Prevalence and clinical outcomes of germline variants among patients with myeloid neoplasms.

AIMS: Myeloid neoplasms (MNs) with germline predisposition have been recognised as a distinct entity. Emerging evidence suggests that sporadic myelodysplastic syndromes may also harbour undetected germline predispositions. We investigated germline alterations in a cohort of 122 adult Thai MNs. METHODS: MN patients were recruited and tested for germline variants using deep targeted next-generation sequencing. The germline variant was filtered using American College of Medical Genetics classifications and then evaluated for the association with clinical characteristics and outcomes. RESULTS: Our findings revealed pathogenic/likely pathogenic germline alterations in 12 (10%) of the patients. These germline lesions were commonly found in the DNA damage response pathway (n=6, 50%). We also identified novel deleterious FANCA A1219GfsTer59 variants in two patients diagnosed with secondary acute myeloid leukaemia (sAML) from aplastic anaemia and AML with myelodysplasia related. Among sAML, individuals with germline mutations had inferior overall survival compared with those with wild-type alleles (2 months vs 12 months) with HR 4.7 (95% CI 1.0 to 20), p=0.037. Therefore, the presence of pathogenic or likely pathogenic mutations may be linked to inferior survival outcomes. CONCLUSIONS: Our study highlighted that the prevalence of germline predisposition in Southeast Asian populations is comparable to that in Caucasians. This underscores the importance of germline genetic testing within the Asian population.

Good prognosis of adult hemophagocytic lymphohistiocytosis associated with the germline HAVCR2 mutation.

Hemophagocytic lymphohistiocytosis (HLH) in adults may be idiopathic or secondary to various conditions. Recent studies identified germline hepatitis A virus-cellular receptor 2 (HAVCR2) mutations in subcutaneous panniculitis-like T-cell lymphoma (SPTCL) with HLH. The roles of this mutation in HLH, especially in idiopathic group, have never been explored. Of the 65 HLH cases, we detected germline HAVCR2Y82C mutations in nine (13.8%) cases (five SPTCL and four idiopathic HLH). Other causes of HLH were hematologic malignancies excluding SPTCL (32.3%), idiopathic HLH without HAVCR2 mutation (29.2%), infections (15.3%), and autoimmune diseases (9.2%). Germline HAVCR2 mutation was significantly associated with less anemia and better survival. This defines a distinct subgroup of HLH.

Germline HAVCR2 mutations and their relation to the clinical spectrum of subcutaneous panniculitis-like T-cell lymphoma and hemophagocytic lymphohistiocytosis: results from a multicenter study and meta-analysis.

Germline HAVCR2 mutations are frequently detected in subcutaneous panniculitis-like T-cell lymphoma (SPTCL) patients with/without hemophagocytic lymphohistiocytosis (HLH) but factors associated with variable manifestations remain undetermined. To evaluate clinical variations and associated factors in SPTCL and/or HLH with/without HAVCR2 mutations, we performed direct sequencing of HAVCR2 exon 2 using DNA from patients with SPTCL or idiopathic HLH/HLH-like systemic illnesses, defined by HLH alone without secondary causes. The systematic review and individual patient data (IPD) level meta-analysis which included the present and previously published studies reporting HAVCR2 mutations in SPTCL with/without HLH populations was subsequently conducted using random-effects meta-analysis and multivariate logistic regression. Among 34 patients enrolled, ten of 28 SPTCL patients developed HLH/HLH-like systemic illnesses. Six cases with HAVCR2Y82C mutation manifested with HLH without panniculitis. Male sex (P=0.03) and age <18 years (P=0.04) were associated with HLH, corresponding to the inverse correlation between age and HLH-2004 score (r=-0.40; P=0.02). Homozygous HAVCR2Y82C mutation was more common in the presence of HLH compared with the absence (75.0% vs. 44.4%; P=0.02). Using IPD from the present and the other three eligible cohorts (N=127), male sex, heterozygous and homozygous/compound heterozygous HAVCR2 mutations were associated with HLH by the adjusted odds ratio of 2.93 (95% confidence interval [CI]: 1.22-7.06), 4.77 (95% CI: 1.05-21.63) and 8.48 (95% CI: 2.98-24.10), respectively. Patients with male sex and/or germline HAVCR2 mutations showed an increased risk of developing HLH. Younger patients tended to manifest with HLH, while older patients typically presented with SPTCL with less frequent HLH/HLH-like systemic illnesses.

