Congenital fibrinogen disorders: a retrospective clinical and genetic analysis of the Prospective Rare Bleeding Disorders Database.

ABSTRACT: Congenital fibrinogen deficiency (CFD) is a rare bleeding disorder caused by mutations in FGA, FGB, and FGG. We sought to comprehensively characterize patients with CFD using PRO-RBDD (Prospective Rare Bleeding Disorders Database). Clinical phenotypes, laboratory, and genetic features were investigated using retrospective data from the PRO-RBDD. Patients were classified from asymptomatic to grade 3 based on their bleeding severity. In addition, FGA, FGB, and FGG were sequenced to find causative variants. A total of 166 CFD cases from 16 countries were included, of whom 123 (30 afibrinogenemia, 33 hypofibrinogenemia, 55 dysfibrinogenemia, and 5 hypodysfibrinogenemia) were well characterized. Considering the previously established factor activity and antigen level thresholds, bleeding severity was correctly identified in 58% of the cases. The rates of thrombotic events among afibrinogenemic and hypofibrinogenemic patients were relatively similar (11% and 10%, respectively) and surprisingly higher than in dysfibrinogenemic cases. The rate of spontaneous abortions among 68 pregnancies was 31%, including 86% in dysfibrinogenemic women and 14% with hypofibrinogenemia. Eighty-six patients received treatment (69 on-demand and/or 17 on prophylaxis), with fibrinogen concentrates being the most frequently used product. Genetic analysis was available for 91 cases and 41 distinct variants were identified. Hotspot variants (FGG, p.Arg301Cys/His and FGA, p.Arg35Cys/His) were present in 51% of dysfibrinogenemia. Obstetric complications were commonly observed in dysfibrinogenemia. This large multicenter study provided a comprehensive insight into the clinical, laboratory, and genetic history of patients with CFDs. We conclude that bleeding severity grades were in agreement with the established factor activity threshold in nearly half of the cases with quantitative defects.

Ideal marker for targeted axillary dissection (IMTAD): a prospective multicentre trial.

BACKGROUND: Targeted axillary dissection (TAD) is an established method for axillary staging in patients with breast cancer after neoadjuvant chemotherapy (NAC). TAD consists of sentinel lymph node biopsy and initially pathological lymph node excision, which must be marked by a reliable marker before NAC. METHODS: The IMTAD study is a prospective multicentre trial comparing three localisation markers for lymph node localisation (clip + iodine seed, magnetic seed, carbon suspension) facilitating subsequent surgical excision in the form of TAD. The primary outcome was to prospectively compare the reliability, accuracy, and safety according to complication rate during marker implantation and detection and marker dislodgement. RESULTS: One hundred eighty-nine patients were included in the study-in 135 patients clip + iodine seed was used, in 30 patients magnetic seed and in 24 patients carbon suspension. The complication rate during the marker implantation and detection were not statistically significant between individual markers (p = 0.263; p = 0.117). Marker dislodgement was reported in 4 patients with clip + iodine seed localisation (3.0%), dislodgement did not occur in other localisation methods (p = 0.999). The false-negativity of sentinel lymph node (SLN) was observed in 8 patients, the false-negativity of targeted lymph nodes (TLN) wasn´t observed at all, the false-negativity rate (FNR) from the subcohort of ypN + patients for SLN is 9.6% and for TLN 0.0%. CONCLUSION: The IMTAD study indicated, that clip + iodine seed, magnetic seed and carbon suspension are statistically comparable in terms of complications during marker implantation and detection and marker dislodgement proving their safety, accuracy, and reliability in TAD. The study confirmed, that the FNR of the TLN was lower than the FNR of the SLN proving that the TLN is a better marker for axillary lymph node status after NAC. TRIAL REGISTRATION: NCT04580251. Name of registry: Clinicaltrials.gov. Date of registration: 8.10.2020.

Myeloid lineage cells evince distinct steady-state level of certain gene groups in dependence on hereditary angioedema severity.

