Splenectomy at early stage of autoimmune arthritis delayed inflammatory response and reduced joint deterioration in mice.

The spleen plays a role in innate and adaptive immunity, and autoimmune diseases like rheumatoid arthritis (RA). We investigated the effect of splenectomy in early and moderate stages of autoimmune arthritis in a mouse model. To induce recombinant human G1-induced arthritis (GIA), BALB/c mice were immunized intraperitoneally three times in 4-week intervals with the rhG1 antigen. Mice were splenectomized on day 7 (SPE1) or day 35 (SPE2) after the initiation of immunization; tested for clinical severity, joint radiological and histological changes, serum levels of inflammatory cytokines and autoantibodies, and rhG1-specific immune responses; and compared to those in control mice with spleen left intact. Circulating Tregs and T-helper subset ratios in the spleen and inguinal lymph nodes (LNs) were also examined using flow cytometry. The onset of severe inflammatory response was significantly delayed in SPE1 and SPE2 groups compared to control mice at early stages of GIA, which was associated with increased circulating Tregs. After the third immunization, as disease progressed, the severity scores were robustly increased in all mice. Nevertheless, in splenectomized mice, we observed reduced joint deterioration and cartilage damage, more Th2 cells in LNs, and reduced levels of pro-inflammatory cytokines and autoantibodies in their sera. Mesenteric LN cells of splenectomized mice exhibited weaker response in vitro against the rhG1 antigen compared to control mice spleen. In conclusion, splenectomy in the early stages of GIA delayed the inflammatory response, suggesting a protective effect against the development and progression of severe destructive arthritis.

On the Boundary of Exploratory Genomics and Translation in Sequential Glioblastoma.

OMICS methods brought significant advancements to the understanding of tumor cell biology, which transformed the treatment and prognosis of several cancers. Clinical practice and outcomes, however, changed significantly less in the case of glioblastoma (GBM). In this study, we aimed to assess the utility of whole exome (WES) sequencing in the clinical setting. Ten pairs of formalin-fixed, paraffin-embedded (FFPE) GBM specimens were obtained at onset (GBM-P) and at recurrence (GBM-R). Histopathological and molecular features of all samples supported the diagnosis of GBM based on WHO CNS5. WES data were filtered, applying a strict and custom-made pipeline, and occurrence of oncogenic and likely oncogenic variants in GBM-P, GBM-R or both were identified by using the VarSeq program version 2.5.0 (Golden Helix, Inc.). Characteristics and recurrence of the variants were analyzed in our own cohort and were also compared to those available in the COSMIC database. The lists of oncogenic and likely oncogenic variants corresponded to those identified in other studies. The average number of these variants were 4 and 5 out of all detected 24 and 34 variants in GBM-P and GBM-R samples, respectively. On average, one shared oncogenic/likely oncogenic variant was found in the pairs. We assessed the identified variants' therapeutic significance, also taking into consideration the guidelines by the Association for Molecular Pathology (AMP). Our data support that a thorough WES analysis is suitable for identifying oncogenic and likely oncogenic variants in an individual clinical sample or a small cohort of FFPE glioma specimens, which concur with those of comprehensive research studies. Such analyses also allow us to monitor molecular dynamics of sequential GBM. In addition, careful evaluation of data according to the AMP guideline reveal that though therapeutic applicability of the variants is generally limited in the clinic, such information may be valuable in selected cases, and can support innovative preclinical and clinical trials.

Glioblastoma epigenomics discloses a complex biology and potential therapeutic targets.

Background and purpose:

Glioblastoma (GBM), a highly aggressive form of brain tumors, has been extensively studied using OMICS methods, and the most characteristic molecular determinants have been incorporated into the histopathological diagnosis. Research data, nevertheless, only partially have been adopted in clinical practice. Here we aimed to present results of our epige­no­mic GBM profiling to better understand early and late determinants of these tumors, and to share main elements of our findings with practicing professionals.