Comparison of Molecular International Prognostic Scoring System (M-IPSS) and Revised International Prognostic Scoring System (R-IPSS) in Thai patients with myelodysplastic neoplasms.

INTRODUCTION: Risk stratification is essential for treatment decision in myelodysplastic neoplasms (MDS). Molecular international prognostic scoring system (M-IPSS) has been recently developed combining somatic mutations and clinical information being used in the revised international prognostic scoring system (R-IPSS). OBJECTIVE: We aimed to explore the performances of M-IPSS and R-IPSS in Thai patients with MDS. METHOD: MDS patients were stratified into risk categories using R-IPSS and M-IPSS scores. The performance of both models were evaluated for prognostic prediction. RESULTS: One hundred and sixty-two MDS patients were recruited from the multicenter study. Survival analysis revealed that both R-IPSS and M-IPSS were good prediction models with the Concordance Index (C-index) of 0.71 (95% Confidence interval [CI] 0.64-0.78) and 0.75 (95% CI 0.69-0.80), respectively (p = 0.22). Comparing the two, 13 of 162 (8%) cases were re-staged between lower and higher risks which would have affected treatment decisions. CONCLUSION: Our study showed that R-IPSS score can be used for risk stratification in most Thai patients. A prediction model using somatic mutations specifically in Asian patients should be formulated in the future.

Genetic mutations associated with blood count abnormalities in myeloid neoplasms.

INTRODUCTION: Myelodysplastic syndromes (MDS) predominantly present with varying degrees of cytopenia, while myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) exhibit proliferative features. Genetic defects underlying different complete blood count (CBC) alterations remain to be defined. OBJECTIVE: We aimed to evaluate mutations and impacts on abnormal blood counts in MDS and MDS/MPN. METHOD: MDS and MDS/MPN patients were recruited and sequenced by targeted next-generation sequencing. Clinical parameters, especially CBC, were evaluated for the association with genetic abnormalities and clinical outcomes. RESULTS: A total of 168 patients with myeloid neoplasms were recruited (92 cases of low-risk MDS, 57 cases of high-risk MDS and 19 cases of MDS/MPN). Compared to low-risk MDS and MDS/MPN, patients with high-risk MDS were presented with more severe neutropenia with 17.5% showing absolute neutrophil counts (ANC) lower than 0.5 × 109/L. Patients with MDS/MPN more commonly harboured mutations and had a higher number of mutations per case than low-risk MDS (94.7% vs. 56.5%; p < 0.001 and 3 vs. 1; p < 0.001, respectively). Patients with SF3B1 mutations showed lower haemoglobin levels than wild-type (7.9 vs. 8.4 g/dL, p = 0.02), but were associated with normal platelet counts (286 vs. 93 × 109/L; p < 0.001). Patients with U2AF1 mutations were associated with more severe leukopenia than wild-type (3 vs. 4.18 × 109/L; p = 0.02). KRAS mutations were associated with monocytosis (p < 0.001). Multivariate analysis revealed high-risk MDS, MDS/MPN, severe neutropenia (ANC < 0.5 × 109/L), and mutations in ASXL1 and SETBP1 were associated with inferior survival outcomes. CONCLUSION: Certain mutations were related to more severe anaemia, lower white blood cell count or monocytosis in Asian MDS and MDS/MPN patients.

Detection of Red Blood Cell Membrane Proteins in Myelodysplastic Syndromes Using Eosin-5-Maleimide (EMA) Staining by Flow Cytometry.

