The Reduction of the Cell Nuclear Size in the Cell Body Space During the Differentiation Might Be Cell Lineage Specific (A Retrospective Morphological Note).

The cell body space occupied by the nucleus decreased during the cell differentiation of the granulocytic cell lineage in CML (Chronic Myeloid Leukemia) patients. In contrary, in patients suffering from CLL (Chronic Lymphocytic Leukemia), the cell body space occupied by the nucleus during the cell differentiation of the lymphocytic lineage did not decrease despite the reduction of the cell size. Thus, the cell body space occupied by the cell nucleus during the differentiation was characteristic for each of these cell lineages.

The Morphology of Cell Differentiation, Terminal Differentiation and Ageing Seems To Reflect the Same Process: a Short Note.

Based on simple microscopic cell morphology in blood and bone marrow smear preparations, it seems to be likely that the cell differentiation and terminal differentiation in human blood cells, and particularly in erythroid or granulocytic lineages, simultaneously reflect ageing of the lineage progenitors and terminal differentiation steps. The terminal differentiation stages of both these lineages actually appear as senescent cells. Abnormal ageing of progenitor cells may represent one of the "dysplastic" phenomena of the premature terminal differentiation state. Such state is characterized by heterochromatin condensation and nucleolar morphology similar to that in fully differentiated terminal cells of granulocytic or erythroid lineages. It should also be mentioned that in some known erythropoietic disorders, less differentiated erythroblasts may lose nuclei similarly as "normal" fully terminally differentiated cells of the erythroid cell lineage. It seems to be clear that cells in both abnormal less differentiated and terminally differentiated stages of erythroid or granulocytic lineages lose the ability to multiply similarly as senescent cells. On the other hand, the background of cell ageing and differentiation is very complicated and requires a different approach than the simple microscopic morphology at the single cell level. However, the morphology and clinical cytology at the single cell level might still contribute with complementary data to more sophisticated complex studies of that topic. In addition, the morphological approach facilitates the study of the main components of single cells in various states, including the differentiation steps or ageing.

The cell body space occupied by the nucleus during the cell differentiation in human lymphocytic, granulocytic and erythroid cell lineages.

The present nuclear and cell body diameter measurements demonstrated size differences of the approximate cell space estimate occupied by the cell nucleus during the cell differentiation in lymphocytic, granulocytic and erythroid cell lineages. These lineages were used as convenient models because all differentiation steps were easily identified and accessible in diagnostic peripheral blood or bone marrow smears of blood donors (BDs), patients suffering from chronic lymphocytic leukemia (CLL), patients with chronic myeloid leukemia (CML) and refractory anemia (RA) of the myelodysplastic syndrome (MDS). The cell space occupied by the nucleus was constant and did not change during the cell differentiation in the lymphocytic cell lineages of BDs and CLL patients despite the decreased cell size. In contrary, the cell space occupied by the nucleus markedly decreased in differentiating cells of granulocytic and erythroid lineages of patients suffering from CML. In the erythroid cell lineage in patients with RA of MDS the small reduction of the cell space occupied by the nucleus during the differentiation was not significant. The measurements also indicated that in progenitor cells of all studied cell lineages nuclei occupied more than 70 % of the cell space. Thus, the nucleus-cytoplasmic morphological and functional equilibrium appeared to be characteristic for each differentiation step and each specific cell lineage.

Dominant Nucleolus in the Progenitor Cell Using Human Bone Marrow Erythroid and Granulocytic Cell Lineages as a Model. A Morphological and Cytochemical Note.

Progenitor cells of the human erythroid and granulocytic cell lineages are characterized by the presence of several nucleoli. One of these nucleoli is larger and possesses more fibrillar centres than others. Such nucleolus is apparently dominant in respect of both size and main nucleolar function such as nucleolar-ribosomal RNA transcription. Such nucleolus is also visible in specimens using conventional visualization procedures, in contrast to smaller nucleoli. In the terminal differentiation nucleated stages of the erythroid and granulocytic development, dominant nucleoli apparently disappeared, since these cells mostly contained very small nucleoli of a similar size with one fibrillar centre. Thus, the easily visible dominant nucleoli appear to be useful markers of the progenitor cell state, such as proliferation, and differentiation potential.

