MethScore as a new comprehensive DNA methylation-based value refining the prognosis in acute myeloid leukemia.

BACKGROUND: Changes in DNA methylation are common events in the pathogenesis of acute myeloid leukemia (AML) and have been repeatedly reported as associated with prognosis. However, studies integrating these numerous and potentially prognostically relevant DNA methylation changes are lacking. Therefore, we aimed for an overall evaluation of these epigenetic aberrations to provide a comprehensive NGS-based approach of DNA methylation assessment for AML prognostication. RESULTS: We designed a sequencing panel targeting 239 regions (approx. 573 kb of total size) described in the literature as having a prognostic impact or being associated with AML pathogenesis. Diagnostic whole-blood DNA samples of adult AML patients divided into a training (n = 128) and a testing cohort (n = 50) were examined. The libraries were prepared using SeqCap Epi Enrichments System (Roche) and sequenced on MiSeq instrument (Illumina). Altogether, 1935 CpGs affecting the survival (p < 0.05) were revealed in the training cohort. A summarizing value MethScore was then calculated from these significant CpGs. Patients with lower MethScore had markedly longer overall survival (OS) and event-free survival (EFS) than those with higher MethScore (p < 0.001). The predictive ability of MethScore was verified on the independent testing cohort for OS (p = 0.01). Moreover, the proof-of-principle validation was performed using the TCGA dataset. CONCLUSIONS: We showed that comprehensive NGS-based approach of DNA methylation assessment revealed a robust epigenetic signature relevant to AML outcome. We called this signature MethScore and showed it might serve as a strong prognostic marker able to refine survival probability of AML patients.

A validation study of potential prognostic DNA methylation biomarkers in patients with acute myeloid leukemia using a custom DNA methylation sequencing panel.

BACKGROUND: Multiple studies have reported the prognostic impact of DNA methylation changes in acute myeloid leukemia (AML). However, these epigenetic markers have not been thoroughly validated and therefore are still not considered in clinical practice. Hence, we aimed to independently verify results of selected studies describing the relationship between DNA methylation of specific genes and their prognostic potential in predicting overall survival (OS) and event-free survival (EFS). RESULTS: Fourteen studies (published 2011-2019) comprising of 27 genes were subjected to validation by a custom NGS-based sequencing panel in 178 newly diagnosed non-M3 AML patients treated by 3 + 7 induction regimen. The results were considered as successfully validated, if both the log-rank test and multivariate Cox regression analysis had a p-value ≤ 0.05. The predictive role of DNA methylation was confirmed for three studies comprising of four genes: CEBPA (OS: p = 0.02; EFS: p = 0.03), PBX3 (EFS: p = 0.01), LZTS2 (OS: p = 0.05; EFS: p = 0.0003), and NR6A1 (OS: p = 0.004; EFS: p = 0.0003). For all of these genes, higher methylation was an indicator of longer survival. Concurrent higher methylation of both LZTS2 and NR6A1 was highly significant for survival in cytogenetically normal (CN) AML group (OS: p < 0.0001; EFS: p < 0.0001) as well as for the whole AML cohort (OS: p = 0.01; EFS < 0.0001). In contrast, for two studies reporting the poor prognostic effect of higher GPX3 and DLX4 methylation, we found the exact opposite, again linking higher GPX3 (OS: p = 0.006; EFS: p < 0.0001) and DLX4 (OS: p = 0.03; EFS = 0.03) methylation to a favorable treatment outcome. Individual gene significance levels refer to the outcomes of multivariate Cox regression analysis. CONCLUSIONS: Out of twenty-seven genes subjected to DNA methylation validation, a prognostic role was observed for six genes. Therefore, independent validation studies are necessary to reveal truly prognostic DNA methylation changes and to enable the introduction of these promising epigenetic markers into clinical practice.

Successful early use of anti-SARS-CoV-2 monoclonal neutralizing antibodies in SARS-CoV-2 infected hematological patients - A Czech multicenter experience.