Hereditary angioedema (HAE) is a rare genetic disorder with variable expressivity even in carriers of the same underlying genetic defect, suggesting other genetic and epigenetic factors participate in modifying HAE severity. Recent knowledge indicates the role of immune cells in several aspects of HAE pathogenesis, which makes monocytes and macrophages candidates to mediate these effects. Here we combined a search for HAE phenotype modifying gene variants with the characterization of selected genes' mRNA levels in monocyte and macrophages in a symptom-free period. While no such gene variant was found to be associated with a more severe or milder disease, patients revealed a higher number of dysregulated genes and their expression profile was significantly altered, which was typically manifested by changes in individual gene expression or by strengthened or weakened relations in mutually co-expressed gene groups, depending on HAE severity. SERPING1 showed decreased expression in HAE-C1INH patients, but this effect was significant only in patients carrying mutations supposedly activating nonsense-mediated decay. Pro-inflammatory CXC chemokine superfamily members CXCL8, 10 and 11 were downregulated, while other genes such as FCGR1A, or long non-coding RNA NEAT1 were upregulated in patients. Co-expression within some gene groups (such as an NF-kappaB function related group) was strengthened in patients with a severe and/or mild course compared to controls. All these findings show that transcript levels in myeloid cells achieve different activation or depression levels in HAE-C1INH patients than in healthy controls and/or based on disease severity and could participate in determining the HAE phenotype.

Comparison of 3 Different Systems for Non-wire Localization of Lesions in Breast Cancer Surgery.

PURPOSE: Localizing breast lesions by marking tumors and their detection using probes during surgery is a common part of clinical practice. Various nonwire localization systems were intended to be compared from different perspectives. METHODS: Various measurement experiments were performed. Localization techniques, including radioactive seed (RSLS), magnetically guided (MGLS), or radar (SLS), were compared in signal propagation in water and tissue environments, signal interference by surgical instruments, and the practical experience of surgeons. Individual experiments were thoroughly prospectively planned. RESULTS: The RSLS signal was detectable at the largest evaluated distance, ie, 60 mm. The SLS and MGLS signal detection was shorter, up to 25 mm to 45 mm and 30 mm, respectively. The signal intensity and the maximum detection distance in water differed slightly depending on the localization marker orientation to the probe, especially for SLS and MGLS. Signal propagation in the tissue was noted to a depth of 60 mm for RSLS, 50 mm for SLS, and 20 mm for MGLS. Except for the expected signal interferences by approaching surgical instruments from any direction for MGLS, the signal interruption for RSLS and SLS was observed only by inserting instruments directly between the localization marker and probe. Moreover, the SLS signal interference by instrument touch was noted. Based on surgeons' results, individual systems did not differ significantly for most measurement condition settings. CONCLUSION: Apparent differences noted among localization systems can help experts choose an appropriate system for a specific situation or reveal small nuances that have not yet been observed in clinical practice.

Analysis of von Willebrand Disease in the "Heart of Europe".

Background von Willebrand disease (VWD) is a genetic bleeding disorder caused by defects of von Willebrand factor (VWF), quantitative (type 1 and 3) or qualitative (type 2). The laboratory phenotyping is heterogenic making diagnosis difficult. Objectives Complete laboratory analysis of VWD as an expansion of the previously reported cross-sectional family-based VWD study in the Czech Republic (BRNO-VWD) and Slovakia (BRA-VWD) under the name "Heart of Europe," in order to improve the understanding of laboratory phenotype/genotype correlation. Patients and Methods In total, 227 suspected VWD patients were identified from historical records. Complete laboratory analysis was established using all available assays, including VWF multimers and genetic analysis. Results A total of 191 patients (from 119 families) were confirmed as having VWD. The majority was characterized as a type 1 VWD, followed by type 2. Multimeric patterns concordant with laboratory phenotypes were found in approximately 83% of all cases. A phenotype/genotype correlation was present in 84% (77% type 1, 99% type 2, and 61% type 3) of all patients. Another 45 candidate mutations (23 novel variations), not found in the initial study, could be identified (missense 75% and truncating 24%). An exon 1-3 gene deletion was identified in 14 patients where no mutation was found by direct DNA sequencing, increasing the linkage up to 92%, overall. Conclusion This study provides a cross-sectional overview of the VWD population in a part of Central Europe. It is an addition to the previously published BRNO-VWD study, and provides important data to the International Society of Thrombosis and Haemostasis/European Association for Haemophilia and Allied Disorders VWD mutation database with identification of novel causal mutations.