Background: Eosin-5-Maleimide (EMA)-based flow cytometry binds to red blood cell (RBC) membrane-associated proteins which can be used to detect red blood cell (RBC) membrane disorders. Myelodysplastic syndromes (MDS) are stem cell disorders resulting in ineffective hematopoiesis which is commonly present with anemia and erythroid dysplasia. Objectives: We aimed to study RBC membrane defects in MDS using flow cytometry for EMA staining. Methods: We enrolled anemic patients who were diagnosed with low-risk MDS (R-IPSS score ≤ 3.5), RBC membrane disorders [hereditary spherocytosis (HS) and Southeast Asian ovalocytosis (SAO)], and normal controls. Complete blood count (CBC) and flow cytometry for EMA staining were performed. Results: There were 16 cases of low-risk MDS, 6 cases of RBC membrane disorders, and 15 control cases. Mean fluorescence intensity (MFI) of EMA binding test in the RBC membrane disorders was significantly lower than controls (17.6 vs. 24.3, p < 0.001), but the EMA binding test in the low-risk MDS was not significantly different than the controls (26.5 vs. 24.3, p = 0.08). Conclusion: the RBC membrane defect in low-risk MDS was not demonstrated as having detection ability using EMA binding test with flow cytometry.

Characteristics and clinical correlation of TIM-3 and PD-1/PD-L1 expressions in leukemic cells and tumor microenvironment in newly diagnosed acute myeloid leukemia.

Dual targeting of TIM-3 and PD-1/PD-L1 pathways is currently under investigation for cancer immunotherapy. The interaction of these immune checkpoints remains unclear in the leukemic microenvironment of acute myeloid leukemia (AML). We performed an immunophenotypic study of bone marrow in 37 newly diagnosed AML patients. High levels of TIM-3 expression on AML blasts were correlated with first 7 + 3 induction failure (CR 16.2% vs. non-CR 36.4%, p = .038). In contrast, high TIM-3 levels on natural killer (NK) cells were associated with complete remission (CR) status after induction (CR 24.7% vs. non-CR 6.5%, p = .035). Few PD-L1 positive AML blasts and PD-1 or PD-L1 positive NK cells were observed. Although the exhausted PD-1 expressing T cells were detected in 28.3% of T cells, the double positive of PD-1 and TIM-3 T cells were rarely detected. In summary, the TIM-3 levels on AML blasts and NK cells are potentially the prognostic biomarkers in AML.

Excellent Prognosis of Low-Risk Myelodysplastic Syndromes (MDS) Without Detectable Myeloid-Related Mutations.

BACKGROUND: Unexplained cytopenia (UC) and low-risk myelodysplastic syndrome (MDS) are distinguished mainly by morphologic dysplasia, which sometimes shows inter-observer discrepancy. We hypothesized that gene mutations are strong prognostic factors for these low-risk patients. MATERIALS AND METHODS: We enrolled patients from 4 medical centers with unexplained cytopenia of at least 1 lineage. Diagnosis of low-risk MDS was made according to WHO 2016 classification and a revised international prognostic scoring system (R-IPSS) score of ≤ 3.5. DNA was extracted from bone marrow or blood and sequenced by targeted next generation sequencing (NGS). RESULTS: One hundred twenty-one patients were recruited: 25% with UC and 75% with low-risk MDS. Complete blood counts were similar, but low-risk MDS patients carried higher numbers of mutations (1 vs. 0; P = .04) than UC patients. Overall, the most frequent mutated genes were TET2 (14.6%), SF3B1 (12.2%), and ASXL1 (9.7%). Survival rates of low-risk MDS patients versus UC patients were not significantly different. UC patients and low-risk MDS patients without genetic abnormalities showed superior 5-year progression free survival compared to MDS patients with mutations (100% vs. 76.0%; P = .005). Overall, ASXL1 mutations were associated with decreased 4-year overall survival compared to wild-type (59% vs. 31%; P = .01). In a multivariate analysis, ASXL1 and DNMT3A mutations in low-risk MDS patients were associated with a higher risk of disease progression with hazard ratios of 7.88 (95% CI 1.76-35.32, P = .01) and 7.45 (95% CI 1.61-34.46, P = .01), respectively. CONCLUSION: Mutation detection is important for proper risk stratification of patients presenting with idiopathic cytopenia.

