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Bevezetés: A perifériás vérben szabadon keringő tumoreredetű DNS-ek lehetővé teszik cholangiocarcinomákban a molekuláris genetikai eltérések tanulmányozását, akár a kemoterápiára adott válasz hatékony követését is. Célkitűzés: A liquid (’folyékony’) biopszia alkalmazása kedvező megoldás, hiszen a szövetinél sokkal egyszerűbben kivitelezhető, és elkerülhető az ismételt invazív szövettani mintavétel. A liquidbiopszia-alapú szekvenálás hatékonysága a tumor progressziójával és ezáltal a nagyobb mennyiségű szabad DNS felszabadulásával növekszik. Módszer: A jelen vizsgálatban klinikailag releváns pontmutációkat mutattunk ki epeúti tumorok mind szövettani, mind liquid biopsziás mintáiból. Eredmények: Újgenerációs szekvenálás alkalmazásával 33 betegből származó, szövettani és liquid biopszia során nyert DNS-mintákat analizáltunk 67 génes szolidtumor-panelt felhasználva. Megbeszélés: Dolgozatunkban egy minimálisan invazív vizsgálati megközelítést mutatunk be a cholangiocarcinoma és az epehólyag-daganat molekuláris genetikai eltéréseinek azonosítására. Következtetés: A szabad DNS diagnosztikai alkalmazása a tumorok térbeli heterogenitását tükrözi, ezáltal egy új megközelítés a precíziós onkológiai kezelésekben. Orv Hetil. 2022; 163(50): 1982–1991.

Background: Mutation of the TP53 gene is one of the major drivers of myelodysplastic neoplasias (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MR). TP53 mutations present in these hematopoietic malignancies form a distinct molecular genetic cluster with a worse prognosis than without the alteration. However, besides well-characterized hot-spot variants, a significant proportion of TP53 alterations are of uncertain clinical significance. Methods: To enlighten so far unknown aspects, bone-marrow samples from altogether 77 patients are analyzed retrospectively with the diagnosis of AML-MR (26 cases), MDS-IB (12 cases), and MDS-LB (39 cases) according to WHO 2022 guidelines. Next-generation sequencing results are correlated with histological, cytogenetic, and survival data. Results: Twenty out of the 30 TP53 mutation types detected by NGS are not categorized in current public databases; thus, their clinical significance remained mysterious. Because of the interpretation difficulties and the absence of clinical correlations, pathogenicity is established based on in silico approaches. The 12 pathogenicity classification systems, as well as protein stability, protein–DNA, protein–protein interaction, and post-translational modification analyses are applied. We found statistically significant differences between AML/MDS groups considering p53 pathogenicity, protein structural changes, and overall survival. The largest number of abnormalities with the most severe consequences are found in AML-MR cases. Conclusions: These molecular and in silico protein data further support that MDS with increased-blast (MDS-IB) is an intermediate group between AML-MR and MDS with low-blast (MDS-LB) patients, which frequently progresses to AML and is therefore considered a pre-leukemic condition.

Upon the discovery of frequent oncogenic histone alterations in paediatric diffuse high-grade gliomas, the epigenetic and transcriptional landscapes of tumours have become increasingly important aspects of diagnostic and prognostic analysis. The replacement of lysine 27 with methionine in H3 histone variants - H3 p.K28M (K27M) - was the first reported histone mutation associated with human malignancies, seen in up to 80% of paediatric diffuse midline gliomas. This discovery contributed to the updated 2021 World Health Organization (WHO) classification of central nervous system (CNS) tumours in which paediatric diffuse high-grade gliomas were classified into molecular-based categories. Therefore, molecular analysis of tumour cells has become increasingly necessary for determining disease prognosis and potential therapeutic strategies. Although detection of histone alterations is crucial for the diagnosis of specific glioma subtypes, several studies have identified them in other CNS tumours, which may be misleading during routine diagnostic work. While traditional biopsies remain the standard for diagnosis of gliomas, they pose a high risk for surgical complications and patient morbidity. Consequently, this review highlights the importance of the H3 K27-alterations in paediatric gliomas and several other CNS tumours. We also discuss the potential of liquid biopsies as a minimally invasive and highly effective alternative for confirming the diagnosis and potential targeted epigenetic therapies which may improve the survival of patients.

Tumor-derived free-circulating DNA in peripheral blood allows the study of the molecular genetic profile in cholangiocarcinomas and even the effective monitoring of the response to chemotherapy.The use of a liquid biopsy is a favourable solution, as repeated invasive histological sampling is much more practicable and avoidable. The efficiency of liquid biopsy-based sequencing increases with tumor progression and thus with the release of larger amounts of free DNA.In the present study, clinically relevant point mutations were detected from both histological and liquid biopsy specimens of bile duct tumors.During next-generation sequencing, histological and DNA samples obtained during liquid biopsy from 33 patients were analyzed using a 67-gene solid tumor panel.In our prospective study, we present a minimally invasive approach to identify molecular genetic changes in cholangiocarcinoma and gallbladder tumors.The diagnostic application of free DNA reflects the spatial heterogeneity of tumors, making it a new approach to precision oncology treatments. Orv Hetil. 2022; 163(50): 1982-1991.Bevezetés: A perifériás vérben szabadon keringő tumoreredetű DNS-ek lehetővé teszik cholangiocarcinomákban a molekuláris genetikai eltérések tanulmányozását, akár a kemoterápiára adott válasz hatékony követését is. Célkitűzés: A liquid (’folyékony’) biopszia alkalmazása kedvező megoldás, hiszen a szövetinél sokkal egyszerűbben kivitelezhető, és elkerülhető az ismételt invazív szövettani mintavétel. A liquidbiopszia-alapú szekvenálás hatékonysága a tumor progressziójával és ezáltal a nagyobb mennyiségű szabad DNS felszabadulásával növekszik. Módszer: A jelen vizsgálatban klinikailag releváns pontmutációkat mutattunk ki epeúti tumorok mind szövettani, mind liquid biopsziás mintáiból. Eredmények: Újgenerációs szekvenálás alkalmazásával 33 betegből származó, szövettani és liquid biopszia során nyert DNS-mintákat analizáltunk 67 génes szolidtumor-panelt felhasználva. Megbeszélés: Dolgozatunkban egy minimálisan invazív vizsgálati megközelítést mutatunk be a cholangiocarcinoma és az epehólyag-daganat molekuláris genetikai eltéréseinek azonosítására. Következtetés: A szabad DNS diagnosztikai alkalmazása a tumorok térbeli heterogenitását tükrözi, ezáltal egy új megközelítés a precíziós onkológiai kezelésekben. Orv Hetil. 2022; 163(50): 1982–1991.