Biomarker Dynamics and Long-Term Treatment Outcomes in Breast Cancer Patients with Residual Cancer Burden after Neoadjuvant Therapy.

A residual cancer burden after neoadjuvant therapy (NAT) for breast cancer (BC) is associated with worse treatment outcomes compared to patients who achieved pathologic complete remission. This single-institutional retrospective study of 767 consecutive patients, including 468 patients with assessable residual cancer burden (aRCB) after NAT, with a median follow-up of 36 months, evaluated the biomarkers assessed before NAT from a biopsy and after NAT from a surgical specimen, their dynamics, and effect on long-term outcomes in specific breast cancer subtypes. The leading focus was on proliferation index Ki-67, which was significantly altered by NAT in all BC subtypes (p < 0.001 for HER2 positive and luminal A/B HER2 negative and p = 0.001 for TNBC). Multivariable analysis showed pre-NAT and post-NAT Ki-67 as independent predictors of survival outcomes for luminal A/B HER2 negative subtype. For TNBC, post-NAT Ki-67 was significant alone, and, for HER2 positive, the only borderline association of pre-NAT Ki-67 was observed in relation to the overall survival. Steroid and HER2 receptors were re-assessed just in a portion of the patients with aRCB. The concordance of both assessments was 92.9% for ER status, 80.1% for PR, and 92.2% for HER2. In conclusion, these real-world data of a consecutive cohort confirmed the importance of biomarkers assessment in patients with aRCB, and the need to consider specific BC subtypes when interpreting their influence on prognosis.

Deregulation of signaling pathways controlling cell survival and proliferation in cancer cells alters induction of cytochrome P450 family 1 enzymes.

Cytochrome P450 family 1 (CYP1) enzymes contribute both to metabolism of xenobiotics and to the control of endogenous levels of ligands of the aryl hydrocarbon receptor (AhR). Their activities, similar to other CYPs, can be altered in tumor tissues. Here, we examined a possible role of proliferative/survival pathways signaling, which is often deregulated in tumor cells, and possible links with p300 histone acetyltransferase (a transcriptional co-activator) in the control of CYP1 expression, focusing particularly on CYP1A1. Using cell models derived from human liver, we observed that the induction of CYP1A1 expression, as well as other CYP1 enzymes, was reduced in exponentially growing cells, as compared with their non-dividing counterparts. The siRNA-mediated inhibition of proliferation/pro-survival signaling pathway effectors (such as ß-catenin and/or Hippo pathway effectors YAP/TAZ) increased the AhR ligand-induced CYP1A1 mRNA levels in liver HepaRG cells, and/or in colon carcinoma HCT-116 cells. The activation of proliferative Wnt/ß-catenin signaling in HCT-116 cells reduced both the induction of CYP1 enzymes and the binding of p300 to the promoter of CYP1A1 or CYP1B1 genes. These results seem to indicate that aberrant proliferative signaling in tumor cells could suppress induction of CYP1A1 (or other CYP1 enzymes) via competition for p300 binding. This mechanism could be involved in modulation of the metabolism of both endogenous and exogenous substrates of CYP1A1 (and other CYP1 enzymes), with possible further consequences for alterations of the AhR signaling in tumor cells, or additional functional roles of CYP1 enzymes.

Complex Alterations of Fatty Acid Metabolism and Phospholipidome Uncovered in Isolated Colon Cancer Epithelial Cells.