To the approximate size of the nuclear region occupied by nucleolar bodies during cell differentiation and maturation using the human leukemic granulocytic lineage as a convenient model.

The present study was undertaken to estimate the approximate size of nuclear regions occupied by nucleolar bodies during the cell differentiation and maturation. The differentiation and maturation of human leukemic granulocytic cells in patients suffering from the chronic phase of the chronic granulocytic leukemia (CML) represented a convenient model for such study because of the large number of cells for the diameter measurements at the single cell level. Early and advanced differentiation or maturation stages of these cells are well defined and nucleolar bodies and nuclear outlines are easily seen by simple cytochemical methods for the visualization of RNA and silver stained proteins in smear preparations. During the cell differentiation and maturation, the estimated size of the nuclear region occupied by nucleolar bodies decreased in both untreated and treated patients with the anti-leukemic therapy. However, the size reduction of nucleolar bodies in differentiated and mature cells was larger than that of the nucleus. In addition, the results also indicated that the nuclear region occupied by nucleolar bodies was characteristic for each differentiation and maturation stage of the granulocytic cell lineage and was not substantially influenced by the anti-leukemic therapy of CML patients.

Morphometric and Densitometric Analysis of Heterochromatin during Cell Differentiation Using the Leukaemic Granulocytic Lineage as a Convenient Model.

Granulocytic early progenitors and terminally differentiated - mature granulocytes with segmented nuclei were studied using computer-assisted diameter and heterochromatin optical image densitometry to provide more information on the nuclear size and heterochromatin condensation state. Bone marrow smears of patients suffering from chronic myeloid leukaemia untreated as well as treated with "specific" anti-leukaemic therapy with imatinib mesylate are a convenient model for such study because they possess a satisfactory number of cells for diameter and optical density measurements. In addition, the identification of developmental stages of granulocytes is very easy and the morphology is not different from that in not-leukaemic persons. As it was expected, the mean diameter of nuclear segments in fully differentiated and mature granulocytes was much smaller than that in non-segmented nuclei of early granulocytic precursors. Therefore, no wonder that the heterochromatin condensation state in nuclear segments of mature granulocytes was much larger than in non-segmented nuclei of granulocytic progenitors. On the other hand, the sum of mean diameters of all nuclear segments per cell was close to the mean nuclear diameter of early granulocytic progenitors. The heterochromatin condensation state in granulocytic progenitors or fully differentiated mature granulocytes exhibited marked stability and did not change after the anti-leukaemic therapy. In addition, Barr bodies of characteristic drumstick appearance bearing inactive X chromosome in interphase nuclei of mature granulocytes in fertile female patients exhibited a heterochromatin condensation state similar to nuclear segments. This heterochromatin condensation state was also stable and constant, and was not apparently influenced by the anti-leukaemic therapy.

The Relationship of Kynurenine and Neopterin Levels and Their Association with a Selection of Other Immune Markers in Chronic Myeloid Leukaemia Patients.

Among malignant diseases, chronic myeloid leukaemia (CML) is one of the best suited candidates for immunotherapy. For this purpose it is necessary to broaden the present knowledge on the immunology of this disease. As a part of such a project, the levels of kynurenine (KYN) and neopterin (NPT) were studied in 28 CML patients and in the same number of healthy subjects. At diagnosis, both KYN and NPT levels were found to be elevated in a significant portion of the patients and dependent on their leukocyte count. As in the case of KYN, increased NPT levels dropped after achieving remission. When correlating KYN and NPT levels with a selection of other markers tested, significant association was revealed only in the case of CRP and IL-6. However, there were several patients with increased KYN levels in whom NPT was not detected, and vice versa. The relapse of the disease observed in two patients was accompanied by an increased level of NPT in both cases, but by an increased level of KYN in only one of them. No significant correlation was found between KYN and NPT levels in sera taken at diagnosis. However, when the whole set of sera was taken into consideration, the association became statistically significant. Although the data obtained revealed a number of similarities between KYN and NPT production in CML patients, it also suggested a difference in the kinetics of these two biomarkers' production.

