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ABSTRACT: Multiple types of mutations in the BCR-ABL1 kinase domain have been reported. We previously reported a common alternatively spliced BCR-ABL mRNA with a 35-nucleotide insertion (35INS). We report three novel alternative splicing mutants expressed as the dominant transcripts in patient with chronic myelogenous leukemia and resistance to kinase inhibitors.
We screened RNA from more than 200 patients with resistance to more than one of the three kinase inhibitors for ABL1 kinase domain mutations by direct sequencing.
We found three not previously described splice mutants. All three showed >90% mutant transcript. The first resulted from the insertion of 79 nucleotides into the ABL1 exon 8-9 junction. The inserted sequence contained a sequence from regions of intron 8, located 120 bp apart: the 35-nucleotide sequence previously described, and an additional 44-nucleotide segment downstream from 35INS. The combined 79-nucleotide insertion splice mutant showed the same protein change as 35INS (p C475YfsX11). The second splice mutation comprised an 84-nucleotide sequence from intron 7 inserted into the ABL1 exon7-8 junction, also causing a frameshift and protein truncation (p A424EfsX18). The third splice derived from a 231-nucleotide sequence from intron 4 retained in the ABL1 exon 4-5 junction adding 40 intron-encoded amino acids and leading to a frameshift and early termination (p E275LfsX41).
These findings, when combined with the data on 35INS, support the concept that loss of the C-terminus of BCR-ABL1 is associated with significant resistance to kinase inhibitors; this mechanism appears to be a major source of resistance to kinase inhibitors.
International journal of laboratory hematology 01/2011; 33(3):326-31. · 1.30 Impact Factor
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ABSTRACT: The association of V617F JAK2 expression levels with disease behavior has not been studied in patients with nonchronic myelogenous leukemia (CML) myeloproliferative disease (MPD). We found plasma levels of total JAK2 mRNA to be higher in patients with non-CML MPD (n=175) than in CML patients (n=45) and normal controls (n=58) (each P<0.001). Overall survival was studied in 68 patients and showed positive correlation with levels of total and mutant JAK2 mRNA in patients with the V617F mutation, but not those without the mutation. These findings suggest that total JAK2 expression levels play a role in the biology of the disease in V617F-positive patients, and a therapy aiming at downmodulating the expression of the total JAK2 mRNA should be considered. In conclusion, we studied JAK2 total and V6217F mutant mRNA levels in plasma. We show high levels of JAK2 expression in MPD patients and these levels correlate with survival.
International journal of laboratory hematology 03/2009; 32(1 Pt 2):95-102. · 1.30 Impact Factor
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I Jilani,
C Wei,
B N Bekele,
Z J Zhang,
M Keating,
W Wierda,
A Ferrajoli,
Z Estrov,
H Kantarjian,
S M O'Brien,
F J Giles, M Albitar
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ABSTRACT: Syndecan-1 (sCD138) is a transmembrane heparan sulfate-bearing proteoglycan expressed in epithelial cells as well as hematopoietic cells that demonstrate plasmacytoid differentiation. Higher levels of sCD138 correlate with poor outcome in myeloma. We examined the association of circulating sCD138 levels in plasma with clinical behavior in 104 patients with chronic lymphocytic leukemia. sCD138 levels were significantly higher in patients (median, 52.8 ng/ml; range, 13.4-252.7 ng/ml) than in healthy control subjects (median, 19.86; range, 14.49-33.14 ng/ml) (P < 0.01). Elevated sCD138 (>median, 52.8 ng/ml) was associated with significantly shorter survival (P = 0.0004); this association was independent of IgVH mutation status, beta2-microglobulin (beta2-M) level, and treatment history. Patients with mutated IgVH but high sCD138 levels (>52.8 ng/ml) had significantly shorter survival than those with mutated IgVH and lower levels of sCD138. Similarly, patients with unmutated IgVH but high sCD138 levels had significantly shorter survival than those with lower sCD138 levels and unmutated IgVH (P = 0.007). In a multivariate Cox regression model, only Rai stage, beta2-M, and sCD138 remained predictors of survival. These data suggest that sCD138 when combined with beta2-M and Rai stage, may replace the need for testing IgVH mutation status.
