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.

Telomere length, molecular cytogenetic findings, and immunophenotypic features in previously untreated patients with B-chronic lymphocytic leukemia.

UNLABELLED: Telomere length was evaluated by terminal repeat fragment method in 66 previously untreated patients with B-chronic lymphocytic leukemia (B-CLL) to ascertain whether telomere shortening was associated with genomic aberrations, immunoglobulin variable heavy chain (IgVH) mutational status, CD38 and ZAP-70 expression, and telomerase activity. Chromosomal aberrations were present in peripheral blood cells of 73% patients (48/66), no difference in telomere length between patients with good and intermediate prognosis according to cytogenetics was found. Association between telomere length and IgVH mutational status, ZAP-70 and CD38 expression was proved as significantly shorter telomeres in patients with unmutated IgVH status (p=0.01) and ZAP-70 positivity (p=0.01) and CD38 positivity (p=0.05) were detected. Telomerase activity was positive in 11 patients out of 21 examined, correlation between telomere length and telomerase activity was found (p=0.05). Telomere length and telomerase activity in combination with other prognostic parameters complete the risk profile of B-CLL patients and might serve for an easy decision on optimal treatment strategy. KEYWORDS: B-chronic lymphocytic leukemia, telomere length, telomerase activity, chromosomal aberrations, prognosis.

Clonal evolution in chronic lymphocytic leukemia studied by interphase fluorescence in-situ hybridization.

The results of repeated interphase fluorescence in-situ hybridization (I-FISH, FISH) examination of 97 CLL patients and correlation of these findings with IgVH hypermutation status, ZAP-70 and CD38 expression are presented. The appearance of new, FISH-detectable, genomic aberrations during disease course, described as clonal evolution (CE), was observed in 26% of patients. The most frequent newly acquired cytogenetic abnormality was 13q deletion in 64% (16/25). In contrast to earlier studies, there was no correlation found between CE and either one of single negative prognostic factors (unmutated IgVH; CD38 positivity; ZAP-70 positivity). However, the combination of all three negative factors correlated with CE highly significantly (p=0.005) and moreover, also with a shift from lower to higher FISH risk category (p=0.010). As the prognostic data were known in all patients, this study represents the complete insight on the association of CE and other risk parameters in CLL.

Combined molecular biological and molecular cytogenetic analysis of genomic changes in 146 patients with B-cell chronic lymphocytic leukemia.

Interphase fluorescence in situ hybridization was used to detect common deletions in B-CLL patients as well as trisomy 12 and aberrations of IgH gene complex at 14q32.33 where we evaluated not only translocation-like signal pattern but also deletions. 120 (82%) patients showed genetic changes - del(13)(q14) 95 (62%), deletion of ATM gene 22 (15%), deletion of p53 gene 25 (17%) and trisomy 12 was proved in 18 (12%) cases. IgH rearrangements were detected in 45 (31%), split of the signals in 11 (8%), deletion of 3' segment flanking IgH gene in 5 (3%) and deletions of variable segment in 29 (20%) patients. Although deletions of 3' segment flanking IgH gene complex are supposed to have an adverse prognostic impact and the genetic background of variable segment deletions is believed to be most probably physiological, we assumed a detailed mapping of the 14q32.33 region will be needed to unravel these mysteries.

[Lymphocyte immunophenotyping and cytogenetics for more precise prognosis in patients with type B chronic lymphatic leukemia]. / Pouzití imunofenotypizace lymfocytu a cytogenetiky pri zpresnení prognózy u pacientu s chronickou lymfatickou leukémií B typu.

