Mordechai Shtalrid

Molecular characteristics of chronic myelogenous leukemia in blast crisis

We have studied the expression of c-abl and c-myc in leukemic cells of patients in all clinical phases of chronic myelogenous leukemia. We demonstrate that an aberrant 8-Kb c-abl related transcript is present in the RNA of the leukemic cell from all patients with Ph+ CML and that the loss of both normal chromosome #9 is associated with the loss of the normal c-abl related transcripts. This represents direct evidence that the normal c-abl related transcripts derive from the normal c-abl gene locus on the normal chromosome #9, while the aberrant c-abl related transcript in Ph+ CML derives from the hybrid bcr-abl gene formed as a result of the t(9;22). We further demonstrate that trisomy 8 in some instances is associated with enhanced expression of the c-myc oncogene.

Philadelphia-negative chronic myelogenous leukemia with breakpoint cluster region rearrangement: molecular analysis, clinical characteristics, and response to therapy.

We have detected rearrangement in the breakpoint cluster region (bcr) on chromosome 22 in cells derived from seven chronic myelogenous leukemia (CML) patients who had no cytogenetic evidence of a chromosome abnormality. These Philadelphia (Ph)-negative, bcr rearrangement-positive CML patients had clinical features and laboratory parameters that bore a strong resemblance to those of Ph-positive CML; all patients have shown a favorable response to hydroxyurea, busulphan, or alpha interferon (IFN-alpha) therapy. In one patient, because of the deletion of distal 3 sequences, detection of bcr rearrangement required a large probe that recognized proximal 5 sequences. Cells obtained from five patients were studied by Northern blotting and showed an aberrant 8 kilobase (kb) mRNA indistinguishable from the bcr-abl transcript that is felt to be a pathogenetic factor in Ph-positive CML. In three patients with a normal karyotype, bcr rearrangement was detected at the time of hematologic remission, and represented the only evidence for persistent malignancy. Our results suggest that: (1) the presence of bcr rearrangement in CML is associated with clinical features of Ph-positive disease, even in the absence of the Ph chromosome; (2) deletions occur within bcr and necessitate the use of probes covering both 5 and 3 DNA segments for accurate diagnosis; (3) molecular analysis may provide a useful approach to the follow-up of leukemia therapy in some patients; and (4) these patients respond to hydroxyurea, busulphan, and IFN-alpha therapy.

Conventional dose fludarabine-based regimens are effective but have excessive toxicity in elderly patients with refractory chronic lymphocytic leukemia.

The best approach to elderly patients with relapsing chronic lymphocytic leukemia (CLL) or disease refractory to conventional therapy with alkylating agents has not yet been established. Fludarabine and its combination with mitoxantrone and/or cyclophosphamide, which is the most effective treatment in younger patients, has not been extensively utilized in the elderly CLL. Here we report our results with fludarabine-based chemotherapy in 32 previously treated patients over the age of 65 years. The overall response rate was 59% with no complete remission, 3 nodular partial remissions and 16 partial remissions. The median time to progression of disease was 7 months. Only 10 patients completed the entire treatment program, because of poor compliance due to toxicity. Eight patients developed neutropenic fever, 14 severe bacterial infections and 2 patients showed progressive encephalopathy. For comparison, in a younger group of patients with refractory CLL (<65 years), 38 of 50 patients completed the treatment plan, and the ORR was 80% (10 CR, 11 PR-nodular, 19 PR) with a median response of 12 months. Neutropenic fever was diagnosed in 10 and severe bacterial infection in 4 patients. In conclusion, fludarabine-based chemotherapy is effective for refractory CLL, however, excessive toxicity such as severe infections and neurological complications, do not allow completion of treatment in the majority of the elderly patients. Because maintenance of a good quality of life should be the main goal in the elderly CLL population, dose reduction of fludarabine and the appropriate use of myeloid growth factors and prophylactic antibiotics appear mandatory in this group of patients.

Significance and correlations of molecular analysis results in patients with philadelphia chromosome-negative chronic myelogenous leukemia and chronic myelomonocytic leukemia

PURPOSEnSeveral investigators have documented a rearrangement of the breakpoint cluster region (bcr) in selected patients with a morphologic diagnosis of chronic myelogenous leukemia (CML) but no abnormality of the Philadelphia chromosome (Ph) by cytogenetic studies. Our intention was to systematically investigate the incidence of the bcr rearrangement in such patients, and to correlate the findings with patient characteristics, response to therapy (especially alpha interferon treatment), and overall prognosis.nnnPATIENTS AND METHODSnMolecular analysis studies were performed in 40 patients with Ph-negative CML (23 patients) and myelomonocytic leukemia (CMML; 17 patients).nnnRESULTSnRearrangement of the breakpoint cluster region (bcr) was detected in 11 of the 23 patients with Ph-negative CML (48 percent), indicating the presence of the abnormal molecular events in Ph-positive CML without documentation of the Ph cytogenetic abnormality. None of the 17 patients with CMML had the bcr rearrangement. Patients with Ph-negative CML and the bcr rearrangement had characteristics similar to those of patients with Ph-positive disease. These included a younger age, higher white blood cell counts, a higher incidence of thrombocytosis and basophilia, and a lower occurrence of thrombocytopenia. The leukocyte alkaline phosphatase score was not a helpful distinguishing feature. Among 21 patients receiving alpha interferon-based regimens, response to therapy was significantly better among patients with Ph-negative disease and the bcr rearrangement (seven of seven, 100 percent), compared with those without the bcr rearrangement (one of six, 17 percent), or patients with CMML (two of eight, 25 percent) (p less than 0.01). At this time of follow-up, only one of the 11 patients with Ph-negative CML and the bcr rearrangement had died from complications of allogeneic bone marrow transplantation, compared with three deaths among the 12 patients with Ph-negative CML and no bcr rearrangement, and 11 deaths among the 19 patients with CMML.nnnCONCLUSIONnWe conclude that molecular studies help in better understanding the nosology of Ph-negative CML, and define a subgroup of patients with clinical, therapeutic, and prognostic correlations similar to those of patients with Ph-positive CML.