COVID-19 significantly impairs survival rates among hematological patients when compared to the general population. Our prospective multicentre project analyzed early administration of anti-SARS-CoV-2 spike protein neutralizing monoclonal antibodies (NmAbs) - bamlanivimab (72%) and casirivimab/imdevimab (28%) - efficacy among hematological patients with early-stage COVID-19. Mortality rate was compared to a control cohort of 575 SARS-CoV-2 positive hematological patients untreated with any specific anti-COVID-19 therapy. 88 hematological patients with lymphomas, acute leukemias, and myeloma as their most frequent underlying diagnoses (72%) were evaluated with a 97 days median follow-up after NmAb administration. One third of patients (32%) were treated with an anti-CD20 monoclonal antibody before COVID-19 diagnosis. Median time between first COVID-19 symptom and NmAb administration was 2 days. When administering NmAb, 29%, 57%, 11%, 2%, and 1% of our patients had asymptomatic, mild, moderate, severe, and critical degrees of COVID-19, respectively. 80% of baseline asymptomatic patients remained asymptomatic following NmAb administration. Median duration of COVID-19 symptoms after NmAb administration was 2.5 days. Progression to severe/critical COVID-19 occurred among a total of 17% (15/88) of our cases and numerically higher with bamlanivimab versus casirivimab/imdevimab (21% vs. 8%; p = 0.215), and myelomas (29%), lymphomas (17%) and acute leukemias (18%), respectively. During final follow-up, nine deaths (10%) were recorded - all after bamlanivimab (p = 0.056) with 8% attributed to COVID-19. Regarding "remdesivir/convalescent plasma naïve" patients, COVID-19 mortality rates were significantly lower in our NmAbs treated cohort compared to the control cohort of untreated SARS-CoV-2 positive hematological patients (6% vs. 16%, p = 0.020), respectively. Our study validated the safety and efficacy of NmAbs early use among hematological patients with newly diagnosed early-stage COVID-19 in terms of alleviating infection course and decreasing mortality. Results confirmed a more positive effect of a casirivimab/imdevimab combination versus bamlanivimab monotherapy.

Characteristics and outcome of patients with core-binding factor acute myeloid leukemia and FLT3-ITD: results from an international collaborative study.

The aim of this study was to evaluate the prognostic impact of FLT3-ITD in core-binding factor acute myeloid leukemia (CBFAML) in an international, multicenter survey of 97 patients of whom 52% had t(8;21)(q22;q22) and 48% had inv(16)(p13q22)/t(16;16)(p13;q22). The median age of the patients was 53 years (range, 19-81). Complete remission after anthracycline-based induction (n=86) and non-intensive therapy (n=11) was achieved in 97% and 36% of the patients, respectively. The median follow-up was 4.43 years (95% confidence interval [95% CI]: 3.35-7.39 years). The median survival after intensive and non-intensive treatment was not reached and 0.96 years, respectively. Among intensively treated patients, inv(16) with trisomy 22 (n=11) was associated with a favorable 4-year relapse-free survival rate of 80% (95% CI: 59-100%) as compared to 38% (95% CI: 27-54%; P=0.02) in all other patients with CBFAML/ FLT3-ITD (n=75). Overall, 24 patients underwent allogeneic hematopoietic cell transplantation (HCT), 12 in first complete remission and 12 after relapse. Allogeneic HCT in first complete remission was not beneficial (P=0.60); however, allogeneic HCT seemed to improve median survival in relapsed patients compared to that of patients treated with chemotherapy (not reached vs. 0.6 years, respectively; P=0.002). Excluding patients with inv(16) with trisomy 22, our data indicate that compathe outcome of CBF-AML patients with FLT3-ITD may be inferior to that of patients without FLT3-ITD (based on previously published data), suggesting that prognostically CBF-AML patients with FLT3-ITD should not be classified as favorable-risk. FLT3-inhibitors may improve the outcome of these patients.

Low levels of minimal residual disease after induction chemotherapy for BCR-ABL1-negative acute lymphoblastic leukaemia in adults are clinically relevant.

