Morfometrijski i kinetički parametri kao dijagnostički i prognostički čimbenici leukemijskih oblika kroničnih limfoproliferativnih bolesti

The clinical and morphological picture of the leukemic types of chronic lymphoproliferative disorders (CLLPD) shows considerable heterogeneity. Due to the unpredictable course of the disease, the clinical stages and other parameters used to date have been unable to identify patients at a high risk of disease progression. Therefore, the aim of the study was to find new morphometric, proliferative and/or kinetic parameters in different tumor mass compartments, i.e. bone marrow, peripheral blood and lymph node, which would be able to predict survival and disease progression, in order to define the proliferative kinetic index (PKI). In addition, the aim was to evaluate, in terms of survival, the diagnostic and prognostic value of the variables characterizing disease subtypes, clinical stages, tumor mass, time to tumor mass doubling, and lymphocyte count in total CLLPD population and its subgroups. Then, the proliferative and kinetic parameters were used in an attempt to assess the origin and distribution of malignant lymphatic cells in various clinical and morphological entities. Patients, materials and methods. The study included 155 patients diagnosed with CLLPD. A total of 657 puncture smears of bone marrow (n=236), lymph nodes (n=146) and peripheral blood (n=275) were analyzed. The analysis consisted of morphometric studies, assessment of the nucleolar organization region (AgNOR) characteristics, and image cytometry (ICM), performed in 71895 cells on a SFORM PC (VAMSTEC, Zagreb). The univariate and bivariate analyses were used on statistical data processing, and Kaplan-Meier method for the analysis of survival (Statistica 7.1). Results. Prognostic potential was demonstrated for many clinical, hematological, biochemical, immunophenotypic parameters, morphological characteristics of differential blood count and bone marrow, factors of disease progression, and morphometric, proliferative and kinetic parameters. The "single" and "multiple” programmed stops in the development of typical forms of leukemias and lymphomas, subacute and subchronic leukemias were hypothesized. Differentiation impairment may occur at any stage, and different "stop" locations result in different morphology and affinity to accumulation in bone marrow, peripheral blood and lymph nodes. The new parameters of modified analysis of diploid histogram were found to be appropriate for kinetic analysis by the method of image DNA cytometry. Also, the newly characterized types of AgNOR points, homogeneous, inhomogeneous, and annular, yielded a statistically significant correlation with DNA histogram properties and morphometric characteristics of the cell and nucleus, and were additionally found to play a role in the survival, type of tumor mass distribution, biological behavior of tumor disease, and morphological characteristics of lymphatic cells in bone marrow and peripheral blood. Correlation analysis of the morphometric, proliferative and kinetic characteristics revealed the low proliferative cells to possess small, homogeneous AgNOR, with the majority of cells in the peak of DNA histogram. The high proliferative cells had inhomogeneous AgNOR, mostly containing greater DNA amount than peak cells, or pathologic mitoses (DNA >4N), or the majority of cells being in the S-phase of the cell cycle. Cells with medium proliferative activity and annular AgNOR were in-between. Analysis of different tumor mass compartments showed the lymphatic cells with affinity to accumulation in bone marrow ("damaged" naïve B-lymphocytes) to regularly exhibit low proliferative activity (a lower percentage of cells in SFC and highest percentage of cells in the peak of the G0/G1 phase). The "programmed" cells with affinity to accumulation in lymph nodes migrated to lymph nodes, where they transformed to the stage of "programmed stop", exhibiting the characteristics of proliferative cells (an increased number of AgNOR, larger more proliferative inhomogeous AgNOR and lowest percentage of cells in the G0/G1 phase). The migration of cells from bone marrow to lymph nodes and between lymph nodes occurs in peripheral blood (a mixture of cells with low and high proliferative activity: a higher proportion of cells in SFC and at the same time in the G0/G1 phase of the cell cycle). Analysis of cell size and proliferative activity in different compartments of tumor mass revealed a regular pattern within total CLLPD population, and in the subgroups of B-chronic lymphocytic leukemia with variants (B-CLL+V) and typical B-chronic lymphocytic leukemia (B-CLL). Whereas the cells in bone marrow and peripheral blood did not differ substantially according to size and proliferative activity, an inverse pattern was observed between peripheral blood and lymph node. As small cells are inactive and larger cells more proliferative, the analysis quite unexpectedly showed the peripheral blood cells to be largest and most inactive, in contrast to lymph node where the cells were smallest and most active. Based on the most representative AgNOR and DNA characteristics (related to survival) in various tumor mass compartments, the PKI score was calculated for the CLLPD population as a whole and for B-CLL+V in separate. The CLLPD (p=0,00118) and B-CLL+V (p=0,03589) patients had a statistically significantly better prognosis when score of PKI was less than 4. Conclusion. Peripheral blood sample is inadequate and not representative for the study of disease progression or stability. The morphometric, proliferative and kinetic properties of tumor cells, now for the first time analyzed simultaneously in different tumor mass compartments, provide better assessment of the disease dissemination and progression. The morphometric characteristics of homogeneous, inhomogeneous and annular AgNOR, and characteristics of diploid histogram have confirmed the hypothesis that prognostically unfavorable/favorable subgroups could be identified and the course of the disease predicted even within the groups of neoplasms of relatively low malignancy such as CLLPD with subgroups

