[Malignant Melanoma - from Classical Histology towards Molecular Genetic Testing]. / Maligní melanom - od klasické histologie k molekulárne genetickému testování.

BACKGROUND: Malignant melanoma is - in comparison with other skin tumors - a relatively rare malignant neoplasm with highly aggressive biologic behavior and variable prognosis. Recent data in pathology and molecular diagnostics indicate that malignant melanoma is in fact not a single entity but a group of different neoplasms with variable etiopathogenesis, biologic behavior and prognosis. New therapeutic options using targeted treatment blocking MAPK signaling pathway require testing of BRAF gene mutation status. This helps to select patients with highest probability of benefit from this treatment. AIM: This article summarizes information on the correlation of morphological findings with genetic changes, discusses the representation of individual genetic types in various morphological subgroups and deals with the newly proposed genetic classification of melanoma and the current possibilities, pitfalls and challenges in BRAF testing of malignant melanoma. It also describes the current testing situation in the Czech Republic - the methods used, the representation of BRAF mutations in the tested population and the future of testing. It also shows the limitations of the BRAF and MEK targeted treatment concept resulting from the heterogeneity of the tumor population. Mechanisms of acquired resistance to MAPK pathway inhibitors, possibilities of their detection, and issues of combination of targeted therapy and immunotherapy are discussed.Key words: malignant melanoma - BRAF - mutation - molecular targeted therapy - tumor microenvironment - tumor heterogeneity This work was supported by projects PROGRES Q40/11, BBMRICZ LM2015089, SVV 260398 and GACR 17-10331S. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 28. 3. 2017Accepted: 16. 5. 2017.

Gastrointestinal stromal tumors - quantitative detection of the Ki-67, TPX2, TOP2A, and hTERT telomerase subunit mRNA levels to determine proliferation activity and a potential for aggressive biological behavior.

Gastrointestinal stromal tumors (GISTs) have an unpredictable biological potential ranging from benign to malignant. Molecular markers involved in the mechanisms of proliferation and cellular senescence may provide additional information about biological behavior of the tumor. The aim of the present study was to investigate Ki-67, TPX2, TOP2A and hTERT mRNA expression levels in specimens from patients with GISTs to define relationships between proliferation activity and biological potential and progression of the disease. We measured Ki-67, TPX2, TOP2A and hTERT mRNA levels using quantitative real-time reverse transcription PCR (RQ RT PCR). The highest Ki-67, TPX2, TOP2A and hTERT mRNA expression levels were found in the highly proliferative BLs (18 specimens), in comparison with GISTs (137 specimens) and LMSs (9 specimens). Patients with GISTs and adequate information about mitotic activity, tumor size and anatomical site (84 specimens) were divided into two groups - GISTs with benign (29 patients) and with malignant (55 patients) potential. We observed association between higher Ki-67, TPX2 and hTERT mRNA levels and the GISTs with malignant potential. Univariate analysis (57 patients with available follow-up information) of survival (Kaplan Meier curves method) revealed a correlation between higher levels of TPX2, Ki-67 and hTERT markers and shorter event-free survival (EFS) or poorer overall survival (OS). The results demonstrate the importance of quantitative assessment of the proliferation activity in GISTs. Proliferation markers of Ki-67, TPX2, TOP2A and hTERT are suitable markers for detection the proliferation activity and telomerase activity of these tumors. Furthermore, the assessment of TPX2, Ki-67 and hTERT expression levels is appropriate for determination of malignant potential of GISTs.

[Histiocytic necrotizing lymphadenitis / Kikuchi-Fujimoto disease (HNL/K-F) and its differential diagnosis: analysis of 19 patients]. / Histiocytární nekrotizující lymfadenitida /Kikuchiho-Fujimotova choroba (HNL/K-F) a její diferenciální diagnostika: analýza 19 prípadu

