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

Simultaneous Occurrence of Acute Myeloid Leukemia and Chronic Lymphocytic Leukemia: A Case Report

Concurrent development of acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) is very rare. We present a case of simultaneous CLL and AML in a patient with no prior exposure to cytotoxic agents or irradiation

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

Monocytes in Metabolic Disorders – Opportunities for Flow Cytometry Contributions

Chronic inflammation has arised as a major underlying cause of atherosclerosis, obesity and diabetes. It is mediated by cells of innate immune system like macrophages but also by their antecedents, circulating monocytes. Roles of monocyte subsets and different markers of monocyte activation in the context of metabolic disorders have been reviewed. Applying cell based approach through flow cytometry in this field has resulted with new understanding of pathophysiologic mechanisms. Possible implications of these insights in diagnosis, prognosis and revealing of therapeutic targets in metabolic disorders remain a challenge for future

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

Incidence and mortality trends of leukemia and lymphoma in Croatia, 1988-2009

Aim To investigate the time trends of leukemia and lymphoma in Croatia from 1988-2009, compare them with trends in other populations, and identify possible changes. Methods The data sources were the Croatian National Cancer Registry for incidence data, Croatian Bureau of Statistics for the numbers of deaths, and United Nations population estimates. Joinpoint regression analysis using the age-standardized rates was used to analyze incidence and mortality trends. Results Acute lymphoblastic leukemia and chronic lymphocytic leukemia incidence did not significantly change. Acute myeloid leukemia incidence significantly increased in women, with estimated annual percentage change (EAPC) of 2.6% during the whole period, and in men since 1998, with EAPC of 3.2%. Chronic myeloid leukemia incidence significantly decreased in women (EAPC -3.7%) and remained stable in men. Mortality rates were stable for both lymphoid and myeloid leukemia in both sexes. Hodgkin lymphoma non-significantly increased in incidence and significantly decreased in mortality (EAPCs of -5.6% in men and -3.7% in women). Non-Hodgkin lymphoma significantly increased in incidence in women (EAPC 3.2%) and non-significantly in men and in mortality in both men (EAPC 1.6%) and women (EAPC 1.8%). Conclusion While Croatia had similar leukemia and lymphoma incidence trends as the other countries, the mortality trends were less favorable than in Western Europe. The lack of declines of leukemia incidence and non-Hodgkin lymphoma mortality could be attributed to late introduction of optimal therapies. As currently the most up-todate diagnostics and treatments are available and covered by health insurance, we expect more favorable trends in the future

Detection of t(14;18) by PCR of IgH/BCL2 Fusion Gene in Follicular Lymphoma from Archived Cytological Smears

According to WHO classification follicular lymphoma (FL) is a neoplasm composed of follicle centre (germinal centre) B-cells, which usually has at least a partially follicular pattern. Bone marrow (BM) infiltration by lymphoma occurs in 40-70% of cases at the time of diagnosis. The characteristic chromosomal translocation of follicular lymphoma is t(14;18)(q32;q21) with transposition of BCL2 oncogene to the regulatory region of immunoglobulin heavy chain gene IgH. Aim of this study was to determine the frequency of PCR detection of IgH/BCL2 in DNA samples isolated from archival cytological slides of lymph node aspirates, bone marrow and/or peripheral blood (PB) obtained from patients with histologicaly confirmed follicular lymphoma using primers and protocol proposed by BIOMED-2 consortium. We also compared molecular with cytomorphological findings in bone marrow/peripheral blood and tested this method of detection of IgH/BCL2 molecular marker in monitoring minimal residual disease (MRD) in routine clinical setting. DNA was successfully isolated from all archival cytological slides obtained by fine needle aspiration of lymph nodes as well as from 75% of smears of bone marrow aspirates from 19 patients. Fusion oncogene was detected in 10 of 19 patients (52%). For patients with PCR IgH/BCL2 positive lymph nodes, molecular test found BM infiltration in 5 cases (83%), while cytomorphology detected infiltration in three of eight cases (37%) available for comparison. May-Grünwald-Giemsa stained cytological smears can be used for PCR-based ancillary methods and the rate of detection of IgH/BCL2 rearrangement is similar to results reported for paraffin-embedded tissues. For patients with detectable baseline molecular marker, PCR is a highly suitable method for detection of bone marrow involvement and monitoring MRD

Canonical Wnt/β-catenin signaling pathway is dysregulated in patients with primary and secondary myelofibrosis

INTRODUCTION: β-Catenin is a central effector molecule of the canonical wingless-related integration site (Wnt) signaling pathway. It is important for maintenance of stem cell homeostasis and its aberrant activation has been implicated in a wide array of malignant hematological disorders. There are few reports suggesting its dysregulation in Philadelphia chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs). ----- PATIENTS AND METHODS: We analyzed β-catenin mRNA expression in bone marrow (BM) aspirates of 29 patients with primary (PMF) and 4 patients with secondary, post Ph- MPN, myelofibrosis (SMF) using quantitative real-time polymerase chain reaction (qRT PCR). The control group consisted of 16 BM aspirates from patients with limited-stage aggressive non-Hodgkin lymphoma without BM involvement. We compared relative gene expression with clinical and hematological parameters. ----- RESULTS: Relative expression of β-catenin differed significantly among groups (P = .0002), it was significantly higher in patients with PMF and SMF than in the control group, but did not differ between patients with PMF and SMF. A negative correlation was found regarding hemoglobin level in PMF (P = .017). No association according to Janus kinase 2 (JAK2) V617F mutational status or JAK2 V617F allele burden was detected. ----- CONCLUSION: Our results show for the first time that β-catenin mRNA expression is increased in patients with PMF and SMF and its upregulation might potentiate anemia. A number of inflammatory cytokines associated with PMF are capable of mediating their effects through increased β-catenin expression. Accordingly, β-catenin can induce expression of a number of genes implicated in processes of cell cycle control, fibrosis, and angiogenesis, which are central to the PMF pathogenesis. Therefore, β-catenin might represent an interesting new therapeutic target in these diseases