Acquired von Willebrand syndrome (AVWS) is a rare, frequently underdiagnosed and underestimated bleeding disorder. Careful personal and family history and late-onset mucocutaneous bleeding could help clarify the etiology of bleeding deficiency.An 82-year-old male patient was admitted to our clinic with a severe nosebleed on 30.05.2018. Laboratory results revealed thrombocytosis, elevated white blood cell count and high LDH. Basic coagulation parameters were normal. He was referred to our clinic, where a bone marrow biopsy was taken. His personal and family history had no mention of bleeding disorders, nor was he on anticoagulant therapy. We detected elevated VWF antigen and decreased VWF ristocetin cofactor activity. Loss of high molecular weight multimers was detected by using agarose gel electrophoresis. These laboratory results were indicative of AVWS. Hydroxyurea treatment was initiated, leading to a gradual decrease in platelet count. The histological examination revealed essential thrombocytosist while mutation analysis was JAK2/CALR/MPL negative. However, due to severe nosebleeds, the patient was hospitalized and needed blood transfusion. A cardiological check-up revealed the progression of aortic valve stenosis. After, balloon-dilation a transcatheter aortic valve implantation was performed. As a result, VWF activity and activity to antigen ratio returned to normal as did multimeric structure. In July 2019, the follow-up examination showed that the patient was in a satisfactory condition, with normal hematological parameters, and no new nosebleed episode occurred.The patient complained of recurring nosebleeds, which stopped completely after the resolution of both underlying conditions successful cytoreductive treatment of triple-negative ET and transcatheteric aortic valve replacement.

Biliary tract cancer (BTC) is a rare malignancy with a long disease course and an overall poor prognosis. Despite multiple chemotherapy agents, there is no defined second-line treatment opportunity for advanced BTCs. In the era of precision oncology, NGS plays an important role in identifying mutations that may predict the molecular pathomechanism and manage the BTC therapy. The peripheral blood liquid biopsy (LB) of cancer patients represents variable amounts of cell-free DNA (cfDNA) released from tumor foci of any anatomical location. Our study aimed to identify somatic mutations and tumor variant burden (TVB) in cell-free and matched tumor DNA. We found a positive correlation between the estimated tumor volume and cfDNA yield (r = 0.9326, p < 0.0001). Comparing tissue and LB results, similar TVB was observed. SNVs were proven in 84% of the cases, while in two cases, only the LB sample was informative for molecular analysis. The most important aberrations in BTCs, such as FGFR2, IDH1, IDH2, KRAS, and TP53, could be detected in matched LB samples. Our prospective study demonstrates a minimally invasive testing approach to identify molecular genetic alterations in cholangiocarcinoma and gallbladder cancers. Clinical applications of cfDNA reflect by capturing the outstanding spatial tumor heterogeneity and guarantee novel aspects for the precision oncology treatment.

Simple Summary In the era of personalized oncology, next-generation sequencing plays an important role in identifying mutations that may predict the molecular pathomechanism and manage biliary tract cancers (BTC) therapy. The peripheral blood of cancer patients represents variable amounts of cell-free DNA (cfDNA) released from the tumor. Tumor-derived cfDNA in BTCs also allows the effective monitoring of the molecular genetic profile and the response to chemotherapy. Our study aimed to identify genetic aberrations in cell-free and matched tumor DNA in BTCs. We assume that the efficacy of the LB-based sequencing provides a novel perspective for BTCs therapy. Abstract Biliary tract cancer (BTC) is a rare malignancy with a long disease course and an overall poor prognosis. Despite multiple chemotherapy agents, there is no defined second-line treatment opportunity for advanced BTCs. In the era of precision oncology, NGS plays an important role in identifying mutations that may predict the molecular pathomechanism and manage the BTC therapy. The peripheral blood liquid biopsy (LB) of cancer patients represents variable amounts of cell-free DNA (cfDNA) released from tumor foci of any anatomical location. Our study aimed to identify somatic mutations and tumor variant burden (TVB) in cell-free and matched tumor DNA. We found a positive correlation between the estimated tumor volume and cfDNA yield (r = 0.9326, p < 0.0001). Comparing tissue and LB results, similar TVB was observed. SNVs were proven in 84% of the cases, while in two cases, only the LB sample was informative for molecular analysis. The most important aberrations in BTCs, such as FGFR2, IDH1, IDH2, KRAS, and TP53, could be detected in matched LB samples. Our prospective study demonstrates a minimally invasive testing approach to identify molecular genetic alterations in cholangiocarcinoma and gallbladder cancers. Clinical applications of cfDNA reflect by capturing the outstanding spatial tumor heterogeneity and guarantee novel aspects for the precision oncology treatment.

