Unmutated IgVH Research Articles

Surface Antigens Identified by Antibody Microarray That Correlate with IgVH Mutational Status and ZAP70 Expression in CLL.

A new method has been developed to evaluate leukemias based on binding of cells in suspension to a microarray of cluster of differentiation (CD) monoclonal antibodies (mAbs) immobilized on nitrocellulose film on a microscope slide. Cell binding is proportional to antigen expression. This method allows simultaneous evaluation of 88 different CD antigens and is feasible with homogeneous populations of mononuclear cells obtained from blood of patients with leukemia. This methodology is potentially useful in diagnosis and classification of different leukemias and lymphomas and it may further aid in identifying and distinguishing subgroups of patients within a particular diagnosis. We utilized this methodology to assess its effectiveness in distinguishing prognostic subgroups in patients with chronic lymphocytic leukemia (CLL), those with mutated versus unmutated IgVH genes and those with <20% ZAP70+ versus >20% ZAP70+ cells. Unmutated IgVH genes were defined as >98% homologous to germline. To do this we identified 101 unselected samples from CLL patients for which IgVH mutational status and/or ZAP70 expression were known. Both IgVH status and ZAP70 expression were known for 70, in 6 only IgVH status, and in 25 only ZAP70 was known. IgVH mutational status and ZAP70 expression were concordant in 62/70 cases. Patient characteristics (median and range) were as follows: age=59yrs(37–82); abs lym=23k/μL(3.6–123); β2M=2.3mg/L(1.2–9.5); # prior treatments= 0(0–6). 20 had Rai low-risk; 67 had Rai-intermediate-risk, and 14 had Rai high-risk disease; 78 were previously untreated. Reduced space linear discrimination analysis and empirical Bayes moderated t-test was used to evaluate relationships between microarray binding and patients with “good prognosis” (IgVH mutated or ZAP70-) and “poor prognosis” (IgVH unmutated or ZAP70+). Empirical Bayes analysis was used to identify CD antigens with significant differential expression between the “good prognosis” and “poor prognosis” groups. Although there was not a significant difference in overall expression between the two groups, there were antibodies that had significantly different levels of binding. For the IgVH unmutated group, increased binding was noted with CD95, CD38, CD2, CD13, CD11a, CD86, CD25, CD9, CD22 (p<.05; FDR-adjusted). For the ZAP70+ group, increased binding was noted for CD38, CD2, CD95, CD49d, CD79b, kappa, and CD11a (pFDR<.05). In the IgVH/ZAP70 concordant cases, the following had increased binding with the “poor prognosis” group (IgVH unmutated/ZAP70+): CD38, CD95, CD2, CD13, CD24, CD9, and CD11a (pFDR<.05). There is clear evidence of differential binding on the mAb microarray between prognostic groups, although it is not yet possible to accurately predict ZAP70 or IgVH mutation status based on microarray results. Work continues to correlate these findings with flow cytometry and with other meaningful prognostic factors such as β2M; Rai stage, etc. In addition, follow-up continues to assess correlations between microarray results and significant clinical endpoints including survival. This is a simple immunomicroarray method that may be useful and important to enhance the classification and prognostic assessment of patients with lymphoid malignancies, including CLL, based on correlations between an extensive immunophenotype and clinical endpoints.

Allogeneic Transplant with Reduced Intensity Conditioning Regimens may Overcome the Poor Prognosis of B-Cell Chronic Lymphocytic Leukemia with Unmutated Immunoglobulin Variable Heavy-Chain Gene and Chromosomal Abnormalities (11q− and 17p−)

To evaluate the efficacy of reduced intensity conditioning (RIC) allogeneic transplant in 30 patients with poor-prognosis chronic lymphocytic leukemia (CLL) and/or high-risk molecular/cytogenetic characteristics. Eighty-three percent of patients had active disease at the moment of transplant. That is, 14 of the 23 patients analyzed (60%) had unmutated immunoglobulin variable heavy-chain gene (IgV(H)) status; 8 of 25 patients (32%) had 11q-, with four of them also displaying unmutated IgV(H); and six (24%) had 17p- (five were also unmutated). After a median follow-up of 47.3 months, all 22 patients alive are disease free; overall survival and event-free survival (EFS) at 6 years were 70% and 72%, respectively. According to molecular/cytogenetic characteristics, overall survival and EFS for unmutated CLL and/or with 11q- aberration (n = 13) were 90% and 92%, respectively, not significantly different to those with normal in situ hybridization, 13q- and +12, or mutated CLL (n = 7). All six patients with 17p deletion were transplanted with active disease, including three with refractory disease; all except one reached complete remission after the transplant and two are alive and disease free. Nonrelapse mortality (NRM) was 20%; more than two lines before transplant is an independent prognostic factor for NRM (P = 0,02), EFS (P = 0.02), and overall survival (P = 0.01). Patients older than 55 years have a higher risk of NRM (hazard ratio, 12.8; 95% confidence interval, 1.5-111). Minimal residual disease was monitored by multiparametric flow cytometry in 21 patients. Clearance of CD79/CD5/CD19/CD23 cells in bone marrow was achieved in 68% and 94% of the patients at days 100 and 360, respectively. According to these results, RIC allogeneic transplant could overcome the adverse prognosis of patients with unmutated CLL as well as those with 11q- or 17p-.