The aim of the present study was to evaluate the significance of low-level minimal/measurable residual disease (MRD) during early consolidation treatment in adult BCR-ABL1-negative acute lymphoblastic leukaemia. The MRD load was monitored by immunoglobulin/T-cell receptor rearrangements and assessed as negative [complete MRD response (CMR)], positive non-quantifiable (MRDnq) and positive quantifiable (MRDq). MRDnq before the first and second consolidation blocks had a comparable negative effect on survival as MRDq. The 5-year overall survival for CMR, MRDnq and MRDq at week 11 was 74·0%, 42·3% and 35·0% respectively. No central nervous system infiltration and MRD at week 11 were independent prognostic factors for survival on multivariate analysis (hazard ratios 0·32 and 2·25).

Characteristics and outcome of patients with acute myeloid leukaemia and t(8;16)(p11;p13): results from an International Collaborative Study.

In acute myeloid leukaemia (AML) t(8;16)(p11;p13)/MYST3-CREBBP is a very rare abnormality. Previous small series suggested poor outcome. We report on 59 patients with t(8;16) within an international, collaborative study. Median age was 52 (range: 16-75) years. AML was de novo in 58%, therapy-related (t-AML) in 37% and secondary after myelodysplastic syndrome (s-AML) in 5%. Cytogenetics revealed a complex karyotype in 43%. Besides MYST3-CREBBP, whole-genome sequencing on a subset of 10 patients revealed recurrent mutations in ASXL1, BRD3, FLT3, MLH1, POLG, TP53, SAMD4B (n = 3, each), EYS, KRTAP9-1 SPTBN5 (n = 4, each), RUNX1 and TET2 (n = 2, each). Complete remission after intensive chemotherapy was achieved in 84%. Median follow-up was 5·48 years; five-year survival rate was 17%. Patients with s-/t-AML (P = 0·01) and those with complex karyotype (P = 0·04) had an inferior prognosis. Allogeneic haematopoietic cell transplantation (allo-HCT) was performed in 21 (36%) patients, including 15 in first complete remission (CR1). Allo-HCT in CR1 significantly improved survival (P = 0·04); multivariable analysis revealed that allo-HCT in CR1 was effective in de novo AML but not in patients with s-AML/t-AML and less in patients exhibiting a complex karyotype. In summary, outcomes of patients with t(8;16) are dismal with chemotherapy, and may be substantially improved with allo-HCT performed in CR1.

WT1 Gene Expression in Peripheral Blood Before and After Allogeneic Stem Cell Transplantation is a Clinically Relevant Prognostic Marker in AML - A Single-center 14-year Experience.

BACKGROUND: This work summarizes our experience with WT1 monitoring before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). PATIENTS AND METHODS: The expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood according to the European Leukemia Net recommendations. Between May 2005 and August 2019, we analyzed 147 consecutive patients with acute myeloid leukemia with high WT1 expression at diagnosis, transplanted in first (CR1) or second (CR2) complete remission. RESULTS: At the time of allo-HSCT, 107 patients had WT1-normal expression (WT1 ≤ 50 copies), and 40 patients had WT1-high expression. The median follow-up was 21 months. The estimated 5-year overall survival and event-free survival was significantly better in the WT1-normal cohort (65% and 57% vs. 37% and 25%; P = .0003 and P < .0001, respectively) and 5-year cumulative incidence of relapse was significantly lower in the WT1-normal group (19% vs. 53%; P < .0001). Five-year non-relapse mortality was not significantly different (20% and 23%). Multivariate analysis revealed WT1-high expression and acute graft-versus-host disease grade 3/4 as significantly negative prognostic factors for OS. Overall, 49 patients developed WT1 molecular relapse in the post-transplant period; in 14 cases, the therapeutic intervention was done. In all but 1 relapsed patient where WT1 minimal residual disease (MRD) was monitored (38 patients), we detected WT1-high levels (sensitivity of 97%). CONCLUSION: The results of the analysis confirmed our previous experience that WT1 status before allo-HSCT is a strong prognostic factor for both OS and relapse risk. In addition, we confirmed the usefulness of this marker for MRD monitoring after allo-HSCT. The main advantage is the possibility of frequent MRD monitoring in peripheral blood and early bone marrow examination based on WT1-high expression.

