Severe haemorrhages leading to a diagnosis of rare bleeding disorder occur at a very young age: A study from the FranceCoag network.

INTRODUCTION: In the context of severe unexplained haemorrhage (SH), it is usual to seek haematological evaluation and investigate for an inherited rare bleeding disorder (IRBD). In such circumstances, appropriate screen can discriminate between IRBD and suspected child abuse. Yet, little information is available about the frequency of SH in the population of patients with IRBD. AIM: To collect epidemiologic data about SH and IRBD. METHOD: The database of the FranceCoag network has collected information about IRBD since January 2004. Based on data gathered up to 16 March 2022, a retrospective search was conducted for of SH events having occurred before or at the time of IRBD diagnosis. Demographics and diagnosis circumstances were retrieved, as well as information about SH, defined as any life-threatening bleeding or intracranial haemorrhage. RESULTS: Among the 13,433 patients of the database, 109 (0.8%) fulfilled inclusion criteria including a known date of IRBD diagnosis, haemophilia A or B (HA/HB) being the most frequent (82.5%). IRBD was discovered as a consequence of an SH event in 82.6% of the cases while CNS was involved in 55%. Severe and moderate HA/HB and other severe IRBD presented significantly more intracranial haemorrhage (p < .02) and a lower age at diagnosis (p = .03). CONCLUSIONS: These data support that any unusual SH should raise a suspicion of IRBD. Particularly before 1-year of age, it is suggested to first confirm moderate or severe haemophilia and severe IRBD by standard coagulation tests (APTT, PT and fibrinogen), combined with a clotting FXIII assay as first-line investigation. Subsequent assays of coagulation factors should be performed in the case of abnormal values, in second-line investigation.

Venous thromboembolism during systemic inflammatory and autoimmune diseases associated with myelodysplastic syndromes, chronic myelomonocytic leukaemia and myelodysplastic/myeloproliferative neoplasms: a French multicentre retrospective case-control study.

OBJECTIVES: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML) are associated with systemic inflammatory and autoimmune diseases (SIADs) in 10-30% of cases. The aims of this study were (i) to evaluate the prevalence of venous thromboembolism VTE in patients presenting with both MDS/CMML and SIADs, (ii) to describe risk factors associated with thrombosis, and (iii) to analyse the impact of VTE on overall survival and transformation to acute myeloid leukaemia in comparison to patients with MDS/CMML-associated SIADs without VTE. METHODS: This retrospective multicentre case-control study was conducted among patients with MDS/CMML and dysimmune disorders and featured in the French retrospective database of the French Network of Dysimmune Disorders Associated with Hemopathies (MINHEMON), diagnosed with MDS/CMML and dysimmune disorders. RESULTS: During a median follow-up of 16 months (5-48) VTE occurred in 35 patients (21.6 %) whereas 127 patients did not. Among those with VTE, 8 patients (22.9%) experienced two or more VTE. Common prothrombotic risk factors were not significantly different in patients with or without VTE. CMML was more frequent in patients without VTE (37 % vs. 14.3%, p=0.01), whereas myelodysplasic/myeloproliferative neoplasm (MDS/MPN) was higher in VTE patients (20 % vs. 5.5 %, p=0.01). In a multivariate analysis, only MDS/CMML progression at the time of VTE (odds ratio 28.82, 95 % CI (5.52-530.70) was significantly associated with VTE. When treated with an anticoagulation therapy, bleeding occurred in 19.4% of cases (6/31). Overall survival was not significantly different between patients with and without VTE (p=0.68). Leukaemia-free survival between groups was not significantly different (p=0.83). CONCLUSIONS: VTE is a common complication in MDS/CMML-associated SIADSs with an increased risk of bleeding when treated by anticoagulants. In the MDS/CMML subgroup, SIADS flares and MDS/CMML progression seem to be prothrombotic risk factors.

Emicizumab does not interfere with the activated clotting time.