The World Health Organization classification recommends follicular lymphoma (FL) grading (G1-3) by considering centroblast number, while also suggesting its influence on disease outcome. As centroblast counting and other proliferation markers have limitations, we looked for more specific measures of cellular activity in FL. Phosphorylated histone H3 (pHH3) was widely applied for the objective detection of mitotic activity in different tumors. The aim was to evaluate the utility of pHH3 protein in FL grading and compare its value with the classical features of cell proliferation. Representative samples from 48 FL patients and 9 samples with follicular hyperplasia were examined. Hematoxylin-eosin-based mitosis index (HE-MI), number of mitotic figures based on anti-pHH3 immunohistochemical staining (pHH3-MI), and percentage of Ki-67-positive cells [proliferation index (PI)] were determined and compared with centroblast-based histologic grade. PHH3-MI showed significant correlation with HE-MI (r=0.85, P

LB

Apoptosis and the proper clearance of apoptotic cells play a central role in maintaining tissue homeostasis. Previous work in our laboratory has shown that when a high number of cells enters apoptosis in a tissue, the macrophages that engulf them produce retinoids to enhance their own phagocytic capacity by upregulating several phagocytic genes. Our data indicated that these retinoids might be dihydroretinoids, which are products of the retinol saturase (RetSat) pathway. In the present study, the efferocytosis of RetSat-null mice was investigated. We show that among the retinoid-sensitive phagocytic genes, only transglutaminase 2 responded in macrophages and in differentiating monocytes to dihydroretinol. Administration of dihydroretinol did not affect the expression of the tested genes differently between differentiating wild type and RetSat-null monocytes, despite the fact that the expression of RetSat was induced. However, in the absence of RetSat, the expression of numerous differentiation-related genes was altered. Among these, impaired production of MFG-E8, a protein that bridges apoptotic cells to the αvβ3/β5 integrin receptors of macrophages, resulted in impaired efferocytosis, very likely causing the development of mild autoimmunity in aged female mice. Our data indicate that RetSat affects monocyte/macrophage differentiation independently of its capability to produce dihydroretinol at this stage.

Upregulation of carbonic anhydrase IX (CAIX) was found to be associated with unfavorable prognosis and resistance to treatment in a broad spectrum of malignancies, recently also in classical Hodgkin&rsquo, s lymphoma (cHL). As demonstrated, variable CAIX expression in a significant number of cHL cases was associated with poor treatment response. The current study focused on the quantification CAIX immunopositivity and its relative expression compared to the total CD30+ neoplastic pool using digital image analysis. One hundred and one lymph node samples featuring cHL histology were analyzed for both CD30 and CAIX by immunohistochemistry. Whole histological slides were scanned and immunopositivity was determined as the histoscore (H-score) using the DensitoQuant software module (3DHistech Kft., Budapest, Hungary). CAIX positivity was observed in the HRS-cells of 56/101 cases (55.44%) and frequently observed in the proximity of necrotic foci. CAIX H-scores were highly variable (range: 2.16&ndash, 90.36, mean 18.7 &plusmn, 18.8). Individual CAIX values were independent of the much higher CD30 values (range 3.46&ndash, 151.3, mean 52.37 &plusmn, 30.74). The CAIX/CD30 index proved to be the highest in the aggressive lymphocyte-depleted (LD) subtype (CAIX/CD30: 0.876). The CAIX expression and the CAIX/CD30 relative index can be precisely determined by image analysis, and values reflect the extent of a tumor mass undergoing hypoxic-stress-related adaptation in the most aggressive forms of cHL.

Thymic epithelial tumors (TETs) are uncommon neoplasms of the mediastinum. The gold standard treatment is complete surgical resection which can be followed by radio/chemotherapy in selected cases. Targeted tyrosine kinase inhibition can be considered in only a limited number of aggressive or metastatic tumors as EGFR, BRAF, or c-kit mutations are rare. However, previous studies have demonstrated the efficacy of immune checkpoint inhibitors in epithelial neoplasias, such as in programmed cell death ligand 1 (PD-L1) expressing nonsmall cell lung carcinoma. Because of their rare occurrence the data on PD-L1 distribution in thymic neoplasias are limited. PD-L1 and PD-1 expression in tumor cells and tumor infiltrating immune cells was determined in TETs according to criteria published for lung carcinomas. Comparison with major clinical, pathologic, and biological features was also done. In total, 36 TETs (29 thymomas and 7 thymic carcinomas) were analyzed. PD-L1 immunohistochemical staining (Ventana PD-L1 clone SP142) was performed in all cases. The percentage of the positive tumor cells (TC value), the percentage of tumor area occupied by positive immune cells (IC value) was evaluated. Evaluation of PD-L1 expression in tumor cells showed a good reproducibility (κ-value: 0.840; Spearman r=0.966; P