Molecular analysis of chromosome 22 breakpoints in adult Philadelphia‐positive acute lymphoblastic leukaemia

Summary. The Philadelphia (Ph) translocation. t(9;22)(q34:q11). is found in the majority of patients with chronic myelogenous leukaemia (CML) as well as in approximately 20% of adult acute lymphoblastic leukaemia (ALL) patients. The chromosome 22 breakpoint in CML has been localized within a restricted 5.8 kb segment of DNA known as the breakpoint cluster region (bcr). To investigate the chromosome 22 breakpoint in ALL. we analysed five adult Ph‐positive ALL patients for bcr rearrangement. Rearrangement was detected within bcr in two patients. However, in one patient the break occurred 5’to the first exon of bcr and in two patients the bcr region was not involved. We conclude that the identical cytogenetic marker, t(9;22), may yield a different genomic configuration in ALL and CML.

Acute basophilic leukaemia: eight unsuspected new cases diagnosed by electron microscopy.

Summary. We report eight new patients with de novo acute basophilic leukaemia (ABL) diagnosed by electron microscopy (EM) in 184 patients with poorly differentiated AML who were selected for ultrastructural analysis between the years 1989 and 2002. Morphology by light microscopy, cytochemistry, immunophenotyping and cytogenetics did not enable an accurate diagnosis in any of these patients. In almost all the patients, the blasts showed reactivity for HLA‐DR and CD34. EM studies demonstrated the presence of basophilic granules in the leukaemic blasts. These granules were membrane bound and their contents varied in appearance from uniformly electron dense to partially speckled or electron lucent. Theta granules were present in only three patients and no mast‐cell type granules were observed. By light microscopy, the myeloperoxidase reaction was positive in three patients in an unusual coarse granular pattern. Ultrastructural demonstration of peroxidase in the granules, nuclear membrane and profiles of endoplasmic reticulum was observed in all eight patients. The reaction in the granules showed a particular speckled pattern. The outcome was unfavourable in six of our eight patients. As a definitive diagnosis of ABL may be made only by EM, we suggest including such studies as an integral part of the diagnostic work‐up of acute leukaemia cases that lack differentiation markers.

COMPLEX IGA GAMMOPATHY IN GAUCHER'S DISEASE

A 55-year-old Jewish patient was simultaneously diagnosed as having Gauchers disease and IgA multiple myeloma. Serum protein electrophoresis and immunoelectrophoresis showed two different IgA kappa type monoclonal spikes. After four years of observation, a rapid fatal course of disease developed together with expression of J chain protein and an additional IgA lambda paraprotein. Although dysproteinaemias and multiple myeloma have occasionally been reported in Gauchers patients, such a complex gammopathy has to the best of our knowledge not yet been described.

Ultrastructural characteristics and lysozyme content in hypergranular and variant type of acute promyelocytic leukaemia

We investigated the electronmicroscopic (EM) features and cellular lysozyme (LZ) content in 16 cases of acute promyelocytic leukaemia (APL): 11 cases of the hypergranular form (M3) and five cases of the microgranular variant (M3‐V). The main EM features in all cases were: irregular, folded or bilobed nuclei, many cytoplasmic granules, distended rough endoplasmic reticulum (RER) cisternae which, in some cases, presented as stellate forms (more frequent in M3‐V), and bundles of cytoplasmic microfilaments. Many Auer rods were present in M3 cases and few in M3‐V; most of these disclosed parallel tubular arrays (PTA) with a varied periodicity ranging from 13 to 26u2003nm. There was a significant difference between M3 and M3‐V (P<0.0001) in both the number of granules per cell section (62.9u2003±u200334.5 v 38.0u2003±u200323.6) and in the granule section area (0.044±0.033 v 0.026±0.015u2003μm2). In some cases, mainly in M3‐V, we found cells with large granules containing PTA which probably represent poorly developed Auer rods. Intracellular LZ content assayed by a post‐embedding immunogold method, showed high granular LZ density (in the range of that found in M4 and M5) in M3 cells and very low granular LZ content in M3‐V. This study adds new objective parameters for the diagnosis of these two types of APL and provides new information on their LZ pattern of expression.

Clinical significance of serologic markers related to red blood cell autoantibodies production after red blood cell transfusion―severe autoimmune hemolytic anemia occurring after transfusion and alloimmunization: successful treatment with rituximab

BACKGROUND: The association of autoantibody formation with blood transfusion was previously noted. Severe autoimmune hemolytic anemia (AIHA) diagnosed after red blood cell (RBC) transfusion determined us to undertake this study and investigate the incidence and clinical significance of autoantibodies occurring after transfusion by a retrospective review of blood bank and medical records.

Advantage of MIDAM Protocol in Treatment of Elderly Patients With Refractory and Relapsing Acute Myeloid Leukemia

regarding treatment of refractory and relapsing acute myeloid leuke-mia (AML) with the intermediate-dose cytarabine and mitoxantrone(MIDAM) regimen. As is well known, the complete response rates(CRR) for these patients are low and the remission duration is short.The MIDAM regimen consisting of cytarabine 1g/m