International Journal of Laboratory Hematology 01/2008; 31(1):97-105. · 1.18 Impact Factor
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F Ravandi,
I Jilani,
E Estey,
H Kantarjian,
A Dey,
C Aguilar,
C Jitkaroon,
F Giles,
S O'Brien,
M Keating, M Albitar
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ABSTRACT: FLT3 ligand (FL) has a significant role in the proliferation and differentiation of hematopoietic cells. Mutations in the FLT3 receptor gene have been reported in 30% of patients with AML. We investigated whether abnormal phosphorylation of FLT3 may be more common in AML. We evaluated FLT3 protein and its phosphorylation in the plasma from 85 patients with AML, 16 patients with myelodysplastic syndrome (MDS) and 5 patients with acute lymphoblastic leukemia (ALL). There were no significant differences in the level of plasma FLT3 protein level in the different diseases (p=0.57). AML patients had a significantly higher level of phospho-FLT3:FLT3 ratio (p=0.02). FLT3-ITD and FLT3 point mutations were present in 27 (32%) of the AML patients. Phosphorylated FLT3 was significantly higher in the plasma from patients with FLT3 mutation (p=0.002). Overall, there was no correlation between survival and the plasma level of FLT3 protein or its phosphorylated form. However, amongst the patients without FLT3 mutations, those with a higher level of phosphorylated FLT3 had a significantly shorter duration of remission (p=0.04). Other mechanisms may be responsible for abnormal phosphorylation of FLT3 and inhibitors of FLT3 should also be investigated in patients without mutations.
Leukemia Research 07/2007; 31(6):791-7. · 2.92 Impact Factor
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M Albitar,
M Johnson,
K A Do,
A Day,
I Jilani,
S Pierce,
E Estey,
H Kantarjian,
M Keating,
S Verstovsek,
S O'brien,
F J Giles
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ABSTRACT: beta-2 Microglobulin (beta2M), a subunit of human leukocyte antigen-class I (HLA-I), is well established as a marker of prognosis in various solid tumors and hematologic malignancies. The prognostic role of intact free-circulating HLA-I (sHLA-I) is less well understood. We compared the clinical relevance of plasma levels of sHLA-I and beta2M in patients with acute myeloid leukemia (AML; n=209) or advanced myelodysplastic syndrome (MDS; n=98). sHLA-1 and beta2M levels were significantly higher in AML and MDS patients than in control subjects, but did not differ significantly between the two disease groups. In AML patients, multivariate analysis showed both sHLA-1 and beta2-M to be highly predictive of complete remission (CR), survival and duration of complete response (CRD). In MDS, the predictive value of the two markers differed substantially from one another: beta2M was associated with survival, CR and CRD, whereas sHLA-I was not. These findings not only establish the role of sHLA-I as a tumor marker in AML but also support that MDS is clinically and biologically distinct from AML. sHLA-I has been reported to be an immunomodulator inhibiting the cytotoxic effects of T-lymphocytes, which may offset its predictive value for disease aggressiveness in patients with MDS.
Leukemia 04/2007; 21(3):480-8. · 9.56 Impact Factor
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ABSTRACT: Most studies test for mutations in the kinase domain of the abl gene in chronic myeloid leukemia (CML) using peripheral blood (PB) cells. Frequently, progression of the disease manifests with increased blasts in bone marrow (BM) and not in PB. Simultaneous analysis of plasma, PB cells and BM cells from 41 imatinib-resistant CML patients showed mutations in 63% of PB cells and 68% of plasma or BM cells (P = 0.04). In discordant patients, 13 mutations were detected in plasma, 11 in BM cells and 9 in PB cells. The T315I mutation was detected in plasma and BM but not PB cells in one patient. We detected no mutations in the plasma of 45 previously untreated CML patients, but two of these patients showed mutations in plasma and not cells by 9 months on therapy. Circulating plasma mRNA is a reliable alternative to BM mRNA for detecting ABL mutations.