[Tyrosine kinase inhibitors -  major change in the prognosis of chronic myeloid leukaemia]. / Inhibitory tyrozinových kináz -  zásadní zmena v prognóze chronické myeloidní leukemie.

Chronic myeloid leukaemia (CML) is an example of a tumour disease, in which the discovery of its molecular principle resulted in the development of the specific targeted molecular treatment with the first representative being imatinib. The introduction of other tyrosine kinase inhibitors, nilotinib and dasatinib, into the clinical practice meant not only a revolutionary change in the treatment approach to CML but literally an epochal improvement of the prognosis and quality of life. The disease with an originally fatal prognosis in patients diagnosed in the chronic stage has changed into a disease with the expected survival rate median estimated to be more than 25 years.

Microscopic notes on the perinucleolar chromatin region in immature and mature human B-leukemia lymphocytes.

Perinucleolar region was studied in lymphocytes of patients suffering from chronic B lymphocytic leukemia to provide more information on the perinucleolar-condensed chromatin - heterochromatin - during the maturation of these cells. The perinucleolar heterochromatin of lymphocytes in smear preparations was visualized using a simple, but sensitive cytochemical method for the demonstration of DNA. The perinucleolar heterochromatin was also easily visible as unstained perinucleolar regions in specimens stained for RNA. In addition, the perinucleolar heterochromatin of lymphocytes was distinct and apparent in the transmission electron microscope using conventional as well as cytochemical methods for visualization of chromatin structures. Despite the great variability, the maturation of leukemic lymphocytes was accompanied by an increased width of the perinucleolar heterochromatin shell. It seems to be also interesting that the perinucleolar region of both immature as well as mature leukemia lymphocytes contains heterochromatin bodies about 2µm in diameter. They appeared to be a regular component of the perinucleolar heterochromatin shell and were apparently different from other nuclear bodies present at the nucleolus. In contrary to other known nuclear bodies, perinucleolar heterochromatin bodies in leukemia lymphocytes consisted only of conglomerates of DNA containing chromatin fibrils and did not contain other structural components including RNA. The presence of perinucleolar heterochromatin bodies in the perinucleolar region of leukemia lymphocytes is not contradictory with the present knowledge on that nuclear territory. They might be associated with presumed special perinucleolar DNA loci, which according some previous studies were more expressed in malignant cells.

Heterochromatin density (condensation) during cell differentiation and maturation using the human granulocytic lineage of chronic myeloid leukaemia as a convenient model.

The present study was undertaken to provide complementary data on the heterochromatin condensation in both central and peripheral nuclear regions during the cell differentiation and maturation using computer-assisted density measurements at the single-cell level. The lineage of neutrophilic granulocytes in the bone marrow of patients suffering from chronic myeloid leukaemia was very convenient for such study because the increased number of granulocytes in all developmental stages was satisfactory for heterochromatin density measurements. The morphology of leukaemic and non-leukaemic neutrophilic granulocytes is similar and each differentiation or maturation stage is easily identified. A markedly increasing heterochromatin density--condensation--in the peripheral nuclear region at the nuclear envelope accompanied both the differentiation and maturation of these cells. Thus, peripheral chromosomal territories at the nuclear envelope are important for both the differentiation and maturation process. In contrast, the heterochromatin density of nuclear central regions was already high in early differentiation stages and exhibited a less distinct increase during the differentiation, but was more apparent in late maturation stages representing the terminal differentiation. A limited number of maturing cells with persisting large heterochromatin density in central nuclear regions without markedly increased heterochromatin condensation at the nuclear periphery might represent a further maturation abnormality--asynchrony--during the granulocytic development. From the methodological point of view, both, the cytochemical method for the DNA demonstration and the panoptic May-Grünwald-Giemsa staining, are convenient for computer-assisted chromatin densitometry at the single-cell level.