AIMS The present study evaluates the impact of hypoxia-related carbonic anhydrase IX and XII isoenzyme expression as a basic adaptive mechanism to neutralise intracellular acidosis in classical Hodgkin's lymphoma (cHL). METHODS AND RESULTS Eighty-one primary biopsies and 15 relapsed tissue samples diagnosed with cHL were analysed for necrosis, CAIX and CAXII expression and cell proliferation to compare hypoxia-related histological and functional data with survival characteristics. Variable, but highly selective cell membrane CAIX expression could be demonstrated in Hodgkin-Reed-Sternberg (HRS) cells in 39 of 81 samples (48.1%), while virtually no staining presented in their microenvironment. In contrast, CAXII expression in HRS cells could be demonstrated in only 18 of 77 samples (23.4%), with significant stromal positivity (50 of 77, 64.9%). The CAIX+ positive phenotype was strongly associated with lymphocyte depletion (four of four, 100%) and nodular sclerosis (29 of 51, 56.9%) subtypes. CAIX/Ki-67 dual immunohistochemistry demonstrated suppressed cell proliferation in CAIX+ positive compared to CAIX- negative HRS cells (P

Moderate thrombocytosis can accompany several diseases (bleeding, inflammation, iron deficiency, or autoimmune diseases), but hematologic examination is strongly recommended in a patient with persistent platelet count above 450 G/L unless reactive origin can be confirmed. The 47-year-old woman's medical history included hypertonia, asthma bronchiale, and endometriosis. In March 2015, she underwent laboratory examination due to weight loss and lack of appetite. Her results showed elevated thrombocyte count (617 G/L), but no iron deficiency. She presented in our clinic on 07. 04. 2015 with acute pain below her left hypochondrial region, but simple imaging examinations showed no difference to explain it. Abdominal CT revealed a 4.5 cm thrombus which protruded into the left renal artery, blocking it. We started APTI- (activated partial thromboplastin time) monitored continuous intravenous treatment with unfractionated heparin. The JAK2V617F mutation analysis came back positive. Subsequent bone marrow examination revealed prefibrotic/early stage myelofibrosis, prompting treatment with hydroxyurea. The applied treatments led to the disappearance of the patient's symptoms accompanied by the gradual normalisation of the thrombocyte count. Moderate thrombocytosis is often secondary, but if it persists and is accompanied by mainly thromboembolic events, the risk of diseases of the haematopoietic system, primarily Philadelphia chromosome negative chronic myeloproliferative disease should also be considered. Clinically, essential thrombocythaemia and the prefibrotic/early stage of myelofibrosis can be very similar. Differential diagnosis is only possible through the histological examination of the bone marrow, which becomes indispensible due to the difference in prognosis and treatment options. Orv Hetil. 2018; 159(15): 603-609.

Abstract: Moderate thrombocytosis can accompany several diseases (bleeding, inflammation, iron deficiency, or autoimmune diseases), but hematologic examination is strongly recommended in a patient with persistent platelet count above 450 G/L unless reactive origin can be confirmed. The 47-year-old woman’s medical history included hypertonia, asthma bronchiale, and endometriosis. In March 2015, she underwent laboratory examination due to weight loss and lack of appetite. Her results showed elevated thrombocyte count (617 G/L), but no iron deficiency. She presented in our clinic on 07. 04. 2015 with acute pain below her left hypochondrial region, but simple imaging examinations showed no difference to explain it. Abdominal CT revealed a 4.5 cm thrombus which protruded into the left renal artery, blocking it. We started APTI- (activated partial thromboplastin time) monitored continuous intravenous treatment with unfractionated heparin. The JAK2V617F mutation analysis came back positive. Subsequent bone marrow examination revealed prefibrotic/early stage myelofibrosis, prompting treatment with hydroxyurea. The applied treatments led to the disappearance of the patient’s symptoms accompanied by the gradual normalisation of the thrombocyte count. Moderate thrombocytosis is often secondary, but if it persists and is accompanied by mainly thromboembolic events, the risk of diseases of the haematopoietic system, primarily Philadelphia chromosome negative chronic myeloproliferative disease should also be considered. Clinically, essential thrombocythaemia and the prefibrotic/early stage of myelofibrosis can be very similar. Differential diagnosis is only possible through the histological examination of the bone marrow, which becomes indispensible due to the difference in prognosis and treatment options. Orv Hetil. 2018; 159(15): 603–609.

Disseminating cells of a primary solid tumor may represent the origin of metastases and relapses. We aimed at comparing the diagnostic efficacy of multicolor flow cytometry (MFC) and morphology/immunohistochemistry (IHC) in the detection of disseminated tumor cells in the bone marrow (BM) and body fluids of patients with solid tumors, and in pediatric neuroblastoma cases. We investigated 72 samples retrospecively from 50 patients by MFC. Morphology/IHC data were available in 48 cases. In the first cohort, 36 samples derived from 34 patients with various forms of suspected and proven solid tumors and in the second cohort, 36 samples of 16 children with suspected and proven neuroblastoma were analyzed at diagnosis or during follow-up in a 4-color setting by MFC, and the results were compared with those obtained by IHC. In the group of various solid tumors, we found 91% concordance between IHC and MFC, and it was 65% in the neuroblastoma group, and 77% overall. Detection of disseminated tumor cells was found to be more effective by MFC in de novo neuroblastoma samples (100% vs. 86%). The advantage of MFC was even more pronounced when minimal residual disease was evaluated (efficacy, 92% vs. 68%). In contrast, efficacy of IHC was 100% in the group of various solid tumors, whereas it was 91% for MFC. We conclude that MFC and IHC are both essential tools for examining infiltration of BM and body fluids by disseminating solid tumor cells. In the case of neuroblastoma, however, minimal residual disease detection by MFC in a hypoplastic/aplastic BM environment was more effective than IHC, as considerably more cells could be analyzed.

