Marker In Chronic Lymphocytic Leukemia Research Articles

Combination of LPL/ADAM29 Ratio and ZAP Expression Can Replace IGVH Sequencing in the Majority of CLL.

Background: The mutational status of immunoglobulin VH genes (IgVH) is an important prognostic marker in chronic lymphocytic leukemia (CLL), but its determination remains unadapted to routine practice. Several reports have showed that ZAP-70, whose expression can be detected by flow cytometry, can be considered as a reliable surrogate marker of IgVH mutational status. We recently conducted a gene expression profiling study of 18 cases of CLL, which pointed out 2 other genes which might also discriminate CLL groups: the lipoprotein lipase (LPL) and the disintegrin and metalloprotease 29 (ADAM29) genes which were overexpressed preferentially in unmutated (UM) and mutated (MT) cases respectively.Methods: We analyzed frozen cells obtained at diagnosis for 127 CLL patients (87 Binet stage A, 40 stage B or C). LPL, ADAM29 and house keeping GAPDH gene expression were measured by real time quantitative polymerase chain reaction in 111 cases, and ZAP-70 protein by flow cytometry in 107 cases, both analyses being performed in 93 cases. LPL and ADAM29 were also quantified in peripheral blood mononuclear cells (PBMC, n=4) and purified B cells (n=3) of healthy individuals. We correlated the results with the previously determined IgVH mutational status and clinical outcome.Results: With a cut-off value determined to be 1 for the LPL/GAPDH copy number ratio, LPL expression had a positive predictive value (PPV) of 68% for UM cases and a negative predictive value (NPV) of 85% for MT patients. Alternatively ADAM29 expression (ADAM29/GAPDH > 3) had a PPV of 77% for MT cases and of 86% for UM cases. Combining LPL and ADAM29 RNA quantifications by a simple 1:1 ratio (L/A ratio; threshold=1) provided slightly better results than those obtained with ZAP-70 (positivity threshold = 20%) for PPV of UM status (90% vs 76%) and similar results for NPV of MT status (90% vs 91%). Simultaneaous usage of L/A ratio and ZAP-70 expression allowed an almost perfect (73/74) assessment of the IgVH status in 80% (74/93) of patients with concordant results (L/A+, ZAP-70+ or L/A-, ZAP-70-). Serial measurements of L/A ratio and ZAP-70 expression showed that both parameters can change over time (median follow-up 38 months, range 6–159) in a fraction of patients (5/25 tested). ADAM29 was not detected while LPL was present at very low levels or absent in PMBC or purified B cells from healthy donors. The IgVH mutational status, ZAP-70 and L/A ratio were predictive of event-free survival for the whole cohort and for stage A patients. However only the L/A ratio was significantly associated with a shorter survival (p=0.03) for stage B/C patients.Conclusions: Combination of LPL and ADAM29 mRNA quantification provides a good surrogate marker of the IgVH mutational status in CLL patients. Used in association with ZAP-70, it represents an reliable alternative to the sequencing of IgVH genes. In addition, it might constitute a more powerful prognostic marker than IgVH mutational status or ZAP-70.

Thrombopoietin as an Independent Prognostic Marker in Chronic Lymphocytic Leukemia.

Several cytokines and growth factors are involved in the regulation of megakaryocytopoieses and platelet formation. Interleukin-11 (IL-11), IL-6, IL-3, IL-1b, and thrombopoietin (TPO) act synergistically to promote proliferation and maturation of megakaryocytes. Recombinant IL-11 and TPO are under clinical investigation as supplements to stimulate thrombopoiesis in patients with cancer. We investigated the plasma levels of TPO in 127 patients with chronic lymphocytic leukemia (CLL) and correlated these levels with platelet counts and various laboratory and clinical characteristics. TPO levels were significantly higher in patients with CLL (median, 232 pg/mL; range, 26.4–2714.5 pg/mL) than in healthy control subjects (P <0.01). More importantly, higher levels of TPO were associated with advanced Rai stage (P <0.0001), higher b-2-microglublin (b2-M) levels (P<0.001), and the presence of mutation in the IgVH gene (P <0.001). TPO levels were inversely correlated with platelet count (P = 0.002). Univariate Cox proportional hazard models demonstrated that high TPO levels in CLL patients were associated with shorter survival (P <0.0001); multivariate analysis demonstrated that this association was independent of the IgVH mutation status, b2-M levels, and Rai stage. Recursive partitioning showed that a cut-point of 639.4 pg/mL separated CLL patients into two major groups with a significant difference in survival (P <0.0001). When b2-M level was added to this model, its effect was masked by the effects of TPO in patients with TPO >639.4 pg/mL. However, among patients with TPO levels =639.4 pg/mL, those with b2-M levels >4.95 mg/L had significantly shorter survival than those with lower b2-M levels. These data not only emphasize the importance of TPO in reflecting the aggressiveness of CLL, but also suggest that TPO and b2-M can be used to predict clinical behavior in this disease and may replace the need for determining IgVH mutation status. Further studies are needed to determine if the prognostic role of TPO is due to its direct effects on the growth of the leukemic clone or simply reflects a general failure in the homeostatic regulation of various cytokines. [Display omitted]