Mutations in the ATM gene lead to impaired overall and treatment-free survival that is independent of IGVH mutation status in patients with B-CLL

AbstractThe ataxia telangiectasia mutated (ATM) protein is the principal activator of the p53 protein in the response to DNA double-strand breaks. Mutations in the ATM gene have been previously found in B-cell chronic lymphocytic leukemias (B-CLLs) but their clinical significance is unknown. We analyzed 155 CLL tumors and found 12% with ATM mutations and 4% with TP53 mutations; 2 tumors contained mutations in both genes. Retrospective analysis on selected samples indicated that the ATM mutations were usually present at diagnosis. Compared with patients with wild-type ATM/TP53 genes, patients with ATM mutations had statistically significantly reduced overall and treatment-free survival. Although present in both IGVH mutation subgroups, ATM mutations were associated with unmutated IGVH genes and they provided independent prognostic information on multivariate analysis. Mutations in the ATM gene resulted in impaired in vitro DNA damage responses. Tumors with ATM mutations only partially correlated with tumors with loss of an ATM allele through an 11q deletion and, interestingly, those 11q-deleted tumors with a second wild-type ATM allele had a preserved DNA damage response. The majority of patients with ATM mutations were refractory to DNA damaging chemotherapeutic drugs and as such might benefit from therapies that bypass the ATM/p53 pathway.

Differential expression of CD180 and IgM by B‐cell chronic lymphocytic leukaemia cells using mutated and unmutated immunoglobulin VH genes

We have studied the surface expression of the Toll-like receptor family member CD 180 on cells from 78 patients with B-chronic lymphocytic leukaemia (B-CLL). B-CLL cells had variable levels of CD 180 expression, but this was always less than that expressed by normal blood B cells and was stable for 24 months. Significantly higher levels of CD 180 were expressed by B-CLL cells with mutated IGVH genes compared with those using unmutated IGVH genes. This was in contrast to the higher levels of expression of surface immunoglobulin M by B-CLL cells using unmutated, rather than mutated IGVH genes. CD 180 was functional on B-CLL cells from some of the patients, as shown by the increased expression of CD 86 following incubation in vitro with anti-CD 180. The differential expression of CD 180 amongst B-CLL patients is one more marker that may define more precisely the different biological properties of this heterogeneous disease.

The LPL/ADAM29 expression ratio is a novel prognosis indicator in chronic lymphocytic leukemia

AbstractAlthough the zeta-associated protein of 70 kDa (ZAP-70) is overexpressed in patients with chronic lymphocytic leukemia (CLL) displaying unmutated IGVH genes and poor prognosis, a previous microarray study from our group identified overexpression of LPL and ADAM29 genes among unmutated and mutated CLL, respectively. To assess the prognostic value of these genes, we quantified their expression by real-time quantitative polymerase chain reaction (PCR) in a cohort of 127 patients with CLL and correlated this with clinical outcome, IGVH mutational status, and ZAP-70 protein expression. IGVH mutational status, ZAP-70, and the LPL and ADAM29 mRNA ratios (L/A ratio) were predictive of event-free survival for the whole cohort and for patients with stage A disease. In patients in stage B and C, the L/A ratio was an independent prognostic factor, whereas ZAP-70 did not predict survival. Simultaneous usage of the L/A ratio and ZAP-70 expression allowed an almost perfect (99%) assessment of the IGVH status in the 80% of patients with concordant results (L/A+, ZAP-70+ or L/A-, ZAP-70-). LPL and ADAM29 gene expression could also be determined by a simple competitive multiplex reverse transcription PCR assay. Overall, quantification of LPL and ADAM29 gene expression is a strong prognostic indicator in CLL, providing better prognostic assessment than ZAP-70 in advanced stages of the disease. (Blood. 2005;106:650-657)

In B-CLL, time to first treatment is dependant on the IgVH gene mutations and expression of CD38 on leukemic cells

6555 Background: Clinical outcome in B-CLL is correlated IgVH gene mutations and CD38 expresion A recent study repoted that expression of ZAP-70 and IgVH gene mutations could be correlated with time from diagnosis to treatment which could be used as a reliable end point for progression. We report the time to first treatment based on IgVH mutation status and CD38 expression. Methods: Clinical histories were obtained from the patient charts. Analyses of IgVH gene mutations and CD38 expression were done on frozen blood samples from these patients as described elsewhere(Fais et al. J Clin Invest 102:1515,1998 and Damle et al. Blood 94:1840, 1999, respectively). A cut-off of 30% was used to define CD38- vs. CD38+ cases. Mutation was defined as 2% or higher change as compared with germline. Time-to-first treatment was analyzed using Kaplan-Meier curves, comparing these curves using the Log Rank test and Bonferroni-corrected pairwise comparisons performed as needed. Results: A total of 115 CLL patients were analyzed. Conclusions:B-CLL patients with either unmutated IgVH genes and/or CD38+ status (either 1 or both adverse factors) require chemotherapy significantly sooner than those with mutated IgVH genes and CD38- status. The patients in the worse group/s may be followed more closely and also may be enrolled to clinical trials studying early treatment vs. late treatment as the survivals of these patients is also significantly shorter. The following table 1 summarises the results: Table 2 shows the differences in treatment at 2 fixed time points of 2 and 5 years: No significant financial relationships to disclose.