BACKGROUND: The B-chronic lymphocytic leukemia (B-CLL) has highly variable prognosis. Possibility of more relevant prognosis has a great impact for the beginning and mode of therapy. METHODS AND RESULTS: One hundred patients diagnosed as having B-CLL were included into the study. Beside usual examinations necessary to establish the diagnosis, cytogenetic examination for the detection of deletion 13q14, 17p13, 11q23 and trisomy 12 and immunophenotyping was done. The mean age of the patients was 58.2 years, there were 62 men and 38 women. 91 evaluated patients were divided into two groups--those with the steady disease (55 pts) and those with progressive disease (36 pts). No relation between the number of cytogenetic abnormalities and the Rai stage of the disease was found. We identified a relation between the bone marrow infiltration pattern (diffuse) and the number of cytogenetic abnormalities and the del 13q14 (p.05). Using the immunophenotyping of the lymphocytes we found a relation between the expression of CD38 and CD11c and the disease progression (p < 0.05). Neither of the method (FISH and immunophenotyping) revealed differences between results from bone marrow samples and those from peripheral blood. CONCLUSIONS: Though the cytogenetic (FISH) and immunophenotype evaluation at the time of diagnosis did not improve the ability to define better the clinical course of the B-CLL, we suggest to use both methods routinely as an important tool to identify patients who would develop the progressive disease.

[The International Prognostic Scoring System for primary myelodysplastic syndrome]. / Mezinárodní prognostický bodovací systém u pacientu s primárním myelodysplastickým syndromem.

A number of prognostic scoring systems for patients with myelodysplastic syndrome (MDS) have been introduced since FAB classification of the MDS in 1982. Recently, the International Prognostic Scoring System (IPSS), published in 1997 by Greenberg et al. [9] is based on the percentage of bone marrow (BM) blasts, cytogenetic abnormalities and number of cytopenias. We applied criteria of the IPSS on 205 patients (pts) with primary MDS (RA = 82, RARS = 49, RAEB = 42, RAEB-t = 8, CMML = 24 pts). IPSS discriminated within each of the FAB-subgroups: RA pts were present in low risk and intermediate (Int) I and II risk subgroups, RARS pts were separated into low and Int I, RAEB were distributed predominantly between Int I and Int II risk groups, RAEB-t in high-risk group, and CMML pts were distributed in all groups. In contrary to Greenberg's group of the MDS patients there are only three risk-groups in our study: low risk (score 0-0.5), intermediate (1-2) and high risk (> 2); the median survival and the risk of the evolution to the acute leukemia (p = 0.0001) are significantly different.

[Importance of prognostic factors in patients with chronic B-lymphocytic leukemia at the time of diagnosis]. / Význam prognostických faktoru pacientu s B-chronickou lymfatickou leukemií v dobe diagnózy.

BACKGROUND: B-chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia in the Western countries. Any routinely used staging system does not distinguish exactly the probable course of the disease at the time of diagnosis. Therefore the new prognostic factors, which help to assess the optimal therapeutic plan of patients, are searched intensively. METHODS AND RESULTS: We evaluated 154 patients with the B-CLL at the time of diagnosis--133 of them were retrospectively divided into two groups--one with stable form and the other with progressive form of the disease. We compared these two groups of patients with some of the prognostic factors (absolute lymphocyte count, the level of C-reactive protein, lactatdehydrogenase, beta-2-microglobulin, tumour necrosis factor, the immunoglobulin levels, the expression of CD38, FMC7, surface immunoglobulins, the type of bone marrow infiltration, and the cytogenetic abnormalities (trisomy 12, del(13)(q14), del(17)(p13) a del(11)(q23)). We found higher absolute lymphocyte count, level of beta-2-mikroglobulin, tumour necrosis factor, expression of CD38, light chains lambda, lower expression of FMC7 and less frequent nodular type of bone marrow infiltration by the patients with progressive disease. The correlation of the cytogenetic abnormalities and the course of the disease or stage according to the RAI et al. [27] staging system were not significant may be due to the small number of evaluated patients and short period of follow up. CONCLUSION: The routine evaluation of some risk factors in patients with B-chronic lymphocytic leukemia at the time of diagnosis helps to distinguish those with the probable more aggressive course of the disease and have the implication for the design of risk-adapted treatment strategies. The prognostic impact of the cytogenetic abnormalities and other risk factors has to be evaluated on larger group of patients during longer follow up period and repeated evaluations.

[The interphase fluorescence in situ hybridization (I-FISH) technique in patients with chronic lymphatic leukemia (CLL)]. / Význam interfázické fluorescencní in situ hybridizace (I-FISH) pro vysetrení nemocných s chronickou lymfatickou leukémií (CLL).

