IgVH Gene Mutational Status Research Articles

R-(−)-Gossypol (AT101) Binds to Bcl-2 Family Proteins and Induces Apoptosis in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B cells in the blood, secondary lymphoid tissues, and marrow. The leukemia cells primarily are arrested in the G0/G1 phase of the cell cycle and appear resistant to programmed cell death. Several anti-apoptotic proteins are over expressed in CLL and this correlates with resistance to treatment, disease progression and overall poor prognosis.Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L), Bcl-2, and Mcl-1, are overexpressed in many cancers including CLL and contribute to tumour initiation, progression and resistance to therapy. Mcl-1 is of particular interest because this molecule appears to be regulated by Nurse-like Cells and other stromal cells that promote survival of CLL cells in vitro and very likely also in vivo.These proteins enhance the resistance of CLL cells to spontaneous and/or drug-induced apoptosis primarily by interacting with, and antagonizing the activity of mitochondria membrane pro-apoptotic proteins such as Bax and Bak. The protein-protein interaction of Bcl-2 family members is critical for their activity, and these interactions are governed by binding to the BH3 domain. Racemic gossypol is found in cotton seeds and has been studied as a cytotoxic agent in cancer cell lines and in clinical studies in patients with a large variety of cancers. The antitumor activity of racemic gossypol appears to reside principally in the R-(−)-enantiomer (AT101), with reduced activity observed for the S-(+)-enantiomer. AT101 is an antagonist of the BH3-binding groove of the Bcl-2 family of proteins that can inhibit the interactions of these proteins with pro-apoptotic molecules.We examined whether AT101 could induce apoptosis in Chronic Lymphocytic Leukemia (CLL) and its ability to bind in vitro anti-apoptotic molecules from the Bcl-2 family. Using a Fluorescence Polarization Assay (FPA) we studied the competitive binding affinity of AT101 to Bcl-2 family member proteins. We observed that both, racemic gossypol and AT101 had comparable affinity for Bck-2, Bcl-B, Bfl-1 with EC50=0.6 to 10 μM range. AT101 had a stronger binding affinity to Bcl-X(L) (EC50=0.998 μM vs. 3.084 μM for racemic gossypol), and to Mcl-1 (EC50= 0.52μM vs. 1.07μM for racemic gossypol).CLL cells were incubated with AT101 for 48 hrs at different concentrations. We observed that leukemia cells were induced to undergo apoptosis in a time and dose dependent manner and that this effect was independent of ZAP-70 expression or IgVH gene mutational status (IC50= 2μM). Cells undergoing apoptosis showed PARP-1 cleavage and upregulation of pro-apoptotic molecules such as Bid, p53, as well as downregulation of Mcl-1.These results indicate that AT101 has stronger pan-specific binding affinity for Bcl-2 family proteins than racemic gossypol, in particular to Mcl-1 and Bcl-X(L), and that this compound induces apoptosis in CLL B cells independently of ZAP-70 expression or IgVH gene mutational status. Because of these encouraging results a clinical trial using AT101 in CLL patients with high-risk features is currently open at our institution.

Evaluation of the Prognostic Implications of Pro- and Anti-Angiogenic Cytokines in 311 Patients with Untreated Chronic Lymphocytic Leukemia.