Incidence of second malignancies during treatment of chronic myeloid leukemia with tyrosine kinase inhibitors in the Czech Republic and Slovakia.

Tyrosine kinase inhibitors (TKI) have completely changed the prognosis of patients with Ph+ chronic myeloid leukemia (CML). The occurrence of a second malignancy (SM) in CML patients successfully treated with TKI may significantly affect their prognosis. In a retrospective study of 1,038 patients with CML treated at 10 centers in the Czech Republic and Slovakia between 2000 and 2009, SM was detected in 35 (3.37%) patients after TKI therapy was initiated. The median intervals from the diagnosis of CML and from the start of TKI therapy to the diagnosis of SM were 58 months (range 2 - 214) and 32 months (range 1 - 102), respectively. The observed age-standardized incidence of SM after the start of TKI therapy was 8.95 / 1,000 person-years. Comparison of the incidence of SM in CML patients with population data was performed only for patients from the Czech Republic. The age-standardized incidence rate of all malignant tumors except non-melanoma skin cancers was 6.76 (95% CI: 6.74; 6.78) / 1,000 person-years in 2000 - 2007 while the incidence rate of SM in 708 CML patients from the Czech Republic treated with TKI was 9.84 (95% CI: 6.20; 13.48) / 1,000 person-years, i.e. 1.5-fold higher, although the difference was statistically insignificant. The distribution of SM types in CML patients treated with TKI was similar to that in the age-standardized general Czech population. The median overall survival (OS) of patients treated with TKI who also developed SM (57 months) was shorter than the OS of patients treated with TKI but not suffering from SM (median OS not reached, log rank test p < 0.001. Prospective long-term population-based studies in CML patients treated with TKI as first-line therapy are needed to determine the relationship of SM to KTI therapy.

Dasatinib in imatinib-resistant or -intolerant CML patients: data from the clinical practice of 6 hematological centers in the Czech Republic.

Dasatinib is effective second line treatment for patients with chronic myeloid leukemia (CML) resistant or intolerant to imatinib. We report here the first experiences with dasatinib therapy in 71 CML patients resistant or intolerant to imatinib from the real clinical practice of 6 hematological centers in the Czech Republic. Dose 100 mg daily and 70 mg twice daily was administered to patients with chronic phase (CP) and advanced phases (AP) CML. In chronic phase (n=46), complete hematological reponse (CHR) was achieved in 97%, major cytogenetic reponse (MCgR) in 77% and complete cytogenetic response (CCgR) in 67%. Major molecular reponse (MMR) was achieved in 19/31 patients in median of 10 months. In advanced phase (n=25), CHR was attained in 77%, MCgR in 39%, CCgR in 33% and MMR in 2/18 patients. Eleven different baseline mutations were followed up in 15 patients. Dasatinib eliminated mutations in most of the patients, but 3 patients acquired a new one. Novel mutations were detected under dasatinib therapy in 2 patients. Dasatinib was well tolerated, cytopenias were common and was managed by dose modification. The estimated progression free survival (PFS) at 12 months was 97+/-3% in CP and 62+/-21% in AP. The median time to treatment failure was 605 days in AP while it was not reached in CP patients. Our clinical experiences, described here, confirmed that dasatinib is associated with high response rates especially in imatinib resistant or intolerant CML patients in chronic phase.

A short cytochemical note on the nucleolar and cytoplasmic RNA concentration in differentiating cells represented by human erythroblasts.