Prognostic Impact of VH Gene Mutational Status and CD38 Expression in Japanese CLL Patients.

In the western countries, it has been well established that both immunoglobulin VH gene mutational status and CD38 expression are useful prognostic markers in chronic lymphocytic leukemia (CLL). However, it is not clear whether it is also true in other regions, especially such as Japan where CLL incidence is not so frequent as other countries. Therefore, we investigated the prognostic impact of VH gene mutational status and CD38 expression in Japanese B-CLL. The subjects of this study were 44 patients (29 males and 15 females) referred to our institutions between March 1999 and March 2004. The median age at the time of diagnosis was 68 years (37–92 years). The diagnosis was based on immunophenotypic analysis and cell morphology analyzed on Wright's–stained peripheral blood and bone marrow smears. Median follow-up period of these patients was 4.0 years (0.5–34.2 years).cDNA mainly prepared from peripheral blood samples of the CLL patients was amplified using VH family-specific framework region primers or leader primers, and CH primers. PCR products were sequenced directly or after TA-cloning using the BigDye Terminator Cycle Sequencing FS Ready Reaction kit on a 310 Genetic Analyzer. Nucleotide sequences were compared to the nearest germ line VH genes in databases: IMGT, V-QUEST or IgBLAST. Of 44 B-CLL patients, IgH variable region genes could be sequenced from their cDNA in 43 patients; no amplified band was obtained in 1 patient. The usage of the seven VH gene families in the 43 B-CLL patients were as following: VH 1, 4/43 (9.3%); VH 2, 2/43 (4.6%); VH 3, 23/43 (53.5%); VH 4, 12/43 (27.9%); VH 5, 1/43 (2.3%); VH 6, 1/43 (2.3%); VH 7, 0/43 (0%). Eighteen cases (41.9%) displayed unmutated V H genes, defined as the sequences having more than or equal to 98% homology with nearest germ line gene, and 25 cases (58.1%) showed somatically mutated, defined as less than 98% homology. The proportion of unmutated cases in this study was almost comparable to previous reports, which showed a range of 30% to 50%. It have been uniformly reported that prognostic difference is apparent between unmutated (bad) and mutated (good) groups. Also in this study, the overall survival, defined as the time from diagnosis to death from any cause or to last contact, was significantly shorter for unmutated cases compared to mutated cases estimated by the Kaplan-Meier method as previous reports: predicted 50% survival rate for the unmutated cases was 9.1 years, but that for mutated cases did not reach the median survival. The difference was significant (p=0.029, log-rank test).Cell surface CD38 expression, which has been reported to correlate with a poor prognosis, was analyzed by flow cytometry in all patients. With the cut off level of 30% in CD19+CD5+ lymphocytes, 13 patients (29.5%) were estimated to be CD38 positive and 31 patients (70.5 %) to be negative. There was no significant difference in overall survival between those 2 groups (p=0.519, log-rank test).In conclusion, VH gene mutational status is a strong prognostic indicator whereas CD38 expression is not in our B-CLL cohort.

ZAP-70 Expression, VH-Mutation Status, Genomic Aberrations and Prognosis in Chronic Lymphocytic Leukemia.