The efficacy of non-myeloablative allogeneic stem cell transplantation in ZAP-70 positive/chemo-antibody refractory patients with chronic lymphocytic leukemia (CLL)

6559 Background and Purpose: In aggressive CLL, the presence of an unmutated IgVH gene is strongly associated with the expression of ZAP-70. We used immunohistochemical techniques and routinely fixed, paraffin-embedded tissue to survey ZAP-70 expression in chemo-refractory patients with CLL who received a non-myeloablative allogeneic transplantation (NMT). Methods: The methodology for the ZAP-70 immunostaining was previously described (Modem Pathology 17:954, 2004). Tissues were scored in evenly labeled areas. All cases were reviewed by two authors. Nuclear staining of non-neoplastic, reactive T cells served as an internal control in each case. Neoplasms demonstrating nuclear staining in greater than 20% of tumor cells were considered positive. Patients were eligible for NMT if they have failed chemo-antibody combination of therapy. The conditioning regimens consisted of fludarabine, cyclophosphamide and rituximab (Blood 98:3595, 2001). Results: Twenty five consecutive pts were treated. ZAP-70 information was available for 22. Median age of these 22 pts was 54 yrs (range, 36–73), and 16 (73%) were males. Median time from diagnosis to NMT was 69 mos. Median # of prior chemoregimens received was 4. Ten (45%) had a B2-microglobulin level of >3, and 7 (32%) had evidence of large cell transformation. Seventeen of the 22 pts (77%) tested positively for ZAP-70 and five were negative. All 22 pts had evidence of active disease prior NMT by morphological examination as well as by flow cytometry. With a median follow-up time of 20 months, overall survival at 2-years was 63% and 80% for the ZAP (+) and the ZAP(-) groups, respectively (P=0.6). Patients with ZAP(+) and who had evidence of large cell transformation faired worse: only 2 of 6 pts (33%) were alive at 9+, and 18+ mos; whereas 10 of 11 pts (91%) who were ZAP(+) but with no evidence of transformation remained alive at a median of 23+ mos post NMT (range, 7+ to 45+ mos) (P = 0.001). Conclusions: These data suggest that NMT may overcome the negative prognostic impact of ZAP-positivity in CLL, and that transplantation should be considered earlier in the course of the disease, prior to large cell transformation. No significant financial relationships to disclose.

ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia

AbstractChronic lymphocytic leukemia (CLL) B cells that express unmutated immunoglobulin heavy-chain variable region genes (IgVH) generally express ZAP-70, in contrast to normal B cells or most CLL cases with mutated IgVH. Following IgM ligation, ZAP-70+ CLL cells had significantly higher levels of phosphorylated p72Syk, BLNK, and phospholipase-Cγ (PLCγ) and had greater[Ca2+]i flux than did ZAP-70–negative CLL cases, including unusual ZAP-70–negative cases with unmutated IgVH. IgM ligation of ZAP-70–negative CLL B cells infected with an adenovirus vector encoding ZAP-70 induced significantly greater levels of phosphorylated p72Syk, BLNK, and PLCγ and had greater[Ca2+]i flux than did similarly stimulated, noninfected CLL cells or CLL cells infected with a control adenovirus vector. We conclude that expression of ZAP-70 in CLL allows for more effective IgM signaling in CLL B cells, a feature that could contribute to the relatively aggressive clinical behavior generally associated with CLL cells that express unmutated IgVH.

CD38 as a prognostic marker in CLL

Cell-surface expression of CD38 in CLL has been recognised recently as a marker of progressive disease and poor outcome. In contrast to traditional staging systems, CD38 is able to identify progressive cases at an early stage. Measurement of CD38, in conjunction with other novel prognostic factors such as p53 and ZAP-70 helps to identify patients who might benefit from early and more intensive therapy. In addition, CD38 positivity can predict unmutated IgVH gene mutation status in most cases. These features, together with its easy applicability, render CD38 a valuable tool in the routine diagnostics of CLL. Questions remaining to be clarified about CD38 include the incidence and significance of its variations during the course of the disease, the optimal method to define CD38 positivity and the impact of different methodologies on results. Only after these issues are resolved can the definitive place of CD38 be defined in the diagnostics of CLL.

Allogeneic Transplant with Reduced Intensity Conditioning Regimens (RIC) Could Reverse Poor Prognosis of Unmutated Igvh, 11q- or 17p- Abnormalities in B-Cell Chronic Lymphocytic Leukemia (B-CLL).

In the present study we have evaluated the outcome of reduced intensity conditioning allogeneic transplant (RIC) in 31 patients with symptomatic B-CLL. 74% of the patients were older than 50 years, and 70% had previously received purine analogues, with 38% of them relapsing or progressing after treatment. At transplant, 83% had active disease. Data from VH homology, FISH analysis, CD38 expression and ZAP 70 expression was available in 21, 27,12 and 2 patients, respectively. 21 out of 31 were conditioned with melphalan plus fludarabine according to our GETH 99–051 protocol; the other patients received other fludarabine-containing regimens. All patients engrafted. Early complete donor chimerism (CDC) (days +28) in Bone Marrow (BM) was observed in 67% of patients; all except two relapsing patients remained CDC after day +180. With a median follow up of 36 months (1–63), 9 patients have died, two of them due to disease progression and seven (22%) due to TRM. In the univariate analysis age older than 55 years, aGVHD and more than two lines of previous chemotherapy significantly influenced TRM (p<0,05). On multivariate analysis only the latter variable remained as an independent prognostic factor (p=0,02).After a median follow up of 36 months (1–63), 9 patients have died, two of them due to disease progression and seven (22%) due to TRM, with a probability for TRM of 26%; Overall survival (OS) and event free survival (EFS) are 64 and 68%, respectively.23 patients are evaluable for response: CR ( no CD19/CD5/CD23 + cells in BM) was 90% in transplanted patients with active or progressive disease −21 cases−. In 68% of patients, the response was time related to the development of GVHD; with the two latest responses observed at days +240 and +360. Regarding biological characteristics, 12 of the analyzed patients showed unmutated IgVH. The CR rate, EFS and OS of these patients did not differ from that of mutated IgVH patients . In this series 11 patients showed poor prognosis cytogenetics abnormalities (11q- in 7 and 17 p- in 4) and 5 of them had also unmutated IgVH. This group of patients showed a similar outcome to that of patients with normal cytogenetics. CD38 overexpression was detected in 7 out of 10 patients and only one has relapsed. Two patients died due to progression, one was unmutated with normal FISH and the other was 17 p- and CD38+; eight patients had at least two of the above mentioned abnormalities, including 3 with 17p-, and 7 of them, remained in CR between 20 and 63 months. According to this data, a GVL effect associated with GVHD appears to exist in CLL patients receiving RIC allograft Our data demonstrates the efficacy of RIC related allogeneic transplantation in patients with B-CLL and adverse prognostic features. Moreover, our data suggests that CLL could be cured with RIC allograft, avoiding the high TRM of myeloablative regimens.