Abstract: Enormous airway secretion after lung resection is common and can lead to dyspnea and radiological anomalies. Depending on the patient’s medical history, it can lead to heart failure, which appears in arrythmia or pulmonary edema. An inflammatory event with high level of proinflammatory cytokines can also lead to arrythmia. The stretch of the wall of vessels can also cause a cytokine release, and the thromboembolic event is a possible common cause of this stretch in the perioperative period. Orv Hetil. 2018; 159(28): 1158–1162.

Myocyte enhancer binding factor 2 B (MEF2B) is a member of the evolutionary conserved transcription family MEF2. MEF2B has been shown to directly control biological activity of the B cell lymphoma 6 (BCL6) gene in germinal center (GC) B cells. To validate MEF2B as an immunohistochemical marker, we studied a large consecutive series of hyperplastic lymphoid tissues (n = 38) and malignant lymphoproliferative conditions (n = 471), including all major categories of B and T cell neoplasms. In hyperplastic lymphoid tissues, MEF2B staining revealed intense and crisp nuclear expression confined to GC B cells. Unlike BCL6, MEF2B was not detected in follicular T cells. In addition, weak nuclear staining of plasma cells was noted. MEF2B staining labeled neoplastic cells of follicular lymphoma both in common and variant cases as well as in bone marrow biopsies with high sensitivity, while it was almost consistently negative in marginal zone lymphoma. Consistent MEF2B expression was found in Burkitt lymphoma and nodular lymphocyte predominant Hodgkin lymphoma as well as in the large majority of cases of mantle cell lymphoma and diffuse large cell B cell lymphoma. MEF2B protein expression showed a statistically significant association with that of BCL6 in cases of diffuse large B cell lymphoma, not otherwise specified. We conclude that MEF2B is a valuable marker of normal GC B cells, potentially useful in differential diagnosis of small B cell lymphomas.

Item does not contain fulltext AIMS: Myelofibrosis is the result of aberrant stromal activity which is determined routinely by reticulin staining in bone marrow biopsies. As matrix fibres are the product of activated fibroblasts, we analysed fibre accumulation compared to stromal cell activity during myelofibrosis progression using the fibroblast activation marker platelet-derived growth factor receptor beta (PDGFRbeta) by immunohistochemistry. METHODS AND RESULTS: Initial and follow-up bone marrow biopsies from 84 patients with myeloproliferative neoplasia, including 55 cases with primary myelofibrosis, were evaluated from five haematopathology centres. The stromal mass was measured by conventional reticulin staining [myelofibrosis (MF) grade, 0-3] and PDGFRbeta-positive cells using a novel PDGFRbeta scoring system (0-3). Results were correlated for prediction of progression. The MF grade and the PDGFRbeta score showed excellent correlation (Spearman's r = 0.83, P < 0.0001). Elevated PDGFRbeta scores (higher than MF-grade) predicted myelofibrosis progression in total with 43% sensitivity and 57% specificity, and short-term (within 1 year) progression with 82% sensitivity and 53% specificity. Progression of prefibrotic disease to manifest myelofibrosis could be forecast with 90% sensitivity and 75% specificity. CONCLUSION: PDGFRbeta highlights stromal cell activation in marrow fibrosis, which is closely related to matrix accumulation, indicating a direct clinical impact especially in prefibrotic myeloproliferative disorders.

Objectives: Clonal selection in the follicular germinal centers in lymphatic tissues is accompanied by an intense proliferation of polyclonal B cells in a precisely regulated fashion. In contrast, B-cell neoplasias proliferate autonomously due to endogenous stimuli. The cell kinetic activity is obvious at many levels including progressive chromatin modification and elevated mitotic rates. We asked if there are differences in the kinetics of histone H3S10 phosphorylation required for mitotic entry between highly proliferating B cells of reactive germinal centers and in B-cell lymphomas with different proliferative capacity. Material and Methods: Phospho-H3 histone (pH3S10)-specific immunohistochemistry was applied to cultivated cell, reactive and selected indolent and aggressive lymphoma samples (diffuse large B-cell lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, follicular lymphoma and small lymphocytic lymphoma). Microscopic quantification of the “dot-type” (representing late G2 phase) and “mitotic” immunolabeling patterns per field of view was performed and compared with classical cell proliferation markers. Results: In addition to the dense homogeneous chromatin labeling highlighting mitotic figures, we stated a selective dot-type nuclear labeling representing ongoing chromatin condensation in premitotic G2 phase cells. While cell proliferation and mitotic counts correlated in general with histology, statistical analysis indicated an accumulation of G2 phase pH3S10 pattern in the reactive germinal centers in contrast to lymphomas. The dot-type G2 staining pattern was surprisingly overrepresented (1,321.7 ± 356.5/10 HPF) in the reactive germinal centers compared to aggressive lymphomas (101.3 ± 33.1) (p < 0.005). The relative G2/M value was significantly higher (4.6 ± 0.6) in reactive germinal center B cells than in any lymphoma entity evaluated (0.7 ± 0.2 in Burkitt lymphoma, 0.9 ± 0.4 in grade 3b follicular lymphoma, 1.3 ± 1.1 in diffuse large B-cell lymphoma, 1.5 ± 0.6 in lymphoblastic lymphoma, and 0.9 ± 0.2 in small lymphocytic lymphoma). Conclusions: pH3S10 immunohistochemistry enabled the presentation of significant differences in the cell cycle kinetics between reactive and neoplastic B-cell lymphoproliferations. Accumulation of G2 phase B cells in reactive folliculi directs to physiological G2/M checkpoint blockade. In contrast, accelerated G2/M transition in lymphomas is potentially associated with impaired genomic repair and cell death mechanisms.