Histiocytic necrotizing lymphadenitis / Kikuchi-Fujimoto disease (HNL/K-F) is being recognized with an increasing frequency not only in the East Asia but also on the American continents and in the Europe. Still the diagnostics of HNL/K-F is not easy and difficulties with its proper classification persist. In a group of 19 patients diagnosed primarily or as consults at our department there were 12 woman and 7 men. An average age at diagnosis was 28 years, median 25 years. Cervical lymph nodes were involved in 18 patients. Bilateral lymphadenopathy was present in one patient, the remaining 17 were unilateral. Inguinal lymph node was affected in one patient. In one other patient there were enlarged retroperitoneal lymph nodes simultaneously with a cervical lymphadenopathy. The size of the lymph nodes varied between 5 mm to 32 mm. The subclassification showed the necrotizing type in 14 patients, in one there was a predominant xanthomatous tissue reaction around the necrotic areas (xanthomatous type), and in 4 patients the disease was recognized as the proliferative type without necrosis (in two with a variously intense apoptosis of the proliferating lymphocytes). Of 10 consult cases the tumor was primarily evaluated as B cell lymphoma not otherwise specified (1x), peripheral T cell lymphoma (1x), classical Hodgkin lymphoma of mixed cellularity (1x); two patients were submitted with a differential diagnosis between peripheral T cell lymphoma and HNL/K-F; in one diagnosis of probable EBV lymphadenitis and in one diagnosis HNL/K-F was made. There were no data submitted in the remaining three cases. The authors stress diagnostic features which should lead to the diagnosis of the disease and should prevent unnecessary oncological staging investigations and potential chemotherapy for a lymphoma. Among diagnostic features of HNL/K-F identification of the proliferating cells - CD8 activated lymphocytes with apoptotic decay prevail, there are frequent plasmacytoid monocytes and a striking reaction of macrophages which are CD68/myeloperoxidase positive. There are virtually no neutrophil granulocytes and there is a miminal participation of plasma cells. In case of necrotizing and xanthomatous type infectious causes are to be ruled out as well. In case we still need to distinguish HNL/K-F from a lymphoma PCR analysis of a rearrangement of the immunoreceptor gene in T cell population should be investigated.

Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: a European multicenter study.

A standardized, sensitive and universal method for minimal residual disease (MRD) detection in acute myeloid leukemia (AML) is still pending. Although hyperexpression of Wilms' tumor (WT1) gene transcript has been frequently proposed as an MRD marker in AML, wide comparability of the various methods used for evaluating WT1 expression has not been given. We established and standardized a multicenter approach for quantifying WT1 expression by quantitative reverse transcriptase PCR (qRT-PCR), on the basis of a primer/probe set combination at exons 6 and 7. In a series of quality-control rounds, we analyzed 69 childhood AML samples and 47 normal bone marrow (BM) samples from 4 participating centers. Differences in the individual WT1 expressions levels ranged within <0.5 log of the mean in 82% of the cases. In AML samples, the median WT1/1E+04 Abelson (ABL) expression was 3.5E+03 compared with that of 2.3E+01 in healthy BM samples. As 11.5% of childhood AML samples in this cohort harbored WT1 mutations in exon 7, the effect of mutations on WT1 expression has been investigated, showing that mutated cases expressed significantly higher WT1 levels than wild-type cases. Hence, our approach showed high reproducibility and applicability, even in patients with WT1 mutations; therefore, it can be widely used for the quantitation of WT1 expression in future clinical trials.

Wilms' tumor gene 1 (WT1) expression in childhood acute lymphoblastic leukemia: a wide range of WT1 expression levels, its impact on prognosis and minimal residual disease monitoring.

Wilms' tumor gene 1 (WT1) is overexpressed in the majority (70-90%) of acute leukemias and has been identified as an independent adverse prognostic factor, a convenient minimal residual disease (MRD) marker and potential therapeutic target in acute leukemia. We examined WT1 expression patterns in childhood acute lymphoblastic leukemia (ALL), where its clinical implication remains unclear. Using a real-time quantitative PCR designed according to Europe Against Cancer Program recommendations, we evaluated WT1 expression in 125 consecutively enrolled patients with childhood ALL (106 BCP-ALL, 19 T-ALL) and compared it with physiologic WT1 expression in normal and regenerating bone marrow (BM). In childhood B-cell precursor (BCP)-ALL, we detected a wide range of WT1 levels (5 logs) with a median WT1 expression close to that of normal BM. WT1 expression in childhood T-ALL was significantly higher than in BCP-ALL (P<0.001). Patients with MLL-AF4 translocation showed high WT1 overexpression (P<0.01) compared to patients with other or no chromosomal aberrations. Older children (> or =10 years) expressed higher WT1 levels than children under 10 years of age (P<0.001), while there was no difference in WT1 expression in patients with peripheral blood leukocyte count (WBC) > or =50 x 10(9)/l and lower. Analysis of relapsed cases (14/125) indicated that an abnormal increase or decrease in WT1 expression was associated with a significantly increased risk of relapse (P=0.0006), and this prognostic impact of WT1 was independent of other main risk factors (P=0.0012). In summary, our study suggests that WT1 expression in childhood ALL is very variable and much lower than in AML or adult ALL. WT1, thus, will not be a useful marker for MRD detection in childhood ALL, however, it does represent a potential independent risk factor in childhood ALL. Interestingly, a proportion of childhood ALL patients express WT1 at levels below the normal physiological BM WT1 expression, and this reduced WT1 expression appears to be associated with a higher risk of relapse.

