[Efficacy and safety analysis of venetoclax combined with hypomethylating agents for the treatment of higher-risk myelodysplastic syndromes in the real world].

Objective: To investigate the efficacy and safety of combining venetoclax (VEN) with hypomethylated drugs (HMA) in the treatment of higher-risk (IPSS-R score >3.5) myelodysplastic syndromes (MDS) . Methods: From March 2021 to December 2022, forty-five MDS patients with intermediate and high risk were treated with VEN in combination with HMAs. Clinical data were collected and analyzed retrospectively, including gender, age, MDS subtype, IPSS-R score, treatment regimen, and efficacy, etc. Kaplan-Meier method and Cox regression model were used to analyze univariate and multivariate of survival prognosis. Results: ①Forty-five patients with MDS, including ninety-one percent were classified as high or very high risk. According to the 2023 consensus proposal for revised International Working Group response criteria for higher-risk MDS, the overall response rate (ORR) was 62.2% (28/45), with the complete response rate (CR) was 33.3% (15/45). For twenty-five naïve MDS, the ORR was 68% (17/25) and the CR rate was 32% (8/25). In nonfirst-line patients, the ORR and CR were 55% (11/20) and 35% (7/20) respectively. The median cycle to best response was 1 (1-4). ②With a median followup of 189 days, the median overall survival (OS) time was 499 (95% confidence interval, 287-711) days, and most patients died from disease progression. Responders had a significantly better median OS time than nonresponders (499 days vs 228 days, P<0.001). Multifactor analysis revealed that IPSS-R score and response to treatment were independent prognostic factors for OS; the presence of SETBP1 gene mutations was associated with a longer hospital stay (51.5 days vs 27 days, P=0.017) . Conclusions: There is clinical benefit of venetoclax in combination with hypomethylated agents in patients with higher-risk MDS, but adverse events such as severe hypocytopenia during treatment should be avoided.

[Molecular features of 109 patients with chronic myelomonocytic leukemia in a single center].

Objective: To explore the molecular features of chronic myelomonocytic leukemia (CMML) . Methods: According to 2022 World Health Organization (WHO 2022) classification, 113 CMML patients and 840 myelodysplastic syndrome (MDS) patients from March 2016 to October 2021 were reclassified, and the clinical and molecular features of CMML patients were analyzed. Results: Among 113 CMML patients, 23 (20.4%) were re-diagnosed as acute myeloid leukemia (AML), including 18 AML with NPM1 mutation, 3 AML with KMT2A rearrangement, and 2 AML with MECOM rearrangement. The remaining 90 patients met the WHO 2022 CMML criteria. In addition, 19 of 840 (2.3%) MDS patients met the WHO 2022 CMML criteria. At least one gene mutation was detected in 99% of CMML patients, and the median number of mutations was 4. The genes with mutation frequency ≥ 10% were: ASXL1 (48%), NRAS (34%), RUNX1 (33%), TET2 (28%), U2AF1 (23%), SRSF2 (21.1%), SETBP1 (20%), KRAS (17%), CBL (15.6%) and DNMT3A (11%). Paired analysis showed that SRSF2 was frequently co-mutated with ASXL1 (OR=4.129, 95% CI 1.481-11.510, Q=0.007) and TET2 (OR=5.276, 95% CI 1.979-14.065, Q=0.001). SRSF2 and TET2 frequently occurred in elderly (≥60 years) patients with myeloproliferative CMML (MP-CMML). U2AF1 mutations were often mutually exclusive with TET2 (OR=0.174, 95% CI 0.038-0.791, Q=0.024), and were common in younger (<60 years) patients with myelodysplastic CMML (MD-CMML). Compared with patients with absolute monocyte count (AMoC) ≥1×10(9)/L and <1×10(9)/L, the former had a higher median age of onset (60 years old vs 47 years old, P<0.001), white blood cell count (15.9×10(9)/L vs 4.4×10(9)/L, P<0.001), proportion of monocytes (21.5% vs 15%, P=0.001), and hemoglobin level (86 g/L vs 74 g/L, P=0.014). TET2 mutations (P=0.021) and SRSF2 mutations (P=0.011) were more common in patients with AMoC≥1×10(9)/L, whereas U2AF1 mutations (P<0.001) were more common in patients with AMoC<1×10(9)/L. There was no significant difference in the frequency of other gene mutations between the two groups. Conclusion: According to WHO 2022 classification, nearly 20% of CMML patients had AMoC<1×10(9)/L at the time of diagnosis, and MD-CMML and MP-CMML had different molecular features.

[Clinical characteristics and prognostic factors of patients with Philadelphia-negative myeloproliferative neoplasm accelerated/blast phase].

