Acute myeloid leukemia (AML) is a malignant hematopoietic disease with poor clinical course and outcome. There is a constant need for new prognostic factors that could facilitate patient risk ...stratification. The aim of our research was to determine the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways in leukemic cells, their relation to P-glycoprotein (P-gp) expression/activity and their prognostic significance in adult de novo AML. A total of 118 patients with AML were enrolled in the study. In a multivariate Cox regression analysis we found that P-gp activity and Akt phosphorylation were independent poor prognostic factors of overall survival (OS). In contrast, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) represented a favorable prognostic factor of OS and relapse-free survival (RFS). A negative correlation between P-gp activity and p38 phosphorylation level was found, implying a possible role of this MAPK pathway in P-gp regulation. In addition, we found correlation between Akt and p38 phosphorylation levels, indicative of co-activation of two signaling cascades in AML.
Klasifikacija tumora hematopoetskog i limfocitnog tkiva prema Svjetskoj zdravstvenoj organizaciji (engl. World Health Organization; WHO) objedinila je podatke dobivene citomorfološkim, citokemijskim, ...imunofenotipskim, citogenetičkim i molekularnim analizama s kliničkim obilježjima pojedinih mijeloidnih novotvorina i uvrstila ih u dijagnostički algoritam kojim se pojedini entiteti definiraju. Osnovu dijagnoze čini citomorfološko određivanje postotka blasta unutar 200 stanica u razmazu periferne krvi ili 500 stanica u koštanoj srži bojenim prema May-Gruenwald Giemsi (MGG), gdje se nalaz više od 20 % blasta smatra akutnom leukemijom. Uz citomorfološku analizu konačna dijagnoza postavlja se uz nalaze imunofenotipizacije, citogenetike i molekularne dijagnostike, a početak liječenja temelji se na procjeni kliničkih parametara progresije bolesti.
Fine-needle aspiration (FNA) biopsy has become a well established technique in the diagnosis, staging, and follow-up of patients with head and neck lesions. As in lymphoma diagnostics, FNA serves as ...a screening method in evaluating potentially affected lymph node for open or core biopsy. According to the World Health Organization classification of lymphoid neoplasms, today it is important to recognize cell morphology and reveal its phenotype, then combine it with different genotypic information and clinical data to provide appropriate therapy. The aim of this study was to assess the efficacy of FNA and immunocytochemistry based lymphoma diagnostic in head and neck region. We conducted a retrospective study during a period of three years where cases with either FNA diagnosis or clinical suspicion of newly recognized or relapsing lymphoma were reviewed. In the study were included patients that were referred to our laboratory from hematology department, in whom head and neck lymphadenopathia was found and lymph node FNA preceded other procedures. Two hundred eighty-five aspirations from 248 patients fulfilled study criteria. Adequate specimens were diagnosed as lymphoma in 100 cases (36%), in 65 male and 35 female patients, 76 in patients with newly discovered disease and 24 in patients with prior lymphoma diagnosis. Overall sensitivity of FNA specimens in the diagnosis of head and neck lymphomas was 90%, specificity 88%, predictive value of a positive result 97%, and predictive value of negative result 61%. Based on our results FNA corroborated with immunophenotyping by immunocytochemistry can be method of choice in primary lymphoma diagnosis as a method complementary to histopathology in lymphoma diagnostics.
Lymphomas represent the third most common group of cancers in childhood and adolescence, mature B non Hodgkin's lymphoma (B-NHL) accounting for up to 60% of newly diagnosed patients. The diagnosis of ...specific entities of B-NHL is based on well-defined morphologic analysis, immunophenotyping, cytogenetics and molecular genetics, which determine the optimal treatment strategy. In adult population a major turning point in treatment of B-NHL has been achieved since rituximab, in combination with CHOP has improved the survival rate up to 19%. Rituximab is a chimeric monoclonal antibody that targets CD20, a transmembrane calcium channel expressed on normal and malignant B-cells that mediates cytotoxic, apoptotic and anti-proliferative effects. The effect of rituximab in pediatric population is still not well enough investigated. Based on morphology and immunophenotype of malignant cells, seven children with B-NHL in our institution were eligible for treatment with modified B-NHL-Berlin-Frankfurt-Münster (BFM)-95-based protocol with rituximab administered on day -5. The complete remission was achieved in all seven patients. Six patients are still in complete remission at least 12 months after having finished chemotherapy and one patient relapsed two months after the last cycle and subsequently died. Major adverse effects observed during treatment were prolonged B-cell depletion and myelosuppression. Rituximab in combination with B-NHL-BFM-95 protocol was otherwise well tolerated and proved to be effective in children and adolescents with B-NHL. The number of our patients is too small and the follow-up of a larger group of patients will help in defining the role of rituximab in the treatment of childhood B-NHL.