Upregulation of asparagine synthetase fails to avert cell cycle arrest induced by L-asparaginase in TEL/AML1-positive leukaemic cells.

L-Asparaginase is a standard component in chemotherapy of childhood acute lymphoblastic leukaemia (ALL). Leukaemic cells carrying TEL/AML1 fusion gene are more sensitive to treatment with L-asparaginase compared to other subtypes of ALL. We demonstrate in vitro the prolonged growth suppression of TEL/AML1[+] cells compared to TEL/AML1[-] leukaemic cells after L-asparaginase treatment simulating treatment protocol. Cell cycle analysis revealed TEL/AML1[+] cells to accumulate in G1/G0 phase (81-98%) compared to TEL/AML1[-] cells (47-60%). Quantitative analysis of asparagine synthetase (AsnS) expression showed the ability of TEL/AML1[+] cells to increase AsnS mRNA levels after L-asparaginase treatment to the same extent as TEL/AML1[-] leukaemic and nonleukaemic lymphoid cells. We hypothesise that TEL/AML1[+] cells are unable to progress into the S phase of cell cycle under nutrition stress caused by L-asparaginase, despite the ability of AsnS upregulation. Significantly higher expression of AsnS was found in untreated leukaemic cells from children with TEL/AML1[+] ALL (n=20) in comparison with the group of age-matched children with ALL bearing no known fusion gene (n=25; P=0.0043). Interestingly, none of the TEL/AML1[+] patients with high AsnS level relapsed, whereas 10/15 patients with AsnS below median relapsed (P=0.00028). Therefore, high AsnS levels in TEL/AML1[+] patients correlate with better prognosis, possibly reflecting the stretched metabolic demand of the lymphoblast.

[Follicular lymphomas: molecular diagnosis using t(14;18)(q32;q21)--fluorescence in situ hybridization, qualitative and quantitative PCR]. / Folikulární lymfomy: molekulární diagnostika t(14;18)(q32;q21)--fluorescencní in situ hybridizace, kvalitativní a kvantitativní PCR.

Diagnosis of follicular lymphoma (FL) is based on histology and immunohistochemical profile (CD20+, CD79alfa+, CD10+, BCL-2+, CD5-). A chromosomal marker--translocation t(14;18)(q32;q21) supporting the tumor diagnosis and useful for monitoring bone marrow or peripheral blood infiltration by the tumor cells is also used. The BCL2 gene (18q21) is controlled by an enhancer of the IGH gene (14q32) resulting in BCL-2 protein overexpression. The translocation is present in the majority of patients with FL. The aim of the study was to introduce the quantitative PCR (RQ-PCR, real-time quantification) method for the assessment of the quantity of cells bearing the translocation t(14;18) in patients with FL. The fluorescence in situ hybridization on interphasic nuclei (I-FISH) in histologic sections was used for screening of patients with the t(14;18). A search for the break of the BCL2 gene at the major breakpoint region (mbr) was performed by means of qualitative PCR. We determined the relative number of the tumor cells bearing t(14;18) translocation (mbr) in patients with FL by the RQ-PCR. The relative quantity of these cells was significantly higher in the lymph nodes than in the bone marrow or peripheral blood. The RQ-PCR is a tool of choice to monitor the activity of the disease in individual patients, and to detect an early disease relapse before its manifestation at the level diagnosed by morphology.

Real-time quantitative PCR detection of WT1 gene expression in children with AML: prognostic significance, correlation with disease status and residual disease detection by flow cytometry.