The development of colon cancer, one of the most common malignancies, is accompanied with numerous lipid alterations. However, analyses of whole tumor samples may not always provide an accurate description of specific changes occurring directly in tumor epithelial cells. Here, we analyzed in detail the phospholipid (PL), lysophospholipid (lysoPL), and fatty acid (FA) profiles of purified EpCAM+ cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients. We found that a number of FAs increased significantly in isolated tumor cells, which also included a number of long polyunsaturated FAs. Higher levels of FAs were associated with increased expression of FA synthesis genes, as well as with altered expression of enzymes involved in FA elongation and desaturation, including particularly fatty acid synthase, stearoyl-CoA desaturase, fatty acid desaturase 2 and ELOVL5 fatty acid elongase 5 We identified significant changes in ratios of specific lysoPLs and corresponding PLs. A number of lysophosphatidylcholine and lysophosphatidylethanolamine species, containing long-chain and very-long chain FAs, often with high numbers of double bonds, were significantly upregulated in tumor cells. Increased de novo synthesis of very long-chain FAs, or, altered uptake or incorporation of these FAs into specific lysoPLs in tumor cells, may thus contribute to reprogramming of cellular phospholipidome and membrane alterations observed in colon cancer.

The reliability of bone marrow cytology as response criterion in metastatic neuroblastoma.

BACKGROUND: The quantitative assessment of neuroblastoma cell content in bone marrow aspirates for response evaluation has been introduced recently. Data on the concordance of interobserver reports are lacking so far. METHODS: Investigators of seven European countries representing national reference or large oncological centers convened in 2016. They agreed to quantitatively assess routine bone marrow smears of the participating institutions and to discuss the discrepant results in joint meetings. RESULTS: From 2017 through 2019, three cytology rounds with 24, 28, and 28 bone marrow samples were run evaluating the representativity of the smears (yes/[restricted]/no) and the presence of tumor cells (yes/no and %). The comparison of the reports using κ (Fleiss) and α (Krippendorff) statistics demonstrated no robust reliabilities. The agreement on the representativity was moderate to poor, on the presence of tumor cells moderate to good, and on the percentage of tumor cells slight to moderate. Though the value of cytology is unquestioned to detect even tiny metastatic cells in bone marrow, the investigators unanimously agreed that a reliable quantification of the tumor cell content in bone marrow smears is unrealistic. For the key issue of representativity, a new practical definition was developed. CONCLUSION: For any work with bone marrow aspirates, the representativity of the material is of paramount importance. A practical definition is proposed. A reliable quantitative cytological assessment of tumor cell content in bone marrow aspirates is not feasible in metastatic neuroblastoma. Therefore, its use as response criterion should be reconsidered.

Safety of dabigatran etexilate for the secondary prevention of venous thromboembolism in children.

This open-label, single-arm, prospective cohort trial is the first phase 3 safety study to describe outcomes in children treated with dabigatran etexilate for secondary venous thromboembolism (VTE) prevention. Eligible children aged 12 to <18 years (age stratum 1), 2 to <12 years (stratum 2), and >3 months to <2 years (stratum 3) had an objectively confirmed diagnosis of VTE treated with standard of care (SOC) for ≥3 months, or had completed dabigatran or SOC treatment in the DIVERSITY trial (NCT01895777) and had an unresolved clinical thrombosis risk factor requiring further anticoagulation. Children received dabigatran for up to 12 months, or less if the identified VTE clinical risk factor resolved. Primary end points included VTE recurrence, bleeding events, and mortality at 6 and 12 months. Overall, 203 children received dabigatran, with median exposure being 36.3 weeks (range, 0-57 weeks); 171 of 203 (84.2%) and 32 of 203 (15.8%) took capsules and pellets, respectively. Overall, 2 of 203 children (1.0%) experienced on-treatment VTE recurrence, and 3 of 203 (1.5%) experienced major bleeding events, with 2 (1.0%) reporting clinically relevant nonmajor bleeding events, and 37 (18.2%) minor bleeding events. There were no on-treatment deaths. On-treatment postthrombotic syndrome was reported for 2 of 162 children (1.2%) who had deep vein thrombosis or central-line thrombosis as their most recent VTE. Pharmacokinetic/pharmacodynamic relationships of dabigatran were similar to those in adult VTE patients. In summary, dabigatran showed a favorable safety profile for secondary VTE prevention in children aged from >3 months to <18 years with persistent VTE risk factor(s). This trial was registered at www.clinicaltrials.gov as #NCT02197416.

