Soluble syndecan-1 (sCD138) as a prognostic factor independent of mutation status in patients with chronic lymphocytic leukemia.

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.

Levels of soluble HLA-I and beta2M in patients with acute myeloid leukemia and advanced myelodysplastic syndrome: association with clinical behavior and outcome of induction therapy.

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.

Soluble phosphorylated fms-like tyrosine kinase III. FLT3 protein in patients with acute myeloid leukemia (AML).

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.

Simplified sensitive method for the detection of B-cell clonality in lymphoid malignancies.

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.

Variations in the detection of ZAP-70 in chronic lymphocytic leukemia: Comparison with IgV(H) mutation analysis.

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.

Heterogeneity in detecting Abl kinase mutations and better sensitivity using circulating plasma RNA.

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.

Having a higher blast percentage in circulation than bone marrow: clinical implications in myelodysplastic syndrome and acute lymphoid and myeloid leukemias.

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.

Better detection of FLT3 internal tandem duplication using peripheral blood plasma DNA.

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.

More cell death in refractory anemia with excess blasts in transformation than in acute myeloid leukemia.

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.

Higher levels of surface CD20 expression on circulating lymphocytes compared with bone marrow and lymph nodes in B-cell chronic lymphocytic leukemia.

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.

CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia.

CD38 is a transmembrane glycoprotein expressed on the surface of leukemic cells in a significant percentage of patients with B-cell chronic lymphocytic leukemia (B-CLL). A recent study suggested that CD38 expression has prognostic value in CLL. Peripheral blood samples from 218 patients with B-CLL were analyzed by flow cytometry for CD38 expression on CD5/19(+) leukemic cells. Various patient characteristics were studied including age, sex, Rai and Binet stages, splenomegaly, hepatomegaly, hemoglobin (Hgb) level, beta-2 microglobulin (beta2M) level in the serum, number of nodal sites involved with disease, and length of survival. The Kaplan-Meier method was used to construct survival curves, and the log-rank statistic was used to compare these curves. CD38 was expressed in 20% or more of leukemic cells in 43% of the patients. Patients with high CD38 expression (20% or more) had significantly shorter survival times (P =.00005). Multivariate analyses showed that CD38 expression is an important prognostic factor associated with high incidence of lymph node involvement (P =.004), lower hemoglobin level (P =.001), hepatomegaly (P =.05), and high beta2M level (P =.00005). CD38 expression identified a group of patients with aggressive disease that was considered by Rai staging to be early-stage disease (Rai stages 0-II). Patients with CD38(+) samples have significantly aggressive disease regardless of their clinical stage. Measurement of CD38 expression by flow cytometry should become a routine test in the evaluation of patients with CLL.