The clinical significance of WT1 gene expression at diagnosis and during therapy of AML has not yet been resolved. We analysed WT1 expression at presentation in an unselected group of 47 childhood AML patients using real-time quantitative reverse-transcription PCR. We also showed that within the first 30 h following aspiration RQ-RT-PCR results were not influenced by transportation time. We observed lower levels of WT1 transcript in AML M5 (P = 0.0015); no association was found between expression levels and sex, initial leukocyte count and karyotype-based prognostic groups. There was significant correlation between very low WT1 expression at presentation and excellent outcome (EFS P = 0.0014). Combined analysis of WT1 levels, three-colour flow cytometry residual disease detection and the course of the disease in 222 samples from 28 children with AML showed remarkable correlation. Fourteen patients expressed high WT1 levels at presentation. In eight of them, who suffered relapse or did not reach complete remission, dynamics of WT1 levels clearly correlated with the disease status and residual disease by flow cytometry. We conclude that very low WT1 levels at presentation represent a good prognostic factor and that RQ-RT-PCR-based analysis of WT1 expression is a promising and rapid approach for monitoring of MRD in approximately half of paediatric AML patients.

[Monitoring minimal residual disease in pediatric patients with acute lymphoblastic leukemia]. / Sledování minimální reziduální nemoci u detských pacientu s akutní lymfoblastickou leukémií.

The level of minimal residual disease is an important prognostic factor in childhood acute lymphoblastic leukaemia. The end of induction therapy is the most significant time-point for prediction of treatment outcome. Within a pilot study covered by the Paediatric Haematology Working Group in the Czech Republic 51 childhood patients were analysed at diagnosis of acute lymphoblastic leukaemia and at the end of induction using method based on detection of clonal rearrangements of immuno-receptor genes. The majority of tested patients (32/51, 63%) had a low or non-detectable levels of residual disease, a group of patients with the highest levels and thus the highest risk of relapse included 10% of patients (5/51). Within each of three risk groups one patient has relapsed so far. Therefore, the relapse rate in particular subgroups is 3% (1/32), 7% (1/14) and 20% (1/5) to date, respectively. The results are compared with these published by the BFM group (van Dongen et al., Lancet 1998). The pilot phase of a new BFM treatment protocols includes examination of residual disease for stratification of patients into the different risk groups.

TEL/AML1 positivity in childhood ALL: average or better prognosis? Czech Paediatric Haematology Working Group.

The presence of TEL/AML1 fusion gene in childhood acute lymphoblastic leukaemia (ALL) defines a subgroup of patients with better than average outcome. However, the prognostic significance of this aberration has recently been disputed by the Berlin-Frankfurt-Münster (BFM) study group due to its relatively high incidence found in relapsed patients (19.6% and 21.9%, in two cohorts). In contrast, only four out of 45 (8.9%) unselected relapsed patients (all of whom had been treated according to BFM protocols) in the Czech Republic carry this fusion. From March 1995 to June 1998, 41 out of 190 (21.6%) newly diagnosed children with ALL were TEL/AML1-positive. There is a statistically significant difference between the incidence of TEL/AML1 fusion at diagnosis and at relapse within our group (P = 0.035). Interim analysis of the minimal residual disease (MRD) detection shows heterogeneity within the group of newly diagnosed TEL/AML1-positive leukaemias--10 out of 24 patients tested at the end of induction therapy had detectable levels of MRD. However, only one of these patients reached relapse-predictive level (10(-3)) of MRD. In conclusion, we corroborate low frequency of TEL/AML1 positivity among relapsed patients with ALL among Czech children who are treated by the BFM protocols. Moreover, we demonstrate different patterns of bone marrow clean-up in TEL/AML1-positive patients.

[Detection of BCR/ABL, MLL/AF4 and TEL/AML1 hybrid genes and monitoring of minimal residual disease in pediatric patients with acute lymphoblastic leukemia]. / Detekce hybridních genu BCR/ABL, MLL/AF4 a TEL/AML1 a sledování minimální reziduální nemoci u detských pacientu s akutní lymfoblastickou leukémií.