Differential Expression of CD180 and sIgM by B Chronic Lymphocytic Leukaemia Cells (B-CLL) Using Mutated and Unmutated IgVh Genes.

Introduction. B cell chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease as shown by differential expression of a variety of surface and cytoplasmic markers. In a search for markers that could define biological activity of different B-CLL subsets, we have studied the surface expression of the Toll-like receptor (TLR) family member CD180 in relation to other surface markers and mutation status of IgVh genes.Methods. Seventy eight B-CLL patients (68 untreated and 10 treated) and 15 age-matched controls were studied in three different clinics. CD19+ B cells were stained using indirect immuno fluorescence for CD180, surface IgM (sIgM), CD79b and CD38, analysed by flow cytometry and data expressed as the relative antibody binding sites (RBS)/cell for each marker. Monoclonal anti-CD5 antibodies were also used with anti CD180 to determine levels of expression of CD180 in control CD5+ cells. IgVh mutation was determined for 47 patients.Results B-CLL cells had variable levels of CD180 expression, but this was always less (1036 ± 935 RBS/cell) than that expressed by normal blood B cells (5548 ± 2271 RBS/cell) and was stable for up to 18 months. Significantly higher levels of CD180 were expressed by B-CLL cells with mutated (M) compared with those using unmutated (UM) IgVh genes. This was in contrast to the higher levels of expression of sIgM by B-CLL cells using UM than M IgVh genes (Figure).Conclusions. CD180 is expressed at higher levels on B-CLL cells using M than those using UM IgVh genes and is in contrast to the level of expression of sIgM which is higher on B-CLL cells using UM versus M genes.This differential expression of CD180 supports the notion that B-CLL cells using UM IgVh genes represent a population of cells actively responding to signals (perhaps to self antigens) via their surface IgM. [Display omitted]

Prognostic Relevance of Lipoprotein Lipase (LPL) Expression in B-CLL.

We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic and potential functional relevance. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 55 CLL patients differed by 2 orders of magnitude between cases with mutated (N=28) or unmutated (N=27) IGVH (median: 1.62 vs. 100.43 compared to normal PBMNC). LPL expression correlated strongly with IGVH mutational status (R=0.78; P<0.0001). High LPL expression (≥ 10) predicted unmutated IGVH status with an odds ratio of 128.71 (P < 0.0001) and discriminated between mutated and unmutated cases in 51 of 55 patients (93%). In an extended cohort of 104 patients, a trend for LPL as a prognostic factor regarding overall survival was found: While the median overall survival (OS) in patients with LPL expression of more than 10 was 105 months, the median OS was not reached in the low LPL group. Importantly, high LPL expression (≥ 10) was significantly associated with a short treatment-free survival (median 40 vs. 96 months, P = 0.001). By indirect immunofluorescence, LPL was detected in the cytoplasm of leukemic B-CLL cells and showed intense, large granular and punctate distribution. Fine granular and diffuse staining was observed in monocytes but no immunoreactivity was detected in T- cells. In order to compare the LPL protein expression unmutated (LPL mRNA high) and mutated (LPL mRNA low) samples from 10 patients (5 mutated and 5 unmutated) were prepared and stained under identical conditions.The intensity of intracellular immunoreactivity to LPL antibody in B-CLL cells was higher in each unmutated compared to the mutated samples. In order to investigate the association of LPL with other genes we performed gene expression profiling on 12 patient samples selected for high or low LPL yielding 251 differentially expressed genes: 171 genes associated with LPL only (group 1), 58 genes with LPL and mutational status (group 2) and 22 genes with mutational status only (group 3). While group 2 and 3 contained a number of genes previously described by Rosenwald and Klein*, a considerable number of genes associated with lipid metabolism were clustered in group 1, indicating a functional role of LPL in B-CLL cells. Our data establish LPL as a prognostic marker and suggest functional consequences of LPL overexpression in patients with B-CLL.

Gene Expression in CLL Cells Associated with Lymphadenopathy and Chromosome 11q23 Deletion.

