von Willebrand disease: proposing definitions for future research

Thrombosis and Hemostasi

A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples

The bisulfite genomic sequencing protocol is a widely used method for analyzing DNA methylation. It relies on the deamination of unmethylated cytosine residues to uracil; however, its high rates of DNA degradation and incomplete cytosine to uracil conversion often lead to failed experiments, uninformative results, and false positives. Here, we report the addition of a single-step multiple restriction enzyme digestion (MRED) designed to differentially digest polymerase chain reaction products amplified from unconverted DNA while leaving those of converted DNA intact. We show that for our model system, RARB2 P2 promoter, use of MRED increased informative sequencings ninefold, and MRED did not alter the clonal representation in one fully methylated cell line, H-596, treated or not with 5-azadeoxycytidine, a methylation inhibitor. We believe that this method may easily be adapted for analyzing other genes and provide guidelines for selecting the most appropriate MRED restriction enzymes

Surgical management of patients with von Willebrand disease: summary of 2 systematic reviews of the literature

von Willebrand disease (VWD) is the most common inherited bleeding disorder. The management of patients with VWD who are undergoing surgeries is crucial to prevent bleeding complications. We systematically summarized the evidence on the management of patients with VWD who are undergoing major and minor surgeries to support the development of practice guidelines. We searched Medline and EMBASE from inception through October 2019 for randomized clinical trials (RCTs), comparative observational studies, and case series that compared maintaining factor VIII (FVIII) levels or von Willebrand factor (VWF) levels at >0.50 IU/mL for at least 3 days in patients undergoing major surgery, and those with options for perioperative management of patients undergoing minor surgery. Two authors screened and abstracted data and assessed the risk of bias. We conducted meta-analyses when possible. We evaluated the certainty of the evidence using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) approach. We included 7 case series for major surgeries and 2 RCTs and 12 case series for minor surgeries. Very-low-certainty evidence showed that maintaining FVIII levels or VWF levels of >0.50 IU/mL for at least 3 consecutive days showed excellent hemostatic efficacy (as labeled by the researchers) after 74% to 100% of major surgeries. Low- to very-low-certainty evidence showed that prescribing tranexamic acid and increasing VWF levels to 0.50 IU/mL resulted in fewer bleeding complications after minor procedures compared with increasing VWF levels to 0.50 IU/mL alone. Given the low-quality evidence for guiding management decisions, a shared-decision model leading to individualized therapy plans will be important in patients with VWD who are undergoing surgical and invasive procedures

Novel retinoic acid metabolism blocking agents have potent inhibitory activities on human breast cancer cells and tumour growth

Antitumour effects of retinoids are attributed to their influence on cell proliferation, differentiation, apoptosis and angiogenesis. In our effort to develop useful agents for breast cancer therapy, we evaluated the effects of four representative retinoic acid metabolism blocking agents (RAMBAs, VN/14-1, VN/50-1, VN/66-1 and VN/69-1) on growth inhibition of oestrogen receptor positive (ER +ve, MCF-7 and T-47D) and oestrogen receptor negative (ER −ve, MDA-MB-231) human breast cancer cells. Additionally, we investigated the biological effects/molecular mechanism(s) underlying their growth inhibitory properties as well as their antitumour efficacies against MCF-7 and MCF-7Ca tumour xenografts in nude mice. We also assessed the effect of combining VN/14-1 and all-trans-retinoic acid (ATRA) on MCF-7 tumuor xenografts. The ER +ve cell lines were more sensitive (IC50 values between 3.0 and 609 nM) to the RAMBAs than the ER −ve MDA-MB-231 cell line (IC50=5.6–24.0 μM). Retinoic acid metabolism blocking agents induced cell differentiation as determined by increased expression of cytokeratin 8/18 and oestrogen receptor-α (ER-α). Similar to ATRA, they also induced apoptosis via activation of caspase 9. Cell cycle analysis indicated that RAMBAs arrested cells in the G1 and G2/M phases and caused significant downregulation (>80%) of cyclin D1 protein. In vivo, the growth of MCF-7 mammary tumours was dose-dependently and significantly inhibited (92.6%, P<0.0005) by VN/14-1. The combination of VN/14-1 and ATRA also inhibited MCF-7 breast tumour growth in vivo (up to 120%) as compared with single agents (P<0.025). VN/14-1 was also very effective in preventing the formation of MCF-7Ca tumours and it significantly inhibited the growth of established MCF-7Ca tumours, being as effective as the clinically used aromatase inhibitors, anastrozole and letrozole. Decrease in cyclin D1 and upregulation of cytokeratins, Bad and Bax with VN/14-1 may be responsible for the efficacy of this compound in inhibiting breast cancer cell growth in vitro and in vivo. Our results suggest that our RAMBAs, especially VN/14-1 may be useful novel therapy for breast cancer

