, p<0.0001). In addition, increased CD26 expression was associated with advanced stage disease (31.6% versus 19.9%) requiring chemotherapeutic treatment (47.4% versus 29.1%). No association was observed between CD26 membrane expression and age at diagnosis nor with cytogenetic abnormalities commonly observed in B-CLL. However, somatic hypermutation of the IGVH gene was significantly more frequent in the low CD26 sub-group (p<0.0001). Preliminary results also indicate that IGVH 4–34 gene usage is associated with low CD26 membrane expression. In addition, IGHV 3–21 gene usage (n=19) was not detected in the high CD26 sub-group.Summary / Conclusions In our B-CLL cohort, increased CD26 B-cell membrane expression was associated with the male gender, un-mutated IGHV gene status and more advanced Binet stage requiring treatment, irrespective of age upon presentation. Interestingly, CD26 expression has previously been associated with unfavorable prognosis in T-cell malignancies and poor response to 2′-deoxycoformycin, an inhibitor of the CD26 ligand ADA, although there have been some reports of good responses in CD26 positive T-PLL. Furtheremore, CD26-positive hairy cell leukaemia responds favorably to 2′-deoxycoformycin treatment. The significance of CD26 expression in B-cell malignancies remains to be fully elucidated. The possibility that it may be linked to responsiveness to treatment with the ADA inhibitor 2′-deoxycoformycin is intriguing and warrants further investigation.

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.

High-Level Expression of the T Cell Chemokines CCL3 and CCL4 by Chronic Lymphocytic Leukemia B Cells in Nurselike Cell Co-Cultures and in Response to BCR Stimulation.

Despite their apparent longevity in vivo, isolated Chronic Lymphocytic Leukemia (CLL) B cells generally undergo spontaneous apoptosis in vitro when cultured under conditions that support the growth of human B cell lines. This suggests that interactions between CLL cells and a distinct tissue microenvironment in the marrow and the lymphatic tissues, where CLL cells are in close contact with accessory cells (mesenchymal stromal cells, and CD68+ “nurselike cells”/NLC), are critical for the progression of the disease. NLC can be detected in secondary lymphoid tissues from CLL patients and appear to be an integral part of the CLL microenvironment, comparable to lymphoma-associated macrophages in follicular lymphoma. The molecules involved in CLL-NLC cross talk are only partially understood. Therefore, we examined the gene expression profile of purified CLL B cells using Affymetrix U133 Plus 2.0 Arrays to define distinct expression profiles induced in purified CLL B cells by co-culture with NLC. When compared to freshly isolated blood CLL B cells, we found that CLL B cells co-cultured for 14 days with NLC displayed high level expression of two T cell chemoattractants, macrophage inflammatory protein-1a and b (MIP-1 a, MIP-1b, also called CCL3 and CCL4). CCL3 and CCL4 expression levels correlated with ZAP-70 expression by the CLL cells, suggesting that B cell receptor signaling is involved in inducing the expression of these chemokines. Supernatants from CLL-NLC co-cultures harvested 7 and 14 days after initiation of the cultures revealed high CCL3 and CCL4 protein levels (up to >30 ng/ml) by ELISA, predominantly in the same CLL cases that displayed high CCL3, CCL4 and ZAP-70 expression by expression profiling. Moreover, serum samples from CLL patients were tested for CCL3 and CCL4 protein expression by ELISA. These studies demonstrated higher CCL3 and CCL4 serum levels in CLL patients when compared to healthy volunteers. CCL3 and CCL4 serum levels were found to be higher in CLL patients that were CD38+, displayed non-mutated IgVH genes, and b2 microglobulin levels that are > 4 mg/L. In vitro, B cell receptor (BCR) triggering of CLL cells, using anti-IgM antibodies, induced a rapid and robust induction of CCL3 and CCL4 protein production by CLL B cells. In contrast, CD40 triggering did not induce expression of these chemokines. These studies reveal a novel mechanism of cross-talk between CLL B cells and their microenvironment, namely the induction of two T cell chemokines, CCL3 and CCL4, by CLL-NLC interaction. Thus, we provide the first evidence that neoplastic B cells are an important source of T cell chemokines, that can induce recruitment of T cells of helper/effector phenotype to sites of cognate T cell-CLL interactions. Besides inducing the outgrowth of NLC, this is another mechanism how CLL cells actively create a microenvironment that favors their growth and survival. [Display omitted]

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.

Percentage of Smudge Cells on Blood Smear Predicts Prognosis in Chronic Lymphocytic Leukemia: A Multicenter Study.