BACKGROUND: Trisomy 12 was found to be the most frequent chromosomal aberration identified by conventional cytogenetic studies of bone marrow cells and peripheral lymphocytes of patients with CLL. Molecular-cytogenetic techniques which enable examination of dividing and/or non-diving interphase nuclei (I-FISH), proved existence of other chromosomal abnormalities, mainly deletions, which could have in CLL patients relation to the origin, course and prognosis of the disease. METHODS AND RESULTS: During the last two years bone marrow chromosomes of all patients with CLL were examined by G-banding and by I-FISH. The numerical changes of chromosome 12 were followed by centromeric DNA probe in dividing and non-dividing cells. The small deletions were ascertained by locus specific probes for 13q14 (Rb gene), 17p13 (p53 protein) and 11q23 (MLL gene). These genes are responsible for cell division and their function is probably in connection with neoplastic process. It is of interest whether numerical and structural chromosomal rearrangements are primary or secondary changes and what is their impact on etiology of CLL. 93 patients were examined by DNA prove CEP12 and trisomy 12 was found in 24 of them (25.8%), the range of the clone was 2.5-75.5% of the screened cells. Deletion del(13)(q14) was examined by probe D13S319 in 73 patients and proved in 24 of them (32.8%), pathological clone ranged 2.5-80.0% of the cells. Deletion del(17)(p13) was found in 14 patients out of 61 examined by probe LSI p53 (22.9%). The extent of the clone was 2.5-34.0% of examined cells. Deletion 11q23 was not ascertained in any of 11 patients by means of probe LSI 11q23 (MLL). All probes used for FISH were manufactured by VYSIS. CONCLUSIONS: FISH is very sensitive method, suitable for molecular-cytogenetic examination of leukemic patients. With I-FISH the deletion of 13q14 was ascertained as the most frequent chromosomal aberration in series of 73 patients with CLL. We continue to increase the number of patients screened by I-FISH with all eligible DNA probes and start the prospective study on patients with chromosomal pathology. We will correlate the immunophenotype, morphology, clinical course and prognosis with karyotypic findings.

[Normal blood values in the adult population in the Czech Republic]. / Normální hodnoty krevního obrazu u dospelé populace CR.

In a group of 2033 healthy subjects-1475 men, mean age 36 years and 558 women mean age 51 years-normal values of the haemogram of the present healthy Czech population were assessed. Men: Hb 135-174 g/l, haematocrit 0.39-0.51, Ery 4.19-5.75 x 10(12)/l, MCV 82.6-98.4 fl, Leuco 4.1-10.2 x 10(9)/l, thrombocytes 142-327 x 10(9)/l. Women: Hb 116-163 g/l, haematocrit 0.33-0.47, Ery 3.54-5.18 x 10(12)/l, MCV 82.3-100.6 fl, Leuco 4.0-10.7 x 10(9)/l, thrombocytes 131-364 x 10(12)/l. When examining the haemogram it is necessary, if possible, to adhere to a standard procedure. Results must be always evaluated in conjunction with anamnestic data, the physical finding, and possible slightly abnormal values may not be always evaluated as pathological.

[Administration of granulocyte-macrophage colony growth factors in the treatment of postcytostatic myelosuppresion in oncohematologic patients]. / Pouzití rustového faktoru pro granulocyto-makrofágovou radu v lécbe postcytostatické myelosuprese u onkohematologických nemocných.