Previously we have demonstrated that peripheral blood samples with B-cell Chronic Lymphocytic Leukemia (CLL) have different gene expression profiles associated with chromosome aberrations detected by fluorescence in situ hybridization (FISH). In particular, the vast majority of differentially expressed genes were related to the presence of the 11q23 deletion. The 11q23 deletion has previously been shown to be correlated with shortened over-all survival and extensive/bulky lymphadenopathy. In this study we sought to identify genes whose expression may play role in the progression of CLL in patients that carry the 11q23 deletion. Gene expression from 10,700 human gene specific 50-mer oligos (MWG Biotech, Ebersberg, Germany) was compared between two groups of peripheral blood CLL samples. The first group consisted of CLL patients with the 11q23 deletion detected by FISH as well as with the presence of abdominal/mediastinal lymphadenopathy. The second group consisted of CLL patients without the 11q23 deletion and without the presence of known abdominal/mediastinal lymphadenopathy. The non-11q deletion group included CLL patients with the 13q14 deletion, trisomy 12, 17p13 deletion, as well as several without any detectable abnormality. Immunoglobulin heavy chain variable region (IgVH) mutational status was compared in CLL samples in both groups to ensure that resulting expression differences were not the result of this known prognostic marker. Median of ratios was compared between the two groups. Eighty-eight (87) genes had a p-value < 0.01. Gene function was classified at the European Molecular Biology Laboratory (EMBL) Bioinformatic Harvester website. Twenty of these differentially expressed genes were cell cycle/cell signaling related genes that were over-expressed in the 11q23 deletion group. Examples include: activating transcription factor 4, rho-associated coiled-coil containing protein kinase 2, fibroblast growth factor 21 precursor, signal transducing adaptor molecule 1, mad2-like 1, interferon receptor 1, pim-2 oncogene, and zw10 interactor. In comparison, using the same peripheral blood CLL samples, 78 genes were differentially expressed (p < 0.01) between those samples that had a mutated IgVH versus those that had an unmutated IgVH. Therefore, the presence of both the 11q23 deletion and bulky abdominal/mediastinal lymphadenopathy significantly alters the gene expression profile of peripheral blood CLL cells, particularly genes related to the cell cycle and cell signaling related processes. Biological roles of some of these genes may help further elucidate the basis of the clinical behavior of CLL patients

ICOS Is Induced by B Cell Receptor Signalling on Chronic Lymphocytic Leukemia B Cells.

Patients with chronic lymphocytic leukemia (CLL) cells that express unmutated immunoglobulin (Ig) heavy chain variable region genes (IgVH genes) generally have a more aggressive clinical course than do patients with leukemia cells that express mutated IgVH. The reason(s) accounting for this are not known. Microarray gene expression analyses revealed that CLL cells that express unmutated IgVH could be distinguished from the leukemia cells that express mutated IgVH via the differential expression of a relatively small number of genes, one of which encodes the zeta-associated chain of 70kD (ZAP-70), which generally is expressed by CLL cells that express unmutated IgVH. Although the expression of ZAP-70 is associated with expression of unmutated IgVH in CLL, this association is not absolute. This was the case for a pair of monozygotic twins who both developed CLL at age 57. Although each of the twins had leukemia cells that expressed mutated IgVH, only one of the twins had leukemia cells that lacked expression of ZAP-70 protein and has indolent, non-progressive disease (Blood 100: 4609–14, 2002). We performed microarray analysis using Affymetrix HG-U133A array on the isolated leukemia cells of each twin to define the genes that were differentially expressed between the two. In addition to ZAP-70, we found that the CLL cells of the twin with progressive disease also expressed the inducible co-stimulatory molecule (ICOS), a member of the CD28/CTLA-4 family of immune accessory co-stimulatory molecules that ordinarily only is expressed by activated T cells. Expression of ICOS protein by this leukemia B cell population, but not by the CLL B cells population of the other twin, was confirmed using fluorochrome-labeled anti-ICOS mAb and flow cytometry. We examined the CLL B cells from 58 additional patients for expression of ICOS by flow cytometry and found that 16 (28%) also expressed ICOS. We found that expression of ICOS was associated with expression of ZAP-70, as assessed via flow cytometry and immunoblot analyses. Whereas 14 of the 29 ZAP-70+ cases expressed ICOS, only 2 of the 29 ZAP-70-negative cases expressed this immune co-stimulatory molecule. Nevertheless, we found that nearly all of the 56 of the 58 cases expressed B7h, the ligand for ICOS. The two cases that did not express detectable B7h expressed ZAP-70 and were ICOS+. In preliminary studies, we found that treatment of ICOS-negative, ZAP-70+ CLL cells (n = 2) with goat anti-human Ig could induce expression of ICOS, suggesting that, as on T cells, this molecule also might be inducible in some cases of B cell CLL. Culture of ICOS+ CLL cells with an anti-B7h mAb capable of blocking ICOS-B7h interactions significantly enhanced ICOS surface expression, as assess by flow cytometry, suggesting that B7h may down-modulate ICOS through paracrine/autocrine receptor-ligand interactions. Because of this we evaluated for functional expression of ICOS on CLL B cells. We found that ligation of ICOS could induce enhanced signaling via the PI3K/Akt pathway in isolated CLL B cells, resulting in enhanced phosphorylation and activation of Akt. As such, we speculate that the expression of ICOS and its ligand in B cell CLL may enhance leukemia cell survival and/or proliferation, potentially contributing to the more aggressive disease observed in some patients with this disease.

Serum Thymidine Kinase Levels, lgVH Mutational Status and Cytogenetic Aberrations in B-CLL.