Leukemia 12/2006; 20(11):1989-91. · 9.56 Impact Factor
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ABSTRACT: Molecular response and monitoring of minimal residual disease (MRD) is becoming an essential part of most protocols for treating leukemia and lymphoma patients. Detection of abnormal clones by PCR analysis of complementarity determining regions (CDRs) in immunoglobulin genes is currently standard practice for diagnosis, but is not widely used to monitor MRD because of the low sensitivity of assays that use consensus primers. Use of specific primers can improve the sensitivity of the assay, but is a cumbersome, expensive, and time-consuming process. We developed a simple and cost-effective approach to detect MRD in B-cell malignancies that is usable in clinical laboratories. The new assay uses ligase chain reaction (LCR) to detect clonality. The sensitivity of the LCR assay is 1 per 500,000 cells, and it can detect all subclones that were present in the pretherapy diagnostic sample.
Clinical & Laboratory Haematology 11/2006; 28(5):325-31. · 1.11 Impact Factor
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British Journal of Haematology 09/2006; 134(3):341-3. · 4.94 Impact Factor
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ABSTRACT: Lack of immunoglobulin heavy chain genes (IgV(H)) mutation in patients with chronic lymphocytic leukemia (CLL) is associated with rapid disease progression and shorter survival. The zeta-chain (T-cell receptor) associated protein kinase 70 kDa (ZAP-70) has been reported to be a surrogate marker for IgV(H) mutation status, and its expression in leukemic cells correlates with unmutated IgV(H). However, ZAP-70 detection by flow cytometry varies significantly dependant on the antibodies used, the method of performing the assay, and the condition of the cells in the specimen. The clinical value of ZAP-70 testing when samples are shipped under poorly controlled conditions is not known. Furthermore, testing in a research environment may differ from testing in a routine clinical laboratory. We validated an assay for ZAP-70 by comparing results with clinical outcome and the mutation status of the IgV(H). Using stored samples, we show significant correlation between ZAP-70 expression and clinical outcome as well as IgV(H) mutation at a cut-off point of 15%. While positive samples (>15% positivity) remain positive when kept in the laboratory environment for 48 h after initial testing, results obtained from samples from CLL patients tested after shipping at room temperature for routine testing showed no correlation with IgV(H) mutation status when 15% cut-off was used. In these samples, cut-point of 10% correlated with the IgV(H) mutation (P = 0.0001). This data suggests that although ZAP-70 positivity correlates with IgV(H) mutation status and survival, variations in sample handling and preparation may influence results. We show that IgV(H) mutation results, unlike ZAP-70 remain correlated with CD38 expression and beta-2 microglobulin in shipped samples, and ZAP-70 testing should not be used as the sole criterion for stratifying patients for therapy.
Cytometry Part B Clinical Cytometry 08/2006; 70(4):270-5. · 2.53 Impact Factor
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British Journal of Haematology 04/2006; 133(6):690 - 692. · 4.94 Impact Factor
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ABSTRACT: Determining the percentage of peripheral blood (PB) and bone marrow (BM) blasts is important for diagnosing and classifying acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Although most patients with acute leukemia or MDS have a higher percentage of BM blasts than PB blasts, the relative proportion is reversed in some patients. We explored the clinical relevance of this phenomenon in MDS (n = 446), AML (n = 1314), and acute lymphoblastic leukemia (ALL) (n = 385). Among patients with MDS or ALL, but not AML, having a higher blast percentage in PB than in BM was associated with significantly shorter survival. In multivariate analyses, these associations were independent of other relevant predictors, including cytogenetic status. Our findings suggest that MDS and ALL patients who have a higher percentage of PB blasts than BM blasts have more aggressive disease. These data also suggest that MDS classification schemes should take into account the percentage of blasts in PB differently from the percentage of blasts in BM.
