Jan Stritesky

Introduction. Fli1 (Friend leukemia virus integration 1) together with other transcription factors induces the megakaryocytic differentiation of MEP (megakarycytic and erythroid progenitor). Refractory anemia and thrombocythemia is typical for 5q- syndrome. We found increased mRNA level of Fli1 in mononuclear bone marrow cells of 5q- syndrome patients in comparison with healthy controls (Neuwirtova et al., Ann Hematol 2013). The reason of the elevated Fli1 in 5q- syndrome is haploinsufficiency of microRNA-145, which targets Fli1 mRNA (Kumar et al., Blood 2011). Due to haploinsufficiency of RPS14 in 5q- syndrome non-consumed ribosomal proteins cause ribosomal stress and inactivate HDM2 in erythroblasts. E3 ubiquitin ligase HDM2 regulates p53 level by p53 degradation in proteasome. Inactivated HDM2 in erythroid precursors of 5q- syndrome leads to apoptosis of erythroblasts and to anemia. Why ribosomal stress does not cause thrombocytopenia and ineffective megakaryopoiesis as well? Our previous results support significant role of Fli1 in this process. Fli1 binds to promoter of the HDM2 gene and increases its transcription (Truong et al., Oncogene 2005). The increased activity of HDM2 in megakaryocytes inspite of ribosomal stress maintains p53 regulation and its degradation in proteasome. Megakaryopoiesis remains effective. Why it is not the case in erythroid precursors? To answer this question it was necessary to detect Fli1 as the protein and to determine in which cells Fli1 is present. Material and Methods. Twenty-three control representative bone marrow trephine biopsies of patients from controls (8 negative staging biopsies in lymphoma) and of patients with various hematological diagnoses (7 MDS with normal chromosome 5, 4 MPN, 3 AML and 1 RARS-T) and from 15 patients with 5q- syndrome were examined. In 13 patients with 5q- syndrome, samples taken before and 6 months after lenalidomide (Revlimid) therapy were available. The expression of Fli1 protein was investigated by immunohistochemistry (IHC). Expression of Fli1 on erythroid precursors was studied by double staining IHC procedure utilizing antibodies against Fli1 and either glycophorin A or E-cadherin known as reliable markers for erythroid precursors. Results. Nuclear expression of Fli1 was demonstrated in normal as well as in dysplastic megakaryocytes, in most cells of granulocytic series and lymphocytes. No staining for Fli1 was seen in erythroblasts and proerythroblasts visualized by expression of either glycophorin A or E-cadherin both in 5q- syndrome and controls. There were no significant differences in Fli1 expression between samples taken before and after lenalidomide treatment.The used IHC technique does not permit quantitative analysis of Fli1 protein levels. This fact could explain why we did not find any difference in Fli1 protein labeling in megakaryocytes before and after lenalidomide treatment while Fli1 mRNA level was decreased in majority of 5q- syndrome patients after...

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of a metabolic syndrome. To date, liver biopsy has been the gold standard used to differentiate between simple steatosis and steatohepatitis/fibrosis. Our aim was to compare the relevance of serum non-invasive parameters and scoring systems in the staging of liver fibrosis and non-alcoholic steatohepatitis (NASH) in patients with NAFLD. A total of 112 consecutive patients diagnosed with NAFLD were included. A liver biopsy was performed on 56 patients. The Kleiner score was used for the staging and grading of the histology. Non-invasive parameters for fibrosis (hyaluronic acid; AST/ALT; fibrosis scoring indexes OELF, ELF, BARD score, APRI, NAFLD fibrosis score); and inflammation (M30 and M65 cytokeratin-18 fragments) were measured and calculated. The same analyses were performed in 56 patients diagnosed with NAFLD, who were not indicated for liver biopsy. Based on the liver histology, NASH was diagnosed in 38 patients; simple steatosis in 18 patients. A cut-off value of 750 U/L of serum M65 discriminated patients with and without NASH with a 80% sensitivity and 82% specificity (95% CI:57–95). Fibrosis stage F0–F2 was present in 39 patients; F3–F4 in 17 patients. Serum concentrations of hyaluronic acid were higher in patients with advanced fibrosis (p<0.01); a cut-off value of 25 µg/l discriminated patients with F3–F4 with a 90% sensitivity and 84% specificity from those with F0–F2 (95% CI:59–99). When applying the non-invasive criteria to those patients without a liver biopsy, NASH could only be diagnosed in 16%; however, advanced fibrosis could be diagnosed in 35% of them. In patients with NAFLD, non-invasive serum parameters with a high accuracy can differentiate those patients with NASH and/or advanced fibrosis from those with simple steatosis. A substantial portion of those patients not indicated for liver biopsy might have undiagnosed advanced fibrosis.