Introduction We created a simple and effective flow cytometry scoring system (FCSS) for suspected Myelodysplastic syndromes (MDS) samples and evaluated its diagnostic and prognostic potential. Methods Besides evaluating the four parameters suggested by Ogata, we investigated erythroid precursors and mast cells. We evaluated the six-parameter FCSS in a four-color setting (test cohort: 51 patients; 25 controls), then we implemented it into an eight-color setting and tested it on a validation cohort of patients with MDS (n=31). Results When we compared MDS cases to non-MDS samples in the test cohort, we detected significant differences regarding not only the four major parameters but also two additional ones, namely CD71 rCV% of erythroid precursors (P=.004) and mast cell percentage (MC%) (P=.001). The utilization of the modified six-parameter FCSS provided high sensitivity and specificity both in the four color (84% and 80%, respectively) and in the eight color (81% and 100%, respectively) setting, with an excellent discriminative power between MDS and non-MDS samples. Furthermore, we found significant difference in event-free survival between the risk groups based on the modified six-parameter FCSS (P=.001). Conclusion We evaluated and validated a single-tube flow cytometric procedure for a simple six-parameter FCSS which has not only high diagnostic but also prognostic power.

Background Myelofibrosis (MF) is characterized by accumulation of stromal cells and extracellular matrix. Progression of fibrosis is an important clinical issue and monitoring is required for new therapeutic approaches. Currently, the quantification is based on semiquantitative evaluation of reticulin silver stained slides. We recently reported that platelet derived growth factor receptor beta (PDGFRβ) expression in fibroblasts is a useful marker of stromal activation. PDGFRβ expression based scores represent significant differences in different MF grade which provides optimal source of quantification. In this study, slide-based measurements were performed to support correlations of PDGFRβ expression with MF grade. Methods Scanned image tiles from 79 bone marrow samples (BM) with different MF grades were evaluated for PDGFRβ-related IHC parameters. Following the determination of immunopositive (brown component) and total area (region of interest) of the BM, PDGFRβ related image parameters were defined and evaluated in comparison with the classical reticulin based grading. Results Eight PDGFRβ expression related image parameters showed excellent correlation with the MF grade (correlation coefficient ranging between 0.79 and 0.83) and with PDGFRβ score (0.76–0.87). Despite the significant sample heterogeneity, the parameters showed significant differences between fibrotic and nonfibrotic cases and between mild and advanced fibrosis. Distribution of values within a particular specimen emphasizes the heterogeneity of bone marrow involvement which may cause difficulties in semiquantitative methods. Conclusions Our results clearly demonstrated the correlation between MF and PDGFRβ expression considering all relevant areas in BM samples. This method provides good basis for follow-up comparison of the fibrotic samples. © 2014 International Clinical Cytometry Society

Polyclonal mesenchymal cells (fibroblasts, endothelial cells, pericytes, osteoblasts, reticular cells, adipocytes, etc.) of the bone marrow create a functional microenvironment, which actively contributes to the maintenance of hemopoesis. This takes place through cellular interactions via growth factors, cytokines, adhesion molecules and extracellular matrix components, as well as through the control of calcium and oxygen concentration. Inflammatory and neoplastic diseases of the bone marrow result in pathologic interaction between hemopoietic progenitors and stromal cells. This may lead to the activation and expansion of the stroma and to the accumulation of reticulin and collagen fibers produced by mesenchymal cells. Clinically relevant fiber accumulation, termed as myelofibrosis accompanies many diseases, although, the extent and the consequence of myelofibrosis are variable in different disorders. The aim of this review is to summarize basic features of the normal bone marrow mesenchymal environment and the pathological process leading to myelofibrosis. In addition, the special features of myelofibrosis in bone marrow diseases, including myeloproliferative neoplasia, myelodysplastic syndrome and other neoplastic conditions are discussed.A csontvelőben található polyclonalis mesenchymalis sejtek növekedési faktorok, citokinek, extracelluláris mátrix termelésével, adhéziós molekulák expressziójával, a kalciumion- és oxigénkoncentráció szabályozásával egy olyan funkcionális mikrokörnyezetet hoznak létre, amely aktívan részt vesz a haemopoesis fenntartásában. Számos gyulladásos és neoplasticus folyamat során jöhet létre kóros kölcsönhatás a haemopoeticus progenitor sejtek és a csontvelői stroma között. Ennek hatására sokszor a stroma aktivációja, expansiója és a mesenchymalis sejtek által termelt reticulin- és kollagénrostok akkumulációja figyelhető meg. Az így kialakult myelofibrosis számos, csontvelőt érintő elváltozás kísérő jelensége lehet, megítélése azonban kórképről kórképre eltérő. Jelen összefoglalás célja a normális és kóros csontvelői mesenchymalis környezet bemutatása, a myelofibrosis kimutatásának szemléltetése. Tanulmányukban a szerzők részletesen jellemzik a myelofibrosis szerepét myeloproliferativ neoplasiákban, myelodysplasiás szindrómákban, illetve kitérnek a rostszaporulat jelentőségére egyéb neoplasticus folyamatokban. Orv. Hetil., 2014, 155(10), 367–375.