Leukemia 10/2005; 19(9):1567-72. · 9.56 Impact Factor
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Leukemia 12/2004; 18(11):1912-4. · 9.56 Impact Factor
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ABSTRACT: Alemtuzumab is effective in reducing the risk of acute graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (ASCT). Alemtuzumab may also delay immune reconstitution and reduce graft-versus-leukemia effects. The optimal dose has not been established. We investigated engraftment, acute GVHD incidence and severity, and pharmacokinetics of alemtuzumab associated with the use of low-dose alemtuzumab/cyclophosphamide/total body irradiation and ASCT for patients with aggressive CD52-positive hematologic malignancies. In all, 12 patients were treated. Alemtuzumab 10 mg daily on days -7 to -3 was given intravenously. Tacrolimus and methotrexate were used for GVHD prophylaxis. Alemtuzemab was not detected in any of the 36 sequential serum samples tested between days -1 and +21 of transplant. All patients engrafted rapidly; the median time to an absolute neutrophil count >0.5 x 10(9)/l was 14 days (range 11-17 days), and the median time to a platelet count >20 x 10(9)/l was 16 days (range 6-30 days). By 1 month after transplant, nine patients had 100% donor chimerism, while three had mixed donor chimerism. At 3 months, 11 had achieved 100% donor chimerism. No cases of grade III/IV acute GVHD occurred. At a median follow-up interval of 14.7 months (range 4-24), seven patients remained alive, and five remained free of disease.
Bone Marrow Transplantation 04/2004; 33(8):833-7. · 3.75 Impact Factor
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ABSTRACT: Mitochondrial DNA (mtDNA) codes for 13 respiratory chain subunits and is more vulnerable to damage than nuclear DNA due, in part, to a lack of histone protection and a weak repair capacity. While mtDNA alterations have been observed in human cancer, their roles in oncogenesis and chemosensitivity remain unclear. We investigated the relationship between mtDNA mutations, reactive oxygen species (ROS) generation, and clinical outcomes in chronic lymphocytic leukemia (CLL) patients. An analysis of mtDNA from 20 CLL patients revealed that primary CLL cells from patients with prior chemotherapy had a significantly higher frequency of heteroplasmic mutations than did those from untreated patients. Overall, mtDNA mutations appeared to be associated with increased ROS generation. Patients refractory to conventional therapeutic agents tended to have higher mutation rates than patients who responded to treatment. Analysis of paired blood samples from the same patient led to the identification of a heteroplasmic mutation in the cytochrome c oxidase II gene several months after chemotherapy. The mutation was associated with increased ROS generation. Our results suggest for the first time that chemotherapy with DNA-damaging agents may cause mtDNA mutations in primary leukemia cells, which often exist in heteroplasmy, and are associated with increased ROS generation.
Leukemia 09/2003; 17(8):1437-47. · 9.56 Impact Factor
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A-M Tsimberidou,
H M Kantarjian,
E Estey,
J E Cortes,
S Verstovsek,
S Faderl,
D A Thomas,
G Garcia-Manero,
A Ferrajoli,
J T Manning,
M J Keating, M Albitar,
S O'Brien,
F J Giles
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ABSTRACT: Granulocytic sarcoma (GS) is an extramedullary tumor composed of immature myeloid cells. The objectives of this study were to describe the frequency, presenting characteristics, and survival in patients with nonleukemic GS by conducting a review of all untreated patients presenting to the MD Anderson Cancer Center between January 1990 and June 2002. In all, 21 patients with nonleukemic GS, 1520 patients with acute myeloid leukemia (AML), and 402 patients with high-risk myelodysplastic syndrome (MDS) were identified. GS occurred in 1.4% of patients with AML, and 1.1% of patients with AML or high-risk MDSs. The median patient age was 57 years (range, 7-81). Among 20 patients with available cytogenetics in tissue and/or bone marrow, six had chromosome 8 abnormalities. The median follow-up of surviving patients is 12 months (range, 7-75). In all, 20 patients were treated. Patients were treated with AML-type chemotherapy (n=16), chemotherapy and radiotherapy (n=3), or radiotherapy alone (n=1). A total of 13 patients (65%) achieved complete remission and one patient (5%) achieved partial remission. The median overall survival was 20 months (range, 1-75), median overall failure-free survival was 12 months (range, 1-75). The median survival of patients with chromosome 8 abnormalities was 12 months compared with 40 months of those without (P=0.17). Novel therapies for patients with GS are required.