Chronic Lymphocytic Leukemia: Insights from Lymph Nodes & Bone Marrow and Clinical Perspectives

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by highly variable distribution of tumor mass between peripheral blood, bone marrow and lymphoid organs which is important for staging, classification and prognosis. These clinical findings with novel data about importance of B-cell receptor and its stimulation with the support of microenvironment indicate important role of tissues (lymphoid organs and bone marrow) in the pathogenesis of B-CLL. Here is presented the novel approach of simultaneous characterization of B-CLL cells form peripheral blood, bone marrow and lymph nodes by flow cytometry and immunocytochemistry, defining inter- and intraclonal diversity with respect to various molecules. These include adhesion molecules (integrins, immunoglobulins, selectins), chemokine receptors (including CXCR-4), signaling molecules and prognostic factors (CD38 and ZAP-70), proliferation and apoptosis markers (including Ki67, AgNORs with PK index, survivin, bcl-2) and therapeutic targets (CD20 and CD52) and residual hematopoietic stem cells. A number of interesting significant interactions have been discovered, pointing to the important role of neoplastic cell microenvironment. These may in addition to insights in pathogenesis and roles of different microenvironments add to diagnosis, prognosis and treatment of B-CLL patients

Chronic lymphocytic leukemia: insights from lymph nodes & bone marrow and clinical perspectives [Kronična limfocitna leukemija: uvidi iz limfnog čvora i koštane srži i kliničke perspektive]

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by highly variable distribution of tumor mass between peripheral blood, bone marrow and lymphoid organs which is important for staging, classification and prognosis. These clinical findings with novel data about importance of B-cell receptor and its stimulation with the support of microenvironment indicate important role of tissues (lymphoid organs and bone marrow) in the pathogenesis of B-CLL. Here is presented the novel approach of simultaneous characterization of B-CLL cells form peripheral blood, bone marrow and lymph nodes by flow cytometry and immunocytochemistry, defining inter- and intraclonal diversity with respect to various molecules. These include adhesion molecules (integrins, immunoglobulins, selectins), chemokine receptors (including CXCR-4), signaling molecules and prognostic factors (CD38 and ZAP-70), proliferation and apoptosis markers (including Ki67, AgNORs with PK index, survivin, bcl-2) and therapeutic targets (CD20 and CD52) and residual hematopoietic stem cells. A number of interesting significant interactions have been discovered, pointing to the important role of neoplastic cell microenvironment. These may in addition to insights in pathogenesis and roles of different microenvironments add to diagnosis, prognosis and treatment of B-CLL patients

Multimodal Image Analysis of Chronic Leukemic Lymphoproliferative Disorders and the Hypothesis of »Single« and »Multiple« Programmed Stops in the Development of Typical and Atypical Forms of Leukemias and Lymphomas