Novel SAMD9 Mutation in a Patient With Immunodeficiency, Neutropenia, Impaired Anti-CMV Response, and Severe Gastrointestinal Involvement.

Mutations in the Sterile alpha motif domain containing 9 (SAMD9) gene have been described in patients with severe multisystem disorder, MIRAGE syndrome, but also in patients with bone marrow (BM) failure in the absence of other systemic symptoms. The role of hematopoietic stem cell transplantation (HSCT) in the management of the disease is still unclear. Here, we present a patient with a novel mutation in SAMD9 (c.2471 G>A, p.R824Q), manifesting with prominent gastrointestinal tract involvement and immunodeficiency, but without any sign of adrenal insufficiency typical for MIRAGE syndrome. He suffered from severe CMV (cytomegalovirus) infection at 3 months of age, with a delayed development of T lymphocyte functional response against CMV, profound T cell activation, significantly reduced B lymphocyte counts and impaired lymphocyte proliferative response. Cultured T cells displayed slightly lower calcium flux and decreased survival. At the age of 6 months, he developed severe neutropenia requiring G-CSF administration, and despite only mild morphological and immunophenotypical disturbances in the BM, 78% of the BM cells showed monosomy 7 at the age of 18 months. Surprisingly, T cell proliferation after CD3 stimulation and apoptosis of the cells normalized during the follow-up, possibly reflecting the gradual development of monosomy 7. Among other prominent symptoms, he had difficulty swallowing, requiring percutaneous endoscopic gastrostomy (PEG), frequent gastrointestinal infections, and perianal erosions. He suffered from repeated infections and periodic recurring fevers with the elevation of inflammatory markers. At 26 months of age, he underwent HSCT that significantly improved hematological and immunological laboratory parameters. Nevertheless, he continued to suffer from other conditions, and subsequently, he died at day 440 post-transplant due to sepsis. Pathogenicity of this novel SAMD9 mutation was confirmed experimentally. Expression of mutant SAMD9 caused a significant decrease in proliferation and increase in cell death of the transfected cells. Conclusion: We describe a novel SAMD9 mutation in a patient with prominent gastrointestinal and immunological symptoms but without adrenal hypoplasia. Thus, SAMD9 mutations should be considered as cause of enteropathy in pediatric patients. The insufficient therapeutic outcome of transplantation further questions the role of HSCT in the management of patients with SAMD9 mutations and multisystem involvement.

Analysis of von Willebrand Disease in the South Moravian Population (Czech Republic): Results from the BRNO-VWD Study.

BACKGROUND: von Willebrand disease (VWD) is an inherited bleeding disorder caused by a quantitative (type 1 and 3) or qualitative (type 2) defect of von Willebrand factor (VWF). The heterogeneity of laboratory phenotyping makes diagnosing difficult. OBJECTIVE: A cross-sectional, family-based VWD study in a collaboration between University Hospital Brno (Czech Republic) and Antwerp University Hospital (Belgium) to improve the understanding of laboratory phenotype/genotype correlation. PATIENTS AND METHODS: A total of 205 patients with suspected VWD were identified from historical records. Complete laboratory analysis was established using all available VWD assays including VWF multimers and genetic analysis. RESULTS: Based on the current International Society of Thrombosis and Haemostasis (ISTH) - Scientific and Standardization Committee VWD classification and type 2A sub-division into 2A/IIA, IID, IIC and IIE, the majority was characterized as a type 1 VWD, followed by type 2. Proposed laboratory phenotypes were confirmed by their multimeric pattern within 98% of this cohort. All type 2, 3 and 75% of type 1 VWD patients were confirmed by underlying causative mutations. Forty-six different causal mutations (117 not previously described in the literature) could be identified. Fifty per cent of all cases was represented by eight individual mutations, mainly p.Pro812ArgfsX31. Thirteen patients had a large heterozygous gene alteration. CONCLUSION: Although an extensive panel of tests was used, VWD classification and (sub)typing remains difficult and fluid. This study provides a cross-sectional overview of the VWD population in the Czech Republic and provides important data to the ISTH/European Association for Haemophilia and Allied Disorders VWD mutation database in linking causal mutations with unique VWD (sub)types. It also identifies new, as not previously described in the literature, causal mutations.