GBM specimens were surgically obtained after first diagnosis (GBM1) and at recurrence (GBM2). DNA was extracted from 24 sequential pairs of formalin-fixed, paraffin-embedded tumor tissues. The Reduced Representation Bisulfite Sequencing kit was used for library preparation. Pooled libraries were sequenced on an Illumina NextSeq 550 instrument. Methylation controls (MC) were obtained from a publicly available database. Bioinformatic analyses were performed to identify differentially methylated pathways and their elements in cohorts of MC, GBM1 and GBM2.

Several differentially methylated pathways involved in basic intracellular and brain tissue developmental processes were identified in the GBM1 vs. MC and GBM2 vs. MC comparisons. Among differentially me­thylated pathways, those involved in immune regulation, neurotransmitter (particularly dopaminergic, noradrenergic and glutaminergic) responses and regulation of stem cell differentiation and proliferation stood out in the GBM2 vs. GBM1 comparisons.

Our study revealed biological complexity of early and late gliomagenesis encompassing mechanisms from basic intracellular through distorted neurodevelopmental processes to more specific immune and highjacked neurotransmitter pathways in the tumor microenvironment. These findings may offer considerations for therapeutic approaches.

PersonALL: a genetic scoring guide for personalized risk assessment in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Recurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations. METHODS: Disease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment. RESULTS: CNAs were detected in 93.8% (n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status (IKZF1normal, IKZF1del and IKZF1plus) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low (n = 215): 86.3%, IKAROS-medium (n = 27): 57.4% and IKAROS-high (n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients (n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients (n = 103) showed high (n = 74) or ultra-poor (n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively). CONCLUSIONS: PersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

Parallel testing of liquid biopsy (ctDNA) and tissue biopsy samples reveals a higher frequency of EZH2 mutations in follicular lymphoma.

BACKGROUND: Recent genomic studies revealed enhancer of zeste homolog 2 (EZH2) gain-of-function mutations, representing novel therapeutic targets in follicular lymphoma (FL) in around one quarter of patients. However, these analyses relied on single-site tissue biopsies and did not investigate the spatial heterogeneity and temporal dynamics of these alterations. OBJECTIVES: We aimed to perform a systematic analysis of EZH2 mutations using paired tissue (tumor biopsies [TB]) and liquid biopsies (LB) collected prior to treatment within the framework of a nationwide multicentric study. METHODS: Pretreatment LB and TB samples were collected from 123 patients. Among these, 114 had paired TB and LB, with 39 patients characterized with paired diagnostic and relapse samples available. The EZH2 mutation status and allele burden were assessed using an in-house-designed, highly sensitive multiplex droplet digital PCR assay. RESULTS: EZH2 mutation frequency was found to be 41.5% in the entire cohort. In patients with paired TB and LB samples, EZH2 mutations were identified in 37.8% of the patients with mutations exclusively found in 5.3% and 7.9% of TB and LB samples, respectively. EZH2 mutation status switch was documented in 35.9% of the patients with paired diagnostic and relapse samples. We also found that EZH2 wild-type clones may infiltrate the bone marrow more frequently compared to the EZH2 mutant ones. CONCLUSION: The in-depth spatio-temporal analysis identified EZH2 mutations in a considerably higher proportion of patients than previously reported. This expands the subset of FL patients who most likely would benefit from EZH2 inhibitor therapy.

Screening and monitoring of the BTKC481S mutation in a real-world cohort of patients with relapsed/refractory chronic lymphocytic leukaemia during ibrutinib therapy.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has revolutionised the therapeutic landscape of chronic lymphocytic leukaemia (CLL). Acquired mutations emerging at position C481 in the BTK tyrosine kinase domain are the predominant genetic alterations associated with secondary ibrutinib resistance. To assess the correlation between disease progression, and the emergence and temporal dynamics of the most common resistance mutation BTKC481S , sensitive (10-4 ) time-resolved screening was performed in 83 relapsed/refractory CLL patients during single-agent ibrutinib treatment. With a median follow-up time of 40 months, BTKC481S was detected in 48·2% (40/83) of the patients, with 80·0% (32/40) of them showing disease progression during the examined period. In these 32 cases, representing 72·7% (32/44) of all patients experiencing relapse, emergence of the BTKC481S mutation preceded the symptoms of clinical relapse with a median of nine months. Subsequent Bcl-2 inhibition therapy applied in 28/32 patients harbouring BTKC481S and progressing on ibrutinib conferred clinical and molecular remission across the patients. Our study demonstrates the clinical value of sensitive BTKC481S monitoring with the largest longitudinally analysed real-world patient cohort reported to date and validates the feasibility of an early prediction of relapse in the majority of ibrutinib-treated relapsed/refractory CLL patients experiencing disease progression.