The biological heterogeneity of chronic lymphocytic leukemia (B-CLL) is well recognised. Clinical staging schemes of Rai or Binet do not identify those patients, presenting with early clinical stage, who nevertheless will have progressive disease. The presence or absence of somatic hypermutation in the variable region of the immunoglobulin heavy chain gene (IgVH) identifies two major disease subsets. Patients with unmutated IgVH genes have significantly shorter survivals than do patients with mutated IgVH genes. Cytogenetic aberrations have also been identified, associated with significantly different outcomes. Thus, 17p deletions or mutations and 11q23 deletions have been associated with particularly poor outcomes, trisomy12 with intermediate outcomes and patients with del13q or no detectable abnormality usually have indolent disease. Thymidine kinase (TK) is a cellular enzyme involved in DNA synthesis by a salvage pathway, where it catalyses the conversion of deoxythymidine to deoxythymidinemonophosphate. Serum TK activity is thought to reflect the proliferation activity of the tumour in patients with cancer. Its measurement has been proposed as a prognostic marker in Binet stage A B-CLL patients, with levels over 7.0 U/l identifying those previously untreated Binet Stage A patients with significantly shorter survivals. This study was designed to investigate possible associations between serum TK levels and IgVH mutational status or cytogenetic aberrations. Thirty-four recently diagnosed, previously untreated, B-CLL patients (all Binet stage A) were recruited and gave written informed consent to participate in the study, which was approved by the local research ethics committee. IgVH mutational status and gene usage investigations were performed using a multiplex PCR method and BIOMED-2 standardised primers and protocol, and the sequences analysed using the NIH igblast database. A cut-off at 98% homology to the germ-line sequence was used to classify patients as IgVH mutated or unmutated. FISH analysis was carried out using CLL Set (Abbott Diagnostics). Serum TK levels were determined by batch analysis of previously collected serum samples, which had been stored at minus 80 degrees Celcius until testing, using a radio-enzyme assay (Cambridge Life Sciences, UK). Statistical analysis was performed using SPSS software. Significant differences were found between serum TK levels in patients with mutated (N=20, TK 13.9 U/l) and unmutated (N=14, TK 22.64 U/l) IgVH genes (p 0.012). Twelve out of 14 patients with unmutated IgVH genes, and 11 out of 20 patients with mutated IgVH genes, had serum TK levels >7 U/l. Serum TK levels were significantly higher in patients with intermediate or poor prognosis cytogenetic aberrations (del17p, del11q23 or trisomy12) (25.5 U/l) compared to patients with del13q or no detectable abnormality (15.79) (p0.030). No associations were found between serum TK levels and total leukocyte count or absolute lymphocyte count. These findings identify a significantly higher proliferation activity, measured by serum TK levels, in B-CLL patients with unmutated, compared to mutated, IgVH genes, and in patients with cytogenetic aberrations associated with poorer outcomes. Furthermore, the finding that all but two patients with unmutated IgVH genes had levels >7 U/l is in keeping with previous reports that a level >7 U/l in newly diagnosed, Binet stage A patients, was associated with progressive disease.

Activation and Proliferation of B Cell Chronic Lymphocytic Leukaemia (B-CLL) Cells through Anti-CD180, Anti-CD40 and IL-4.

Introduction: We have previously shown that Toll-like receptor RP105 (CD180) is heterogeneously expressed on B-CLL cells and that the ligation of CD180 by monoclonal antibodies (mAb) on CD180+ B-CLL cells resulted in delineation of responder and non-responder B-CLL clones [1]. In this study we have examined the role of IL-4 together with CD180 and CD40 as activation signals.Methods: Blood mononuclear cells were separated from 7 responder B-CLL patients with both mutated and unmutated Ig Vh genes and 11 controls and were cultured for 72 hours in optimum concentrations of anti-CD180 (G28-8) or anti-CD40 mAb or both in presence and absence of 15 ng/ml of IL-4. CD19+ B cells were stained with mAb to the activation marker CD86 or cell cycle protein Ki-67, measured by flow cytometry and expressed as Mean Fluorescence Intensity (MFI) or % of Ki-67+ cells.Results: B-CLL cells and normal control B cells responded to CD180-ligation by activation and proliferation (Table). Higher levels of CD86 and Ki67 were detected when both anti CD40 mAb and anti CD180 mAb were added (p<0.05) compared with either alone. IL4 alone induced both activation and proliferation of control cells and this was even higher with the leukemic cells (p<0.01) confirming that IL-4 also provides a strong survival/activatory stimulus for B-CLL cells. Addition of IL-4 had no significant enhancing effect on normal B-cells stimulated with both anti-CD180 and anti-CD40, although IL-4 synergised with anti-CD40 in B cell activation (p=0.026) and with CD180 in B cell proliferation (p=0.044).Conclusion: CD180 had an additive effect with CD40 ligation in activation and proliferation of both B-CLL cells and normal control B cells. IL-4 provides a strong additional stimulus for B-CLL cells.CD86 and Ki-67 expression by CD19+ cellsCD86Ki67B-CLLControlB-CLLcontrolSpontaneous−IL-47.2±4.14.0±1.08.1±2.717.4±2.3+IL-422.4±11.811.2±4.217.0±12.823.6±14.8CD180−IL-414.7±5.826.9±13.017.1±10.928.1±12.0+IL-435.4±14.530.0±14.626.9±25.648.3±9.7CD40−IL-419.4±8.814.8±5.214.9±9.734.0±5.1+IL-4286±145.544.0±19.452.7±22.841.0±19.8CD180+CD40−IL-432.5±12.697.0±26.228.0±10.465.5±26.6+IL-4299±163.263.5±27.449.3±14.655.5±26.4

Reversal of Acquired von Willebrand Disease after Allogeneic Hematopoietic Stem Cell Transplantation in a Patient with High Risk Chronic Lymphocytic Leukemia.