The effect of body weight on altered expression of nuclear receptors and cyclooxygenase-2 in human colorectal cancers

<p>Abstract</p> <p>Background</p> <p>Epidemiological studies on risk factors for colorectal cancer (CRC) have mainly focused on diet, and being overweight is now recognized to contribute significantly to CRC risk. Overweight and obesity are defined as an excess of adipose tissue mass and are associated with disorders in lipid metabolism. Peroxisome proliferator-activated receptors (PPARs) and retinoid-activated receptors (RARs and RXRs) are important modulators of lipid metabolism and cellular homeostasis. Alterations in expression and activity of these ligand-activated transcription factors might be involved in obesity-associated diseases, which include CRC. Cyclooxygenase-2 (COX-2) also plays a critical role in lipid metabolism and alterations in COX-2 expression have already been associated with unfavourable clinical outcomes in epithelial tumors. The objective of this study is to examine the hypothesis questioning the relationship between alterations in the expression of nuclear receptors and COX-2 and the weight status among male subjects with CRC.</p> <p>Method</p> <p>The mRNA expression of the different nuclear receptor subtypes and of COX-2 was measured in 20 resected samples of CRC and paired non-tumor tissues. The association between expression patterns and weight status defined as a body mass index (BMI) was statistically analyzed.</p> <p>Results</p> <p>No changes were observed in PPARγ mRNA expression while the expression of PPARδ, retinoid-activated receptors and COX-2 were significantly increased in cancer tissues compared to normal colon mucosa (<it>P </it>≤ 0.001). The weight status appeared to be an independent factor, although we detected an increased level of COX-2 expression in the normal mucosa from overweight patients (BMI ≥ 25) compared to subjects with healthy BMI (<it>P </it>= 0.002).</p> <p>Conclusion</p> <p>Our findings show that alterations in the pattern of nuclear receptor expression observed in CRC do not appear to be correlated with patient weight status. However, the analysis of COX-2 expression in normal colon mucosa from subjects with a high BMI suggests that COX-2 deregulation might be driven by excess weight during the colon carcinogenesis process.</p

ASH ISTH NHF WFH 2021 guidelines on the management of von Willebrand disease

Background: Von Willebrand disease (VWD) is a common inherited bleeding disorder. Significant variability exists in management options offered to patients. Objective: These evidence-based guidelines from the American Society of Hematology (ASH), the International Society on Thrombosis and Haemostasis (ISTH), the National Hemophilia Foundation (NHF), and the World Federation of Hemophilia (WFH) are intended to support patients, clinicians, and health care professionals in their decisions about management of VWD. Methods: ASH, ISTH, NHF, and WFH formed a multidisciplinary guideline panel. Three patient representatives were included. The panel was balanced to minimize potential bias from conflicts of interest. The University of Kansas Outcomes and Implementation Research Unit and the McMaster Grading of Recommendations Assessment, Development and Evaluation (GRADE) Centre supported the guideline development process, including performing and updating systematic evidence reviews (through November 2019). The panel prioritized clinical questions and outcomes according to their importance to clinicians and patients. The panel used the GRADE approach, including GRADE Evidence-to-Decision frameworks, to assess evidence and make recommendations, which were subject to public comment. Results: The panel agreed on 12 recommendations and outlined future research priorities. Conclusions: These guidelines make key recommendations regarding prophylaxis for frequent recurrent bleeding, desmopressin trials to determine therapy, use of antiplatelet agents and anticoagulant therapy, target VWF and factor VIII activity levels for major surgery, strategies to reduce bleeding during minor surgery or invasive procedures, management options for heavy menstrual bleeding, management of VWD in the context of neuraxial anesthesia during labor and delivery, and management in the postpartum setting

Outcomes of long-term von Willebrand factor prophylaxis use in von Willebrand disease: A systematic literature review