The VH status is a strong prognostic marker in chronic lymphocytic leukemia (CLL). ZAP-70, a zeta associated tyrosine kinase physiologically expressed by T-cells, is overexpressed in VH unmutated CLL and could therefore serve as a surrogate marker for the VH status. We analyzed ZAP-70 expression (n=96), the VH status (n=75) and genomic aberrations (n=84) in a single center CLL cohort to study associations among these parameters and to assess their relative prognostic value. ZAP-70 expression was measured by 4-colour flow cytometry (CD5, CD19, CD3/56, ZAP-70) applying an unconjugated anti-ZAP-70-antibody (Upstate, clone 2F3.2) according to Crespo et al., NEJM 2003. ZAP-70 expression was positive (cut-off 20%) in 67% and negative in 33% of cases. VH was mutated in 33% and unmutated in 67% of cases. Unfavorable genomic aberrations (17p−, 11q−) were more frequently observed in cases with unmutated VH (46 vs. 9%) and in ZAP-70 positive cases (39 vs. 20%), while favorable genomic aberrations (13q− as single aberration) occurred more frequently in VH mutated (48 vs. 17%) and ZAP-70 negative subgroups (50 vs. 18%). ZAP-70 expression predicted the VH status in 84% of cases. At a median follow up time of 47 months (m), the median treatment free survival (TFS) of ZAP-70 positive and negative cases was 31 and 86 m (p=.057). The median TFS of the VH unmutated and VH mutated subgroups were 24 and 172 m (p<.001). Within the follow up time 10 deaths occurred. Of these, 8 cases exhibited high ZAP-70 expression and an unmutated VH, whereas 2 cases showed discordant results. Overall, discordant results for ZAP-70 expression and VH status were identified in 12 cases (ZAP-70 positive/VH mutated, 8 cases; ZAP-70 negative/VH unmutated, 4 cases). Of the 8 VH mutated cases with high ZAP-70 expression, only 1 case exhibited unfavorable genomic aberrations, 4 remained in stable disease, 4 developed progressive disease, 3 patients required therapy, and 1 of these 3 died within follow up time. Two of the 3 patients who required therapy, including the patient who died, showed a mutated V3-21 gene rearrangement, associated with an unfavorable outcome. Among the 4 cases with an unmutated VH and low ZAP-70 expression, 2 cases exhibited unfavorable genomic aberrations, 3 cases required therapy, 1 of these 3 died, and for one patient no clinical data were available. In summary, the imbalanced distribution of high risk genomic aberrations was similar when comparing the subgroups according to ZAP-70 expression and VH status. In our series an unmutated VH status predicted for shorter TFS, whereas high ZAP-70 expression did not reach significance. ZAP-70 expression was associated with unmutated VH, but a substantial number of cases showed discordant results for ZAP-70 expression and VH status. The pattern of genomic aberrations and the clinical course of the discordant cases were typical for their respective VH status. Compared to ZAP-70 expression the VH status appeared to be more informative in the prediction of the clinical course in our series of CLL patients.

ZAP-70 Expressed in CLL Is an Hsp90 Client Protein and Its Level of Expression Can Be Downregulated by Specific Hsp90 Inhibitors.

Hsp90 is a ubiquitous molecular chaperone protein that is involved in the folding, activation and assembly of many client proteins, including mediators of signal transduction, cell-cycle control and transcriptional regulation. In order to exert its function on client proteins, Hsp90 requires the formation of an active protein complex composed of cochaperone molecules and an active ATP binding site. Proteins identified as Hsp90 client proteins include transmembrane tyrosine kinases [HER-2/neu, epidermal growth factor receptor (EGFR), MET and insulin-like growth factor-1 receptor (IGF-1R)], metastable signaling proteins (Akt, Raf-1 and IKK), mutated signaling proteins (p53, Kit, Flt3 and v-src), chimeric signaling proteins (NPM-ALK, Bcr-Abl), steroid receptors (androgen, estrogen and progesterone receptors), cell-cycle regulators (cdk4, cdk6) and apoptosis related proteins. It has been postulated that malignant progression and cancer prognosis may be associated with the presence of activated Hsp90 which exists in heightened complexes with cochaperone proteins.The zeta-associated protein of 70 kD (ZAP-70), is a protein kinase found in a subset of patients with chronic lymphocytic leukemia (CLL) and its expression correlates with disease progression, poor clinical outcome, decreased overall survival and early requirement for treatment. ZAP-70 promotes phosphorylation of downstream signaling molecules after engagement of the B cell receptor (BCR). Since ZAP-70 is a marker of disease progression in CLL we hypothesized that its abnormal over-expression and function in CLL B cells may depend on activated Hsp90. To test this, we performed immunoprecipitation, western blot and flow cytometry analyses on CLL samples from patients that differ in their level of ZAP-70 expression (ZAP-70 positive and negative based on a cutoff expression of 20% by flow cytometry). We observed that in ZAP-70 positive CLL samples (n=4) the majority of Hsp90 present in the cytoplasm was in a complexed-activated form, whereas in ZAP-70 negative samples (n=4) most Hsp90 was found to be non-complexed. In addition, we found that ZAP-70 co-immunoprecipitated with Hsp90, suggesting that it is an Hsp90 client protein. In addition, the level of ZAP-70 expression was decreased 30–40% in cells treated for 24 hours with Hsp90 specific inhibitors at nanomolar concentrations.Together, our data show that CLL B cells that express ZAP-70 have larger amounts of activated Hsp90 than those with negligible expression of this tyrosine kinase protein. In addition, we found that ZAP-70 is an Hsp90 client protein and its level of expression could be downregulated by using specific Hsp90 inhibitors. These findings suggest an important role for Hsp90 as a prognostic marker in CLL and provide the rationale to investigate, in clinical trials, the activity of Hsp90 inhibitors and their effect on ZAP-70 expression.