The clinical course and therapy of mantle cell lymphoma (MCL) are heterogeneous and often unsatisfactory. Prognostic factors are needed to stratify the patients. Microvessel density (MVD) has prognostic significance in some malignancies. There is little information about the vasculature of MCL, although some antiangiogenic drugs are in use. We studied MVD using systematic uniform random sampling and unbiased counting frames in immunohistochemical reactions with anti-CD34 antibody in pre-therapeutic extramedullary MCL samples of 177 patients. We analyzed the relationship of MVD to overall survival (OS) and progression-free survival (PFS), as well as to proliferative activity (Ki-67), mantle cell lymphoma prognostic index (MIPI), morphological variant, pattern of growth, and localization. MVD varied widely: range 54.6–503.6 vessels/mm², median 158.2 vessels/mm². Higher MVD was associated with bone marrow infiltration at the time of diagnosis (P = 0.001). High MVD was associated with significantly worse OS (P = 0.04) only in patients treated with non-intensive (conventional) therapy. MVD correlated positively with MIPI scores but not with the proliferation, morphological variant, growth pattern, or localization. Univariate analysis identified a prognostic influence of morphological variant, MIPI, and proliferative activity on OS and PFS and a prognostic influence of bone marrow infiltration at the time of diagnosis on PFS. Multivariate analysis showed prognostic influence of MIPI and proliferative activity on OS and PFS only. In conclusion, this is the first clinicopathological study of MVD of MCL with long-term follow-up showing negative prognostic trends of high MVD in MCL and positive correlation of MVD and MIPI.

Introduction: Friend leukemia virus integration 1 (Fli1) is one of the factors involved in the differentiation of megakaryocytic and erythroid progenitor (MEP). Fli1 plays an important role in the development of megakaryocytes (MGC) (Doré and Crispino 2011). We demonstrated the increased level of Fli1 mRNA in bone marrow mononuclear cells of MDS patients (pts) with 5q- syndrome (Ann Hematol 2013). Kumar et al. (2011) explained the Fli1 increase as a consequence of miR-145 haploinsufficiency in 5q- syndrome. Fli1 is one of the target genes of miR-145. Our presumption is that the interaction between Fli1, MDM2 and p53 is important for the magakaryopoiesis in 5q- syndrome. Truong et al. (2005) demonstrated that Fli1 activates E3-ubiquitin ligase MDM2, which plays an important role in p53 degradation in proteasome. Therefore Fli1 probably protects MGC against ribosomal stress contrary to erythroid cells in 5q- syndrome pts. However, Fli1 and p53 mRNA levels, analysed by us in these patients need not mirror protein levels, therefore we decided to analyse Fli1 and p53 protein levels by imunohistochemistry with monoclonal antibodies. Patients and methods: We examined bone marrow histological specimens from 21 5q- syndrome pts, 14 MDS low risk pts with normal chromosome 5 (non5q) and 10 normal bone marrow controls. Fli1 was examined with rabbit monoclonal antibody (clone BV4-Diagnostic BioSystems), p53 with mouse monoclonal antibody clone DO-1, Immunotech). Detection system used was N-Histofine (Nichirei Bio). In five 5q- syndrome pts immunohistochemistry was repeated after lenalidomide treatment. In 2 pts p53 mutation was assessed by next-generation sequencing (GS Junior, Roche). Fli1 and p53 mRNA levels in total mononuclear cell RNA were determined by TaqMan-based quantitative real-time polymerase chain reaction (PCR). Eight 5q- syndrome pts were treated with lenalidomide, bone marrow was reexamined in five pts after the treatment in six pts. Results: We did not find any difference of Fli1 positive MGC, nor in the intensity of Fli1 in nucleai staining (42 - 94 % of Fli1 positive MGC) in both, MDS groups and in controls. p53 was in the majority of MGC negative or only weakly positive (in less than 20% of MGC). In order to quantify p53 staining in other types of mononuclear cells beside MGC, double staining is needed to identify the relevant (erythroid?) type of cells. However, there was qualitatively significant difference of p53 positivity, which was strong in cells of 5q- syndrome pts, weak in non5q MDS pts and very weak in controls. In four successfully treated pts with lenalidomide 20%-50% decrease of Flil positive MGC and marked reduction of p53 positivity in other cells was observed. One patient responded temporarily to lenalidomid, but in 15 months she transformed to RAEB II. In this patient two p53 mutations [p.G245S (23.9%) /p.F134L (3,5%)] were found. In our group of 5q- syndrome pts another woman had p53 mutation [V173M (49.6%)]. She transformed after 3 years in RAEB II. Both pts express strong positivity of p53 staining in non-MGC cells regardless of being now treated by azacitidine. Concerning the comparison of our findings of Fli mRNA and of p53 mRNA levels in mononuclear bone marrow cells with immunohistochemical data, we found partial agreement only in the case of p53. We did not confirm the increase of Fli1mRNA level in bone marrow mononuclear cells (3.5 x in comparison to non5q MDS and controls) of 5q- syndrome pts. Nevertheless, our presumption of the protective Fli1 function against MGC apoptosis remains focus of our further study. Conclusion: Contrary to our findings of the increased Fli1 mRNA level in mononuclear bone marrow cells of 5q- syndrome pts, the protein Fli1 levels did not differ from controls either in 5q- syndrome or in non5q MDS pts. p53 as the protein was negative or weakly positive in MGC in all pts and controls. The protein p53 was weakly positive in mononuclear bone marrow ( erythroid?) cells in controls, slightly positive in non5q MDS pts and differently strong positive in 5q- syndrome pts. The strongest p53 positivity was observed in two 5q- syndrome pts with mutated TP53 clone. Lenalidomide treatment of 5q- syndrome patients substantially decreased both Fli1 and p53 protein levels. In purpose to specify the type of bone marrow mononuclear cells we introduce double staining of these cells. Supported by the research grant NT/13836-4/2012 from MH CR and by PRVOUK-P27/LF1/2 Disclosures: No relevant conflicts of interest to declare.