BACKGROUND: The BCR/ABL and MLL/AF4 fusion genes--resulting from t(9;22)(q34;q11) and t(4;11)(q21;q23) translocations, respectively--are considered as a high risk prognostic factors in children with acute lymphoblastic leukaemia (ALL). Their presence in malignant cells indicates patient for the most intensive antileukaemic therapy regardless of the other criteria. In contrast, the most common non-random chromosomal aberration in paediatric ALL--translocation t(12;21)(q12;q22)--is associated with a favourable prognosis. The examination of these rearrangements is important for the stratification of patients to the risk groups and also provides the most sensitive and specific tool for minimal residual disease (MRD) follow-up. METHODS AND RESULTS: This study comprises 241 patients with ALL from Czech and Slovak Republics younger than 18 years at diagnosis. They were examined for presence of m-RNA of fusion genes BCR/ABL, MLL/AF4 and TEL/AML1 by reverse transcriptase-polymerase chain reaction (RT-PCR) method. Seven out of 197 (3.6%) carried MLL/AF4 fusion gene, but among infants it was 56% (5 out of 9). BCR/ABL positivity was found in 2.5% (7 out of 240) and TEL/AML1 in 21.7% (41 out of 189) cases. Event free survival (EFS) curves demonstrate the clinical impact of these hybrid genes on patients' prognosis. Moreover, we present the possibility of the monitoring of MRD levels in follow-up samples of these patients. CONCLUSIONS: All particular rearrangements were found only in a cohort of patients with B-precursor ALL (or hybrid leukaemia), which constitutes 85% of our group. Presence of BCR/ABL or MLL/AF4 fusion gene is associated with poor prognosis and is indispensable condition for correct stratification of patients to the risk groups according to treatment protocols. Hybrid gene TEL/AML1 defines subgroup of children with better prognosis and due to its high frequency provides us with a very useful tool for MRD detection.

[Extracorporeal elimination of LDL-cholesterol in the treatment of hypercholesterolemia: indications and methods]. / Extrakorporální eliminace LDL-cholesterolu v lécbe hypercholesterolémií: indikace a metody.

Extracorporeal elimination of LDL-cholesterol is at present an important part of comprehensive treatment of patients with very high cholesterol levels. An absolute indication for their use are patients with the homozygous form of familial hypercholesterolaemia. Treatment is, after individual consideration, indicated also patient with severe heterozygous familial hypercholesterolaemia, with a positive family history of IHD, if it is not possible to reduce LDL-cholesterol by diet and hypolipidaemic agents below 5.2 mmol/l; also patients with severe IHD and severe hypercholesterolaemia, included in secondary prevention where it is not possible to reduce LDL-cholesterol by diet and pharmacotherapy below 3,4 mmol/l. Another indication for treatment by LDL apheresis are patients where cardiosurgery cannot be performed because of angiosclerosis. These are patients with severe hypercholesterolaemia which does not respond to drugs and with diffuse changes of the coronary circulation in young age, which cannot be treated by angioplasty or coronary bypass, and also patients after a coronary bypass with a refractory disorder of the lipid metabolism. LDL apheresis is furthermore indicated in patients with severe hyperlipidaemic crises which eventually develop into necrosis of the pancreas. Long-term LDL-apheresis leads to regression of manifestations of xanthomatosis of the skin and tendons, it prevents progression and starts regression of atherosclerosis in patients with severe hypercholesterolaemia. In homozygotes with familial hypercholesterolaemia treatment by LDL-apheresis leads to prolongation of life and improves the quality of life. In heterozygotes neither prolongation of the life span nor a lower incidence of IHD is observed, while the quality of life improves and regression of atherosclerosis occurs. A combination of LDL-apheresis, dietary provisions and hypolipidaemic treatment in heterozygotes is the most effective method to reduce the LDL-cholesterol level. Extracorporeal elimination of LDL-cholesterol can be done by non-selective centrifuging or membrane plasmapheresis. More recent methods of LDL-apheresis are more selective and effective. They use active columns or capsules to remove atherogenic particles from plasma. These methods include cascade filtration, immunoadsorption heparin-induced LDL precipitation, thermofiltration and dextran-induced LDL precipitation.

[The effect of carnitine on the early phase of liver regeneration after partial hepatectomy in rats with experimental diabetes]. / Ucinek karnitinu na casnou fázi regenerace jater po cástecné hepatektomii u potkanu s experimentálním diabetem.

The effect of intraperitoneally administered carnitine (10 or 100 mg/kg) on the early stage of liver regeneration after two-third hepatectomy was studied in rats with experimentally induced alloxan diabetes (60 mg/kg IV). The level of regeneration was assessed mainly on the basis of DNA synthesis in the liver. Untreated diabetic rats exhibited significantly lower values of DNA synthesis determined 24 hours after hepatectomy. Administration of carnitine resulted in a significant increase in DNA synthesis over the given time interval. The stimulatory effect of carnitine in diabetic rats was confirmed also by the recorded values of serum lipids, particularly by the decreased concentration of triacylglycerols and the increased concentration of HDL-cholesterol, as determined in carnitine treated diabetic rats 24 hours following partial hepatectomy. (Tab. 2, Fig. 3, Ref. 34.)