Ljiljana Tukic

[Expression of Bcl-2 protein and the amplification of c-myc gene in patients with chronic myeloid leukemia].

BACKGROUND/AIM Chronic myeloid leukemia (CML) represents a malignant myeloproliferative disease developed out of pluripotent hematopoietic stem cell that contains the fusion bcr-abl gene. Disorders that occur in the process of apoptosis represent one of the possible molecular mechanisms that bring about the disease progress. The aim of our study was to carry out the analysis of the presence of the amplification of the c-myc oncogene, as well as the analysis of the changes in the expression of Bcl-2 in the patients with CML. METHODS Our study included 25 patients with CML (18 in chronic phase, 7 in blast transformation). Using an immunohistochemical alkaline phosphatase-anti-alkaline phosphatase (APAAP) method, we analyzed the expression of cell death protein in the mononuclear bone marrow cells of 25 CML patients. By a differential PCR (polymerase chain reaction) method, we followed the presence of amplified c-myc gene in mononuclear peripheral blood cells. RESULTS The level of the expression of Bcl-2 protein was considerably higher in the bone marrow samples of the patients undergoing blast transformation of the disease. The amplification of c-myc gene was detected in 30% of the patients in blast transformation of the disease. CONCLUSION The expression of Bcl-2 protein and the amplification of c-myc gene are in correlation with the disease progression.

Free Light Chains of Immunoglobulin as a Prognostic Factor for Some Plasmaproliferative Diseases

Free Light Chains of Immunoglobulin as a Prognostic Factor for Some Plasmaproliferative Diseases Quantitation of monoclonal immunoglobulins and their fragments is used for monitoring the plasmaproliferative disease course and the effect of therapy. The aim of free light chains examination was to evaluate the significance of the FLC ratio as a prognostic factor for remission, progression and survival in different disease groups. The concentrations of immunoglobulins and free light chains were measured by an immunonephelometric method on a »SIEMENS« DADE BN II analyser with reagents (Freelite, The Binding Site, UK). In this examination 151 patients from 3 different disease groups: 1. Light chain disease or Bence Jones myeloma (37), 2. Biclonal gammopathy with FLC (23) and 3. Monoclonal gammopathy of undetermined significance (91), were investigated during a period of 7 years. The reference interval for FLC ratio is 0.26-1.65. According to the International Staging System for multiple myeloma, a serum FLC ratio of <0.03 or >32 was taken as abnormal. The patients with light chain disease and biclonal gammopathy with FLC with an abnormal FLC ratio and a combination of adverse risk factors (76.7%) had median survival times of 22-30 months, versus patients with a normal or slightly varied FLC ratio without adverse risk factors (23.3%) with median survival times of 39-51 months. About 38% of patients who had shown lowered free light chains values by more than 50% under therapy, achieved disease remission in the light chain disease and biclonal gammopathy with FLC groups. In the group of patients with monoclonal gammopathy of undetermined significance, 66.0% had a normal or slightly modified FLC ratio which corresponds to low and low-intermediate risk of disease progression, as opposed to 34.0% with an abnormal FLC ratio (<0.25 or >4) which corresponds to high and high-intermediate risk. An abnormal FLC ratio in the examined groups could be an independent risk factor for progression and poorer disease prognosis. Slobodni Laki Lanci Imunoglobulina Kao Prognostički Faktor Kod Nekih Plazmaproliferativnih Bolesti Kvantitativno određivanje monoklonskih imunoglobulina i njihovih fragmenata koristi se za praćenje toka i terapijskog odgovora kod plazmaproliferativnih bolesti. Cilj određivanja slobodnih lakih lanaca imunoglobulina u serumu bolesnika jeste provera značaja njihovog količnika (κ/λ indeks) kao prognostičkog faktora remisije, progresije i preživljavanja. Koncentracije imunoglobulina i slobodnih lakih lanaca određivane su imunonefelometrijskom metodom na analizatoru SIEMENS DADE Behring II sa reagensima (FREELITE, The Binding Site, UK). U ispitivanje je uključen 151 bolesnik tokom perioda od 7 godina, koji su razvrstani u 3 grupe: 1. bolest lakih lanaca ili Bence Jones mijelom (37); 2. biklonalna gamapatija sa slobodnim lakim lancima (23) i 3. monoklonska gamapatija neutvrđenog značaja (91). Referentnim intervalom za κ/λ indeks smatraju se vrednosti 0,26-1,65. Prema Internacionalnom prognoznom indeksu za multipli mijelom, kao patološki uzet je κ/λ indeks <0,03 ili >32. Bolesnici iz prve dve grupe sa patološkim k/λ indeksom i kombinacijom nepovoljnih faktora rizika (76,7%) imali su prosečno vreme preživljavanja 22-30 meseci, nasuprot bolesnicima sa fiziološkim ili neznatno izmenjenim κ/λ indeksom bez nepovoljnih faktora rizika (23,3%), sa prosečnim vremenom preživljavanja 39-51 mesec. Oko 38% bolesnika koji su pod terapijom imali sniženje κ/λ indeksa >50% su ostvarili remisiju bolesti. U grupi ispitanika sa MGNZ, 66,0% je imalo fiziološke ili neznatno izmenjene κ/λ indekse, što odgovara niskom i srednje niskom riziku progresije, nasuprot 34,0% sa patološkim κ/λ indeksom (<0,25 ili >4), što odgovara srednje visokom i visokom riziku progresije. Postojanje patološki značajnog κ/λ indeksa u ispitivanim grupama predstavlja nezavisan faktor rizika za progresiju bolesti i lošiju prognozu.

PCR-based clonality assessment in patients with lymphocytic leukaemias: a single-institution experience

PCR-based clonality testing can be performed in all lymphoproliferations by analysing gene rearrangements of antigen receptors, rearrangements that are unique for each kind of lymphocyte. Reactive lymphoproliferations have polyclonally rearranged Ig/TCR genes, whereas malignant proliferations (leukaemias and lymphomas) show clonal rearrangements. The aim of this study was to assess the clinical benefits of clonality testing with previously evaluated consensus primers in leukaemia patients. The study included peripheral blood and bone marrow samples of 67 leukaemia patients (32 B-CLL, 24 B-ALL and 11 T-ALL). Clonality testing was based on PCR amplification of rearranged IgH and TCR genes. During diagnosis, monoclonal pattern was found in all analysed B-CLL and T-ALL samples. Testing in B-ALL patients showed positive results in all bone marrow and one peripheral blood samples. Results of clonality testing in B-CLL patients during follow-up were concordant between peripheral blood and bone marrow. Obtained results corresponded to clinical course in all but one patient. In B-ALL group, results of molecular testing in peripheral blood and bone marrow confirmed remission estimated according to clinical criteria in all except one patient. Before any clinical sign of relapse, monoclonal pattern was found in six/seven patients by bone marrow and in three/seven patients by peripheral blood analysis, respectively. Results of molecular monitoring in T-ALL patients did not confirme clinical evaluation in two patients. Obtained results indicate high accuracy of re-evaluated primers for clonality assessment in ALL and CLL patients at the time of diagnosis. Results of clonality testing in B-ALL patients indicate that bone marrow analysis has higher sensitivity compared to analysis of peripheral blood.