IL-22-Independent Protection from Colitis in the Absence of Nkx2.3 Transcription Factor in Mice.

The transcription factor Nkx2.3 regulates the vascular specification of Peyer patches in mice through determining endothelial addressin preference and may function as a susceptibility factor in inflammatory bowel diseases in humans. We wished to analyze the role of Nkx2.3 in colonic solitary intestinal lymphoid tissue composition and in colitis pathogenesis. We studied the colonic solitary intestinal lymphoid tissue of Nkx2.3-deficient mice with immunofluorescence and flow cytometry. Colitis was induced in mice using 2.5% dextran sodium sulfate, and severity was assessed with histology, flow cytometry, and quantitative PCR. We found that the lack of Nkx2.3 impairs maturation of isolated lymphoid follicles and attenuates dextran sodium sulfate-induced colitis independent of endothelial absence of mucosal addressin cell-adhesion molecule-1 (MAdCAM-1), which was also coupled with enhanced colonic epithelial regeneration. Although we observed increased numbers of group 3 innate lymphoid cells and Th17 cells and enhanced transcription of IL-22, Ab-mediated neutralization of IL-22 did not abolish the protection from colitis in Nkx2.3-deficient mice. Nkx2.3-/- hematopoietic cells could not rescue wild-type mice from colitis. Using LacZ-Nkx2.3 reporter mice, we found that Nkx2.3 expression was restricted to VAP-1+ myofibroblast-like pericryptal cells. These results hint at a previously unknown stromal role of Nkx2.3 as driver of colitis and indicate that Nkx2.3+ stromal cells play a role in epithelial cell homeostasis.

[A case of destructive cervical spondylarthropathy related to chronic dialysis]. / Krónikus dialízis kapcsán kialakult destruktív cervicalis spondylarthropathia esete.

A case of a 61-year-old male patient suffered chronic renal failure and dialysed for 23 years with destructive cervical spondylarthropathy is presented. The patient presented with sudden onset of cervical pain radiating into his shoulders without neurological deficits. CT and MRI of the cervical and thoracic spine revealed severe destructive changes and compressive fractures of C6 and C7 vertebrae which caused the narrowing of the nerve root canals at these levels. A 360-degree fixation was performed to treat the unstable fracture and the patient's pain (C6 and C7 corpectomy, autolog bone graft replacement of the two vertebral bodies, anterior plate fixation and posterior instrumentation with screws and rods). Postoperatively the patient had no significant pain, no neurological deficit and he was able to manage independent life himself. During the immediate follow-up CT of the neck showed the satisfactory position of the bone graft and the metal implantations. The 6 months follow-up CT revealed the anterior migration of the two screws from the Th1 vertebral body and 2 mm ventral elevation of the caudal end of the plate from the anterior surface of the Th1 vertebral body. The 1-year follow-up could not be performed because the patient died due to cardio-pulmonary insufficiency. This is the second Hungarian report of a chronic dialysis related severe spondylarthropathy which may cause pathologic fractures of the vertebral bodies. The typical radiological and histological findings are discussed. This disease affect patients' quality of life and the conservative treatment alone seems to be ineffective in most cases. Based on the literature and personal experiences, the authors suggest 360-degree fixation of the spine to provide sufficient stability for the vertebrae of "bad bone quality", and early mobilisation of the patient can be achieved.