Background: Smudge cells are ruptured CLL cells seen on blood smears of CLL patients. For over a century, smudge cells were thought to represent an artifact of slide preparation. We recently showed that smudge cell formation was inversely proportional to leukemic B cell vimentin content. Vimentin is a cytoskeletal protein critical for lymphocyte rigidity and migration; high vimentin content is related to poor prognosis in CLL. Concordantly, in an initial small cohort of patients from a single institution, we found that patients with a low (<30%) percentage of smudge cells on a blood smear have a shorter time to treatment (Mayo Clin Proc. 2007; 82:449–53). In the current study, we evaluated the impact of smudge cell percentage on prognosis of patients with CLL seen at member institutions of the CLL Research Consortium (CRC).Methods: Archived blood smears from untreated patients with CLL were evaluated for smudge levels. All blood smears were prepared manually in a standard fashion from blood obtained by CRC Tissue Core and stained with WrightGiemsa stain. Smudge cells were defined as broken cells with no intact cytoplasm and a disrupted nuclear membrane. A total of 200 lymphocytes and smudge cells were counted on each slide and the results were expressed as the percent smudge cells. The association between the percentage of smudge cells, prognostic factors (IgVH status, CD38, ZAP-70 and FISH) and time to initial therapy (TTT) was examined.Results: We calculated smudge cell percentage on blood smears obtained prior to treatment for 337 CLL patients. The median smudge cell percentage was 32 (range 2–95%). The percentage of smudge cells was lower in CD38 positive patients (mean 33% vs. 38% in CD38 negative patients, p=0.04). No difference in smudge cell percentage was observed based on IgVH gene mutation status or Zap70 expression. Smudge cell percentage as a continuous variable was associated with prolonged TTT, exponential coefficient 0.98, p=0.04. Using our previously published cutoff of 30% to stratify patients in low and high risk categories, the estimated median time to first therapy of patients with smudge cell percentage ≤30% (n=178) was 7.8 years versus not reached in patients in patients with > 30% (n=159) of smudge cells, p=0.0036, (Figure 1). Ten years from diagnosis, 62% of patients with ≤30% of smudge cells versus 39% of patients with >30% of smudge cells required therapy. In multivariate analysis, the low percentage of smudge cells (≤30%) was an independent predictor of the shortened time to treatment (HR 1.86, 95%CI 1.09–3.16, p=0.02).Conclusion: This multicenter study confirms our initial finding that the percentage of smudge cells on a blood smear is an independent predictor of clinical outcome in patients with CLL. The estimation of smudge cell percentage on routine blood smear provides a simple and inexpensive prognostic test available to nearly all patients with a diagnosis of CLL worldwide. It also allows reanalysis and risk stratification of previously completed trials provided that blood smears have been archived. Further studies of the role of the cytoskeleton in CLL biology are warranted and ongoing in our laboratory. [Display omitted]

IgVH Mutational Status Does Not Affect Complete Remission Rate but Is Associated with Reduced Remission Duration in CLL Patients Treated with Fludarabine, Cyclophosphamide and Rituximab (FCR)-Based Therapy.

IgVH mutational status is associated with inferior survival in studies of patients with heterogenous genetic background and treatment status. Its significance in patients receiving modern fludarabine- and rituximab- containing chemoimmunotherapy is currently unknown. In order to prospectively evaluate its significance in such patients, we examined the relationship between pre-treatment IGVH mutation status and clinical outcome in patients followed in two independent cohorts at the MD Anderson Cancer Center. Cohort 1 contains 95 uniformly treated patients from the phase II study of FCR as initial therapy for CLL (Keating JCO 23:4079), treated between 1999 to 2004, with unknown FISH status. Cohort 2 contains 95 patients from the Low Risk FISH study (LRF), a prospectively collected set of untreated patients with low risk (not 11q or 17p) FISH findings followed between 2004 to 2007, who started therapy with a FCR-like regimen: FCR as standard treatment (n=30, complete remission (CR) 73%); FCR with thrice weekly rituximab in the first week (n=25, 72% CR); FCR & mitoxantrone (n=19, 84% CR); CFAR (FCR & alemtuzumab, n=9, 78% CR); FCR & GMCSF (n=6, 3 CR); and fludarabine & rituximab (n=2, 2 CR). Mutational status were determined from pre-therapy peripheral blood specimens in all patients from cohort 2, and from 77 patients from cohort 1. Eighteen patients from cohort one without pre-FCR blood samples had mutational status determined from pre-treatment formalin-fixed parafin embedded bone marrow blood clot. Mutational status had no significant impact on the CR/p (complete remission with or without cytopenia) rate of FCR in patients treated in cohort one. Similarly, patients in cohort two who received FCR-like therapy had comparable CR/p rates (Table). The median follow-up for patients in cohort one was sixty-seven months. In this cohort, patients in CR (n=75) with mutated IGVH gene showed a significantly longer time to progression (TTP) compared with unmutated patients: at 5 year follw up, 93% mutated patients (n=29) remained in remission, compared with 46% in unmutated patients (n=46) (p= 0.0006). Amongst those patients in flow cytometry negative CR, 100% mutated patients (n=20) remained progression free at five years, compared with 67% for unmutated patients (n=36) (p=0.006). Despite being associated with shorter remissions, IGVH mutational status has not yet impacted on long-term survival, with 5 year survival rates of 83% and 77% for mutated and unmutated patients in cohort one respectively (p=0.34). The median follow-up was too short in cohort two for evaluation of TTP or survival. This analysis of prospectively collected patients with uniform treatment (cohort one) or similar genetic status (cohort two) showed that IGVH remained an important prognostic factor in the era of highly potent chemoimmunotherapy, with the main impact being reduction in remission duration. Most patients in CR with mutated IGVH remain in remission at five years.NCR/p(%)P-ValueCohort 1, MUT3329 (88%)0.19Cohort 1, UNMUT6246 (74%)Cohort 2, MUT3425 (74%)0.84Cohort 2, UNMUT6146 (75%)