Leukemia 07/2003; 17(6):1100-3. · 9.56 Impact Factor
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ABSTRACT: Somatic mutation of the FLT3 gene as an internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence causes constitutive tyrosine phosphorylation and activation. Tumor-specific DNA has been documented in the sera of patients with solid tumors even when it is in an early stage. We compared the detection of FLT3 ITD in DNA extracted from cells of bone marrow (BM) aspirations with DNA extracted from peripheral blood (PB) plasma in patients newly diagnosed with acute myeloid leukemia (AML; 85 patients), myelodysplastic syndrome (MDS; 16 patients), and acute lymphocytic leukemia (ALL; 16 patients). FLT3 ITD was detected in 18 (21%) AML samples and in one (6%) MDS sample in both cellular and plasma DNA but in none of the ALL samples. Hemizygous/homozygous FLT3 ITD was detected in five (28%) of the FLT3 ITD-positive AML using plasma DNA, whereas only four of these cases showed hemizygous/homozygous FLT3 ITD using cellular DNA. The presence of FLT3 ITD was associated with significantly shorter survival (P = 0.02) when only patients younger than 50 years of age (48 AML+MDS patients) were considered. This finding was independent of cytogenetics in this age group. However, patients with the FLT3 ITD hemizygous/homozygous phenotype had even shorter survival (P = <0.001). As expected, the presence of FLT3 ITD correlated with higher white blood cell (WBC) counts. These data demonstrate that plasma DNA is a reliable alternative resource for detecting FLT3ITD, especially the hemizygous/homozygous genotype. Furthermore, the data derived from this study support the notion that the presence of FLT3 ITD in conjunction with the absence of the wild-type FLT3 allele predicts an especially poor prognosis for patients with AML.
Leukemia 01/2003; 17(1):114-9. · 9.56 Impact Factor
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ABSTRACT: Refractory anemia with excess blasts in transformation (RAEB-T) is a subgroup of myelodysplastic syndrome (MDS) in which the bone marrow blast count ranges from 20% to 30%. The recently proposed World Health Organization Classification of Hematologic Malignancies eliminated this category from MDS by lowering the blast count cutoff for acute myeloid leukemia (AML) from 30% to 20%. However, MDS is distinguished from AML by a significant increase in apoptosis. To investigate the difference in apoptosis between RAEB-T, AML, and other categories of MDS, we prospectively analyzed fresh bone marrow samples using the Annexin V and mitochondrial potential assays. There was a significantly higher level of apoptosis in RAEB-T than in AML according to both assays, while no significant differences between RAEB-T and other categories of MDS were noted. The data suggest that RAEB-T is more likely to be an advanced stage of MDS and biologically different from AML.
