Iman Jilani

Differences in CD33 intensity between various myeloid neoplasms.

We measured the concentration of CD33 antigen on the surface of cells in 315 bone marrow (BM) samples and 114 corresponding peripheral blood (PB) samples from patients with various leukemias (acute myeloid leukemia [AML], chronic myelogenous leukemia [CML], myeloproliferative disorder [MPD] other than CML, myelodysplastic syndrome [MDS]) and from control subjects. Overall CD33 intensity in total CD33+ cells was significantly higher in BM than in PB. CD33 intensity in total BM CD33+ cells differed significantly with the type of disease. The median number of CD33 molecules per cell was highest in AML, followed by MDS, CML, and control subjects and lowest in MPD. When only CD34+/CD33+ cells were examined, CD33 molecules per cell were highest in CD34+ cells in AML and lowest in MPD (P = .027). Patients with AML or MDS younger than 60 years had significantly higher intensity of CD33 expression on CD34+ cells than patients 60 years or older. Levels of CD33 intensity did not correlate with cytogenetics in patients with AML or MDS. There was no correlation between CD33 intensity and response to therapy or overall survival in 35 patients treated with protocols including Mylotarg. These data demonstrate variation in CD33 intensity between various leukemias.

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.

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.

Proliferation and apoptosis in acute and chronic leukemias and myelodysplastic syndrome

Clonal expansion of leukemic cells is thought to be due to proliferation in excess of apoptosis. To define and compare proliferation and apoptosis between various leukemias and myelodysplastic syndrome (MDS), we measured proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation as surrogate markers for proliferation and caspase 3 activity and annexin V surface binding as surrogate markers for activation of the apoptotic cascade in patients with MDS, chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). We found high proliferation in bone marrow cells from MDS and CMML as measured by PCNA and BrdU incorporation. The lowest level of proliferation was found in CLL. Apoptosis was also highest in MDS and CMML as measured by annexin V and caspase 3 activity. Unexpectedly, we found no significant difference in proliferation in bone marrow CD34+ cells from various leukemias or MDS. Apoptosis was significantly higher in bone marrow CD34+ cells from MDS and CML in chronic phase as compared to CD34+ cells from AML patients. Our results illustrate differences in proliferation and apoptosis between acute and chronic leukemias and MDS. These differences may have diagnostic and therapeutic implications.

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.

Proteomic-based prediction of clinical behavior in adult acute lymphoblastic leukemia†

Response in adult acute lymphoblastic leukemia (ALL) can be achieved in a majority of patients. However, unlike pediatric ALL, recurrence is common in adult ALL, and the ability to predict at an early stage which patients are most likely to experience recurrence may help in devising new therapeutic approaches to prevent recurrence.

Enzymatic activity of circulating proteasomes correlates with clinical behavior in patients with chronic lymphocytic leukemia

The ubiquitin‐proteasome pathway has been implicated in the pathogenesis of many hematologic malignancies.

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.

A potential role for HSP90 inhibitors in the treatment of JAK2 mutant-positive diseases as demonstrated using quantitative flow cytometry

The V617F mutation of the JAK2 tyrosine kinase is found in a majority of patients with myeloproliferative disorders. Flow cytometry assays for quantitation of phosphorylated and total protein for JAK2, STAT5, and heat shock proteins (HSPs) were developed to facilitate the study of the JAK/STAT pathway. A cell line homozygous for V617F (HEL) was treated with inhibitors of JAK2 tyrosine kinase activity and the HSP90 inhibitor 17-AAG. 17-AAG reduced HSP90 levels, but increased HSP70 levels. Phospho-STAT5, total STAT5, and total AKT levels were also reduced by17-AAG treatment. Further, phospho-JAK2, total JAK2, and cell viability were reduced to a greater extent by 17-AAG than by the pan-JAK kinase family inhibitor JKII or the JAK2-specific inhibitor AG490, and these inhibitors failed to synergize with 17-AAG. Flow-cytometry-based assays for JAK/STAT signaling pathway and HSPs are likely to have broad clinical utility for monitoring patients with abnormalities in the JAK2 pathway.

Variations in the detection of ZAP‐70 in chronic lymphocytic leukemia: Comparison with IgVH mutation analysis

Lack of immunoglobulin heavy chain genes (IgVH) 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 IgVH mutation status, and its expression in leukemic cells correlates with unmutated IgVH. 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 IgVH. Using stored samples, we show significant correlation between ZAP‐70 expression and clinical outcome as well as IgVH 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 IgVH mutation status when 15% cut‐off was used. In these samples, cut‐point of 10% correlated with the IgVH mutation (P = 0.0001). This data suggests that although ZAP‐70 positivity correlates with IgVH mutation status and survival, variations in sample handling and preparation may influence results. We show that IgVH mutation results, unlike ZAP‐70 remain correlated with CD38 expression and β‐2 microglobulin in shipped samples, and ZAP‐70 testing should not be used as the sole criterion for stratifying patients for therapy.