The present study was undertaken to provide more information on the nucleolar and cytoplasmic RNA concentration in differentiating cells of the erythroid lineage. These cells represent a convenient model to study cell differentiation since all stages are morphologically well characterised. The bone marrow of patients suffering from the chronic phase of chronic myeloid leukaemia without a large increase in the granulocyte to erythroid ratio provided erythroblasts for computer-assisted image density measurements of RNA in nucleoli and cytoplasm at the single cell level. The measurements indicated a significant decrease of the nucleolar and cytoplasmic RNA concentration only in advanced stages of erythroblast differentiation (polychromatic and orthochromatic erythroblasts). The ratio of the nucleolar to cytoplasmic RNA concentration was otherwise very stable and did not change during differentiation, being similar in the early and advanced stages of erythroblastic development. In contrast, the nucleolar size significantly decreased even during the early stages of erythroid development (basophilic erythroblasts). This marked decrease in the nucleolar diameter in differentiating erythroblasts and the less marked decrease in the nucleolar RNA concentration suggest that the amount of RNA in the nucleolus is closely associated with nucleolar size rather than on its concentration within the nucleolar body.

WT1 expression in peripheral leukocytes of patients with chronic myeloid leukemia serves for the prediction of Imatinib resistance.

The determination of patient's resistance to a particular drug contributes to more efficient therapeutical approach. The aim of this study was to evaluate if the responsiveness of Chronic Myeloid Leukemia (CML) patients to Imatinib therapy was predictable from WT1 gene expression in peripheral blood leukocytes. To examine the resistance we implemented an in vitro cultivation of the primary cells of 48 CML patients with Imatinib. The effect of Imatinib was characterized not only by the expression of WT1 but also by BCR-ABL, and proliferative factor Ki-67.
Our results showed that leukocytes of CML patients, clinically responsive to Imatinib treatment, significantly decreased WT1 expression after in vitro incubation with Imatinib. It was accompanied by an inhibition of expression of Ki-67 but not BCR-ABL. In leukocytes of CML patients clinically resistant to Imatinib, the expression of WT1, Ki-67, and BCR-ABL remained unaffected. The presented results showed that in vitro testing using peripheral blood cells enabled clinicians to predict responsiveness of CML patients to Imatinib.

[Therapy of chronic myeloid leukemia. Uncommon view of the modern approaches]. / Lécba chronické myeloidní leukémie--méne bezný pohled na moderní postupy.

Chronic myeloid leukemia has got an outstanding position among the other oncology diseases, particularly due to specific chromosomal abnormality called Philadelphia chromosome, its molecular consequence BCR/ABL fusion gene and its biologic product BCR/ABL tyrosinekinase, which is responsible for the leukemic transformation of the hematopoetic cells. We can see a real revolution in the treatment of the chronic myeloid leukemia in the last few years. The authors pay attention to the developement of the new drugs in the large historical consequences from the beginning in the 19th century (describing leukemia first time) to the introduction of the new small specific molecules into the clinical practice.

To the density and distribution of heterochromatin in differentiating, maturing and apoptotic cells represented by granulocytic, lymphocytic and erythrocytic precursors.

The present study was undertaken to provide more information on the density and distribution of heterochromatin in early and advanced stages of the granulocytic, lymphocytic and erythroid development. Heterochromatin was visualized using a simple cytochemical method for the demonstration of DNA followed by computer-assisted densitometry of the digitized images. The largest heterochromatin density in early proliferating stages of all studied blood cell lineages was noted in the perinucleolar region and centrally located chromocentres. In contrast, the heterochromatin density at the nuclear membrane was significantly lower. In advanced nonproliferating stages or apoptotic cells the heterochromatin density increased and was similar in all nuclear regions, i.e. in the perinucleolar regions, chromocentres, and at the nuclear membrane. Thus, such observations indicated that the heterochromatin condensation in the perinucleolar region and chromocentres, i.e. in "gene-rich nuclear regions", of differentiating and maturing progenitors of blood cells preceded that at the nuclear periphery.