Leukemia 12/2002; 16(11):2249-52. · 9.56 Impact Factor
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ABSTRACT: It has been suggested that the expansion of the leukemic cells in chronic lymphocytic leukemia (CLL) is due to dysregulation of pathways of programmed cell death (apoptosis) rather than cell proliferation, although differences may exist in early vs late and treated vs untreated patients. In the present study, we analyzed the expression of 11 proteins in CLL cells that are implicated in the control of apoptosis, proliferation, and differentiation, and correlated this expression profile with survival. Using a quantitative solid-phase radioimmunoassay (RIA), we measured the cellular protein levels of Bcl-2, cyclin D1, PCNA, ATM, Fas, Bax, retinoic acid receptor alpha (RARalpha), retinoic acid receptor beta (RXRbeta), Flt1, VEGF, and cellular beta2-microglobulin in 230 samples of CLL. Univariate analysis using the Cox proportional hazard model showed a correlation with survival of only the following proteins: Bcl-2 (P < 0.001), cyclin D1 (P = 0.027), Fas (P = 0.055), PCNA (P < 0.001), and ATM (P = 0.028). In a multivariate analysis using classification and regression tree analysis (CART), five groups of patients (nodes) could be generated with significant differences of survival expectation (P < 0.0001) based on levels of expression of the above proteins. Based on CART analysis, Bcl-2 levels emerge as the most important protein in predicting survival between all 11 proteins studied. Patients with marked elevation in Bcl-2 levels had the worst outcome while patients with intermediate levels, but with high levels of PCNA and cyclin D1 or abnormal ATM expression had intermediate survival. These data indicate that intracellular levels of proteins such as Bcl-2, ATM, cyclin D1, and PCNA can be used as markers to predict clinical behavior and survival in patients with CLL. The pathways in which these proteins are involved may also represent possible targets for future therapeutic trials in CLL.
Leukemia 07/2002; 16(6):1045-52. · 9.56 Impact Factor
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ABSTRACT: Patients with chronic lymphocytic leukemia (CLL) are considered in nodular partial remission (nPR) when they are in remission but bone marrow biopsies show rare nodules. The significance of the level of residual disease in nPR is not known. We studied 91 previously untreated CLL patients who were treated with fludarabine alone, fludarabine with prednisone, or fludarabine with cyclophosphamide and achieved nPR at the end of six courses. We compared bone marrow lymphoid infiltration before therapy and at the end of three and six courses of therapy as evaluated by a pathologist in retrospective fashion with that of the routine evaluation at the time of performing bone marrow biopsy. We then compared these results with those obtained by computer-aided histomorphometry in 28 patients in nPR. There was significant correlation (P < 0.05) between pathologists as well as between pathologists and histomorphometry. Upon correlation with clinical characteristics, there was significant correlation (P 0.01) between marrow involvement before therapy and white blood cell counts (wbc), hemoglobin (hgb), absolute lymphocyte counts, and beta2-microglobulin (beta2-m) but none of these parameters correlated with the lymphoid infiltrate at the end of three or six courses of therapy. more importantly, lymphoid infiltration after three and six courses did not correlate with time to progression (ttp) or overall survival (os). however, patients with >70% marrow involvement before therapy had a significantly shorter TTP (P = 0.02). All 91 patients showed similar results. However, we found reverse correlation between marrow lymphoid infiltrate at the end of three courses and OS (P = 0.01).
Leukemia 04/2002; 16(4):632-5. · 9.56 Impact Factor
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ABSTRACT: Differential expression of CD20 surface antigen in B-cell neoplasms at different sites is largely unknown. The number of CD20 antibodies bound per cell (CD20 ABC) in bone marrow (BM), peripheral blood (PB), and lymph node aspirate (LNA) samples from patients with B-cell chronic lymphocytic leukemia (B-CLL) or other B-cell disease was studied using quantitative flow cytometry. CD20 ABC differed significantly with the specimen type in B-CLL, being highest in PB (mean, 9,051) and lower in BM (mean, 4,067) and LNA (mean, 3,951). No difference in CD20 ABC between BM and PB samples was found in splenic lymphoma, mantle cell lymphoma, or follicular lymphoma. Also, we found a significant difference of CD20 ABC by type of disease: lowest in B-CLL; higher in splenic, follicular, and mantle cell lymphoma; and highest in hairy cell leukemia. The lower CD20 surface antigen levels in BM and LNA than in PB in B-CLL may have clinical relevance with regard to the efficacy of rituximab therapy.
American Journal of Clinical Pathology 10/2001; 116(3):437-43. · 2.60 Impact Factor