Comprehensive profiling of disease-relevant copy number aberrations for advanced clinical diagnostics of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is the most common pediatric cancer characterized by a heterogeneous genomic landscape with copy number aberrations occurring at various stages of pathogenesis, disease progression, and treatment resistance. In this study, disease-relevant copy number aberrations were profiled in bone marrow samples of 91 children with B- or T-cell precursor acute lymphoblastic leukemia using digital multiplex ligation-dependent probe amplification (digitalMLPATM). Whole chromosome gains and losses, subchromosomal copy number aberrations, as well as unbalanced alterations conferring intrachromosomal gene fusions were simultaneously identified with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples, and increased numbers of copy number aberrations were detected at the time of relapse as compared with diagnosis. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients revealed three different patterns of clonal relationships with (i) one patient displaying identical copy number aberration profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse, and (iii) four patients displaying conserved as well as lost or gained copy number aberrations at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. A newly introduced risk classifier combining cytogenetic data with digitalMLPATM-based copy number aberration profiles allowed for the determination of four genetic subgroups of B-cell precursor acute lymphoblastic leukemia with distinct event-free survival rates. DigitalMLPATM provides fast, robust, and highly optimized copy number aberration profiling for the genomic characterization of acute lymphoblastic leukemia samples, facilitates the decipherment of the clonal origin of relapse and provides highly relevant information for clinical prognosis assessment.

Upregulation of the TRPA1 Ion Channel in the Gastric Mucosa after Iodoacetamide-Induced Gastritis in Rats: A Potential New Therapeutic Target.

Acute gastritis is often untreatable by acid secretion-inhibiting drugs. Understanding the protective mechanisms including the role of Transient Receptor Potential Ankyrin1 (TRPA1) and Vanilloid1 (TRPV1) channels localized on capsaicin-sensitive afferents and non-neuronal structures might identify novel therapeutic approaches. Therefore, we characterized a translational gastritis model using iodoacetamide (IAA) and investigated TRPA1/V1 expressions. Wistar rats and CD1, C57Bl/6J mice were exposed to IAA-containing (0.05, 0.1, 0.2, 0.3, 0.5%) drinking water for 7 or 14 days. Body weight and water consumption were recorded daily. Macroscopic lesions were scored, qualitative histopathologic investigation was performed, TRPA1/V1 immunopositivity and mRNA expressions were measured. IAA induced a concentration-dependent weight loss and reduced water intake in both species. Hyperemia, submucosal edema, inflammatory infiltration and hemorrhagic erosions developed after 7 days, while ulcers after 14 days in rats. Trpa1 mRNA/protein expressions were upregulated at both timepoints. Meanwhile, TRPV1 immunopositivity was upregulated in the gastric corpus after 0.05% IAA ingestion, but downregulated after 0.2%, whereas Trpv1 mRNA did not change. Interestingly, no macroscopic/microscopic changes were observed in mice. These are the first data for the concentration- and duration-dependent changes in the IAA-induced gastritis in rats accompanied by TRPA1 upregulation, therefore, its therapeutic potential in gastritis should further be investigated.

Immense random colocalization, revealed by automated high content image cytometry, seriously questions FISH as gold standard for detecting EML4-ALK fusion.

EML4-ALK gene fusion (inv2(p21p23)) of non-small cell lung cancer (NSCLC) predisposes to tyrosine kinase inhibitor treatment. One of the gold standard diagnostics is the dual color (DC) break-apart (BA) FISH technique, however, the unusual closeness of the involved genes has been suggested to raise likelihood of random co-localization (RCL) of signals. Although this is suspected to decrease sensitivity (often to as low as 40-70%), the exact level and effect of RCL has not been revealed thus far. Signal distances were analyzed to the 0.1 µm precision in more than 25,000 nuclei, via automated high content-image cytometry. Negative and positive controls were created using conventional DC BA-, and inv2(p21p23) mimicking probe-sets, respectively. Average distance between red and green signals was 9.72 pixels (px) (±5.14px) and 3.28px (±2.44px), in positives and negatives, respectively; overlap in distribution being 41%. Specificity and sensitivity of correctly determining ALK status was 97% and 29%, respectively. When investigating inv2(p21p23) with DC BA FISH, specificity is high, but seven out of ten aberrant nuclei are inevitably falsely classified as negative, due to the extreme level of RCL. Together with genetic heterogeneity and dilution effect of non-tumor cells in NSCLC, this immense analytical false negativity is the primary cause behind the often described low diagnostic sensitivity. These results convincingly suggest that if FISH is to remain a gold standard for detecting the therapy relevant inv(2), either a modified evaluation protocol, or a more reliable probe-design should be considered than the current DC BA one. © 2018 International Society for Advancement of Cytometry.