Acquired von Willebrand (AvWD) disease is a relatively rare bleeding disorder and since the initial description, fewer than 300 cases of AvWD have been reported. The most frequently observed association is with dysproteinemias such as monoclonal gammopathy of undetermined significance (MGUS) and plasma cell proliferative disorders such as Waldenstroem macroglobulinemia and multiple myeloma. Other hematological diseases associated with AvWD have been reported including lymphoproliferative and myeloproliferative disorders. We report the case of a 59 year old male patient with an AvWD associated with a chronic lymphocytic leukemia.In November 2000 this patient was diagnosed with a B cell chronic lymphocytic leukemia (CLL). Because of an initial Binet-Stage A no specific therapy was initiated. In October 2003 the CLL progressed to Binet-Stage B and molecular examinations revealed an unfavorable risk profile with a 11q22.3-q23.1 deletion and an unmutated IgVH gene status. Because of the presence of an HLA-identical brother the patient was enrolled into a pilot study of allogeneic stem cell transplantation following conditioning with fludarabine and cyclophosphamid in patients with high-risk chronic lymphocytic leukemia. Four cycles of fludarabine and cyclophosphamide were given prior to allogeneic stem cell transplantation and induced a partial remission.Since 2000 the patient reported a new clinical bleeding tendency, presenting mainly as frequent epistaxis. Further laboratory investigations showed a prolonged activated partial thromboplastin time (50 sec.) and a reduced plasma von Willebrand factor (vWF) antigen and vWF activity of 6% and 2%, respectively. This was accompanied by a reduced Factor VIII activity of 14% (factor IX-, XI-, XII- activity > 100%, respectively; lupus anticoagulant assay negative; immunofixation negativ). A vWF multimer analysis showed selective decrease in the high-molecular-weight multimers as seen in type II congenital vWD.The reduced intensity conditioning (RIC) regimens consisted the following combination: Fludarabine 30mg/m² /d (=150mg/m² total dose) and Cyclophosphamide 500mg/m² /d (=2500mg/m² total dose) (day -7 through day -3), GVHD prophylaxis with Cyclosporin A (CsA) and mycophenolate mofetil (MMF).A therapeutic trial of desmopressin (DDAVP) was insufficient and therefore a continuous infusion of the factor VIII/vWF concentrate (Haemate® P; Aventis Behring) was administered from the start of conditioning until platelet recovery. Under this regimen no bleeding complication was observed, neither spontaneously nor during intensive procedures. In accordance with previous reports of replacement therapy in AvWD we observed a shortened half-life of the vWF.On day 30 after allogenic hematopoietic stem cell transplantation we observed for the first time a normalization of the activated partial thromboplastin time followed by a gradual increase of the vWF antigen and activity. On day 60 we were able to document the normalization of both the vWF antigen and activity and the vWF multimer analysis. This nomalisation was coincident with the establishment of a complete donor chimerism for the first time.We therefore believe that this case shows a form of AvWD associated with chronic lymphocytic leukemia and its reversal by treatment of the underlying disease by allogeneic hematopoietic stem cell transplantation. [Display omitted]

Unmutated Immunoglobulin Heavy Chain Variable Region Genes and Disease Progression Post Splenectomy Are Poor Prognostic Factors in Splenic Marginal Zone Lymphoma.

Splenic marginal zone lymphoma (SMZL) is generally an indolent disorder which has a median survival of 10 – 13 years, but a minority of patients pursue a more aggressive clinical course. A number of adverse prognostic factors including the level of haemoglobin, lymphocyte count and platelet count, B2 microglobulin, presence of a paraprotein, IgVH gene mutational status and p53 loss or mutation have been identified, but these have not been consistent among recent series. We have studied 89 patients with SMZL, diagnosed on the basis of lymphocyte morphology, immunophenotype and marrow and splenic histology, when available, and have evaluated the impact of presenting Hb, lymphocyte count, spleen size, presence of a paraprotein, cytogenetic abnormalities and IgVH gene mutational status on both the need for treatment and on overall survival. In 43 patients the diagnosis was made following a routine blood count performed for an incidental reason. The M:F ratio was 1:1.78, median age at presentation was 67.5 years (range; 49 – 91) and mean follow-up was 78.1 months. 63/89 (71%)patients presented with palpable splenomegaly, and a further 5 developed splenomegaly during the course of their disease. 42/89 (47%) patients required treatment of whom 29 (32%) underwent splenectomy. 36 patients have not required treatment, 25 have received an alkylating agent, 12 have received fludarabine and 4 were treated with rituximab.15/29 (17%) received chemotherapy for progressive disease post splenectomy. In six patients there was transformation to a high grade lymphoma. 14 patients (16%) died, 11 of whom had a disease-related death. 24/83 patients (29%) had a paraprotein, 67/87 (77%) had an abnormal karyotype of whom 39 (45%) had an abnormality of 7q. Using interphase FISH, 7/78((9%) showed trisomy 3 and 7/74(9%) had p53 loss. Of the patients with high grade transformation, 3/6 had p53 loss. 47/67 patients (70%) had mutated IgVH genes, and 15 cases utilised the VH1-02 gene segment. Only spleen size >10cm (p=0.0002) and Hb <100 gm/l (p=0.008) correlated with the need for treatment. Only unmutated IgVH genes (p=0.032) and the need for further therapy following initial treatment with splenectomy (p=0.034) correlated with overall survival.The median survival of unmutated cases was 113 months and was not reached for mutated cases.The median survival of patients for whom splenectomy has been their only treatment has not been reached, but was 110 months for patients requiring additional treatment post splenectomy. There is a need for larger, multi-centre studies to identify poor risk patients with SMZL.

CD38 Compared with ZAP-70 or Immmunoglobulin Mutation Status as Predictor of Disease Progression in Chronic Lymphocytic Leukemia.