Immunological profiles of patients with chronic myeloid leukaemia. I. State before the start of treatment.

In view of the increasing interest in the immunotherapy of CML it seems highly desirable to broaden the present knowledge on the immune reactivity of CML patients. A group of 24 patients and 24 healthy controls were studied for the total of 15 immunological parameters, including the prevalence of antibodies against human herpesviruses and papillomaviruses. To clearly discriminate between changes associated with the disease and those induced by the therapy, all patients were enrolled prior to the start of any anti-leukaemic therapy. Statistically significant differences between patients and controls were found in the levels of IgA, C4 component of complement, CRP and IL-6, the production of Th1 cytokines in stimulated CD3 cells and the E. coli stimulatory index. The analysis of the interrelationship between the results obtained in the individual patients presented some unexpected findings, such as the lack of correlation between the CRP and IL-6 levels. It will be the purpose of a follow-up to determine whether and how the immune status of the patients prior to the treatment correlates with their response to therapy and how the individual immunological profiles change in the course of the disease. These observations will be utilized in the future immunotherapeutic studies to constitute the vaccine- and placebo-treated groups.

A karyometric note on nucleoli in human early granulocytic precursors.

The diameter of nucleoli was measured in human bone marrow early granulocytic precursors after visualization by a simple cytochemical method for demonstration of RNA. Such method facilitated to clearly see nucleolar bodies without perinucleolar chromatin, including those of micronucleoli. The bone marrow of patients suffering from chronic myeloid leukaemia (untreated with cytostatics) provided a satisfactory number of both myeloblasts and promyelocytes for nucleolar measurements because of prevailing granulopoiesis. The direct nucleolar measurement was carried out on digitized and processed images on the screen at magnification 4,300x. It seems to be likely that the nucleolar size is directly related to the number of nucleoli per cell. The largest nucleoli were present in both myeloblasts and promyelocytes that possessed a single nucleolus. In contrast, the nucleolar diameter was significantly smaller in cells with multiple nucleoli. However, in cells with small multiple nucleoli, one of them was always larger and dominant with a large number of AgNORs. Such large nucleoli are possibly visible in specimens stained with panoptic procedures or methods staining nuclear chromatin or DNA. It should also be mentioned that both myeloblasts and promyelocytes mostly possessed two nucleoli with the mean diameter close to 1.5 microm. The incidence of early granulocytic precursors classified according to the nucleolar number and size strongly suggested that the various nucleolar number and nucleolar size in these cells might be related to the different stage of the cell cycle and might also explain their heterogeneity.

On the nucleolar size and density in human early granulocytic progenitors, myeloblasts.

Human myeloblasts were studied in bone marrow of patients suffering from chronic phase of chronic myeloid leukaemia to provide more information on the nucleolar diameter in these early granulocytic progenitors. These cells are a convenient model for such study since the number of myeloblasts in diagnostic bone marrow smears of investigated patients is larger than in not-leukemic persons because of the increased granulopoiesis. The nucleolar diameter was measured in myeloblasts after various cytochemical procedures such as methods for visualisation of RNA, DNA and proteins of AgNORs using digitized images and image processing. The results clearly demonstrated that values of the nucleolar diameter depended on the procedures used for visualising nucleoli. It seems to be also clear that a close relationship exists between the diameter of nucleoli and their number since the larger the number of nucleoli per cell the smaller their mean size. However, one of multiple nucleoli present in the nucleus is usually significantly larger. Moreover, the possibility exists that the variability of nucleolar diameter of leukemic myeloblasts and thus the heterogeneity of these cells might depend on various stages of the cell cycle as supported by nucleolar measurements on aging leukemic myeloblasts (K 562 cells) in vitro. Since the staining density of small and large nucleoli did not differ substantially after staining for RNA, it seems to be likely that the nucleolar size is directly related to the total RNA content in myeloblasts. In addition, karyometry combined with RNA cytochemistry still appears to be an useful tool to study nucleoli at the single cell level.