Local upregulation of transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 ion channels in rectosigmoid deep infiltrating endometriosis.

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis ( n = 15), uterine fibroid-induced moderate dysmenorrhoea ( n = 7) and tubal infertility with no pain ( n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease.

[TP53 mutation analysis in chronic lymphocytic leukaemia]. / A TP53-mutáció-analízis jelentosége krónikus lymphocytás leukaemiában.

INTRODUCTION: In recent years much progress has been made in the therapy of chronic lymphocytic leukaemia, as the new innovative medicine proved to be effective in managing patients carrying TP53 abnormalities. To identify all these patients, it is essential to screen for both forms of TP53 defects, including both 17p deletions and TP53 mutations. AIM: The aim of this study was to determine the frequency of TP53 mutations and their association with 17p deletions in a large Hungarian cohort of 196 patients suffering from chronic lymphocytic leukaemia. METHOD: We performed mutation analysis of TP53 (exons 3-10) using Sanger sequencing. RESULTS: TP53 mutations were present in 15.8% of patients, half of which were associated with 17p deletion. By analysing both forms, TP53 defect was identified in 25.4% of the patients. CONCLUSIONS: Our study demonstrates that by performing a TP53 mutation analysis, an additional 10% of high-risk patients can be detected. Orv. Hetil., 2017, 158(6), 220-228.

Sequential and hierarchical chromosomal changes and chromosome instability are distinct features of high hyperdiploid pediatric acute lymphoblastic leukemia.

BACKGROUND: Pathogenesis of the non-random accumulation of extra chromosomes in the low and high hyperdiploid (HeL, HeH) pre-B pediatric acute lymphoblastic leukemia (B-pALL) is largely unknown, and has been clarified with respect only to tetrasomic chromosomes. We analyzed the hierarchy of changes in chromosome number and chromosomal instability, as well as clonal heterogeneity and evolution, in the untreated bone marrow cell samples from 214 B-pALL patients. PROCEDURE: Applying relocation, 2 × 4 color interphase fluorescence in situ hybridization was used to detect copy number alterations (CNAs) of the most commonly involved chromosomes, 4, 6, 10, 14, 17, 18, 21, and X. This approach allowed us to acquire a dataset correlated for all eight parameters. RESULTS: Based on chromosome number, an average of 6.9 and 10.2, whereas according to unique constellation 15.3 and 26.7 subclones could be identified in the HeL and HeH subgroups, respectively. Cluster analysis revealed the order of CNAs to chromosomes was highly conserved, and network analysis indicated changes in chromosome number were sequential for 80-90% of all numerical aberrations. Significant chromosome instability was revealed in both subgroups of leukemia. CONCLUSIONS: Data generated using this new approach indicate that chromosomal instability, which causes heterogeneity in the subclonal landscape, and the sequential changes to chromosome numbers, are both determining factors in the pathomechanism of the hyperdiploid B-pALL. These new observations could prompt research into the mitotic machinery of leukemic cells to identify new therapeutic targets for treating this disease.

Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization are complementary techniques to detect cytogenetic abnormalities in multiple myeloma.

Multiple myeloma (MM) is a genetically heterogeneous disease with diverse clinical outcomes. Interphase fluorescence in situ hybridization (i-FISH) is the most commonly used approach to detect recurrent cytogenetic abnormalities in this malignancy. We aimed to assess the performance of multiplex ligation-dependent probe amplification (MLPA) to reveal copy number abnormalities (CNAs) in MM. Diagnostic bone marrow samples from 81 patients were analyzed using 42 MLPA probes for the following regions: 1p32-31, 1p21, 1q21.3, 1q23.3, 5q31.3, 12p13.31, 13q14, 16q12, 16q23, and 17p13. All samples were also screened by i-FISH for the presence of hyperdiploidy, deletion/monosomy of chromosome 13, deletion of TP53, disruption of the immunoglobulin heavy-chain gene, t(4;14), t(11;14), t(14;16), t(8;14), gain of 5q and abnormalities of chromosome 1. A total of 245 alterations were detected in 79 cases (98%). Investigating the same aberrations, the two methods showed a congruency of higher than 90%. A low proportion of cells with the relevant abnormality, focal CNAs and unmatched probes were responsible for the discrepancies. MLPA revealed 95 CNAs not detected by i-FISH providing additional information in 53 cases (65%). Scrutiny of CNAs on chromosome 1, using more than 20 probes, revealed significant heterogeneity in size and location, and variable intra-chromosomal and intra-clonal rates of loss or gain. Our results suggest that MLPA is a reliable high-throughput technique to detect CNAs in MM. Since balanced aberrations are key to prognostic classification of this disease, MLPA and i-FISH should be applied as complementary techniques in diagnostic pathology.