Background Von Willebrand Disease (VWD) is a common inherited bleeding disorder. Patients with VWD suffering from severe bleeding may benefit from the use of secondary long-term prophylaxis. Aim Systematically summarize the evidence on the clinical outcomes of secondary long-term prophylaxis in patients with VWD and severe recurrent bleedings. Methods We searched Medline and EMBASE through October 2019 for relevant randomized clinical trials (RCTs) and comparative observational studies (OS) assessing the effects of secondary long-term prophylaxis in patients with VWD. We used Cochrane Risk of Bias (RoB) tool and the RoB for Non-Randomized Studies of interventions (ROBINS-I) tool to assess the quality of the included studies. We conducted random-effects meta-analyses and assessed the certainty of the evidence using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. Results We included 12 studies. Evidence from one placebo controlled RCT suggested that VWD prophylaxis as compared to no prophylaxis reduced the rate of bleeding episodes (Rate ratio [RR], .24; 95% confidence interval [CI], .17–.35; low certainty evidence), and of epistaxis (RR, .38; 95%CI, .21–.67; moderate certainty evidence), and may increase serious adverse events RR 2.73 (95%CI .12–59.57; low certainty). Evidence from four before-and-after studies in which researchers reported comparative data suggested that VWD prophylaxis reduced the rate of bleeding (RR .34; 95%CI, .25–.46; very low certainty evidence). Conclusion VWD prophylaxis treatment seems to reduce the risk of spontaneous bleeding, epistaxis, and hospitalizations. More RCTs should be conducted to increase the certainty in these benefits

ASH ISTH NHF WFH 2021 guidelines on the management of von Willebrand disease

Von Willebrand disease (VWD) is a common inherited bleeding disorder. Significant variability exists in management options offered to patients. These evidence-based guidelines from the American Society of Hematology (ASH), the International Society on Thrombosis and Haemostasis (ISTH), the National Hemophilia Foundation (NHF), and the World Federation of Hemophilia (WFH) are intended to support patients, clinicians, and health care professionals in their decisions about management of VWD.ASH, ISTH, NHF, and WFH formed a multidisciplinary guideline panel. Three patient representatives were included. The panel was balanced to minimize potential bias from conflicts of interest. The University of Kansas Outcomes and Implementation Research Unit and the McMaster Grading of Recommendations Assessment, Development and Evaluation (GRADE) Centre supported the guideline development process, including performing and updating systematic evidence reviews (through November 2019). The panel prioritized clinical questions and outcomes according to their importance to clinicians and patients. The panel used the GRADE approach, including GRADE Evidence-to-Decision frameworks, to assess evidence and make recommendations, which were subject to public comment.The panel agreed on 12 recommendations and outlined future research priorities.These guidelines make key recommendations regarding prophylaxis for frequent recurrent bleeding, desmopressin trials to determine therapy, use of antiplatelet agents and anticoagulant therapy, target VWF and factor VIII activity levels for major surgery, strategies to reduce bleeding during minor surgery or invasive procedures, management options for heavy menstrual bleeding, management of VWD in the context of neuraxial anesthesia during labor and delivery, and management in the postpartum setting5130132

Outcomes of long-term von Willebrand factor prophylaxis use in von Willebrand disease: A systematic literature review

Background: Von Willebrand Disease (VWD) is a common inherited bleeding disorder. Patients with VWD suffering from severe bleeding may benefit from the use of secondary long-term prophylaxis. Aim: Systematically summarize the evidence on the clinical outcomes of secondary long-term prophylaxis in patients with VWD and severe recurrent bleedings. Methods: We searched Medline and EMBASE through October 2019 for relevant randomized clinical trials (RCTs) and comparative observational studies (OS) assessing the effects of secondary long-term prophylaxis in patients with VWD. We used Cochrane Risk of Bias (RoB) tool and the RoB for Non-Randomized Studies of interventions (ROBINS-I) tool to assess the quality of the included studies. We conducted random-effects meta-analyses and assessed the certainty of the evidence using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. Results: We included 12 studies. Evidence from one placebo controlled RCT suggested that VWD prophylaxis as compared to no prophylaxis reduced the rate of bleeding episodes (Rate ratio [RR],.24; 95% confidence interval [CI],.17–.35; low certainty evidence), and of epistaxis (RR,.38; 95%CI,.21–.67; moderate certainty evidence), and may increase serious adverse events RR 2.73 (95%CI.12–59.57; low certainty). Evidence from four before-and-after studies in which researchers reported comparative data suggested that VWD prophylaxis reduced the rate of bleeding (RR.34; 95%CI,.25–.46; very low certainty evidence). Conclusion: VWD prophylaxis treatment seems to reduce the risk of spontaneous bleeding, epistaxis, and hospitalizations. More RCTs should be conducted to increase the certainty in these benefits