Free circulating soluble CD52 as a tumor marker in chronic lymphocytic leukemia and its implication in therapy with anti-CD52 antibodies.

The CD52 antigen is a glycoprotein anchored on the cell membrane of mature B and T lymphocytes, monocytes, and eosinophils. Alemtuzumab (CAMPATH-1H; anti-CD52) is currently approved for the treatment of patients with refractory chronic lymphocytic leukemia (CLL). The authors investigated the possibility that CD52 may be shed from cells and, once soluble, may bind to injected alemtuzumab, forming immune complexes. The authors used Western blot analysis, immunoprecipitation, and enzyme-linked immunoadsorbent assay to investigate the presence of soluble CD52 (sCD52) in the plasma specimens of 117 patients with CLL. They also used in vitro mixing experiments to examine the ability of sCD52 to compete with cells and sequester therapeutic alemtuzumab. The authors detected high levels of sCD52 in the plasma specimens of patients with CLL. sCD52 can compete with cells in vitro for binding to alemtuzumab, and can form complexes in patients receiving alemtuzumab. Plasma levels of sCD52 were found to be correlated (r)with Rai stage (P = 0.0001), beta-2-microglobulin (beta-2M) levels (P = 0.00002), soluble CD23 levels (r = 0.42, P < 0.001), and immunoglobulin mutation status (P = 0.003). In the multivariate analysis adjusted for beta-2M level, patients with sCD52 levels > 2336 nM/L had a nearly 4-fold increase in risk of death. Higher levels of plasma alemtuzumab were achieved when levels of sCD52 were lower. These data not only demonstrated that sCD52 was detectable and useful in the staging and monitoring of patients with CLL, but also showed that sCD52 formed immune complexes with alemtuzumab and may influence the efficacy and toxicity of alemtuzumab therapy.

Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients.

Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples.

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.

Differential signaling via surface IgM is associated with VH gene mutational status and CD38 expression in chronic lymphocytic leukemia

AbstractThe mutational status of tumor immunoglobulin VHgenes is providing a powerful prognostic marker for chronic lymphocytic leukemia (CLL), with patients having tumors expressing unmutated VH genes being in a less favorable subset. However, the biologic differences correlating with VH gene status that could determine the clinical course of the disease are unknown. Here we show that differing responses to IgM ligation are closely associated with VH gene status. Specifically, 80% of cases with unmutated VH genes showed increased global tyrosine phosphorylation following IgM ligation, whereas only 20% of samples with mutated VH genes responded (P = .0002). There was also an association between response to IgM ligation and expression of CD38 (P = .015). The Syk kinase, critical for transducing B-cell receptor (BCR)– derived signals, was constitutively present in all CLL samples, and there was a perfect association between global phosphorylation and induction of phosphorylation/activation of Syk. Nonresponsiveness to anti-IgM could be circumvented by ligation of IgD (10 of 15 samples tested) or the BCR-associated molecule CD79α (12 of 15 samples tested). These results suggest that multiple mechanisms underlie nonresponsiveness to anti-IgM in CLL and that retained responsiveness to anti-IgM contributes to the poor prognosis associated with the unmutated subset of CLL. The prognostic power of the in vitro response to IgM ligation remains to be determined in a large series, but the simple technology involved may present an alternative or additional test for predicting clinical course.