Quantitative Analysis of NKX2-3 Expression in Human Colon: An Immunohistochemical Study.

In mice, Nkx2-3 homeodomain transcription factor defines the vascular specification of secondary and tertiary lymphoid tissues of the intestines. In human studies, polymorphisms in NKX2-3 have been identified as a susceptibility factor in inflammatory bowel diseases, whereas in mice, its absence is associated with protection against experimental colitis and enhanced intestinal epithelial proliferation. Here, we investigated the expression of NKX2-3 in normal, polyp, and adenocarcinoma human colon samples using immunohistochemistry and quantitative morphometry, correlating its expression with endothelial and mesenchymal stromal markers. Our results revealed that the expression of NKX2-3 is regionally confined to the lamina propria and lamina muscularis mucosae, and its production is restricted mostly to endothelial cells and smooth muscle cells with variable co-expression of CD34, alpha smooth muscle antigen (αSMA), and vascular adhesion protein-1 (VAP-1). The frequency of NKX2-3-positive cells and intensity of expression correlated inversely with aging. Furthermore, in most colorectal carcinoma samples, we observed a significant reduction of NKX2-3 expression. These findings indicate that the NKX2-3 transcription factor is produced by both endothelial and non-endothelial tissue constituents in the colon, and its expression changes during aging and in colorectal malignancies. (J Histochem Cytochem XX: XXX-XXX, XXXX).

Low-burden TP53 mutations represent frequent genetic events in CLL with an increased risk for treatment initiation.

TP53 aberrations predict chemoresistance and represent a contraindication for the use of standard chemoimmunotherapy in chronic lymphocytic leukaemia (CLL). Recent next-generation sequencing (NGS)-based studies have identified frequent low-burden TP53 mutations with variant allele frequencies below 10%, but the clinical impact of these low-burden TP53 mutations is still a matter of debate. In this study, we aimed to scrutinise the subclonal architecture and clinical impact of TP53 mutations using a sensitive, NGS-based mutation analysis in a 'real-world' cohort of 901 patients with CLL. In total, 225 TP53 mutations were identified in 17.5% (158/901) of the patients; 48% of these alterations represented high-burden mutations, while 52% were low-burden TP53 mutations. Low-burden mutations as sole alterations were identified in 39% (62/158) of all mutated cases with 82% (51/62) of these being represented by a single low-burden TP53 mutation. Patients harbouring low-burden TP53 mutations had significantly lower time to first treatment compared to patients with wild-type TP53. Our study has expanded the knowledge on the frequency, clonal architecture, and clinical impact of low-burden TP53 mutations. By demonstrating that patients with sole low-burden TP53 variants represent more than one-third of patients with TP53 mutations and have an increased risk for treatment initiation, our findings strengthen the need to redefine the threshold of TP53 variant reporting to below 10% in the routine diagnostic setting.

[Practical approach to molecular pathology of central nervous system tumours]. / A központi idegrendszeri daganatok molekuláris patológiája gyakorlati szempontból.

