Mariano Scolnik

CD34 and CD117 are overexpressed in AML and may be valuable to detect minimal residual disease

We estimated by quantitative flow cytometry (FC) the expression of CD13, CD33, CD34 and CD117 antigens in cells from 64 patients with acute myeloid leukaemia (AML) and 22 normal bone marrows (BMs). The method converts fluorescence intensity into number of antigen molecules per cell, measured by antibody binding capacity (ABC). The number of molecules per cell in normal BM was 9.5+/-5.7 for CD13, 7+/-2.3 for CD33, 6+/-0.7 for CD34, and 6.3+/-1.5x10(3) for CD117. AML blasts expressed 11.4+/-12.4 molecules per cell for CD13, 9.5+/-9.7 for CD33, 74+/-2328.5 for CD34 and 12.5+/-33 x 10(3) for CD117. The number of CD34 and CD117 molecules were significantly higher in AML than in normals (P<0.0001 and P<0.05, respectively) while only in a few cases, CD13 and CD33 were abnormally expressed in myeloblasts. Our results indicate that quantitative analysis of CD34 and CD117 may be useful to detect minimal residual disease (MRD) and could be tested in a future to monitor therapy in AML.

Interphase cytogenetic analysis in Argentinean B-cell chronic lymphocytic leukemia patients: association of trisomy 12 and del(13q14)

We have evaluated genomic aberrations by conventional cytogenetics and fluorescence in situ hybridization (FISH) analysis in a series of 57 Argentinean B-cell chronic lymphocytic leukemia (B-CLL) patients. The studies were performed on stimulated peripheral blood lymphocytes. FISH analysis for trisomy 12, 13q14 deletion, and monosomy of TP53 (also known as p53) was performed according to standard protocols. Our results showed 46.3% of patients with clonal chromosomal alterations by conventional cytogenetics and 80.7% by FISH. Trisomy 12 was found in 21.9% of patients by G-banding analysis and in 35% by FISH studies. Allelic loss of 13q14 was observed in 63.2% patients, most of them showing D13S319 and D13S25 deletion; 11% of patients showed TP53 monosomy. Coexistence of trisomy 12 and 13q14 deletion was found in 17.5% of patients. In this group, deletion 13q14 was the prevalent clone, with percentages 25-35% higher than those observed for trisomy 12, suggesting clonal evolution. The coexistence of trisomy 12 with deletion 13q14 was observed in a higher frequency than reported in the literature. A probable adverse prognosis is suggested for this group of patients, likely related to clonal evolution.

Promyelocytic Blast Crisis of Chronic Myelogenous Leukaemia with Translocations (9;22) and (15; 17)

The promyelocytic blast crisis is a rare form of transformation during the evolution of chronic myeloid leukaemia (CML). We report a case of promyelocytic blast crisis with t(15;17) in addition to t(9;22). The morphology and immunophenotype of the blasts were similar to those seen in acute promyelocytic leukaemia (APL). The t(15;17) was confirmed by FISH. The patient had evidence of coagulopathy with clinical and laboratory findings of disseminated intravascular coagulation (DIC). This report highlights the importance of correlating the results of multiple diagnostic methods in order to establish a correct diagnosis of the promyelocytic blast crisis of CML.

Modulator activity of PSC 833 and cyclosporin-A in vincristine and doxorubicin-selected multidrug resistant murine leukemic cells.

Multidrug resistance (MDR) lines from a murine T-cell leukemia were selected in increasing vincristine (VCR) or doxorubicin (DOX) concentrations. Daunorubicin (DNR) efflux was evidenced after 25 additional passages with constant 160 ng ml(-1) of either VCR or DOX, an effect that was inhibited by verapamil, cyclosporin-A (CsA) and PSC 833. The expression of Pgp was not evidenced in the resistant cell lines using anti-human Pgp antibodies. Cell proliferation assay showed that cell lines resistant to VCR (LBR-V160) or DOX (LBR-D160) required higher doses of either drug to produce GI50 compared with control cell line obtained after culture in the absence of VCR or DOX. When resistant cell lines were maintained during 60 days in the absence of either VCR or DOX, MDR phenotype reversal was obtained in LBR-D160 while LBR-V160 remained resistant to the drug, as shown by cell proliferation assays and by drug efflux pump functionality. When VCR or DOX were used together with either CsA or PSC 833, the latter was more effective to produce reversal of resistance than the former, whereas CsA presented greater cytotoxic effect than PSC 833 for sensitive and resistant cells. Cross-resistance was found between VCR, DOX and other antineoplasic agents on murine leukemic cell line. VCR was more effective to induce MDR since the resistant cell lines were more stable to the MDR phenotype.

Growth hormone inhibits normal B-cell differentiation and neutrophils' chemotaxis in vitro.

In acromegalic patients we have previously described a low ability of B-lymphocytes to differentiate into plasma cells under PWM stimulation, and a decreased chemotaxis of polymorphonuclear leukocytes (PMN) towards N-formylmethionylphenilalanine (FMP). In this study we examined the effect of exogenous GH over these immune functions in normal cells. PMN were purified by dextran sedimentation, incubated with recombinant human GH (0 to 20 ng/ml) and subjected to stimulation with FMP. PBMC were cultured with or without PWM, in the presence of GH (between 0 and 100 ng/ml). Plasma cells were determined as hemolysis plaque forming cells and also by immunofluorescence. GH, in a dose-dependent way, decreased directed migration of PMN (5 ng/ml: 1.787 +/- 148 microns; 10 ng/ml: 1.581 +/- 221 microns; 20 ng/ml: 1.569 +/- 149 microns, all as mean +/- S.E.M.), when compared to similar values of untreated PMN (0 ng/ml 2.085 +/- 139 microns). GH treatment did not modify spontaneous migration. Net migration showed the same pattern as directed migration. GH decreased dose-dependently the PWM-driven differentiation of B-lymphocytes into plasma cells to 60% of the basal level. Although not significantly, GH tended to increase spontaneous B-cell differentiation. These results could account for the already described defect in B-cell differentiation and PWN chemotaxis in acromegaly, emphasizing the relationship between the endocrine and immune systems.