Objective: To evaluate the clinical characteristics and prognostic factors of patients with Philadelphia-negative myeloproliferative neoplasm-accelerated phase/blast phase (MPN-AP/BP) . Methods: A total of 67 patients with MPN-AP/BP were enrolled from February 2014 to December 2021 at the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences. Their clinical features and prognostic factors were analyzed retrospectively. Results: ① Sixty-seven patients with MPN-AP/BP with a median age of 60 (range, 33-75) years, including 31 males (46.3% ) and 36 females (53.7% ) , were analyzed. Forty-eight patients progressed from primary myelofibrosis (PMF) , and 19 progressed from other myeloproliferative neoplasms (MPNs) , which included polycythemia vera, essential thrombocythemia, and MPN unclassifiable. Patients who progressed from PMF had higher lactate dehydrogenase (LDH) levels than those who progressed from other MPNs (925.95 vs. 576.2 U/L, P=0.011) , and there were higher proportions of patients who progressed from PMF with splenomegaly (81.4% vs. 57.9% , P=0.05) , a myelofibrosis grade of ≥2 (93.6% vs. 63.2% , P=0.004) , and a shorter duration from diagnosis to the transformation to AP/BP (28.7 vs. 81 months, P=0.001) . ② JAK2V617F, CALR, and MPLW515 were detected in 41 (61.2% ) , 13 (19.4% ) , and 3 (4.5% ) patients, respectively, whereas 10 (14.9% ) patients did not have any driver mutations (triple-negative) . Other than driver mutations, the most frequently mutated genes were ASXL1 (42.2% , n=27) , SRSF2 (25% , n=16) , SETBP1 (22.6% , n=15) , TET2 (20.3% , n=13) , RUNX1 (20.3% , n=13) , and TP53 (17.2% , n=11) . The ASXL1 mutation was more enriched (51.1% vs. 21.1% , P=0.03) , and the median variant allele fraction (VAF) of the SRSF2 mutation (median VAF, 48.8% vs. 39.6% ; P=0.008) was higher in patients who progressed from PMF than those who progressed from other MPNs. ③ In the multivariate analysis, the complex karyotype (hazard ratio, 2.53; 95% confidence interval, 1.06-6.05; P=0.036) was independently associated with worse overall survival (OS) . Patients who received allogeneic stem cell transplantation (allo-HSCT) (median OS, 21.3 vs. 3 months; P=0.05) or acute myeloid leukemia-like (AML-like) therapy (median OS, 13 vs. 3 months; P=0.011) had significantly better OS than those who received supportive therapy. Conclusion: The proportions of patients with PMF-AP/BP with splenomegaly, myelofibrosis grade ≥2, a higher LDH level, and a shorter duration from diagnosis to the transformation to AP/BP were higher than those of patients with other Philadelphia-negative MPN-AP/BP. The complex karyotype was an independent prognostic factor for OS. Compared with supportive therapy, AML-like therapy and allo-HSCT could prolong the OS of patients with MPN-AP/BP.

[Clinical and laboratory features compared between JAK2 exon12 and JAK2 V617F mutated polycythemia vera].

Objective: To compare clinical and laboratory features between JAK2 exon12 and JAK2 V617F mutated polycythemia vera (PV) . Method: We collected data from 570 consecutive newly-diagnosed subjects with PV and JAK2 mutation, and compared clinical and laboratory features between patients with JAK2 exon12 and JAK2 V617F mutation. Results: 543 (95.3%) subjects harboured JAK2 V617F mutation (JAK2 V617F cohort) , 24 (4.2%) harboured JAK2 exon12 mutations (JAK2 exon12 cohort) , and 3 (0.5%) harboured JAK2 exon12 and JAK2 V617F mutations. The mutations in JAK2 exon12 including deletion (n=10, 37.0%) , deletion accompanied insertion (n=10, 37.0%) , and missense mutations (n=7, 25.9%) . Comparing with JAK2 V617F cohort, subjects in JAK2 exon12 cohort were younger [median age 50 (20-73) years versus 59 (25-91) years, P=0.040], had higher RBC counts [8.19 (5.88-10.94) ×10(12)/L versus 7.14 (4.11-10.64) ×10(12)/L, P<0.001] and hematocrit [64.1% (53.7-79.0%) versus 59.6% (47.2%-77.1%) , P=0.001], but lower WBC counts [8.29 (3.2-18.99) ×10(9)/L versus 12.91 (3.24-38.3) ×10(9)/L, P<0.001], platelet counts [313 (83-1433) ×10(9)/L versus 470 (61-2169) ×10(9)/L, P<0.001] and epoetin [0.70 (0.06-3.27) versus 1.14 (0.01-10.16) IU/L, P=0.002] levels. We reviewed bone marrow histology at diagnosis in 20 subjects with each type of mutation matched for age and sex. Subjects with JAK2 exon12 mutations had fewer loose megakaryocyte cluster (40% versus 80%, P=0.022) compared with subjects with JAK2 V617F. The median follow-ups were 30 months (range 4-83) and 37 months (range 1-84) for cohorts with JAK2 V617F and JAK2 exon12, respectively. There was no difference in overall survival (P=0.422) and thrombosis-free survival (P=0.900) . Conclusions: Compared with patients with JAK2 V617F mutation, patients with JAK2 exon12 mutation were younger, and had more obvious erythrocytosis and less loose cluster of megakaryocytes.