A nucleophosmin gén (NPM1) 12-es exonjának mutációja, jelenlegi ismereteink szerint, a leggyakoribb genetikai eltérés akut myeloid leukaemiában. Az eddigi beszámolók alapján kimutatásával a betegség sajátos biológiájú és kedvező lefolyású formája határozható meg, amely a leukaemiák WHO-osztályozásában 2008-tól külön csoportként kerül említésre. A mutáció hatására megváltozik a kódolt fehérje sejten belüli eloszlása, és az NPM fehérje a sejtmag helyett a citoplazmában halmozódik fel (NPMc+). Ez a jelenség, amely a kizárólag mutáns géntermék kapcsán figyelhető meg, szövettani vizsgálat keretében immunhisztokémiával is kimutatható. Jelen tanulmányunkban hazai körülmények között először vizsgáltuk az NPM-mutáció előfordulását felnőttkori AML-ben immunhisztokémia módszerével. A 2005 és 2008 között diagnosztizált összesen 41 eset részletes kiértékelése során 6 esetben (14,6%) észleltünk citoplazmikus reakciót, valamennyi de novo jelentkező és jellegzetes citogenetikai eltérést nem mutató AML-esetek közül került ki (6/23, 26,1%), egy kivételével nőbetegekben. A blasztsejtek alacsony CD34-, c-kit- és HLA-DR-expressziója alapján a NPMc+ AML a nem érintett esetektől jól elkülöníthetőnek bizonyult, ezek a markáns jellegzetességek az eddig közölt külföldi adatokkal teljes mértékben megegyeznek. Tapasztalataink alapján az NPM-mutáció immunhisztokémiai vizsgálata egyszerűen beépíthető a hétköznapi hematológiai diagnosztikai gyakorlatba.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disease originating from cells characterized by antigen-presenting Langerhans cell phenotype. The clinical spectrum of LCH is highly variable including localized and disseminated forms mostly occurring in children. Recently, about 60% of LCHs were reported to carry the activating BRAF mutation V600E. In our retrospective study, we evaluated the occurrence and prognostic impact of the V600E mutation in formaldehyde-fixed, paraffin-embedded samples from 15 pediatric LCH cases treated at our institution. Allele-specific polymerase chain reaction (PCR) and direct sequencing were used to demonstrate the presence of V600E mutation, and immunohistochemistry (IHC) using the mutant protein-specific VE1 antibody clone was performed to confirm mutant BRAF protein expression. Eight of 15 (53.3%) cases proved to be BRAF mutants by any of the methods applied, with a single case showing a discrepancy (PCR negative/IHC positive). Four of the BRAF-mutant cases (50.0%) showed refractory disease and progressed to death within 43 months, whereas the remaining mutant cases were stable and responded well to therapy. Wild-type BRAF cases (7/15, 46.6%) with generally comparable initial presentation were all treated successfully. In conclusion, activating V600E BRAF mutation can be frequently demonstrated in pediatric LCH by both allele-specific PCR and IHC. Unfavorable risk cases potentially also responding to BRAF-inhibitory therapy can be identified by mutation testing using archival formaldehyde-fixed, paraffin-embedded tumor samples.

Mortality of acute myeloid leukemia is still 60-70% in young (60 years) adults and 90% in elderly (≥60 years) patients.The aim of the authors was to analyse the outcome of treatment in their patients with acute myeloid leukemia.From 2007 to 2013, 173 patients with acute myeloid leukemia were treated. Patients were classified according to the European LeukemiaNet prognostic guideline. Association between mortality and the type of acute myeloid leukemia (secondary or primary), dose of daunoblastin at induction of treatment, and the rate of minimal residual disease were investigated.The 5-year survival probability was 25% in young adults and 2% in the elderly. The survival was significantly influenced by these prognostic factors. The 5-year survival rate was 50% in the young, favorable prognostic group. The 90 mg/m2 daunoblastin dose was found to be beneficial. Addition of bortezomib to the standard induction protocol had an additional beneficial effect.The speed and depth of the response to induction therapy, and the initial white blood cell count had an apparent effect on survival.Bevezetés: Heveny myeloid leukaemiában a fiatal felnőttek (60 év) kétharmada, az idősek (≥60 év) mintegy 90%-a hal meg a betegség következtében. Célkitűzés: A szerzők heveny myeloid leukaemiás betegeik kezelésével szerzett tapasztalataik elemzését tűzték ki célul. Módszer: 2007–2013 között 173 heveny myeloid leukaemiás beteget kezeltek. A betegeket a European LeukemiaNet javaslata alapján osztályozták. Vizsgálták az életkor, a „de novo” vagy „szekunder” betegség, a daunoblastin dózisa, a bortezomib, a minimális residualis betegség és a betegség lefolyása közötti összefüggést. Eredmények: A fiatal betegek ötéves túlélése 25%, az időseké 2% volt. A túlélést a prognosztikai tényezők jelentősen befolyásolták: a fiatal, jó prognózisú betegek túlélése 50% volt. Fiataloknál a 90 mg/m2 daunoblastin előnyös volt, míg a 45 mg/m2 és 60 mg/m2 hatékonysága között nem volt jelentős különbség. A bortezomibbal kiegészített standard indukciós kezelés ígéretesnek tűnt, de ennek megítéléséhez nagyobb számú beteg vizsgálata szükséges. Következtetések: A remisszió kialakulásának gyorsasága és mélysége, valamint a kiindulási fehérvérsejtszám jelentősen befolyásolhatják a betegség lefolyását a European LeukemiaNet osztályozás szerinti azonos csoportokon belül is. Orv. Hetil., 2014, 155(17), 653–658.

