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.

[Evolution of chronic lymphocytic leukemia: predictive value of immunophenotype, soluble CD23 and morphology]. / Evolución de la leucemia linfática crónica. Valor predictivo del inmunofenotipo, el CD23 soluble y la morfología.

UNLABELLED: Some prognostic factors are useful in chronic lymphocytic leukemia (CLL): lymphocyte doubling time, clinical stage and bone marrow pattern infiltration, while others, such as the percentage of CD38+ cells, are under study and require confirmation. The objective of this study was to evaluate whether there is an association between morphology, lymphocyte immunophenotype, soluble CD23 (sCD23) and progression free survival (PFS). A total of 36 non-treated patients were enrolled. We analysed prospectively: morphology (typical, mixed and PL-CLL); immunophenotypic profile (Matutes score); sCD23 plasma levels; clinical stage; lymphocyte doubling time; beta 2 microglobulin and karyotype abnormalities. Disease progression (need of treatment, progression to advanced stages, development of bulky organomegaly) and death related to disease were considered as events. Md of follow-up 24 mo. RESULTS: Stage 0: 11/36, PFS 80%; I: 10/36 PFS 90%; II: 13/36; III and IV: 2/36. SLE > or = II PFS 37%. p = 0.023. Lymphocyte doubling time < 12mo. 7/31; > 12mo. 24/31. PFS 28% vs. 80% p < 0.001. Karyotype: normal 13/28, abnormal 15/28. PFS 92% vs. 54% p = 0.053. Trisomy 12: positive 7/30, negative 23/30, PFS 66% vs. 65%. beta 2 microglobulin: normal 9/35; high 26/35. PFS 100% vs. 53% p = 0.006. sCD23 < 350 Ul/ml: 15/32; > 350 Ul/ml: 17/32. PFS 92% vs. 53% p = 0.005. Immunophenotype: Score 5: 15/36, Score 4: 19/36, PFS 64%. Score 3: 2/36. p = 0.516. Morphology: typical 17/35, mixed 17/35, PFS 81% vs. 57%, p = 0.099. PL-CLL 1/35. CONCLUSIONS: sCD23 was suitable to predict PFS, specially useful for early stages without additional markers of active disease. Morphology (excluding PL-CLL) and immunophenotype, two common tools, were not useful for the study purpose.

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

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 (Richter's syndrome). Genetic studies were performed at diagnosis and during the Richter's 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.

Evolucion de la leucemia linfatica cronica: valor predictivo del inmunofenotipo, el CD23 soluble y la morfologia / Evolution of chronic lymphocytic leukemia: predictive value of immunophenotype, soluble CD23 and morphology

Some prognostic factors are useful in chronic lymphocytic leukemia (CLL): lymphocyte doubling time, clinical stage and bone marrow pattern infiltration, while others, such as the percentage of CD38+ cells, are under study and require confirmation. The objective of this study was to evaluate whether there is an association between morphology, lymphocyte immunophenotype, soluble CD23 (sCD23) and progression free survival (PFS). A total of 36 non-treated patients were enrolled. We analysed prospectively: morphology (typical, mixed and PL-CLL); immunophenotypic profile (Matutes score); sCD23 plasma levels; clinical stage; lymphocyte doubling time; beta 2 microglobulin and karyotype abnormalities. Disease progression (need of treatment, progression to advanced stages, development of bulky organomegaly) and death related to disease were considered as events. Md of follow-up 24 mo. RESULTS: Stage 0: 11/36, PFS 80%; I: 10/36 PFS 90%; II: 13/36; III and IV: 2/36. SLE > or = II PFS 37%. p = 0.023. Lymphocyte doubling time < 12mo. 7/31; > 12mo. 24/31. PFS 28% vs. 80% p < 0.001. Karyotype: normal 13/28, abnormal 15/28. PFS 92% vs. 54% p = 0.053. Trisomy 12: positive 7/30, negative 23/30, PFS 66% vs. 65%. beta 2 microglobulin: normal 9/35; high 26/35. PFS 100% vs. 53% p = 0.006. sCD23 < 350 Ul/ml: 15/32; > 350 Ul/ml: 17/32. PFS 92% vs. 53% p = 0.005. Immunophenotype: Score 5: 15/36, Score 4: 19/36, PFS 64%. Score 3: 2/36. p = 0.516. Morphology: typical 17/35, mixed 17/35, PFS 81% vs. 57%, p = 0.099. PL-CLL 1/35. CONCLUSIONS: sCD23 was suitable to predict PFS, specially useful for early stages without additional markers of active disease. Morphology (excluding PL-CLL) and immunophenotype, two common tools, were not useful for the study purpose