Prediction of Response to Erythropoiesis Stimulating Agents in Low-Risk Myelodysplastic Syndromes.

INTRODUCTION: Erythropoiesis stimulating agents (ESAs) represents the principal treatments for anemia in patients with lower-risk myelodysplastic syndromes (MDS). Pre-treatment erythropoietin (EPO) level and previous blood transfusion requirement are the two major predictors for response to ESAs. However, most evidence was derived from Western countries whereas there have been limited data in patients with Asian background. METHODS: We retrospectively collected data on patients with low-risk MDS who received ESAs. Erythroid response was evaluated according to IWG 2006 criteria. MDS subtypes, r-IPSS, baseline hemoglobin (Hb), ESAs dosage and erythropoietin level were reviewed from medical records. Gene mutations were analyzed in patients' blood or bone marrow at diagnosis by 40-gene myeloid panel targeted sequencing. Clinical and laboratory parameters were compared between erythroid responder and non-responder groups. RESULTS: A total of 47 patients were recruited in the study. The median age at diagnosis of the patients in this cohort was 77 years (IQR, 70-83) and 44.7% were male. The median revised international prognostic scoring system (R-IPSS) score of patients was 2.5. Response rate to ESAs was 46.8% (22/47). Median EPO level in responders was significantly lower than non-responders (27.7 vs. 59.1 U/L, p=0.02). Median ESAs dosage in responder group was 30,000 units per week. Cytogenetic abnormalities were detected in 27.3% and 24% of the responder and non-responder groups, respectively. Of 22 patients with available 40 gene mutation targeted sequencing, ASXL1, IDH2 and TET2 represented the 3 most common mutations and were found in 22%, 22% and 17%, respectively. There were no differences in cytogenetic abnormalities and gene mutations between groups. Patients who responded to ESAs showed a higher 5-year overall survival (OS) compared to non-responders (5-year OS 75% vs. 60.9%; p=0.008). CONCLUSION: We conclude that a low serum EPO level is a predictive factor for responsiveness to ESAs in Asian patients with low-risk MDS.

Novel DDX41 variants in Thai patients with myeloid neoplasms.

Germline DDX41 mutations were recently reported to cause MDS/AML and donor-derived leukemia after transplantation. While previously described in Western countries, DDX41 variants have not been reported in a Southeast Asian population. We performed targeted sequencing of blood or bone marrow samples from 109 Thai patients with myeloid malignancies. Among the 109 patients (75 MDS, 8 MPN, 11 MDS/MPN and 15 AML), the most frequent mutations were in ASXL1 (17.4%), TET2 (16.5%) and SRSF2 (12.8%), respectively. DDX41 variants were detectable in six (5.5%) cases. Four patients exhibited three presumable germline DDX41 mutations: p.S21fs (n = 2), p.F235fs (n = 1), and p.R339H (n = 1). While p.S21fs was previously reported in myeloid neoplasm, the latter two variants have not been described. Two of these cases harbored concomitant probable germline/somatic DDX41 mutations (p.S21fs/p.R525H and p.R339H/p.K494T), while the other two patients carried only somatic mutations (p.R525H and p.F438L). The p.K494T and p.F438L variants have not been previously reported. In patients with DDX41 alterations, the diagnoses were MDS with excess blasts (4), secondary AML (1) and low-risk MDS (1). In conclusion, we identified DDX41 variants in Thai patients with myeloid malignancies in which these variants could be used to assess predisposition to MDS in Southeast Asia.