WT1 Expression in Peripheral Blood at Diagnosis and During the Course of Early Consolidation Treatment Correlates With Survival in Patients With Intermediate and Poor-Risk Acute Myeloid Leukemia.

BACKGROUND: Up to 55% of non-APL acute myeloid leukemias (AML) lack a molecular target suitable for standardized disease monitoring. We aimed to evaluate the prognostic significance of WT1 gene expression at early stages of intensive treatment. PATIENTS AND METHODS: A total of 106 consecutive patients with intermediate and high-risk AML who had WT1 expression at diagnosis >500 copies/104ABL and who achieved remission after 1 to 2 cycles of induction treatment were analyzed. WT1 expression was measured in peripheral blood using a standardized European LeukemiaNet method. Overexpression was defined as >50 copies/104ABL. The median follow-up was 30 months. RESULTS: Patients with normal versus high WT1 expression after 2 cycles of chemotherapy had overall survival (OS) at 3 years of 66% versus 41% (P = .01); event-free survival (EFS) 45% versus 22% (P = .01). Prognostic significance of WT1 expression after 2 cycles of treatment was maintained in the group of patients treated with chemotherapy alone without hematopoietic stem cell transplantation in first line treatment (OS 70% vs. 36%, P = .02; EFS 35% vs. 0%, P = .03). Significant prognostic factors for EFS on multivariate analysis were the achievement of molecular remission (<50 copies of WT1) at any time during treatment (hazard ratio [HR] 0.47, P = .04) and increased WT1 expression after 2 cycles of chemotherapy (HR 2.0, P = .03). CONCLUSION: Increased WT1 expression after 2 cycles of chemotherapy is a negative prognostic factor for survival. WT1 remains a valuable molecular marker in AML without any leukemia-specific mutation, especially if next generation sequencing and/or digital polymerase chain reaction are not routinely available.

Prognostic significance of mutation profile at diagnosis and mutation persistence during disease remission in adult acute myeloid leukaemia patients.

In this multi-centre study, we analysed the prognostic impact of mutations in 19 genes associated with myeloid malignancies in 258 newly diagnosed acute myeloid leukaemia patients (aged 19-70 years) undergoing intensive therapy. We identified five patient groups with different prognostic risks and different benefits from allogeneic hematopoietic stem cell transplantation (alloHSCT) within the intermediate cytogenetic risk group patients (n = 184). The most adverse prognosis was observed in patients with DNMT3A and FLT3-ITD co-mutation, whose survival could be significantly improved with alloHSCT. In contrast, the most favourable prognosis without any further benefit from alloHSCT was identified in patients with mutations in NPM1 or CEBPA, after exclusion of the unfavourable prognostic groups defined by mutations in DNMT3A, RUNX1 or genes from chromatin/spliceosome group. An additional analysis of 113 diagnosis-remission paired samples revealed that persistence of non-DNMT3A mutations (above 2% VAF) represented a further negative prognostic factor. The proposed model offers a possible molecular stratification and treatment guidance for intermediate cytogenetic risk group patients.

DNA methylation and hydroxymethylation patterns in acute myeloid leukemia patients with mutations in DNMT3A and IDH1/2 and their combinations.

