Exploring p53 protein expression and its link to TP53 mutation in myelodysplasia-related malignancies-Interpretive challenges and potential field of applications.

AIMS: TP53 alterations have a significant prognostic effect in myeloid neoplasms. Our objective was to investigate the TP53 gene mutation status, p53 protein expression and their relationship in dysplasia-related myeloid neoplasms with varying levels of myeloblast counts. METHODS AND RESULTS: A total of 76 bone marrow biopsy samples with different blast counts were analysed. Total and strong (3+) p53 expression was determined. Dual immunohistochemical staining was performed to determine the cell population associated with p53 expression. NGS analysis was performed using the Accel-Amplicon Comprehensive TP53 panel. Both p53 expression and TP53 VAF showed a significant correlation with the myeloblast ratio (P < 0.0001); however, p53 expression was also present in other cell lineages. The VAF value exhibited a significant correlation with p53 expression. A high specificity (0.9800) was observed for TP53 mutation using the ≥ 10% strong (3+) p53 cut-off value, although the sensitivity (0.4231) was low. CONCLUSIONS: Strong (3+) p53 expression using a ≥ 10% cut-off value accurately predicts TP53 mutation but does not reveal the allelic state. The p53 expression is significantly influenced by myeloblast count, and histological interpretation should consider the presence of intermixed non-neoplastic marrow cells with varying physiological p53 expression.

Acute Erythroid Leukemia Post-Chemo-Radiotherapy and Autologous Stem Cell Transplantation Due to Multiple Myeloma: Tracing the Paths to Leukemic Transformation.

The clinical impact of therapy-related acute leukemias is increasing with the extension of cancer-related survival; however, the origins remain largely unknown. Acute erythroleukemia (AEL), a rare unfavorable type of myeloid neoplasia, may also develop secondary to cytotoxic therapy. The disorder is featured by specific genetic alterations, most importantly multi-allelic mutations of the TP53 gene. While AEL might appear as a part of the therapy-related MDS/AML, spectrum information regarding the genetic complexity and progression is largely missing. We present two AEL cases arising after cytotoxic therapy and melphalan-based myeloablation/autologous peripheral stem cell transplantation due to multiple myeloma (MM). As stated, multiple pathogenic TP53 variants were present unrelated to preexisting MM, in parallel with uninvolved/wild-type hemopoiesis. Potential mechanisms of leukemic transformation are discussed, which include (1) preexisting preneoplastic hemopoietic stem cells (HSC) serving as the common origin for both MM and AEL, (2) the generation and intramedullary survival of p53-deficient post-chemotherapy HSCs, (3) reinoculation of mobilized autologous TP53 mutated HSCs, and (4) melphalan treatment-related late-onset myelodysplasia/leukemia with newly acquired TP53 mutations.

Histone H3 K27 alterations in central nervous system tumours: Challenges and alternative diagnostic approaches.

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.

Hypoxia-related carbonic anhydrase IX expression is associated with unfavourable response to first-line therapy in classical Hodgkin's lymphoma.

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 < 0.001). Seventy-two months' progression-free survival (PFS) was significantly lower for the CAIX positive group (0.192) compared with the CAIX negative group (0.771) (P < 0.001), while the overall survival (OS) did not differ (P = 0.097). CONCLUSION: Hypoxic stress-related adaptation - highlighted by CAIX expression - results in cellular quiescence in HRS cells, potentially contributing to the short-term failure of the standard chemotherapy in cHL.

Quantitative Analysis of Carbonic Anhydrase IX Uncovers Hypoxia-Related Functional Differences in Classical Hodgkin Lymphoma Subtypes.

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'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: 2.16-90.36, mean 18.7 ± 18.8). Individual CAIX values were independent of the much higher CD30 values (range 3.46-151.3, mean 52.37 ± 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.

[Investigation and treatment of prefibrotic/early primary myelofibrosis. A case study]. / A korai/praefibroticus primer myelofibrosis kivizsgálása és kezelése egy eset kapcsán.

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.

Distinct Dynamics of Mitotic Transition in B-Cell Lymphoma and Reactive B-Cell Lymphoproliferations Determined by H3S10 Phosphohistone Immunolabeling.

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.

Platelet-derived growth factor receptor ß (PDGFRß) immunohistochemistry highlights activated bone marrow stroma and is potentially predictive for fibrosis progression in prefibrotic myeloproliferative neoplasia.

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 ß (PDGFRß) 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 PDGFRß-positive cells using a novel PDGFRß scoring system (0-3). Results were correlated for prediction of progression. The MF grade and the PDGFRß score showed excellent correlation (Spearman's r = 0.83, P < 0.0001). Elevated PDGFRß 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: PDGFRß highlights stromal cell activation in marrow fibrosis, which is closely related to matrix accumulation, indicating a direct clinical impact especially in prefibrotic myeloproliferative disorders.