INTRODUCTION: The activated clotting time (ACT) is a useful marker of unfractionated heparin (UFH) activity during cardiopulmonary bypass (CPB) or cardiac catheterization. Emicizumab, recently approved for bleeding prevention in haemophilia A patients, acts like FVIII but does not need prior activation; it therefore shortens coagulation times in assays using intrinsic pathway activators and so is expected to shorten the ACT. AIM: To evaluated emicizumab's impact on heparin-induced ACT (Hemochron®) prolongation. METHODS: We measured the high-range (HR) ACT in citrated blood samples from healthy donors (HDs) (n = 9), CPB patients (n = 3) and emicizumab-treated patients (n = 5) after spiking with UFH and/or emicizumab. The low range (LR) ACT was also measured in spiked-samples from HDs and emicizumab-treated patients. RESULTS: In HDs, the median [interquartile range] baseline HR-ACTs were similar with and without emicizumab (129 [123-138] and 136 [115-141] s for 50 µg/ml, respectively); whatever the concentration of emicizumab (10 to 50 µg/ml), increasing the UFH concentration (1-5 UI/ml) prolonged the HR-ACT. In blood from patients undergoing CPB, the HR-ACT prolongation observed during this procedure was not masked by emicizumab at any concentration. Likewise, the addition of increasing concentrations of UFH to blood from emicizumab-treated patients induced a concentration-dependent prolongation of HR-ACT. Baseline LR-ACT were prolonged in emicizumab-treated patients but as for HR-ACT, emicizumab does not prevent heparin-induced prolongation of LR-ACT. CONCLUSION: Emicizumab does not interfere with UFH-induced ACT prolongation. The hemochron® ACT can be used to monitor UFH in patients receiving emicizumab during CPB or cardiac catheterization.

Unique inflammatory signature in haemophilic arthropathy: miRNA changes due to interaction between blood and fibroblast-like synoviocytes.

In haemophilia, the recurrence of hemarthrosis leads to irreversible arthropathy termed haemophilic arthropathy (HA). However, HA is a unique form of arthropathy in which resident cells, such as fibroblast-like synoviocytes (FLS), come into direct contact with blood. Therefore, we hypothesized that FLS in HA could have a unique inflammatory signature as a consequence of their contact with blood. We demonstrated with ELISA and ELISPOT analyses that HA-FLS expressed a unique profile of cytokine secretion, which differed from that of non-HA-FLS, mainly consisting of cytokines involved in innate immunity. We showed that unstable cytokine mRNAs were involved in this process, especially through miRNA complexes as confirmed by DICER silencing. A miRNOME analysis revealed that 30 miRNAs were expressed differently between HA and non-HA-FLS, with most miRNAs involved in inflammatory control pathways or described in certain inflammatory diseases, such as rheumatoid arthritis or lupus. Analysis of transcriptomic networks, impacted by these miRNAs, revealed that protein processes and inflammatory pathways were particularly targeted in LPS-induced FLS, and in particular vascularization and osteoarticular modulation pathways in steady-state FLS. Our study demonstrates that the presence of blood in contact with FLS may induce durable miRNA changes that likely participate in HA pathophysiology.

Synergistic effects of PRIMA-1Met (APR-246) and 5-azacitidine in TP53-mutated myelodysplastic syndromes and acute myeloid leukemia.

Myelodysplastic syndromes and acute myeloid leukemia with TP53 mutations are characterized by frequent relapses, poor or short responses, and poor survival with the currently available therapies including chemotherapy and 5-azacitidine (AZA). PRIMA-1Met(APR-246,APR) is a methylated derivative of PRIMA-1, which induces apoptosis in human tumor cells through restoration of the transcriptional transactivation function of mutant p53. Here we show that low doses of APR on its own or in combination with AZA reactivate the p53 pathway and induce an apoptosis program. Functionally, we demonstrate that APR exerts these activities on its own and that it synergizes with AZA in TP53-mutated myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) cell lines and in TP53-mutated primary cells from MDS/AML patients. Low doses of APR on its own or in combination with AZA also show significant efficacy in vivo Lastly, using transcriptomic analysis, we found that the APR + AZA synergy was mediated by downregulation of the FLT3 pathway in drug-treated cells. Activation of the FLT3 pathway by FLT3 ligand reversed the inhibition of cell proliferation by APR + AZA. These data suggest that TP53-mutated MDS/AML may be better targeted by the addition of APR-246 to conventional treatments.

A phase II study of guadecitabine in higher-risk myelodysplastic syndrome and low blast count acute myeloid leukemia after azacitidine failure.