Polymorphism in the P2X7 receptor gene and survival in chronic lymphocytic leukaemia

The P2X7 receptor is a ligand-gated cation channel that mediates ATP-induced apoptotic death in haemopoietic and chronic lymphocytic leukaemia (CLL) cells. We aimed to investigate the clinical effect of a single nucleotide polymorphism in the P2X7 receptor gene (1513A-->C) and correlate findings with the immunoglobulin heavy chain variable (V(H)) gene mutation status, a prognostic marker in CLL. We investigated tumour DNA in 170 patients with CLL using PCR-RFLP analysis with HhaI restriction enzyme cleavage to screen for the polymorphism in the P2X7 receptor gene. The VH gene mutation status was assessed in 165 patients by PCR amplification and nucleotide sequencing. We correlated the findings of the P2X7 receptor genotype with the V(H) gene mutation data and overall survival and screened for the P2X7 receptor polymorphism in 200 healthy controls. Of the 170 patients, 35 (21%) were heterozygous and one (1%) homozygous for the 1513C allele; whereas 134 (79%) had the 1513A/A genotype of the P2X7 receptor gene. Overall survival was significantly longer for patients with CLL heterozygous for the 1513C allele than those with the 1513A/A genotype. Median survival for heterozygous patients was 104 months (range 33-467) and 72 months (1-190) for homozygous patients (p=0.009). Of the 165 patients with CLL in whom we assessed the V(H) gene mutation status, the V(H) genes were mutated in 71 (43%) patients and unmutated in 94; 18 (25%) of the 71 patients with mutated genes had 1513C allele compared with 17 (18%) of 94 who had unmutated genes. In patients with mutated VH genes, those with CLL who were 1513C positive had 53 months' longer median survival than did those with the 1513A/A genotype (151 vs 98 months, p=0.011). The P2X7 polymorphism could affect clinical outcome in CLL, especially in patients with mutated V(H) genes. Studies are necessary to elucidate the biological role of the P2X7 polymorphism in CLL in vivo.

New monoclonal antibody specific for a 6.5 kDa glycoprotein which presents mainly on a B cell of chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukaemia (CLL) is a most common form of adult leukemia. No specific marker for CLL has been defined until today. We attempt to produce a specific monoclonal antibody (mAb) to CLL B cells. For this purpose, Balb-C mice were immunised with peripheral blood lymphocytes of a patient with CLL. After the fusion, the immunised mouse spleen cells and SP2/0 myeloma cell line, antibody secreting clones were selected by ELISA and specific antibody was determined by flow cytometry. Leukemic cells from different patients, different cell line and lymphoid tissue were tested with this mAb using flow cytometry and immunoperoxidase methods. Ligand of the mAb on cell surface was identified using epitope analysing method. We have shown that this mAb is specific to a molecule with 6.5 kDa molecular weight, which is present mainly on B CLL cells (63.7±16.4%). It has also been shown that this molecule was a glycoprotein. Amongst the different cell lines that were tested, Raji cell, Molt-3 and P3HR-1 cells were expressing this molecule. We, therefore, suggest that it is a novel molecule with unknown function and is mainly present on the B cells of CLL.

Frequent monoallelic loss of D13S319 in multiple myeloma patients shown by interphase fluorescence in situ hybridization.

Deletions or monosomy of chromosome 13 are frequent in multiple myeloma (MM). A candidate tumor suppressor gene might reside telomeric of the retinoblastoma gene (RBl) at band 13q14 and to play a role in B cell neoplasm. The D13S319 locus, between RB1 and D13S25 loci at 13q14 is the most commonly deleted marker in chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL). We evaluated the D13S319 locus in 24 MM cases by fluorescence in situ hybridization (FISH). We observed monosomy for D13S319 in 6/20 (30%) MM patients with an apparently normal karyotype. As expected, in four karyotypically abnormal MM cases with partial or complete monosomy for chromosome 13, all of them had monoallelic loss of D13S319. Our results indicated that the loss of D13S319 is commonly found in MM, even at diagnosis, and is more frequent than predicted based on conventional cytogenetic analysis of metaphase spreads. This finding implicates a candidate tumor suppressor gene at 13q14 in the pathogenesis of MM.

Surface adenosine deaminase: A novel B-cell marker in chronic lymphocytic leukemia

Previous studies found that ADA is present on the surface of mononuclear blood cells from healthy patients. Because the expression of this surface antigen depends upon the cell type, the presence of ADA on the plasma membrane of cells from patients with malignant hematologic diseases was studied by flow cytometry. The highest percentage of expression was found in CLL, whereas the lowest was found in T-cell-derived malignancies. The enzyme expression in immortalized cell lines showed a similar pattern, with the highest expression (95% ± 5%) in the SKW64 B-derived cell line, the lowest (15% ± 5%) in Jurkat T-lymphoma derived cells, and the intermediate (32% ± 8%)in K562 cells derived from a chronic myelogenous leukemia. Double labeling ADA/CD5 and ADA/CD19, as well as the correlation of ADA expression with the expression of other surface markers, indicate that surface ADA might be considered a novel marker for CLL.