[Pathomechanism and clinical impact of myelofibrosis in neoplastic diseases of the bone marrow]. / A csontvelofibrosis patomechanizmusa és elofordulása neoplasticus kórképekben.

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.

[Treatment of acute myeloid leukemia -- a single center experience (2007-2013)]. / Heveny myeloid leukaemiás betegeink kezelésével szerzett tapasztalataink (2007-2013).

INTRODUCTION: Mortality of acute myeloid leukemia is still 60-70% in young (<60 years) adults and 90% in elderly (≥60 years) patients. AIM: The aim of the authors was to analyse the outcome of treatment in their patients with acute myeloid leukemia. METHOD: 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. RESULTS: 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. CONCLUSIONS: The speed and depth of the response to induction therapy, and the initial white blood cell count had an apparent effect on survival.

Extracranial metastatic oligodendroglioma with molecular progression, case presentation.

BACKGROUND: Extraneural metastasis of central nervous system tumors is generally rare and most often reported in glioblastomas and medulloblastomas, whereas oligodendrogliomas seem to have the lowest risk of extracranial metastasis. Given its infrequent occurrence, both the diagnosis and therapy of metastatic oligodendroglioma is often challenging. CASE PRESENTATION: This case study presents an oligodendroglioma, the isocitrate dehydrogenase 1 (IDH1) mutant, 1p/19q-codeleted tumor with bone marrow metastasis. The significance of this case lies in the comprehensive molecular analysis conducted for both the primary tumor and the metastasis. Chromosome 7 trisomy and chromosome 10 monosomy (+ 7/-10) were detected in the metastasis indicating molecular progression, which, to the best of our knowledge, has not been previously documented in metastatic oligodendroglioma. CONCLUSIONS: This case study serves additional information for better understanding of the metastatic capabilities of CNS tumors.

Case report: Targeted treatment strategies for Erdheim-Chester disease.

Introduction: Erdheim-Chester disease (ECD) is a rare disease that belongs to the group of Dendritic and histiocytic neoplasms. Only 2000 cases have been reported worldwide. It can present with a wide range of symptoms, making a differential diagnosis especially difficult. The primary and most important diagnostic tool is a biopsy of the affected organ/tissue. Nowadays the analysis of different mutations affecting the BRAF and MAPK pathways makes it possible to use targeted treatments, such as vemurafenib, dabrafenib, or cobimetinib. Objective: Our aim is to present the results of three male patients treated in our hematology department. Results: Our BRAF mutation-positive patient presented with retroperitoneal tissue proliferation and diabetes insipidus. The initial therapy of choice was dabrafenib. After 3 months of treatment, 18F-fluoro-deoxyglucose positron emission tomography (FDG-PET)/computed tomography (CT) scans showed regression, and after 2 years of treatment, no disease activity was detected. In our second patient, a recurrent febrile state (not explained by other reasons) and diabetes insipidus suggested the diagnosis. A femoral bone biopsy confirmed BRAF-negative ECD. The first-line therapy was interferon-alpha. After 3 months of treatment, no response was observed on 18FDG-PET/CT, and treatment with cobimetinib was started. The control 18FDG-PET/CT imaging was negative. Our third patient was evaluated for dyspnea, and a CT scan showed fibrosis with hilar lymphadenomegaly. A lung biopsy confirmed BRAF-negative ECD. We started treatment with interferon-alpha, but unfortunately, no improvement was observed. Second-line treatment with cobimetinib resulted in a partial metabolic response (PMR) according to control 18FDG-PET/CT. Conclusions: Our results demonstrate that an appropriately chosen treatment can lead to a good therapeutic response, but dose reduction may be necessary due to side effects. With advanced targeted therapeutic treatment options, survival and quality of life are significantly improved.

Carbonic Anhydrase IX (CAIX) Expressing Hypoxic Micro-environment Hampers CD8+ Immune Cell Infiltrate in Breast Carcinoma.