Background: Angiogenesis has been found to be an important regulator in the disease progression of both solid tumors and hematologic malignancies. In solid tumors, the balance of pro and anti-angiogenic factors appears to be a critical factor in tumorigenesis, with disturbance in favor of angiogenesis promoting tumor growth and metastasis. This observation led to the concept of an “angiogenic switch” where pro-angiogenic influences outweigh anti-angiogenic factors. Patients with CLL have detectable levels of plasma and cellular pro and anti-angiogenic cytokines as well as abnormal neovascularization in the marrow and lymph nodes. Limited studies of pro-angiogenic cytokines have suggested that inter-patient variation in serum/plasma and cellular levels of these markers may have prognostic implications. We evaluated pro- and anti-angiogenic cytokines in a large sample of patients with CLL to evaluate the presence of angiogenic switching and its implications for disease progression.Methods: In a prospective, longitudinal study, we analyzed the serum/plasma, intracellular(determined by flow cytometry), and CLL B cell secreted (24 hour medium values) levels of pro-angiogenic cytokines (VEGF, bFGF) and an anti-angiogenic cytokine (Thrombospondin [TSP]) in 311 patients with previously untreated CLL. VEGF:TSP and bFGF:TSP ratios were calculated to evaluate for evidence of angiogenic switching (higher ratio suggests a pro-angiogenic phenotype). The relationship of angiogenic cytokines to Rai stage, IgVH gene mutation status, % CD38 protein expression, and time to treatment (TTT) from the date of sample were evaluated (median follow-up=11 months). Sequential samples were available on >90% of patients and were evaluated to assess if changes in levels of angiogenic cytokines occurred in patients requiring treatment.Results: Serum, cellular, and secreted levels of VEGF, bFGF, and TSP showed no correlation with Rai stage, IgVH gene mutation (continuous variable), or % expression of CD38 (continuous variable). However, among patients with Rai stage 0-II disease, individuals with lower TSP levels (<75 percentile, p=0.007) or higher bFGF:TSP ratios (continuous variable; p=0.003) had shorter TTT (Figure 1). Similar relationships for a shorter TTT were also observed for secreted TSP levels (p=0.007) and the cellular bFGF:TSP ratio (p=0.009). On analysis of sequential samples, no clear difference in serum, cellular, or secreted levels of VEGF, bFGF, TSP, VEGF:TSP, or bFGF:TSP occurred among those who required treatment.Conclusions: Angiogenic cytokines appear to have prognostic significance in patients with previously untreated early stage CLL. Importantly, we report the original observation that the ratio of bFGF (pro-angiogenic) to TSP (anti-angiogenic) correlates with time to treatment as a continuous variable. This result provides further evidence that angiogenic switching relates to clinical progression in CLL. Longer follow-up of this cohort is needed to fully define the prognostic implications of angiogenic cytokines in CLL and determine if integration of these markers with other prognostic parameters improves risk stratification for early stage patients. [Display omitted]

Toll like Receptors (TLRs) in B Cell Chronic Lymphocytic Leukemia (B-CLL).

Introduction: B-CLL is characterised by the accumulation of long-lived CD5+ B cells. Mature B cells can be activated via TLRs to produce immunoglobulin and proliferate. We have previously reported differential surface expression of CD180, TLR-4 associated receptor, by B-CLL cells in relation to IgVh gene status (Porakishvili N et al., 2004; Blood (ASH annual meeting abstracts) 104: 2807). To further characterise subsets of leukemic cells, we assessed B-CLL cells for TLR mRNA expression.Method: B cells from untreated (n=24) and treated (CHOP n=3, Thalidomide n=1 and chlorambucil and prednisolone n=2) CLL patients, and healthy aged matched controls, (n=14) were isolated by CD19+ selection using iMACs columns (Miltenyi Biotec), total RNA extracted (Qiagen) and primers for TLR2, TLR4, TLR9, CD180 and the adaptor protein MYD88 were used for TaqMan PCR analysis (ABI). GAPDH was used for normalization. Data were expressed as mean delta cycle number (ct) ± standard deviation, giving a value inversely related to the level of expression of the specific gene. Patients were separated into groups according to IgVh gene mutation status or level of Zap 70 expression. Statistical analysis was performed using by Mann-Whitney U test and Spearman rank correlation or Pearson's correlation coefficient.Results: Overall, there was a heterogeneous pattern of TLR expression in patients and controls with no significant differences between them. Treated patients were characterised by higher mRNA levels for the all TLRs measured (TLR9 p=0.018) and MYD88 (MYD88 p=0.026) compared with untreated patients, despite no significant difference of their GAPDH levels (p>0.05). Significant correlations were seen between mRNA levels for the TLRs within the age matched control group (all p<0.05). Groups of patients were further analysed according to mutated (mutated >2%) or unmutated IgVh genes (unmutated <2%). No significant correlation in the TLR expression levels was found within the mutated patient group (n=8) or Zap 70 low expression group (<0.7 level) (n=11). Preliminary data from patients with an unmutated IgVh gene status (n=4) or according to levels of ZAP 70high expression (n=5) showed correlation of mRNA levels for TLRs and MYD88 expression (all p<0.05).Conclusion: Our data indicates a direct relationship between MYD88 and TLRs transcription levels in healthy control individuals. Lack of significant correlation amongst TLR mRNA levels in CLL patients with mutated IgVh genes and Zap70low expression levels suggests aberrant regulation of TLRs in this group.