Transformation of leukemic cells is associated with delay in maturation and in apoptosis, and to altered responsiveness to growth factors. However, some studies have revealed that Fas (CD95/APO1) ...which mediates apoptotic signal and decrease of anti-apoptotic Bcl-2 are frequently observed in acute myeloid leukemia (AML) M4/M5 leukemic cells. The aim of the study was to compare cytomorphology and cytochemistry of bone marrow (BM) apoptotic leukemic cells to preserved peripheral blood (PB) leukemic cells in our patient, a 76-year-old man with AML-M5b treated at Zagreb University Hospital Center. BM and PB of the AL patient were analyzed after Pappenheim and cytochemical stainings, and leukemic cells were classified according to FAB and WHO classification. Analysis of PB revealed leukocytosis and 80-90% monocytic cells (46% monoblasts, 29% promonocytes and 11% monocytes). Only a few preserved monoblasts and promonocytes were found in BM, together with numerous morphologically altered cells with characteristic chromatin condensation and pyknosis of nucleus, as well as nuclear fragmentation and formation of apoptotic bodies. Thus, cytomorphology of PB leukemic cells pointed to proliferation of immature monocytic cells, and cytomorphology of BM to cell apoptosis. Cytochemistry of PB monocytic cells and BM apoptotic cells confirmed monocytic cell lineage because esterase was strongly positive in almost all BM apoptotic leukemic cells and PB leukemic cells, and esterase was completely inhibited with sodium fluoride. On the basis of these findings, AML-M5b was diagnosed in our patient. There are many possible explanations for our observation of BM leukemic cell apoptosis in a patient with AML-M5. The most reliable one is that apoptosis was induced ex vivo after BM aspiration in course of the air drying of BM specimen before staining. Mass BM leukemic cell apoptosis that was recorded in contrast to numerous preserved leukemic cells in PK could be probably connected to unfavorable ratio of relatively low concentration of cytokines in relation to high leukemic cell number in BM aspirated cytologic specimen.
Detection of unusual or aberrant cell immunophenotype with flow cytometry is the basis for the immunologic recognition of minimal residual disease (MRD) in patients with acute leukemia (AL). In this ...study, we have shown that the double immunocytochemical alkaline phosphatase antialkaline phosphatase (APAAP) staining technique also makes possible the detection of leukemic cells with unusual (leukemic) combinations of antigens (ULCA) both at diagnosis and during follow-up of patients with ULCA+ AL. The applicability of double APAAP was analyzed on bone marrow (BM) samples obtained from 12 patients (8 with AML, 3 with ALL, and 1 with undifferentiated acute leukemia AUL) randomly chosen from a larger group of 22 ULCA+ patients treated at our center in a 3-year period (22% observed ULCA+ AL frequency). The percentages of ULCA+ BM cells before chemotherapy were in the range of 5%-60%, which dropped to 0%-7% in 10 patients who achieved remission (range 0%-7%, p < 0.01). However, these cells could also be found 60 days after the initiation of therapy, ranging from 0%-2% of all nucleated cells. In 2 of 10 patients who achieved remission, 2% ULCA+ BM cells were found on days 35 and 60 after initiation of chemotherapy, and this finding was followed by relapse on days 110 and 270. However, the other 8 patients remained in remission despite positive finding of ULCA+ BM cells ranging from 0.2%-2% on at least one occasion. In 2 patients with AML FAB-M3 and cytomorphologic remission, the finding of ULCA+ cells by double APAAP correlated with the molecular finding of PML/RARalpha junction. These results indicate that double APAAP staining can identify leukemic cells in samples with a cytomorphologic pattern consistent with remission, but its applicability in detection of MRD awaits additional studies on a larger number of patients with ULCA+ AL.
Continuous human malignant hematopoietic cell lines are invaluable tool for hematological research. Here we report on a new erythroleukemic cell line termed VES that was established from the bone ...marrow mononuclear cells (BMNC) of a 22-year-old woman with Ph+ CML during her second post-transplant period. Actually, after unsuccessful allo-transplantation, the patient received auto-transplant that also failed to engraft. At the time of initiation of BMNC into culture for the analysis of stromal cell capacity, her bone marrow was tested Ph-negative by both FISH and PCR. Instead of establishment of an adherent stromal layer in vitro, blast-like cells appeared in the supernatant on day 27. These blast cells expressed highly amplified bcr/abl locus as shown by FISH, whereas the patient's bone marrow was still Ph-negative at the time. Following a very short period, the cultured cells started to express stable features of erythroleukemia cell line.
Optimal growth was obtained by suspending the cells at concentration of 0.3x106 cells/ml in IMDM containing 10% FBS, 1% penicillin-streptomycin and 1% L-glutamine solution. After 3 days of culture, cell concentration varied between 1.3 and 1.8x106 cells/ml. VES cells were tested negative for the presence of EBV virus. For analysis of clonogenicity, VES cells at concentration of 103/ml were grown in a serum-free methylcellulose medium without cytokines (H4236; StemCell Tech., Canada). Following 7-day culture, 103 VES cells produced 251 ±42 and after 14 days 431 ±30 GF-independent colonies. Furthermore, on day 14 of the cell culture it was possible to observe reddish color of the colonies indicating the presence of hemoglobin. Cytological examination of cytospin preparations indicated homogenous population of leukemia blasts (>80%) the majority of them (>90%) being glycophorinA positive and MPO negative. Imunophenotyping and multiparameter flow cytometry revealed proerythroblastic lineage-associated profile: GlyA/CD235a+, CD11b+, CD15+, CD29+, CD33+, and CD117+. Conventional cytogenetics revealed complex karyotype with multiple numerical/structural abnormalities (MAKA), while metaphase FISH revealed the following aberrations: t(9;22), 5q31, 7q31, +8, +6.
Since FISH analysis detected highly amplified bcr/abl locus, we tested VES cells' sensitivity to imatinib (Gleevec). Treatment with imatinib for 3 days (MTS assay) inhibited proliferation of VES cells with IC50 value of 0.2mM. Following 14 days of culture in methylcellulose medium with addition of 0.2mM imatinib, the clonogenic potential of VES cells was reduced by 55% in relation to untreated control. We demonstrated that imatinib induced apoptosis in VES cells in time- and dose-dependant manner, assessed with AnnexinV and PI staining, while there were no significant changes observed in the cell cycle except for a mild increase in cells in G0/G1 phase. Although further analyses are required, we believe that VES cells represent a new Ph+ erythroleukemia cell line and a suitable model for studying Ph+ malignant hematopoiesis in vitro.