Recent advances in molecular diagnostics are transforming the classification of malignant tumours, it has long played a major role in the field of CNS tumours. Examination of 1p/19q codeletion is indispensable in case of diffuse gliomas. Glioblastoma may be diagnosed even in the absence of characteristic morphological features, when EGFR amplification, TERT promoter mutation or +7/-10 copy number abnormalities are present. The number of entities defined by a genetic abnormality is growing. Comprehensive analysis of DNA methylation may be of considerable help in addition to histology and basic molecular studies, especially in case of small samples. Keeping up with the ever-expanding diagnostic repertoire is difficult, however, advantages and disadvantages of these methods and the context in which they may be useful should be understood by those who are involved in the diagnosis of CNS tumours. This summary provides a general overview of the main methods used in molecular diagnostics.

Clinicopathological analysis of diffuse large B-cell lymphoma using molecular biomarkers: a retrospective analysis from 7 Hungarian centers.

Background: The clinical and genetic heterogeneity of diffuse large B-cell lymphoma (DLBCL) presents distinct challenges in predicting response to therapy and overall prognosis. The main objective of this study was to assess the application of the immunohistochemistry- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in the diagnosis of DLBCL and its prognostic value in patients treated with rituximab-based immunochemotherapy. Methods: This is a multicenter, retrospective study, which analyzed data from 7 Hungarian hematology centers. Eligible patients were adults, had a histologically confirmed diagnosis of DLBCL, were treated with rituximab-based immunochemotherapy in the first line, and had available clinicopathological data including International Prognostic Index (IPI). On the specimens, immunohistochemistry and FISH methods were performed. Germinal center B-cell like (GCB) and non-GCB subtypes were classified by the Hans algorithm. Outcomes included overall survival (OS), event-free survival (EFS), and EFS at 2 years (EFS24). For survival analysis, we used Kaplan-Meier curves with the log-rank test and multivariate Cox regression. Results: A total of 247 DLBCL cases were included. Cases were positive for MYC, BCL2, BCL6, and MUM1 expression in 52.1%, 66.2%, 72.6%, and 77.8%, respectively. BCL6 translocation, BCL2 gene copy number (GCN) gain, IGH::MYC translocation, MYC GCN gain, IGH::BCL2 translocation, and BCL6 GCN gain were detected in 21.4%, 14.1%, 7.3%, 1.8%, 7.3%, and 0.9%, respectively. At a median follow-up of 52 months, 140 patients (56.7%) had disease progression or relapse. The Kaplan-Meier estimate for EFS24 was 56.2% (CI: 50.4-62.8%). In univariate analysis, only IPI and BCL6 expression were significant predictors of both OS and EFS, whereas MUM1 predicted EFS only. In multivariate analysis, the IPI score was a significant independent negative, whereas MIB-1 and BCL6 protein expressions were significant independent positive predictors of both OS and EFS. Conclusion: In our study, we found that only IPI, BCL6 protein expression and MIB-1 protein expression are independent predictors of survival outcomes in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area.

Next-Generation Sequencing-Based Genomic Profiling of Children with Acute Myeloid Leukemia.

Pediatric acute myeloid leukemia (AML) represents a major cause of childhood leukemic mortality, with only a limited number of studies investigating the molecular landscape of the disease. Here, we present an integrative analysis of cytogenetic and molecular profiles of 75 patients with pediatric AML from a multicentric, real-world patient cohort treated according to AML Berlin-Frankfurt-Münster protocols. Targeted next-generation sequencing of 54 genes revealed 17 genes that were recurrently mutated in >5% of patients. Considerable differences were observed in the mutational profiles compared with previous studies, as BCORL1, CUX1, KDM6A, PHF6, and STAG2 mutations were detected at a higher frequency than previously reported, whereas KIT, NRAS, and KRAS were less frequently mutated. Our study identified novel recurrent mutations at diagnosis in the BCORL1 gene in 9% of the patients. Tumor suppressor gene (PHF6, TP53, and WT1) mutations were found to be associated with induction failure and shorter event-free survival, suggesting important roles of these alterations in resistance to therapy and disease progression. Comparison of the mutational landscape at diagnosis and relapse revealed an enrichment of mutations in tumor suppressor genes (16.2% versus 44.4%) and transcription factors (35.1% versus 55.6%) at relapse. Our findings shed further light on the heterogeneity of pediatric AML and identify previously unappreciated alterations that may lead to improved molecular characterization and risk stratification of pediatric AML.