GX15–070MS, a synthetic small molecule induces apoptosis in vitro and in vivo in chronic lymphocytic leukemia

3167 Chronic lymphocytic leukemia (CLL) B cells express high levels of anti-apoptotic proteins of the Bcl-2 family, such as Mcl-1, Bcl-XL, and Bcl-2. These proteins enhance the resistance of CLL cells to spontaneous and/or drug-induced apoptosis primarily by interacting with, and antagonizing the activity of mitochondria membrane pro-apoptotic proteins such as Bax and Bik. These interactions are governed by the binding to anti-apoptotic proteins via the BH3 domains of Bcl-2-family-member proteins. GX15–070MS is a small-molecule antagonist of the BH3-binding groove of the Bcl-2 family of proteins that can inhibit the interactions of Bcl-2 with proapoptotic molecules. We examined the activity of GX15–070MS on CLL cells in vitro and in vivo in a phase I/II clinical study initiated at our institution for patients with intermediate-risk or high-risk disease refractory to alkylating drugs, fludarabine-based regimens, and, in some cases also Alemtuzumab. Patients were treated with the initial escalating doses of 3.5 and 7 mg/m2. Administration of GX15–070 has been well tolerated and patients have not experienced any Grade 3 or Grade 4 toxicity. We have evaluated in vitro and in vivo the pro-apoptotic activity of GX15–070MS. GX15–070MS induced apoptosis in CLL-B cells in a time and dose-dependent manner with an IC50=10nM at 48 hours in culture. GX15–070MS induced apoptosis in CLL B cells independent of IgVH gene mutational status or ZAP-70 expression. In addition, we have analyzed the effect of this compound based on co-immunoprecipitations and Bcl-2/Mcl-1 mitochondrial displacement assays using video-confocal microscopy. These results indicate that GX15–070MS has activity inhibiting Bcl-2 family member proteins and inducing apoptosis in CLL B cells both in vitro and in vivo at dose ranges that are substantially below those estimated to induce toxicity in preclinical animal models. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Gemin X

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.

Zap70 Methylation Correlates with Zap70 Expression in Chronic Lymphocytic Leukemia.

ZAP70 expression in chronic lymphocytic leukemia (CLL) correlates with the presence of unmutated immunoglobulin chain (IgVH) genes and with poor clinical outcome. We hypothesised that expression of ZAP70 may be related to the methylation status of the ZAP70 gene. Bisulphite sequencing of a 5′ region of the ZAP70 gene showed a significant difference in CpG methylation between ZAP70 positive and ZAP70 negative cells. We developed a bisulfite restriction PCR assay, insensitive to 10% T cell contamination, to determine the methylation status of a CpG dinucleotide 332 base pairs downstream of the transcription start site, which represented methylation across the sequenced region. This assay was used to detect ZAP70 methylation in 87 patients with CLL, 12 patients with mantle cell lymphoma (MCL) and 12 patients with splenic marginal zone lymphoma (SMZL). ZAP70 protein expression and IgVH gene mutation status had been previously established in all cases. Global cytosine methylation was also analysed in a subset of 15 of the CLL patients by the capillary electrophoresis with laser-induced fluorescence method. Considering the CLL cases, the ZAP70 gene was methylated in 53 patients, unmethylated in 32 patients and an equivocal result was obtained for 2 patients. Correlating methylation status with ZAP70 expression, 51 of 53 (96%) ZAP70 positive patients were methylated and 30 of 32 (94%) ZAP70 negative patients were unmethylated (p<0.0001). Similarly, 49 of 54 (91%) patients with mutated IgVH genes were methylated and 27 of 31 (87%) unmutated patients were unmethylated (p<0.001). The median survivals of methylated and unmethylated CLL patients were 211 and 85 months respectively (p<0.0001) which was very similar to that found for ZAP70 expression or IgVH gene mutational status. The mean global cytosine methylation level was 3.9±0.15% in the ZAP70 methylated CLL patients and 4.1±0.12% in the ZAP70 unmethylated CLL patients; the difference between the two patient groups was significant (p=<0.001) which implies that if ZAP70 methylation is malignancy-related, then hypomethylation of ZAP70 in a subset of CLL arises as a consequence of gene specific rather than global hypomethylation. Among the 24 MCL and SMZL patients an absolute correlation was seen between ZAP70 expression and methylation. However, in marked contrast to the correlation seen in CLL, all 7 non-CLL patients with unmutated IgVH-genes and 16 of 17 non-CLL patients with mutated IgVH-genes showed methylated ZAP70. Therefore, measurement of ZAP70 gene methylation is a simple and reproducible method for predicting prognosis in CLL, which correlates closely with ZAP70 expression and IgVH gene mutational status. The assay is suitable for stored DNA and histological samples as well as for fresh or DMSO-frozen cells. In addition, the methodology of our assay presents none of the standardisation problems, which are relevant to the current flow cytometric assays for ZAP70.