BACKGROUND: Growth factors for granulocytic and granulocyte-macrophage series (G-CSF and GM-CSF) in the form of recombinant proteins are used in various leukopenias. The aim of this work was to follow the effect of GM-CSF (Leucomax Sandoz) in myelosuppression after the therapy with cytotoxic drugs. METHOD AND RESULTS: Leucomax was given to 22 patients with oncohematological disease (3 patients with acute lymphoblastic leukemia, 5 patients with various myeloid malignacies, 4 myelomas and 10 patients with non-Hodgkin lymphomas). In comparison to the literary data lower doses of Leucomax were used, it is 150-200 micrograms a day and patient. The length of administration of the growth factor was reduced to the shortest possible time according to the leukocyte count monitoring. Leucomax administration was started when leukocytes dropped below 1.10(9), in patients with severe leukopenias after repeated chemotherapy Leucomax was applied earlier. From 17 patients treated for sufficiently long period of time 70% responded to the GM-CSF application without any serious side-effects. In most cases, with the exception of myeloma, the growth factor was applied for 4-10 days. The leukocyte increase above 3 x 10(9) appeared in 3-8 days after the start of the treatment. CONCLUSIONS: Correctly indicated application of growth factors in postcytostatic myelosuppression is a great contribution to antitumor therapy. According to our experience in most cases 5-7 days of the daily dose of 150-200 micrograms of GM-CSF is sufficient for the stimulation of leukopoiesis. In postherapeutic myelosuppression in acute myeloblastic leukemia the growth factor should be used only as a life saving therapy since it can stimulate the leukemic cell population.

[Lymphocyte subpopulations determined by flow cytometry in chronic type B lymphatic leukemia]. / Zastoupení lymfocytárních subpopulací u chronické lymfatické leukémie typu B pri vysetrení prutokovou cytometrií.

BACKGROUND: For examination of surface characteristics of lymphocytes automatic analyzers of flow cytometry are used which make it possible to assess labelling with 2-3 monoclonal antibodies on one examined cell and provide thus more accurate data on the immunophenotype of proliferating cells. The objective of the present work was-using the method of flow cytometry and monoclonal antibodies-to detect surface signs on lymphocytes in the peripheral blood stream of patients with lymphatic leukaemia. METHODS AND RESULTS: In 29 patients with chronic lymphatic leukaemia type B (B-CLL) CD signs were examined, using flow cytometry and the findings were compared with those obtained in a control group of 63 healthy subjects. Evidence of CD 19+ CD5+ lymphocytes (B1 cells) in B-CLL (83.4 +/- 15% vs. 3.5 +/- 1.9% in controls) was of diagnostic importance. These CD5+ B cells are polyreactive, "memory B" lymphocytes which contrary to "conventional" lymphocytes B2 (CD5-B) do not differentiate further to plasma cells. Accumulation of B1 (CD5+ B) lymphocytes was, on the other hand, associated in patients with B-CLL with a decrease of B2 (CD5-B) lymphocytes (2.8 +/- 4.0% vs 9.0 +/- 4.2%) (p < 0.01). This finding can in case of excess of B1 (CD5+ B) lymphocytes explain the tendency of patients with B-CLL to develop autoimmune complications (e.g. AIHA); and conversely a decrease of B2 (CD5-B) lymphocytes can lead to hypogammaglobulinaemia which is also associated with B-CLL. A tendency towards autoimmunity may be also promoted by a decline of so-called inductors of suppressor cells (CD 4+ CD45RA); in patients with B-CLL among CD4 T lymphocytes 32.5 +/- 15% CD 45RA+ cells were found, as compared to 43.0 +/- 14.8% in controls (p < 0.011) and a lower ratio of true suppressor T cells (CD8+ CD11b+) in patients with B-CLL: 47.0 +/- 17.6% CD11b+ from CD8+ cells, as compared with controls 80.4 +/- 13.3 CD11b from CD 8+ cells (p < 0.01). CONCLUSIONS: The method of flow cytometry with double fluorescence thus contributes to the accurate diagnosis of B-CLL, to monitoring of the therapeutic effect but also to knowledge of the immunopathology of the disease.

Problems with prognostic criteria in patients with chronic lymphocytic leukemia with special reference to DPP II assessment.

106 consecutive patients with chronic lymphocytic leukemia (CLL) were entered into prospective study with the aims of finding out whether a detailed clinical, biochemical, immunological and cytochemical examination might help to subclassify CLL with respect to prognosis. None of the biochemical and immunological parameters studied correlated either with the clinical stage of the disease according to Rai, or with further evolution of the disease. On the other hand, the results of cytochemical tests indicated that the reaction for DPP II (dipeptidylaminopeptidase) might be of prognostic value, because in patients with stable disease, significantly lower number of DPP II positive lymphocytes were found when compared with patients suffering from progressive disease. The authors also point out some short-comings of Rai's staging system in respect to its prognostic significance.