We performed immunophenotypic and IgVH mutation analyses on the chronic lymphocytic leukemia (CLL) cells of 307 patients for expression of CD38, ZAP-70, and IgVH mutation status to determine the relative strength of each in predicting the need for early treatment. For this we assessed the time from diagnosis to initial therapy for progressive and/or symptomatic disease. Patients confirmed to have remained untreated were censored for this endpoint. Expression of CD38 was defined as the proportion of CD38-stained CLL cells that had fluorescence intensity above a defined threshold. Similarly, cases were defined as ZAP-70+ when 20% or more of the ZAP-70-stained CLL cells had fluorescence intensity above a defined threshold, as described (NEJM 351:893, 2004). CLL cells were classified as expressing mutated IgVH genes if they expressed IgVH with <98% homology with a known germline IgVH gene. The median percentage of cells expressing CD38 in our cohort was 11.5% (range 0.1% to 99.5%). In patients with multiple samples, the CD38 expression level appeared constant over time, with a median S.D. of 0.8%. We defined patient samples that had CD38 expression levels at or below the median as having low expression. There was a significant association between low expression of CD38 and lack of ZAP-70 or expression of a mutated IgVH (p < 0.0001 for each assessment by the Fisher exact test). Median level of CD38 among cases expressing mutated IgVH was 4.0%, compared to 27.4% among cases expressing unmutated IgVH (p < 0.0001 by the Wilcoxon rank sum test). Median level of CD38 among ZAP-70 negative cases was 5.3%, compared to 27.2% among ZAP-70+ cases (p < 0.0001 by the Wilcoxon rank sum test). Patients with low-level expression of CD38 had a median time to initiation of therapy of 8.2 years; whereas, patients with high-level expression of CD38 had a median time to initiation of therapy of 4.0 years (p<0.0001). Although high-level expression of CD38 was associated with a shorter time from diagnosis to initiation of therapy (p < 0.0001 in a Cox proportional hazards regression), the inclusion of IgVH mutation status (p < 0.0001) or expression of ZAP-70 (p < 0.0001) in these models reduced the contribution of CD38 status to a p-value of 0.051 (ZAP-70 model) or 0.052 (mutation status model). Although ZAP-70 expression and mutation status each contributed significantly to the hazard of initiating therapy in a Cox proportional hazards model, high-level expression of CD38 did not add significantly to the model that already addresses ZAP-70 and mutational status, p=0.425. As such, although knowledge of the CD38 expression level can be used as a predictor of the time from diagnosis to the initiation of therapy it provides only marginal value if either the IgVH mutational status or expression of ZAP-70 is known and no value if knowledge of both ZAP-70 expression and IgVH mutational status is available.

Is Zap-70 Protein Detection by Flow Cytometry a Reliable Tool in CLL Prognostic Evaluation?.

Background : Expression of ZAP-70 protein has been shown to be correlated with mutational status of immunoglobulin heavy-chain variable region (IgVH) genes, a major prognostic factor in CLL. We investigated whether the detection of ZAP-70 protein by flow cytometric analysis using unconjugated and conjugated monoclonal antibodies (mAbs) could be applied securely in the workup of patients with CLL. Methods: Flow cytometric analysis of ZAP-70 protein was performed using the method described by Crespo et al (N Engl J Med 2003; 348:1764) with minor modifications. Both fresh and cryopreserved mononuclear cells from CLL patients and healthy donors were fixed and permeabilized using Fix and Perm kit (Caltag Laboratories), incubated with anti-ZAP-70 mAb (clone 2F3.2, Upstate Biotechnology) and then revealed with goat antimouse FITC mAb (Immunotech). Finally cells were incubated with CD3-APC, CD56-APC and CD19-PC5. We also tested 3 mAbs conjugated to various fluorochromes: 2F3.2-FITC (Upstate), 1E7.2-PE (eBioscience), 1E7.2-PE or -Alexa 488 (Caltag). ZAP-70 protein detection in B-cells was expressed either as a percentage of its expression in the T and NK-cells or as a ratio (R) of T-cell mean cell fluorescence (MCF) to B-cell MCF. Western blotting of protein lysates from purified B-cells was carried out to control results obtained by cytometry in 55 samples. Mutational status was defined using a cutoff of 98%. Results: In 13 healthy donors, the mean percentage of ZAP-70 protein expression obtained by flow cytometry with unconjugated mAb (clone 2F3.2) was 4.69% ± 1.93 [range 2–9%] and the R ratio was 6.64 ± 1.54 and > 4.8. In 83 B-CLL samples, ZAP-70 expression was determined using the same method and compared to IgVH mutational status. Results in table below show a 75% concordance between gene mutations and ZAP-70 expression when considering a percentage of positive B-cells > 20%. A better concordance (81%) is obtained with a threshold T-cell MCF/ B-cell MCF at 4 determined by Youden's index. To note the high concordance (90%) between unmutated status and ZAP-70 + expression (19/21). Comparison with at least 1 of the 3 conjugated mAbs has been performed for 63 samples, with discordant results in our laboratories.62 mutated IgVH samples21 unmutated IgVH samplesZAP-70 B-Cells +≤ 20 % : 43> 20% : 19T-cell MCF/B-cell MCF≥ 4 : 48< 4 : 19Conclusions: Our data document the concordance between IgVH gene mutational status and ZAP-70 protein expression measured by flow cytometry, particularly in ZAP-70 negative samples. We found that the indirect method of labelling with unconjugated anti-ZAP-70 mAb remains until now, in our hands, the gold standard method compared to the available dyes conjugate mAbs.