Background: Myelofibrosis (MF) is characterized by accumulation of stromal cells and extracellular matrix. Progression of fibrosis is an important clinical issue and monitoring is required for new therapeutic approaches. Currently the quantification is based on semi-quantitative evaluation of reticulin silver stained slides. We recently reported that platelet derived growth factor receptor beta (PDGFRβ) expression in fibroblasts is a useful marker of stromal activation. PDGFRβ expression based scores represent significant differences in different myelofibrosis grade which provides optimal source of quantification. In this study slide based measurements were performed in order to support correlations of PDGFRβ expression with MF grade. Methods: Scanned image tiles from 79 bone marrow samples (BM) with different myelofibrosis grades were evaluated for PDGFRβ-related IHC parameters. Following the determination of immunopositive (brown component) and total area (region of interest) of the BM, PDGFRβ related image parameters were defined and evaluated in comparison to the classical reticulin based grading. Results: Eight PDGFRβ expression related image parameters showed excellent correlation with the MF grade (correlation coefficient ranging between 0.79 and 0.83) and with PDGFRβ score (0.76-0.87). Despite the significant sample heterogeneity the parameters showed significant differences between fibrotic and non-fibrotic cases and between mild and advanced fibrosis. Distribution of values within a particular specimen emphasizes the heterogeneity of bone marrow involvement which may cause difficulties in semi-quantitative methods. Conclusions: Our results clearly demonstrated the correlation between myelofibrosis and PDGFRβ expression considering all relevant areas in BM samples. This method provides good basis for follow-up comparison of the fibrotic samples. © 2014 Clinical Cytometry Society.

The most frequent genetic change currently known in acute myeloid leukemia (AML) is the mutation of the nucleophosmin (NPM) gene. Aberrant cytoplasmic accumulation of NPM protein (NPMc+) is the result of this mutation, and it can be demonstrated by immunohistochemistry for the identification of a favorable subgroup within "AML with normal karyotype" according to the World Health Organization classification. NPM staining pattern was defined in 71 AML and 15 control cases by the use of bone marrow smears in order to overcome limitations observed due to immunohistochemistry. In 13/71 cases (18.3%), clear cytoplasmic staining of the leukemic blast cells was detectable that was comparable with the positivity of mitotic figures physiologically lacking nuclear membrane. The biological and genetic characteristics of the NPMc+ cases determined this way were identical with the previously published results including low CD34 and HLA-DR expression and lack of recurrent karyotype abnormalities. Bone marrow smears are well applicable and therefore a real alternative for the determination of NPM with the highest accuracy for optimal risk stratification in AML.

Platelet derived growth factor receptor (PDGFR) is a membrane tyrosine-kinase receptor required for fibroblast activation in stromal proliferations. In order to assess the role of PDGFR in myelofibrosis (MF) we determined in 60 bone marrow biopsies the occurrence and distribution of its α and β subunits in normal and fibrotic bone marrow stroma using immunohistochemistry, and compared this with the grade of MF determined by Gömöri's silver impregnation. PDGF receptor subunits were found to be differentially expressed in the marrow parenchyma. PDGFRα expression identified megakaryocytes, endosteal and endothelial cells while PDGFRβ was virtually absent from inter-trabecular spaces in normal marrow. Activated fibroblasts characteristic for MF intensely expressed PDGFRβ but only a moderate increase in PDGFRα expression was seen. Semi-quantitative PDGFRβ immunoreactivity scores correlated well with the grade of MF in the vast majority of the MF cases (Spearman r= 0.83). Altogether, 21/60 (35.0%) cases showed a relative increase of PDGFRβ expression, compared to the MF grade, suggesting that increased stromal PDGFRβ expression occurs early and precedes reticulin and collagen fiber production during fibroblast activation. In conclusion, bone marrow PDGFRβ expression closely correlates with the grade of MF. Increased immunoreactivity for PDGFRβ occurs already in the prefibrotic stage of the disease and might allow a functional classification of the bone marrow stromal reaction.

The mutation of the nucleophosmin gene (NPM1) is the most frequently occurring genetic aberration in acute myeloid leukemia (AML). Due to the high frequency and the obvious impact on disease outcome, the current WHO classification also defines the new (tentative) entity of "AML with NPM mutation". Mutations of NPM1 exon 12 affect both nuclear complexion and nuclear export signaling (NES) domain resulting in redistribution and accumulation of the NPM protein in the cytoplasm of leukaemic cells. The effect of gene mutation can be directly demonstrated by the occurrence of cytoplasmic NPM using immunohistochemistry (NPMc+). The present study focused on further biological and clinical characterization of NPMc+ AML determined by histological and cytological preparations of the bone marrow. 41 adult AML cases were investigated in our center between 2005 and 2008, 6/41 cases were presented with cytoplasmic NPM immunostaining (14.6%). All but one were female patients, and were diagnosed as de novo AML with no recurrent cytogenetic aberrations (6/23, 26.1%). The NPMc+ group displayed M2 or M4 morphology, low CD34, c-kit and HLA-DR expression making a clear phenotypic distinction from the unaffected cases possible. These results are in agreement with previous studies. In conclusion, immunohistochemistry is well applicable for the identification of NPM mutated AML in the daily hematopathology practice.

In daily routine the reticulin silver staining is used on bone marrow biopsy samples as a gold standard for the characterization of myelofibrosis, however this method does not provide information about the prefibrotic stage. Recently a specific immunohistochemical method was introduced which may overcome these weaknesses of reticulin staining. Activated fibroblasts responsible for stromal proliferation are highlighted by increased PDGFR β expression, which can be presented by immunohistochemistry in bone marrow samples. Using this staining the pre-fibrotic stage can become detectable and we have information about the disease activity. During development of new staining method it is important to prove its reliability and usability. In this paper we introduce a digital image processing method to measure paranchymal damage in digitalized histological slides that can aid correct interpretation of the staining.