[Sternal bone marrow cell morphology evaluation utility for diagnostic categorization in patients with acquired hypocellular bone marrow failure syndromes].

Objective: Diagnostic value assessment of sternal bone marrow cell morphology in patients with acquired hypocellular bone marrow failure syndromes (BMFS) characterized by normal cytogenetics. Methods: A total of 194 eligible patients with an acquired hypocellular BMFS pre-sternum diagnosis in Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College from June 2014 to January 2019 were reviewed. Sternal bone marrow evaluation was performed, and a post-sternum diagnosis was made. Clinical characteristics and overall survival (OS) were then compared among patients with different post-sternum diagnosis. Binary logistic regression was used to develop a predictive scoring system. Results: In 152 patients with pre-sternum AA diagnosis, 29 patients with a pre-sternum idiopathic cytopenia of undetermined significance (ICUS) diagnosis, and 13 patients with a pre-sternum clonal cytopenia of undetermined significance (CCUS) diagnosis, sternal bone marrow evaluation resulted in a change of diagnosis to hypocellular myelodysplastic syndrome (hypo-MDS) in 42.8% (65/152) , 24.1% (7/29) , and 30.8% (4/13) , respectively. Patients with a post-sternum hypo-MDS diagnosis showed a significant difference in OS compared with patients with a post-sternum AA diagnosis (P=0.005) . Patients with ICUS/CCUS showed no difference in OS compared with AA and hypo-MDS (P=0.095 and P=0.480, respectively) . A 4-item predictive scoring system to identify hypocellular BMFS patients that need sternal bone marrow evaluation was developed, including age > 60 years old (OR=6.647, 95% CI 1.954-22.611, P=0.002, 2 points) , neutrophil alkaline phosphatase score ≤ 160 (OR=2.654, 95% CI 1.214-5.804, P=0.014, 1 point) , abnormal erythroid markers evaluated by flow cytometry on iliac bone marrow (OR=6.200, 95% CI 1.165-32.988, P=0.032, 2 points) , and DAT (DNMT3A, ASXL1, TET2) genes mutation (OR=4.809, 95% CI 1.587-14.572, P=0.005, 1 point) . The Akaike information criterin (AIC) was 186.1. Conclusion: Patients with a pre-sternum acquired hypocellular BMFS diagnosis characterized by normal cytogenetics may not reach accurate diagnostic categorization without sternal bone marrow cell morphology evaluation, which could be considered a diagnostic tool for this patient population. A predictive scoring system was developed, and when the total score is ≥ 2 points, sternal bone marrow evaluation should be performed for accurate diagnostic categorization that is critical to optimal patient care.

[Risk factors for leukemia transformation in patients with myelodysplastic syndromes].

Objective: To explore the risk factors in leukemia transformation (LT) in those with myelodysplastic syndromes (MDS) . Methods: From January 2012 to December 2020,data on 320 patients with newly diagnosed primary MDS were gathered from the MDS center. The clinical features and molecular characteristics are explored. Additionally, a retrospective analysis of risk factors for the development of acute leukemia from MDS was done. Results: The median follow-up was13.6 (0.4-107.3) months. 23.4% (75/320) of the MDS patients had LT group. Significant differences between the LT group and non-LT group can be seen in age (P<0.001) , bone marrow blast percentage (P<0.001) , bone marrow fibrosis (P=0.046) , WHO classification (P<0.001) , IPSS-R (P<0.001) and IPSS-R karyotype group (P=0.001) . The median number of mutation of LT group was 1 (1, 3) , that in non-LT group was 1 (0, 2) ,which had a statistical difference (P=0.003) .At the time of the initial diagnosis of MDS, the LT group had higher rates of the TP53 mutation (P=0.034) , DNMT3A mutation (P=0.026) , NRAS mutation (P=0.027) and NPM1 mutation (P=0.017) . Compared with the mutations at first diagnosis and LT of six patients, the number of mutations increased and the variant allele frequencies (VAF) increased significantly in LT patients. Higher bone marrow blast percentage (Refer to <5% , 5% -10% : HR=4.587, 95% CI 2.214 to 9.504, P<0.001, >10% : HR=9.352, 95% CI 4.049 to 21.600, P<0.001) , IPSS-R cytogenetic risk groups (HR=2.603, 95% CI 1.229-5.511, P=0.012) , DNMT3A mutation (HR=4.507, 95% CI 1.889-10.753, P=0.001) , and NPM1 mutation (HR=3.341, 95% CI 1.164-9.591, P=0.025) were all independently associated with LT in MDS patients, according to results of multivariate Cox regression. Conclusion: Bone marrow blast percentage, IPSS-R cytogenetic risk groups, DNMT3A mutation, and NPM1 mutation are independent risk factors in LT for MDS patients.