BACKGROUND: Hypoxia and necrosis are common features of invasive cancer. The dynamic upregulation of carbonic anhydrase IX (CAIX), triggered by hypoxia-inducible factor 1 (HIF-1) is 1 of the mechanisms supporting cellular adaptation to hypoxia in solid tumors, including breast carcinoma. CAIX activity results in extracellular acidosis and in a profound reorganization of the tumor micro-environment, influencing biological behavior and prognosis. The main focus of our study was to evaluate the mass and distribution of the immune infiltrate, more specifically of CD8+ effector T-cells, in relation with tumoral CAIX expression. MATERIALS AND METHODS: Formalin-fixed and paraffin-embedded breast carcinoma sections were analyzed following double immunohistochemical staining for CAIX and CD8. Scanned digital slides were evaluated for both labelings, and CD8-related signal was determined within and outside CAIX-positive tumor areas using the HistoQuant (3DHistech) image analysis software. Statistical analysis was performed using GraphPad Prism software. RESULTS: Of the 34 breast carcinomas, 18 tested partially positive for CAIX. The remaining 16 cases were used as the CAIX-negative control group. Necrotic foci were generally associated with CAIX overexpression, and tumors exhibiting signs of necrosis had a significantly higher rate of relative CAIX expression compared with samples without necrosis (11.47±5.505 vs. without necrosis 3.765±3.5 P-value=0.0216). On the other hand, no statistically significant difference was found when comparing relative CD8+ lymphocyte counts in cases with necrosis as opposed to those where necrosis was absent (134.7±55.7 vs. 97.70±57.25; P value=0.1579). No difference in gross CD8+ T-lymphocyte infiltrate could be measured between CAIX positive and negative samples (98.48±37.32 vs. 95.99±50 P value=0.5928). However, in CAIX-expressing tumors a statistical correlation between the CD8+ T-lymphocyte infiltrate and the extent of CAIX-positive areas was observed. Within the same tumor, CD8+ T-lymphocyte counts showed a significant difference betweeen CAIX+ and CAIX- areas (13.06±9.4 vs. 135.6±62.2 P value <0.0001). CONCLUSION: Our measurements demonstrate for the first time that tumor areas with CAIX expression potentially hamper CD8+ T-lymphocyte infiltration in breast carcinoma. The hypoxia-driven adaptive micro-environment likely interferes with the specific response to biological and immune therapies requiring intact effector T-cell response.

Correlation Analyses between Histological Staging and Molecular Alterations in Tumor-Derived and Cell-Free DNA of Early-Stage Primary Cutaneous Melanoma.

Here, we investigate the correlation and statistical analyses between histological staging and molecular alterations in tumor-derived (tdDNA) and cell-free DNA (cfDNA) obtained from early-stage primary cutaneous melanoma (PCM) patients using digital PCR (dPCR) for the detection of the BRAF p.V600E somatic pathogenic variant. In the prospective study, a total of 68 plasma and paired tdDNA samples, and in the retrospective cohort, a total of 100 tdDNA samples were analyzed using dPCR and reverse hybridization StripAssay. The Breslow depth (BD) and Clark level were applied to categorize the study population. Our results demonstrate that dPCR is a highly sensitive and specific method for the detection of BRAF p.V600E somatic variants in cfDNA samples from PCM patients. A strong correlation was detected between BD and cfDNA concentration in all mutant and negative cases, between the tdDNA concentration and the tumor-derived variant allele frequency (VAF) of BRAF p.V600E, between the tdVAF and the cfVAF in all cases, and between the cfDNA and cfVAF in mutant cases. The tdVAF and cfVAF of BRAF p.V600E and cfDNA concentration were the highest in Clark's V category. The cfDNA concentration was statistically significantly higher in Clark's III, IV, and V groups compared to cases with a better prognosis. It can also be explained by the fact that cases with a more advanced stage classification release more cfDNA into the peripheral circulation.

Circulating Cell-Free DNA-Based Comprehensive Molecular Analysis of Biliary Tract Cancers Using Next-Generation Sequencing.

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.

Deep Molecular and In Silico Protein Analysis of p53 Alteration in Myelodysplastic Neoplasia and Acute Myeloid Leukemia.

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.

[Molecular analysis of cell-free DNA in peripheral blood in biliary tract malignancies]. / A perifériás vérben keringo szabad DNS molekuláris vizsgálata epeúti malignitásokban.

INTRODUCTION: 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. OBJECTIVE: 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. METHOD: In the present study, clinically relevant point mutations were detected from both histological and liquid biopsy specimens of bile duct tumors. RESULTS: During next-generation sequencing, histological and DNA samples obtained during liquid biopsy from 33 patients were analyzed using a 67-gene solid tumor panel. DISCUSSION: In our prospective study, we present a minimally invasive approach to identify molecular genetic changes in cholangiocarcinoma and gallbladder tumors. CONCLUSION: 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.

Differential diagnostic and treatment difficulties in a patient with acquired von Willebrand syndrome.

BACKGROUND: 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. CASE PRESENTATION: 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. CONCLUSIONS: 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.

Programmed Death-ligand 1 (PD-L1) Expression in Thymic Epithelial Tumors.

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<0.0001). About 69% of thymomas (20/29) and 43% of thymic carcinomas (3/7) showed high positivity rate (TC≥50% or IC ≥10%), which may indicate therapeutic advantage similar to nonsmall cell lung cancers defined by the same conditions. PD-L1 expression is common in different epithelial tumors of the thymus, which suggests the potential effectiveness of drugs targeting the PD-1/PD-L1 interactions in these neoplasms.

[Immunohistochemical demonstration of mutant nucleophosmin in acute myeloid leukemia: biological and clinical features related to NPMc expression]. / Mutáns nucleophosmin fehérje kimutatása akut myeloid leukaemiában: az NPMc+ AML biológiai és klinikai jellemzôi.

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.