In vitro and in vivo genotoxicity of oxygenated polycyclic aromatic hydrocarbons.

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are a group of environmental pollutants found in complex mixtures together with PAHs. In contrast to the extensively studied PAHs, which have been established to have mutagenic and carcinogenic properties, much less is known about the effects of oxy-PAHs. The present work aimed to investigate the genotoxic potency of a set of environmentally relevant oxy-PAHs along with environmental soil samples in human bronchial epithelial cells (HBEC). We found that all oxy-PAHs tested induced DNA strand breaks in a dose-dependent manner and some of the oxy-PAHs further induced micronuclei formation. Our results showed weak effects in response to the oxy-PAH containing subfraction of the soil sample. The genotoxic potency was confirmed in both HBEC and HepG2 cells following exposure to oxy-PAHs by an increased level of phospho-Chk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro. We further exposed zebrafish embryos to single oxy-PAHs or a binary mixture with PAH benzo[a]pyrene (B[a]P) and found the mixture to induce comparable or greater effects on the induction of DNA strand breaks compared to the sum of that induced by B[a]P and oxy-PAHs alone. In conclusion, oxy-PAHs were found to elicit genotoxic effects at similar or higher levels to that of B[a]P which indicates that oxy-PAHs may contribute significantly to the total carcinogenic potency of environmental PAH mixtures. This emphasizes further investigations of these compounds as well as the need to include oxy-PAHs in environmental monitoring programs in order to improve health risk assessment.

Butyrate interacts with benzo[a]pyrene to alter expression and activities of xenobiotic metabolizing enzymes involved in metabolism of carcinogens within colon epithelial cell models.

Butyrate helps to maintain colon homeostasis and exhibits chemopreventive effects in colon epithelium. We examined the interactive effects of butyrate and benzo[a]pyrene (BaP), dietary carcinogen, in regulation of expression of a panel of phase I and II xenobiotic metabolizing enzymes (XMEs) in human colon cells. In human colon carcinoma HCT-116 and HT-29 cell lines, butyrate alone increased mRNA levels of some enzymes, such as N-acetyltransferases (in particular NAT2). In combination with BaP, butyrate potentiated induction of cytochrome P450 family 1 enzymes (CYP1A1), aldo-keto reductases (AKR1C1) or UDP-glucuronosyltransferases (UGT1A1). There were some notable differences between cell lines, as butyrate potentiated induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) and UGT1A4 only in HCT-116 cells, and it even repressed AKR1C3 induction in HT-29 cells. Butyrate also promoted induction of CYP1, NQO1, NAT2, UGT1A1 or UGT1A4 in human colon Caco-2 cells, in a differentiation-dependent manner. Differentiated Caco-2 cells exhibited a higher inducibility of selected XME genes than undifferentiated cells. Butyrate increased induction of enzymatic activities of NATs, NQO1 and UGTs by BaP in HCT-116 and HT29 cells, whereas in differentiated Caco-2 cells it helped to increase only enzymatic activity of NQO1 and UGTs. Together, the present data suggest that butyrate may modulate expression/activities of several enzymes involved in metabolism of carcinogens in colon. In some cases (NAT2, UGT1 A1), this was linked to inhibition of histone deacetylases (HDAC), as confirmed by using HDAC inhibitor trichostatin A. These results may have implications for our understanding of the role of butyrate in regulation of XMEs and carcinogen metabolism in colon.