Aplicaciones diagnósticas del estudio por PCR de los reordenamientos de los receptores T y de las inmunoglobulinas en oncohematología / Diagnostic applications of PCR studies of T cell receptor and immunoglobulin gene rearrangements in oncohematology

El trabajo consiste en la puesta a punto de diversas técnicas de biología molecular para el diagnóstico de leucemias y linfomas dentro del Instituto. Por medio de la técnica de PCR, utilizada para detectar reordenamientos de los genes de la cadena pesada de las inmunoglobulinas y en el receptor de los linfocitos T (cadena µ), se determina clonalidad en las LLA y algunos linfomas en los cuales los métodos convencionales ofrecen dificultad diagnóstica. Se han analizado 64 muestras de ADN provenientes de pacientes de la Sección Inmunología Oncológica del Instituto de Investigaciones Hematológicas "Mariano R. Castex" (I.I.HEMA.), 19 biopsias de linfomas incluidas en parafina y 14 cultivos celulares de pacientes del mismo Instituto. Se ha detectado clonalidad B por PCR mediante la identificación de un fragmento de alrededor de 100 bp en una serie de pacientes oncohematológicos. En 81 ensayos se ha detectado reordenamiento VDJ en 52 casos, distribuidos de la siguiente manera: 15/19 biopsias de linfomas incluidas en parafina, 13/14 cultivos de células mononucleares periféricas de pacientes hemofílicos HIV+ y 24/48 pacientes con leucemias y linfomas (material fresco). Se han detectado reordenamientos en las cadenas µ del receptor T por PCR Multiplex con la idea de complementar el diagnóstico en la identificación de la clonalidad T. Se detectaron reordenamientos en el locus TCGR en 23 de las 30 muestras de ADN provenientes de células mononucleares totales de pacientes con linfomas y leucemias. Este es el primer paso para la realización de un diagnóstico preciso y a partir de aquí poder detectar la enfermedad mínima residual en los pacientes post-tratamiento.

Análisis de factores pronósticos en leucemia linfática crónica. Valor predictivo del inmunofenotipo, el CD23 soluble y la morfología / Predictive factors in chronic lymphocytic leukemia. Value of immunophenotype, SCD23 and morphology

En LLC se reconocen factores pronósticos útiles como la duplicación linfocitaria, el estadio clínico y el patrón de infiltración medular. Otros, como el porcentaje de células CD38+, están en estudio y requieren confirmación. El objetivo del presente trabajo fue evaluar si existe asociación entre morfología, inmunofenotipo linfocitario, CD23 soluble (SL) y sobrevida libre de eventos (SLE). Se evaluaron prospectivamente 36 pacientes sin tratamiento. Se determinaron: morfología típica, mixta y LLC-PL; inmunofenotipo linfocitario (score de Matutes); niveles plasmáticos de CD23 SL; estadio clínico, duplicación linfocitaria; ß2 microglobulina y alteraciones citogenéticas. Se consideró evento: progresión de enfermedad (necesidad de tratamiento, evolución a estadios avanzados, desarrollo de organomegalia voluminosa) y muerte por enfermedad. Mediana de seguimiento 24 meses. Resultados: estadio 0: 11/36, SLE 80 por ciento; I: 10/36 SLE 90 por ciento; II: 13/36: III y IV: 2/36. SLE >= II 37 por ciento. p= 0.023. Duplicación linfocitaria: <12m 7/31, >12m 24/31. SLE 28 por ciento vs 80 por ciento p<0.001. Citogenético: normal 13/28; anormal 15/28. SLE 92 por ciento vs 54 por ciento p=0.053. +12 positiva 7/30, negativa 23/30. SLE 65 por ciento vs 66 por ciento. ß2 microglobulina normal 9/35, elevada 26/35; SLE 100 por ciento vs 53 por ciento p=0.006. D23 SL < 350 Ul/ml 15/32, > 350 Ul/ml 17/32. SLE 92 por ciento vs 53 por ciento p=0.005. Inmunofenotipo: Score 5: 15/36, Score 4: 19/36, SLE 64 por ciento. Score 3: 2/36. p=0.516. Morfología típica 17/35, mixta 17/35. SLE 81 por ciento vs. 57 por ciento p=0.099. LLC-PL 1/35. El CD 23 SL resultó adecuado para predecir SLE, particularmente útil en estadios iniciales sin otros marcadores de actividad. La morfología y el fenotipo linfocitario, dos variables accesibles, no fueron útiles para el propósito del estudio.