BACKGROUND: Aberrant epigenetic patterns are a hallmark of acute myeloid leukemia (AML). Mutations in profound epigenetic regulators DNMT3A and IDH1/2 often occur concurrently in AML. OBJECTIVES: The aim was to analyze DNA methylation, hydroxymethylation and mRNA expression profiles in AML with mutations in DNMT3A and IDH1/2 (individually and in combinations). METHODS: Infinium MethylationEPIC BeadChip (Illumina) covering 850,000 CpGs was utilized. The validation of hydroxy-/methylation data was done by pyrosequencing. HumanHT-12 v4 Expression BeadChip (Illumina) was used for expression examination. RESULTS: Hierarchical clustering analysis of DNA hydroxy-/methylation data revealed clusters corresponding to DNMT3A and IDH1/2 mutations and CD34+ healthy controls. Samples with concurrent presence of DNMT3A and IDH1/2 mutations displayed mixed DNA hydroxy-/methylation profile with preferential clustering to healthy controls. Numbers and levels of DNA hydroxymethylation were low. Uniformly hypermethylated loci in AML patients with IDH1/2 mutations were enriched for immune response and apoptosis related genes, among which hypermethylation of granzyme B (GZMB) was found to be associated with inferior overall survival of AML patients (P= 0.035). CONCLUSIONS: Distinct molecular background results in specific DNA hydroxy-/methylation profiles in AML. Site-specific DNA hydroxymethylation changes are much less frequent in AML pathogenesis compared to DNA methylation. Methylation levels of enhancer located upstream GZMB gene might contribute to AML prognostication models.

Hematopoietic Stem Cell Transplantation From Haploidentical Donors in Aplasia After Cladribine/Cytarabine Chemotherapy for Refractory Acute Myeloid Leukemia or Myelodysplastic Syndrome.

INTRODUCTION: Survival rate of patients with chemorefractory acute myeloid leukemia (AML) or myelodysplastic syndrome with excess blasts (MDS-EB) is poor. Allogeneic hematopoietic cell transplantation (HCT) is the only potentially curative therapy in these patients. PATIENTS AND METHODS: We report a retrospective analysis of outcomes of therapy of 24 patients with AML or MDS-EB refractory to high-dose salvage chemotherapy or who had failed previous HCT, who received T-cell-replete HLA haploidentical HCT in aplasia after cladribine/cytarabine-based chemotherapy followed by reduced intensity or myeloablative conditioning. All patients had active disease before commencement of the treatment. RESULTS: Of the patients, 91.7% achieved complete remission (CR), whereas 2 patients (8.2%) died in aplasia. One-year relapse rate was 49.3%. Cumulative incidence of nonrelapse mortality (NRM) was 25.6%. In a subgroup of patients with HCT-comorbidity index score ≤ 3, NRM was 15.4%. Two-year overall survival and relapse-free survival were 30.6% and 22.6%, respectively. Incidence of grade 3 and 4 acute graft versus host disease was 21.3% and 8.3, respectively. CONCLUSION: We found that sequential therapy with HCT in aplasia after cladribine/cytarabine chemotherapy is feasible, results in high CR rates, and has acceptable toxicity profile; however, posttransplant relapse is common in patients treated with active disease.

Analysis of Real-world Data on Postremission Therapy for Acute Myeloid Leukemia With Intermediate Risk Cytogenetics in First Complete Remission.

BACKGROUND: We retrospectively analyzed data from 310 patients with acute myeloid leukemia with intermediate-risk cytogenetics in first complete remission (CR1) to evaluate the usage and efficacy of various types of postremission therapy. PATIENTS AND METHODS: Cox regression with time-dependent covariates, landmark analysis, and competing risk models were used to estimate the outcomes and effects of treatment and patient- and disease-related risk factors. RESULTS: The early relapse rate and early nonrelapse mortality (NRM) were 12.8% and 4.4%, respectively. In our study, 77.2% of patients completed postremission therapy: 44% received allogeneic hematopoietic cell transplantation (HCT), 20% completed treatment with high-dose cytarabine (HIDAC), and 13% completed treatment with intermediate-dose cytarabine. The 3-year overall survival rate was 67.5% for patients treated with HIDAC and 63.4% after HCT (P = .5876). The NRM and relapse rate at 3 years were 0% and 58.9% after HIDAC and 21.9% and 29.3% after HCT, respectively. HCT reduced the risk of relapse (hazard ratio, 0.6; 95% confidence interval, 0.36-0.98). Total body irradiation-based myeloablative conditioning increased NRM compared with busulfan-based conditioning (hazard ratio, 8.33; 95% confidence interval, 2.52-27.45). CONCLUSION: Most patients with acute myeloid leukemia with intermediate-risk cytogenetics received allogeneic HCT, which decreased the risk of relapse but increased NRM, leading to a similar overall survival for patients who received HCT and HIDAC. Our data support the use of allogeneic transplantation for patients in CR1 from a human leukocyte antigen-matched related or unrelated donor after a busulfan-based myeloablative conditioning regimen as a primary strategy of postremission therapy for eligible younger patients.