Aryl Hydrocarbon Receptor-Dependent Metabolism Plays a Significant Role in Estrogen-Like Effects of Polycyclic Aromatic Hydrocarbons on Cell Proliferation.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that interact in a complex manner with both the aryl hydrocarbon receptor (AhR) and estrogen receptors (ER). Their potential endocrine-disrupting activities may depend on both inhibitory AhR-ER cross-talk and on AhR-dependent metabolic production of estrogenic PAH metabolites. Here, we analyzed the impact of AhR on estrogen-like effects of PAHs, such as benzo[a]pyrene (BaP), in particular, on control of cell cycle progression/cell proliferation. Using AhR knockout variant of estrogen-sensitive human breast cancer MCF-7 cells (MCF-7 AhRKO cells), we observed that the AhR-dependent control of cytochrome P450 family 1 (CYP1) expression played a major role in formation of estrogenic BaP metabolites, most notably 3-OH-BaP, which contributed to the ER-dependent induction of cell cycle progression/cell proliferation. Both BaP metabolism and the BaP-induced S-phase transition/cell proliferation were inhibited in MCF-7 AhRKO cells, whereas these cells remained sensitive towards both endogenous estrogen 17ß-estradiol or hydroxylated BaP metabolites. BaP was found to increase the activity of ER-dependent luciferase reporter gene in wild-type MCF-7 cells; however, unlike its hydroxylated metabolite, BaP failed to stimulate luciferase activity in MCF-7 AhRKO cells. Similarly, estrogen-like effects of other known estrogenic PAHs, such as benz[a]anthracene or 3-methylcholanthrene, were diminished in MCF-7 AhRKO cells. Ectopic expression of human CYP1A1 and CYP1B1 enzymes partly restored both BaP metabolism and its effects on cell proliferation. Taken together, our data suggest that the AhR-dependent metabolism of PAHs contributes significantly to the impact of PAHs on cell proliferation in estrogen-sensitive cells.

CEBPE-Mutant Specific Granule Deficiency Correlates With Aberrant Granule Organization and Substantial Proteome Alterations in Neutrophils.

Specific granule deficiency (SGD) is a rare disorder characterized by abnormal neutrophils evidenced by reduced granules, absence of granule proteins, and atypical bilobed nuclei. Mutations in CCAAT/enhancer-binding protein-ε (CEBPE) are one molecular etiology of the disease. Although C/EBPε has been studied extensively, the impact of CEBPE mutations on neutrophil biology remains elusive. Here, we identified two SGD patients bearing a previously described heterozygous mutation (p.Val218Ala) in CEBPE. We took this rare opportunity to characterize SGD neutrophils in terms of granule distribution and protein content. Granules of patient neutrophils were clustered and polarized, suggesting that not only absence of specific granules but also defects affecting other granules contribute to the phenotype. Our analysis showed that remaining granules displayed mixed protein content and lacked several glycoepitopes. To further elucidate the impact of mutant CEBPE, we performed detailed proteomic analysis of SGD neutrophils. Beside an absence of several granule proteins in patient cells, we observed increased expression of members of the linker of nucleoskeleton and cytoskeleton complex (nesprin-2, vimentin, and lamin-B2), which control nuclear shape. This suggests that absence of these proteins in healthy individuals might be responsible for segmented shapes of neutrophilic nuclei. We further show that the heterozygous mutation p.Val218Ala in CEBPE causes SGD through prevention of nuclear localization of the protein product. In conclusion, we uncover that absence of nuclear C/EBPε impacts on spatiotemporal expression and subsequent distribution of several granule proteins and further on expression of proteins controlling nuclear shape.

Butyrate alters expression of cytochrome P450 1A1 and metabolism of benzo[a]pyrene via its histone deacetylase activity in colon epithelial cell models.

Butyrate, a short-chain fatty acid produced by fermentation of dietary fiber, is an important regulator of colonic epithelium homeostasis. In this study, we investigated the impact of this histone deacetylase (HDAC) inhibitor on expression/activity of cytochrome P450 family 1 (CYP1) and on metabolism of carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in colon epithelial cells. Sodium butyrate (NaBt) strongly potentiated the BaP-induced expression of CYP1A1 in human colon carcinoma HCT116 cells. It also co-stimulated the 7-ethoxyresorufin-O-deethylase (EROD) activity induced by the 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical ligand of the aryl hydrocarbon receptor. Up-regulation of CYP1A1 expression/activity corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts. NaBt significantly potentiated CYP1A1 induction and/or metabolic activation of BaP also in other human colon cell models, colon adenoma AA/C1 cells, colon carcinoma HT-29 cells, or in NCM460D cell line derived from normal colon mucosa. Our results suggest that the effects of NaBt were due to its impact on histone acetylation, because additional HDAC inhibitors (trichostatin A and suberanilohydroxamic acid) likewise increased both the induction of EROD activity and formation of covalent DNA adducts. NaBt-induced acetylation of histone H3 (at Lys14) and histone H4 (at Lys16), two histone modifications modulated during activation of CYP1A1 transcription, and it reduced binding of HDAC1 to the enhancer region of CYP1A1 gene. This in vitro study suggests that butyrate, through modulation of histone acetylation, may potentiate induction of CYP1A1 expression, which might in turn alter the metabolism of BaP within colon epithelial cells.