Biphenotypic Acute Leukemia with t(15;17)

Biphenotypic acute leukemias (BAL) represent 5% of all acute leukemias. The most frequent cytogenetic abnormalities described are Philadelphia chromosome and 11q23 involvement. Here we report a BAL case, with blasts showing lymphoblast morphology and positivity for myeloperoxidase (in 6% of the blast cells). Immunophenotype revealed the compromise of myeloid and B-lymphoid lineages. Cytogenetic analysis showed the t(15;17) and 8 trisomy. PML/RARa rearrangement was detected by fluorescent in situ hybridization (FISH) on interphase nuclei while PML/RARa fusion transcript was detected in the bone marrow and peripheral blood by molecular biology studies (RT-PCR). This report describes a BAL case with an unfrequent cytogenetic abnormality, and highlights the importance of correlating the results of multiple diagnostic methods in order to establish a correct diagnosis and treatment in BAL patients.

Cytogenetic, FISH, and molecular studies in a case of B‐cell chronic lymphocytic leukemia with karyotypic evolution

Abstract:  We report the clinical, cytogenetic, fluorescence in situ hybridization (FISH) and molecular findings in a 54‐yr‐old male patient diagnosed with B‐cell chronic lymphocytic leukemia (B‐CLL), who showed progression to a diffuse large B‐cell lymphoma (Richters syndrome). Genetic studies were performed at diagnosis and during the Richters transformation (RT). A clonal karyotype with two dicentric chromosomes, psu dic(12,21)(q24;q10) and dic(17,18)(p11.2;p11.2), was found. Both rearrangements were confirmed by FISH. Molecular cytogenetics analysis using p53 probe showed monoallelic loss of this tumor suppressor gene in 43.8% and 77.3% of cells for the first and the second studies, respectively). In both studies, deletions of D13S319 (18% and 12% of cells) and D13S25 loci (13% and 12% of cells) at 13q14 were found. Polymerase chain reaction analysis showed the MBR/JH rearrangement of the bcl‐2 gene. FISH studies using LSI bcl‐2/IgH probe allowed quantifying the clonal cell population with this rearrangement (4% and 6.6% of cells at diagnosis and RT, respectively). To our knowledge, this is the first case with a psu dic(12,21) described in B‐CLL. The low percentage of cells with the 13q14 deletion and bcl‐2/IgH rearrangement suggests that they were secondary events that resulted from clonal evolution. Our patient had a short survival (9 months) and a clear lack of response to several therapeutic agents, confirming the association of p53 gene deletion and karyotypic evolution with disease progression.

dup(12)(q13–q22) and 13q14 Deletion in a Case of B-Cell Chronic Lymphocytic Leukemia

Cases with partial trisomy 12 have rarely been found in B-cell chronic lymphocytic leukemia (CLL). We report our clinical, cytogenetic and fluorescence in situ hybridization (FISH) findings in a CLL patient with a duplication of part of the long arm of chromosome 12 between bands q13–q22. This patient was the only case with this duplication among the 112 cases (0.9%) of CLL cytogenetically analyzed in our laboratory. FISH studies using unique-sequence specific probes for the RB-1 (retinoblastoma) gene and the D13S319 locus at the 13q14 band showed a monoallelic loss for the D13S319 locus (20% of cells) with a diploid RB-1 gene. Our case showed an atypical morphology (35% prolymphocytes), a high proliferation rate and progression of the disease, indicating that the duplication of this region may be equivalent to complete trisomy 12 in CLL patients.

Trilineage Phenotypic Compromise in Acute Leukemia

The expression of three lineage specific antigens in the leukemic blasts is extremely infrequent. We here report a case of triphenotypic acute leukemia with involvement of the myeloid and B and T lineages. The morphology of the blasts showed promyelocytic features with agranular cytoplasm, suggesting a M3-variant of AML. The blasts were positive for myeloperoxidase PAS and Sudan Black. Immunophenotype and cytogenetics did not confirm M3-AML diagnosis, showing a trilineage compromise (myeloid and T and B lymphoid markers) and the cytogenetic alterations +8 and +11, respectively. This report highlights the importance of correlating the results of multiple diagnostic methods in order to establish a correct diagnosis of mixed lineage acute leukemias, and allows evaluation of the prognostic importance of this subgroup of patients.

CD8 Expression in a Case of Chronic Lymphocytic Leukemia with Trisomy 12

We report a case of B-cell chronic lymphocytic leukemia (B-CLL) with aberrant expression of the T-cell-associated antigen CD8, as revealed by two-color flow-cytometric analysis. DNA studies showed immunoglobulin heavy-chain gene rearrangement, but not of γ-chain T-cell receptor, confirming the B-cell origin of the neoplastic cells. Ploidy analysis showed a tetraploid population and high S-phase fraction. B-CLL cells also carried trisomy 12, detected by fluorescence in situ hybridization. The identification of more cases with the same features would be necessary to establish the prognosis of this subtype of B-CLL.