The Hans algorithm failed to predict outcome in patients with diffuse large B-cell lymphoma treated with rituximab

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common tissue specimen available after microscopic examination. Molecular methods, such as polymerase chain reaction (PCR) and gene expression examination, serve as a source of diagnostic and prognostic information but require high-quality RNA. However, the increasing application of RNA extracted from FFPE tissue frequently results in very small and degraded quantities of nucleic acid. This study targets gene expression analysis from FFPE specimens using real-time quantitative PCR. The whole protocol consists of several steps, that is, RNA extraction and its quality control, reverse transcription, and fluorescence detection during real-time quantitative PCR. We compared several methods in each step, chose the most effective, and with that combination we successfully examined 95% (62 from 65) FFPE samples for our genes of interest. We reached the best results with RNA isolation by using a commercial kit, carefully interpreted UV spectrophotometric values, and meticulously chose reverse transcriptase and TaqMan fluorescence detection. Our protocol improves the utility of FFPE tissue for molecular profiling studies.

Background Iron accumulation as seen in genetic haemochromatosis is a major cause of hepatic fibrogenesis. A link between chronic liver disease and Dupuytren's disease (DD) is well established, especially in alcoholics.Aim  The aim of the present study was to test the hypothesis that iron accumulation might cause fibrosis of the palmar aponeurosis leading to DD.Patients and methods  We examined iron metabolism, mutations of the HFE gene, serum cholesterol, alcohol consumption, presence of chronic liver disease, diabetes and history of severe manual work in a group of 90 patients who had undergone surgery for a severe form of DD. The tissue removed during surgery was histologically examined to confirm the diagnosis of DD. For a control group, we used 33 healthy subjects with similar profiles.Results  The DD group consisted of 82 men and 8 women. Chronic liver disease was found in 27% of DD patients, compared with 6.1% of control subjects (P = 0.013). A history of hand traumatization was present in 33% of DD patients vs. 15% of control subjects (P = 0.048). Excessive alcohol consumption was present in 35.5% of DD patients compared with 15.1% of controls (P = 0.029). None of the other tested parameters, including the prevalence of HFE gene mutations, showed a significant difference between the two groups.Conclusions  Iron accumulation does not play a major role in the pathogenesis of DD. However, sex, age, manual labour and alcohol consumption are risk factors for progression of DD. We observed a high incidence of chronic liver disease in patients with DD.