High-risk myelodysplastic syndrome/acute myeloid leukemia patients have a very poor survival after azacitidine failure. Guadecitabine (SGI-110) is a novel subcutaneous hypomethylating agent which results in extended decitabine exposure. This multicenter phase II study evaluated the efficacy and safety of guadecitabine in high-risk myelodysplastic syndrome and low blast count acute myeloid leukemia patients refractory or relapsing after azacitidine. We included 56 patients with a median age of 75 years [Interquartile Range (IQR) 69-76]. Fifty-five patients received at least one cycle of guadecitabine (60 mg/m2/d subcutaneously days 1-5 per 28-day treatment cycles), with a median of 3 cycles (range, 0-27). Eight (14.3%) patients responded, including two complete responses; median response duration was 11.5 months. Having no or few identified somatic mutations was the only factor predicting response (P=0.035). None of the 11 patients with TP53 mutation responded. Median overall survival was 7.1 months, and 17.9 months in responders (3 of whom had overall survival >2 years). In multivariate analysis, IPSS-R (revised International Prognostic Scoring System) score other than very high (P=0.03) primary versus secondary azacitidine failure (P=0.01) and a high rate of demethylation in blood during the first cycle of treatment (P=0.03) were associated with longer survival. Thus, guadecitabine can be effective, sometimes yielding relatively prolonged survival, in a small proportion of high-risk myelodysplastic syndrome/low blast count acute myeloid leukemia patients who failed azacitidine. (Trial registered at clinicaltrials.gov identifier: 02197676).

Dual origin of relapses in retinoic-acid resistant acute promyelocytic leukemia.

Retinoic acid (RA) and arsenic target the t(15;17)(q24;q21) PML/RARA driver of acute promyelocytic leukemia (APL), their combination now curing over 95% patients. We report exome sequencing of 64 matched samples collected from patients at initial diagnosis, during remission, and following relapse after historical combined RA-chemotherapy treatments. A first subgroup presents a high incidence of additional oncogenic mutations disrupting key epigenetic or transcriptional regulators (primarily WT1) or activating MAPK signaling at diagnosis. Relapses retain these cooperating oncogenes and exhibit additional oncogenic alterations and/or mutations impeding therapy response (RARA, NT5C2). The second group primarily exhibits FLT3 activation at diagnosis, which is lost upon relapse together with most other passenger mutations, implying that these relapses derive from ancestral pre-leukemic PML/RARA-expressing cells that survived RA/chemotherapy. Accordingly, clonogenic activity of PML/RARA-immortalized progenitors ex vivo is only transiently affected by RA, but selectively abrogated by arsenic. Our studies stress the role of cooperating oncogenes in direct relapses and suggest that targeting pre-leukemic cells by arsenic contributes to its clinical efficacy.

Outcome of patients treated for myelodysplastic syndromes with 5q deletion after failure of lenalidomide therapy.

While lenalidomide (LEN) is the standard of care for the lower-risk myelodysplastic syndromes (MDS) patients with deletion 5q, 35% will not respond to or do not tolerate the drug. Moreover, most of the patients will lose their response after a few years. Defining the outcome of patients with LEN failure and determining the impact of subsequent therapies is therefore important to develop alternative strategies. Based on an international collaboration, we were able to compile a total of 392 patient cases of lower-risk MDS patients with 5q deletion and to analyze their outcome after failure of lenalidomide. The median survival following LEN failure was 23 months. We observed a negative impact on survival of advanced age, higher bone marrow blast count at LEN initiation, progression after LEN failure, and unfavorable cytogenetics. Among the treatment strategies, we observed a relatively prolonged survival of patients treated subsequently with hypomethylating agents and only a limited impact on survival of allogeneic transplantation. In conclusion, our work stresses the relatively short survival of this group of patient and defines the expected baseline for the needed future investigations in this group of patients.

Comparison of TP53 mutations screening by functional assay of separated allele in yeast and next-generation sequencing in myelodysplastic syndromes.

TP53 mutations are major prognostic factors in many hematological malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Next-generation sequencing (NGS) has improved the detection of such mutations by identifying small mutated clones but functional method like FASAY (functional assay of separated allele in yeast) may prove interesting. We compared the detection of TP53 mutations by FASAY and NGS in 91 patients with AML or MDS. By FASAY, 91% of assays were evaluable and 47 patients (57%) had a functional and 36 (43%) a non-functional p53 protein. FASAY could not conclude in 8 cases (9%), mainly because of poor RNA quality. No TP53 mutation was found using NGS in 50 cases (55%), and at least one mutation was detected in 41 cases (45%). The p53 status was concordant between FASAY and NGS in 95% (79/83) of cases. The four discordances included mutations detected by FASAY only in two cases, and by NGS only in two cases. Mutations not detected by NGS consisted of insertions in intronic regions, which were not analyzed by this assay. Mutations not detected by FASAY were mutations for which the percentage of mutated allele was less than 10%, including one mutation reported as non-deleterious in the IARC database. Overall, our data suggest that FASAY is an effective and reliable method to detect TP53 mutations in AML and MDS, which allows the assessment of the protein functionality, contrary to a sequencing approach.