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.

Monoclonal B-Cell Lymphocytosis (MBL) Is a Precursor State for Chronic Lymphocytic Leukemia (CLL) with 1% Progression Per Year.

The 2007 IWCLL guidelines indicate that a diagnosis of Chronic Lymphocytic Leukemia (CLL) requires a B-cell count above 5,000/μL in the absence of other features; below this level the diagnosis is Monoclonal B-cell Lymphocytosis (MBL). There is little outcome data for MBL patients and it is not clear whether the detection of low levels of CLL cells, seen in 3% of the general population, is of clinical relevance. We have therefore investigated two hospital populations: the first with normal blood counts and no history of cancer; and the second MBL patients referred for investigation of a current or prior lymphocytosis. Blood samples from 1520 outpatients aged 60–80 with a normal blood count were screened: CLL cells were detected in 78/1520 (5.1%) with a median CLL cell count of 140/μL (range 15 – 1,248). Chromosomal abnormalities were frequently detected in purified CLL-phenotype cells (deletion 13q14 in 15/38, trisomy 12 in 4/22) although poor-risk abnormalities (deletion 11q or 17p) were not detected. The median IgVH mutation was 6.6% (range 0.5 – 13.7%) with 85% of cases showing >2% mutation from germline. The IgVH gene usage was heavily biased with a similar profile to mutated CLL. Detection of CLL cells in individuals with a normal count was not associated with increased mortality (estimated yearly rate 6.2% vs. 8.9% for matched controls, P=0.76) or risk of developing CLL as subsequent lymphocyte counts remained normal in all cases. A diagnosis of MBL was established in 309 of 2228 referrals for investigation of lymphocytosis between 1995 and 2000. A cohort of 185 MBL patients was monitored for a median 6.7 years (range 0.2 – 11.8): the presenting B-cell count was a median 3,100/μL (range 30 – 5,000), age 73 years (range 42 – 96); IgVH mutation rate was 7.1% (range 1.3 – 9.3%) with 96% of cases showing >2% mutation from germline. Progression to a lymphocyte count above 30,000/μL occurred in 15% of cases (28/185) and chemotherapy for progressive CLL was required in 7% (13/185). The absolute B-cell count was the only independent risk factor for an increasing disease levels. Neither IgVH mutation status nor CD38 expression predicted risk of disease progression or requirement for treatment. During follow-up 33% died: age above 70, hemoglobin concentration below 11 g/dL and T-lymphopenia (CD3+ <1,000/μL) predicted shorter survival, whereas patients presenting with a T-lymphocytosis (>2,400μL) had significantly longer survival. Development of progressive disease did not predict overall survival: 7/13 patients requiring therapy remain alive at a median 1.9 years (range 0–8.6 years) after initiation of treatment. The total lymphocyte count had no impact on the risk of disease progression, time to treatment or overall survival. CLL-phenotype cells are genetically equivalent to CLL even when detected in the general population but are not associated with increased mortality or risk of progression to CLL when present below 1,500/μL. MBL patients with higher levels of CLL cells show a steady increase in disease levels over time with 1–2% per year requiring chemotherapy for progressive disease. As such, periodic monitoring is indicated but this should have a minimal impact on lifestyle as MBL patients are often elderly with multiple health issues. MBL is a newly described disorder which is related to CLL in a similar way that MGUS is related to myeloma.