Proven Invasive Pulmonary Aspergillosis in Stem Cell Transplant Recipient Due to Aspergillus sublatus, a Cryptic Species of A. nidulans.

Invasive fungal disease represents one of the severe complications in haematopoietic stem cell transplant recipients. We describe a case of a patient treated for relapse of chronic lymphoblastic leukaemia 6 years after HSCT. The patient was treated for invasive pulmonary aspergillosis but died 3 months later from multiple organ failures consisting of haemorrhagic necrotizing fungal pneumonia, refractory chronic hepatic graft versus host disease and cytomegalovirus hepatitis. Autopsy samples revealed histopathological evidence of fungal hyphae and an unusual Aspergillus nidulans-like species was isolated in pure culture. More precise identification was achieved by using scanning electron microscopy of ascospores and sequencing of calmodulin gene, and the isolate was subsequently re-identified as A. sublatus (section Nidulantes) and showed good in vitro susceptibility against all classes of antifungals. Commonly used ITS rDNA region and ß-tubulin gene fail to discriminate A. sublatus from related pathogenic species, especially A. quadrilineatus and A. nidulans. Although this is the first case of proven IPA attributed to A. sublatus, we demonstrated that at least some previously reported infections due to A. quadrilineatus were probably caused by this cryptic species.

Early MRD response as a prognostic factor in adult patients with acute lymphoblastic leukemia.

OBJECTIVE: To evaluate the prognostic power of minimal residual disease (MRD) monitored by polymerase chain reaction at defined time points during early treatment in adult patients with acute lymphoblastic leukemia (ALL). METHODS: Seventy-one patients were treated according to the GMALL 07/2003 protocol and evaluated for MRD in bone marrow by specific clonal rearrangements of Ig/TCR in BCR-ABL negative ALL or fusion gene transcript in BCR-ABL positive ALL. RESULTS: Three-year overall survival (OS) was 94% in patients with BCR-ABL negative ALL reaching complete molecular response (CMR) after the first course of chemotherapy (vs. 32% if MRD >10(-4) ; P = 0.001). Patients with CMR prior to the start of consolidation chemotherapy at week 11 had 3-yr OS 82% (vs. 18% if MRD >10(-4) ; P = 0.001). Patients with BCR-ABL positive ALL showed slower MRD dynamics. There was a trend to better OS in patients with ≥ 4 log reduction of BCR-ABL transcript prior to HSCT (92% vs. 50%; P = 0.065). None of the patients with detectable MRD (both BCR-ABL positive and negative) after HSCT survived 3 yr. CONCLUSION: Early MRD kinetics is an important tool for new prognostication models with direct clinical impact irrespective of standard prognostic factors in patients with BCR-ABL negative ALL.

Altered HLA Class I Profile Associated with Type A/D Nucleophosmin Mutation Points to Possible Anti-Nucleophosmin Immune Response in Acute Myeloid Leukemia.

Nucleophosmin 1 (NPM1) mutations are frequently found in patients with acute myeloid leukemia (AML) and the newly generated sequences were suggested to induce immune response contributing to the relatively favorable outcome of patients in this AML subset. We hypothesized that if an efficient immune response against mutated nucleophosmin can be induced in vivo, the individuals expressing HLA alleles suitable for presenting NPM-derived peptides should be less prone to developing AML associated with NPM1 mutation. We thus compared HLA class I frequencies in a cohort of patients with mutated NPM1 (63 patients, NPMc+), a cohort of patients with wild-type NPM1 (94 patients, NPMwt) and in normal individuals (large datasets available from Allele Frequency Net Database). Several HLA allelic groups were found to be depleted in NPMc+ patients, but not in NPMwt compared to the normal distribution. The decrease was statistically significant for HLA B(*)07, B(*)18, and B(*)40. Furthermore, statistically significant advantage in the overall survival was found for patients with mutated NPM1 expressing at least one of the depleted allelic groups. The majority of the depleted alleles were predicted to bind potent NPM-derived immunopeptides and, importantly, these peptides were often located in the unmutated part of the protein. Our analysis suggests that individuals expressing specific HLA allelic groups are disposed to develop an efficient anti-AML immune response thanks to aberrant cytoplasmic localization of the mutated NPM protein.