The study consisted of morphometric analysis, assessment of the argyrophilic nucleolar organization region (AgNOR) characteristics, and image cytometry (ICM) in different tumor mass compartments: bone marrow (BM), peripheral blood (PB) and lymph nodes (LN) from patients with chronic leukemic lymphoproliferative disorders. A total of 71895 cells were analyzed on SFORM PC (VAMSTEC, Zagreb). Correlation between morphometric, AgNOR and ICM characteristics revealed the cells with low proliferative activity to possess small, homogeneous AgNOR, with the majority of cells in the peak of DNA histogram. The cells with high proliferative activity had inhomogeneous AgNOR, mostly containing greater DNA content than peak cells, pathologic mitoses (DNA>4N), or the majority of cells were in the S-phase of the cell cycle. Cells with medium proliferative activity and annular AgNOR were in-between. Analysis of different tumor mass compartments showed that lymphatic cells with the affinity to accumulate in BM regularly exhibited low proliferative activity (a lower percentage of cells in SFC and highest percentage of cells in the peak of the G0/G1 phase). The cells in LN exhibited the characteristics of proliferative cells (an increased number of AgNOR, larger and more proliferative inhomogeneous AgNOR, and lowest percentage of cells in the G0/G1 phase). The migration of cells from BM to LN and between lymph nodes occurred through PB (there were cells with low and high proliferative activity: a higher proportion of cells in SFC and at the same time in the G0/G1 phase of the cell cycle). Analysis of cell size and proliferative activity in different compartments of tumor mass revealed that the cells in BM and PB did not differ substantially according to size and proliferative activity, while an inverse pattern was observed between PB and LN. As small cells are inactive and larger cells more proliferative, the analysis quite unexpectedly showed the PB cells to be largest and most inactive, in contrast to LN where the cells were smallest and most active. The »single« and »multiple programmed stops« have been hypothesized in the development of typical forms of leukemias and lymphomas and atypical forms of subacute and subchronic leukemias. Differentiation impairment may occur at any stage, and different »stop« locations result in different morphology and affinity to accumulation in bone marrow, peripheral blood and lymph nodes

Juvenile Myelomonocytic Leukemia with PTPN11 Mutation in a 23-Month-Old Girl

Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder affecting young children. The natural course of JMML is rapidly fatal with 80% of patients surviving less than three years. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment of JMML. We report a case of a 23-month-old girl who presented with an upper respiratory tract infection, fever, rash, diarrhea, hepatosplenomegaly and abdominal distention. Severe elevation of white blood cell count with monocytosis and myeloid progenitors in the peripheral blood was also detected. Bone marrow smear showed morphology suggestive of JMML, an unspecific immune phenotype and a normal karyotype. DNA analysis revealed a mutation in the PTPN11 gene. Therefore, the final diagnosis of JMML with somatic PTPN11 mutation was established. Following three months of cytostatic therapy with 6-mercaptopurine and low doses of cytarabine partial remission was achieved and allogeneic HSCT was successfully performed. Six months after the diagnosis, the girl was in a good condition and in a complete remission of JMML. Early diagnosis and allogeneic HSCT were crucial for successful treatment outcome

Pre-B-cell acute lymphoblastic leukemia with bulk extramedullary disease and chromosome 22 (EWSR1) rearrangement masquerading as Ewing sarcoma

We report a 2-year-old female with a subcutaneous tumor who was initially misdiagnosed as suffering from Ewing sarcoma with a positive EWSR1 rearrangement and EWS/FLI1 transcript. After finding lymphoblasts in peripheral blood, the diagnosis of acute lymphoblastic leukemia was established. This necessitated further analysis of the subcutaneous tumor. The tissue was positive for immature B-cell markers and an immunoglobulin heavy chain gene rearrangement, which confirmed the final diagnosis of common type acute lymphoblastic leukemia with bulk extramedullary disease. The patient was treated with chemotherapy and was in remission 30 months after the diagnosis