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.

Familial Chronic Lymphocytic Leukemia(CLL): The Mayo Clinic Experience.

Background: Over the last decade, increasing numbers of families with at least 2 cases of CLL or other B-cell lymphoproliferative disorders have been described.Methods: Families seen at Mayo Clinic with at least one individual diagnosed with CLL and one or more first or second degree relative diagnosed with CLL or a B-cell lymphoproliferative disorder(LPD) were identified. Clinical characteristics and risk stratification test results (cytogenetic abnormalities by FISH, level of CD38 expression, and IgVH gene mutation status) were extracted and reviewed. Serum, intracellular and CLL B cell secreted levels of pro- and anti-angiogenic cytokines (VEGF, BFGF, and TSP) were also compared between cases of familial and sporadic CLL.Results: Seventy-one families seen at Mayo Clinic were identified meeting criteria for familial CLL. Of the 71 index CLL families, 78.9% (n=56) had at least one other first or second-degree relative with CLL or a B cell LPD, 15.5% (n=11) had 2 other affected members, and 5.6% (n=4) had 3 or more affected members. Of affected family members with B-cell LPDs other than CLL, 64% (n=25) had non-Hodgkin lymphoma, 21% (n=8) had Hodgkin disease, 10% (n=4) had multiple myeloma or Waldenstrom macroglobulinemia, and 5% (n=2) had another lymphoid leukemia (1 ALL, 1 hairy cell leukemia). The median age was 62, with 63% males and 37% females. The majority of individuals had an early Rai stage at diagnosis (stage 0–66%, stage I-19%). Results of cytogenetic testing by FISH, CD38 status, IgVH gene mutation status appear similar in familial and sporadic CLL cases seen at Mayo clinic during the same time interval. No clear pattern of IgVH gene mutation status, IgVH gene family usage, level of CD38 expression, or cytogenetic abnormalities by FISH was seen among multiple affected members from the same family. There were also no significant differences noted in serum, intracellular, and CLL B cell secreted levels of VEGF, BFGF or TSP in the familial samples when compared to a cohort of non-familial samples.Conclusions: Expression of cytogenetics by FISH, immunophenotype, IgVH mutation status and levels of serum, intracellular and cytoplasmic pro- and anti-angiogenic factors(VEGF, BFGF, and TSP) were similar in individuals with familial and sporadic CLL.

Correlative Study of ZAP-70 Gene Expression by Quantitative PCR and CD-38 Surface Marker in Patients with Chronic Lymphocytic Leukemia (CLL).

Patients with chronic lymphocytic leukemia have heterogeneous clinical courses. Many possible prognostic factors have been evaluated as a way to predict each patient's future course. Two such prognostic factors are IgVH gene mutational status and CD38 expression. Recently, ZAP-70 expression has been found to correlate with IgVH gene mutation, but little is known about the correlation between ZAP-70 and CD 38 expression. We sought to evaluate the concordance between the two. METHODS: A procedure was developed at our institution to evaluate ZAP-70 transcripts by PCR. The results were then confirmed by flow cytometry. The blood or bone marrow samples of 14 patients diagnosed with CLL were analyzed by PCR for ZAP-70 transcripts and flow cytometry for ZAP-70 protein expression and CD38 expression. The results of the ZAP-70 transcripts and CD38 expression were then correlated to the clinical courses of the patients. RESULTS: Of the 14 patients analyzed, the majority had Rai Stage I disease at diagnosis (4 Stage 0, 8 Stage I, and 2 Stage II). There was a median follow-up of 28 months. The median number of treatments for these patients was one (range 0–6). Overall, the results for ZAP-70 and CD38 expression were concordant in eight with CD38 data unavailable for 2 patients. In the four patients with discordant results, 3 of them had high CD38 expression and low ZAP-70. However, patients with high ZAP-70 were more likely to require multiple treatments once treatment was indicated. Patients with high CD38 were also more likely to be treated. Cytogenetic data was available for 9 patients with the following results: 5 with normal cytogenetics, 1 with deletion of 11q, 1 with deletion of 13q, and 2 with deletion of 17p. Five of the nine patients had high ZAP-70 expression. CONCLUSIONS: In our institution, the results of ZAP-70 and CD38 expression were concordant 67% of the time. Longer follow-up is necessary and may further delineate the roles of cytogenetics, ZAP-70 expression, and CD38 expression on prognosis in CLL.