HLA-DR-Associated Invariant Chain Is Highly Expressed in Chronic Lymphocytic Leukemia

Total RNA extracted from peripheral blood lymphocytes of a patient with B-cell chronic lymphocytic leukemia (CLL) and the poly (A+) RNA was purified. A cDNA library was constructed and approximately 4,000 clones were screened in order to identify genes preferentially expressed in CLL. A relatively low repetition frequency characterizes the majority of the abundant mRNA species present in CLL lymphocytes. One clone, corresponding to the mRNA encoding the HLA-DR-associated invariant chain, was selected and its expression was examined in different leukemic cell populations and in normal tissues. DNA-RNA hybridization studies showed that the invariant chain mRNA (In-mRNA) is detectable in RNA preparations from human blood cells and their precursors, whereas no In-mRNA is found in several other tissues examined. Among various normal and leukemic leukocyte populations, the highest levels of In-mRNA are found in CLL. Therefore, a role of In-chain mRNA as a marker of CLL is proposed. Our data support a relationship between high levels of invariant chain mRNA and the out of cycle condition of CLL peripheral blood lymphocytes.

HLA-DR-associated invariant chain is highly expressed in chronic lymphocytic leukemia

Total RNA extracted from peripheral blood lymphocytes of a patient with B-cell chronic lymphocytic leukemia (CLL) and the poly (A+) RNA was purified. A cDNA library was constructed and approximately 4,000 clones were screened in order to identify genes preferentially expressed in CLL. A relatively low repetition frequency characterizes the majority of the abundant mRNA species present in CLL lymphocytes. One clone, corresponding to the mRNA encoding the HLA-DR-associated invariant chain, was selected and its expression was examined in different leukemic cell populations and in normal tissues. DNA-RNA hybridization studies showed that the invariant chain mRNA (In-mRNA) is detectable in RNA preparations from human blood cells and their precursors, whereas no In-mRNA is found in several other tissues examined. Among various normal and leukemic leukocyte populations, the highest levels of In- mRNA are found in CLL. Therefore, a role of In-chain mRNA as a marker of CLL is proposed. Our data support a relationship between high levels of invariant chain mRNA and the out of cycle condition of CLL peripheral blood lymphocytes.

Differentiation of chronic lymphocytic leukemia from reactive lymphocytosis by the mouse red cell rosette assay.

The differential diagnosis of chronic lymphocytic leukemia (CLL) from other neoplastic and nonneoplastic lymphocytoses may be difficult, even when optimal clinical and laboratory data are available. The mouse red cell (M) rosette assay has been valuable in distinguishing normal and lymphomatous lymphocytes from those of CLL. Not yet tested is the ability of this test to separate reactive (benign) lymphocytes from morphologically similar but neoplastic cells of CLL. In the present study, lymphocytes of 58 subjects with CLL, reactive lymphocytosis, and healthy controls were evaluated for M rosette formation, in addition to the usual B and T cell markers. Lymphocytes from patients with CLL showed a marked increase in the ability to form M rosettes in comparison to those from normal controls and patients with reactive lymphocytosis. The formation of M rosettes was a more reliable marker for CLL than surface membrane immunoglobulin.

Human Chronic Lymphocytic Leukaemia Surface Markers and Activation of Lymphocytes

Cell surface markers and the responses of lymphocytes to T- and B-cell mitogens were studied in 10 patients with CCL. T cells were identified as cells rosetting with sheep red blood cells (SRBC), and S-Ig was used as a marker for B lymphocytes. Most cells from all patients had a detectable amounts of S-Ig, and the percentage of cells rosetting with SRBC was low in all cases. Of the lymphocytes from these patients, 3-74% (mean 33%) were positive for the acid esterase (ANAE), which has been claimed to be a T-cell marker. However, some patients had cells that were positive for both S-Ig and ANAE. Acid esterase staining is therefore not a valid T-cell marker in chronic lymphocytic leukaemia. In cultures containing the T-cell mitogen leucoagglutinin (LA) and the T- and B-cell mitogen pokeweed mitogen (PWM) the reactivity of the lymphocytes was low. The cells responded vigorously to the T- and B-cell mitogen protein A (PA); however, the response was serum-dependent, being strong in a culture medium containing foetal calf serum (FCS), but impaired in the presence of human AB serum. Only 1 patient had cells that responded to the B-cell mitogen LPS.