Loss of B cells and their precursors is the most constant feature of GATA-2 deficiency in childhood myelodysplastic syndrome.

GATA-2 deficiency was recently described as common cause of overlapping syndromes of immunodeficiency, lymphedema, familiar myelodysplastic syndrome or acute myeloid leukemia. The aim of our study was to analyze bone marrow and peripheral blood samples of children with myelodysplastic syndrome or aplastic anemia to define prevalence of the GATA2 mutation and to assess whether mutations in GATA-2 transcription factor exhibit specific immunophenotypic features. The prevalence of a GATA2 mutation in a consecutively diagnosed cohort of children was 14% in advanced forms of myelodysplastic syndrome (refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and myelodysplasia-related acute myeloid leukemia), 17% in refractory cytopenia of childhood, and 0% in aplastic anemia. In GATA-2-deficient cases, we found the most profound B-cell lymphopenia, including its progenitors in blood and bone marrow, which correlated with significantly diminished intronRSS-Kde recombination excision circles in comparison to other myelodysplastic syndrome/aplastic anemia cases. The other typical features of GATA-2 deficiency (monocytopenia and natural killer cell lymphopenia) were less discriminative. In conclusion, we suggest screening for GATA2 mutations in pediatric myelodysplastic syndrome, preferentially in patients with impaired B-cell homeostasis in bone marrow and peripheral blood (low number of progenitors, intronRSS-Kde recombination excision circles and naïve cells).

Inhibition of ß-catenin signalling promotes DNA damage elicited by benzo[a]pyrene in a model of human colon cancer cells via CYP1 deregulation.

Deregulation of Wnt/ß-catenin signalling plays an important role in the pathogenesis of colorectal cancer. Interestingly, this pathway has been recently implicated in transcriptional control of cytochrome P450 (CYP) family 1 enzymes, which are responsible for bioactivation of a number of dietary carcinogens. In the present study, we investigated the impact of inhibition of Wnt/ß-catenin pathway on metabolism and genotoxicity of benzo[a]pyrene (BaP), a highly mutagenic polycyclic aromatic hydrocarbon and an efficient ligand of the aryl hydrocarbon receptor, which is known as a primary regulator of CYP1 expression, in cellular models derived from colorectal tumours. We observed that a synthetic inhibitor of ß-catenin, JW74, significantly increased formation of BaP-induced DNA adducts in both colorectal adenoma and carcinoma-derived cell lines. Using the short interfering RNA (siRNA) targeting ß-catenin, we then found that ß-catenin knockdown in HCT116 colon carcinoma cells significantly enhanced formation of covalent DNA adducts by BaP and histone H2AX phosphorylation, as detected by (32)P-postlabelling technique and immunocytochemistry, respectively, and it also induced expression of DNA damage response genes, such as CDKN1A or DDB2. The increased formation of DNA adducts formed by BaP upon ß-catenin knockdown corresponded with enhanced production of major BaP metabolites, as well as with an increased expression/activity of CYP1 enzymes. Finally, using siRNA-mediated knockdown of CYP1A1, we confirmed that this enzyme plays a major role in formation of BaP-induced DNA adducts in HCT116 cells. Taken together, the present results indicated that the siRNA-mediated inhibition of ß-catenin signalling, which is aberrantly activated in a majority of colorectal cancers, modulated genotoxicity of dietary carcinogen BaP in colon cell model in vitro, via a mechanism involving up-regulation of CYP1 expression and activity.