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.

Mutational Status of IgVH Genes Consistent with Antigen-Driven Selection but Not Percent of Mutations Has Prognostic Impact in B-Cell Chronic Lymphocytic Leukemia

Mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes, along with CD38 expression, is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL). Configuration of IgVH genes displaying > 2% mismatch has been shown to correlate with longer survivals. In a series of 64 B-CLLs, we failed to confirm the prognostic value of the IgVH gene mutational status by using the suggested cutoff. However, the IgVH mutational status maintained its prognostic value only when evidence of antigen-driven selection could be documented. This was accomplished by applying statistical methods aimed at evaluating a significant skewing of replacement mutations from framework to complementary determining regions, as it occurs during germinal center differentiation of B cells. These data caution against wide application of the 2% somatic mutation cutoff as a prognostic determinant without demonstration of antigen-driven selection.

ZAP-70 expression and prognosis in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease; many patients never need treatment, whereas some have poor outcomes. New treatments, which can induce complete remissions, allow patients with poor outlook to be treated while they are still asymptomatic. Whether or not the IgVH gene is mutated is the best predictor of clinical outcome, but this assay is unsuited to the routine laboratory. The gene coding for ZAP-70, a tyrosine kinase protein normally expressed in T and NK cells, has been shown by gene-expression profiling to be differentially expressed between patients with mutated and unmutated IgVH genes. We assessed whether ZAP-70 could be used as a prognostic marker in CLL. We developed a flow cytometry assay for ZAP-70 protein expression and investigated its concordance with ZAP-70 mRNA expression, IgVH gene mutational status, and clinical outcome in 167 patients with CLL. We showed high concordance between ZAP-70 protein expression and IgVH gene mutations. 108 patients (65%) had mutated IgVH genes and were ZAP-70 negative; 46 (28%) had unmutated IgVH genes and were ZAP-70 positive. Findings were discordant in 13 patients: six (4%) had mutated IgVH genes but were ZAP-70 positive, and seven (4%) had unmutated IgVH genes and were ZAP-70 negative. Expression of mRNA showed 97% concordance with ZAP-70 protein. Median survival was 24.4 years (95% CI 15.1-33.8) in ZAP-70 negative patients and 9.3 years (7.0-11.5) in those who were ZAP-70 positive (hazard ratio 5.5, 2.8-.8). ZAP-70 protein, which can be measured by flow cytometry in the general laboratory, is a reliable prognostic marker in CLL, equivalent to that of IgVH gene mutational status.

Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors

This study evaluates the prognostic significance of genetic abnormalities (detected at or shortly after presentation), clinical stage, lymphocyte morphology, CD38 expression, and IGVH gene status in 205 patients with chronic lymphocytic leukemia (B-CLL). Deletion of chromosome 11q23, absence of a deletion of chromosome 13q14, atypical lymphocyte morphology, and more than 30% CD38 expression are significantly associated with the presence of unmutated IGVH genes. Advanced stage, male sex, atypical morphology, more than 30% CD38 expression, trisomy 12, deletion of chromosome 11q23, loss or mutation of the p53 gene, and unmutated IGVH genes are all poor prognostic factors in a univariate analysis. However, only 98% or more homology of IGVH genes to the germline sequence, loss or mutation of the p53 gene, and clinical stage retain prognostic significance in a multivariate analysis. The median survival of patients with mutated IGVH genes, unmutated IGVH genes, and loss or mutation of the p53 gene regardless of IGVH gene status is 310, 119, and 47 months, respectively. These data should facilitate the design of new trials for the management of patients presenting with advanced disease or poor prognosis early stage disease.