CBFB-MYH11 hypomethylation signature and PBX3 differential methylation revealed by targeted bisulfite sequencing in patients with acute myeloid leukemia.

BACKGROUND: Studying DNA methylation changes in the context of structural rearrangements and point mutations as well as gene expression changes enables the identification of genes that are important for disease onset and progression in different subtypes of acute myeloid leukemia (AML) patients. The aim of this study was to identify differentially methylated genes with potential impact on AML pathogenesis based on the correlation of methylation and expression data. METHODS: The primary method of studying DNA methylation changes was targeted bisulfite sequencing capturing approximately 84 megabases (Mb) of the genome in 14 diagnostic AML patients and a healthy donors' CD34+ pool. Subsequently, selected DNA methylation changes were confirmed by 454 bisulfite pyrosequencing in a larger cohort of samples. Furthermore, we addressed gene expression by microarray profiling and correlated methylation of regions adjacent to transcription start sites with expression of corresponding genes. RESULTS: Here, we report a novel hypomethylation pattern, specific to CBFB-MYH11 fusion resulting from inv(16) rearrangement that is associated with genes previously described as upregulated in inv(16) AML. We assume that this hypomethylation and corresponding overexpresion occurs in the genes whose function is important in inv(16) leukemogenesis. Further, by comparing all targeted methylation and microarray expression data, PBX3 differential methylation was found to correlate with its gene expression. PBX3 has been recently shown to be a key interaction partner of HOX genes during leukemogenesis and we revealed higher incidence of relapses in PBX3-overexpressing patients. CONCLUSIONS: We discovered new genomic regions with aberrant DNA methylation that are associated with expression of genes involved in leukemogenesis. Our results demonstrate the potential of the targeted approach for DNA methylation studies to reveal new regulatory regions.

Economic evaluation of rFVIIa high initial dose compared to rFVIIa standard initial dose in patients with haemophilia with inhibitors using the Czech HemoRec registry.

INTRODUCTION: In the treatment of bleeds in haemophilia patients with inhibitors, a high initial dose of recombinant Factor VIIa (rFVIIa) provides at least equal efficacy and a similar safety profile to a standard initial dose. However, no pharmacoeconomic comparison between these dosing regimens has previously been performed. Here, we assess the pharmacoeconomics of high (>120 µg/kg) versus standard (≤120 µg/kg) initial rFVIIa dose in inhibitor patients and the impact of time to treatment initiation on costs and outcomes. METHODS: In a retrospective analysis, observational data on bleed characteristics, rFVIIa treatment, hospitalizations and outcomes were extracted from the Czech Republic HemoRec registry. Crude comparisons and generalized linear regression modelling (GLM; correcting for patient differences) were performed to compare costs and outcomes between the high and standard initial dosing groups. RESULTS: Of 314 rFVIIa-treated bleeding episodes (12 inhibitor patients), most were spontaneous joint bleeds and 67.5% were treated with a high initial dose. In the crude comparison, high initial rFVIIa dosing was associated with a lower mean number of doses needed to achieve haemostasis compared with standard dosing (p<0.001), but higher total dose and costs (p ≤ 0.008). However, regression analyses revealed that high initial dose was associated with similar costs (p=0.891) and a shorter time to bleeding resolution (p=0.014). Increasing time to treatment initiation increased both time to bleeding resolution and total costs. CONCLUSION: Compared with a standard dose, a high initial rFVIIa dose may improve treatment outcomes without increasing costs. Early treatment initiation may reduce treatment costs.