[The relationship between symptom burden and hematologic responses after treatment with interferon/hydroxyurea in patients with polycythemia vera].

Objective: To explore the relationship between symptom burden and hematologic responses after treatment with interferon and/or hydroxyurea in patients with polycythemia vera (PV) . Methods: Hematologic responses after continuous treatment with interferon and/or hydroxyurea for six months were evaluated in 190 patients with PV using the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (MPN-10 score) . In all patients, the PV diagnosis was based on the 2016 World Health Organization diagnostic definitions. Results: The study cohort comprised 93 (48.9% ) male and 97 (51.1% ) female patients. The median age at the time of MPN-10 assessment was 60 (32-82) years. The median MPN-10 score of the entire cohort was 9 (range, 0-67) . The median MPN-10 score of patients treated with interferon plus hydroxyurea (n=27) was 11 (0-67) , which was significantly higher than those of patients treated with interferon only (n=64) (6[0-56], P=0.019) or hydroxyurea only (n=99) (9[0-64], P=0.047) , whereas the median MPN-10 score was not significantly different between those treated with interferon only and hydroxyurea only (P=0.421) . The rate of severe symptom burden (i.e., any single symptom burden score ≥ 7 and/or total score ≥ 44) was 28.9% (55/190) in the entire cohort, whereas the rate of severe symptom burden was not significantly different among the interferon only (23.4% ) , hydroxyurea only (29.3% ) , and interferon plus hydroxyurea (40.7% ) groups (P>0.05 for all two-group comparisons) . When evaluating MPN-10 score, 37.4% (71/190) of the patients achieved complete hematologic remission (CHR) . Only 28.9% (55/190) patients had adequate disease control, defined as CHR without severe symptom burden. Reasons for inadequate disease control were evaluating blood counts alone, severe symptom burden alone, and evaluating blood counts accompanied with severe symptom burden in 42.1% (80/190) , 8.4% (16/190) , and 20.5% (39/190) of the patients, respectively. Compared to the patients with a platelet count ≤ 400×10(9)/L, those with a platelet count > 400×10(9)/L had a significantly higher rate of severe symptom burden (40.8% [20/49] vs 24.8% [35/141], P=0.044) and a higher median MPN-10 score (14[0-67] vs 7[0-56], P=0.038) . Platelet count > 400×10(9)/L was associated with an increased risk of severe symptom burden (hazard ratio, 2.089; 95% confidence interval, 1.052-4.147, P=0.035) . Conclusions: Symptoms related to disease after treatment with interferon and/or hydroxyurea were rather universal in patients with PV. Some patients still experienced severe symptom burden despite achieving CHR. Platelet count > 400×10(9)/L was associated with an increased risk of severe symptom burden in patients with PV treated with interferon and/or hydroxyurea.

[Prognostic factors of cyclosporine A combined with danazol with or without thalidomide in myelodysplastic syndrome treatment with low-percentage bone marrow blasts].

Objective: To explore the outcome of cyclosporine A (CsA) combined with danazol with or without thalidomide regimen for myelodysplastic syndrome (MDS) with low-percentage bone marrow blasts and predictive factors for treatment response. Methods: Data of 115 subjects who were newly diagnosed with primary MDS with low-percentage bone marrow blasts and were treated with CsA combined with danazol with or without thalidomide from December 2011 to December 2019 in our center were collected. Their clinical features, efficacy, and predictive factors of efficacy were retrospectively analyzed. A model for predicting this response was developed. Results: A total of 55 subjects responded (47.8%) , including 11 complete responses and 44 hematologic improvements. Fifty-two patients (52/105, 49.5%) achieved erythrocyte response; 35 (35/86, 40.7%) , platelet response; and 14 (14/40, 35%) , neutrophil response. Of 29 subjects (24.1%) , 7 who were red blood cell (RBC) transfusion-dependent became independent of transfusion. The median response duration was 20 months (range, 3-84 months) . In the univariate analysis, patients <0 years had a higher response rate than those ≥60 years (52.5% vs 22.2%, P=0.018) . Contrarily, the response rate was substantially decreased in patients with RBC transfusion dependence compared with those without RBC transfusion dependence (24.1% vs 55.8%, P=0.003) , as well as in patients with the mutated U2AF1 compared with those with the wild-type U2AF1 (26.1% vs 53.2%, P=0.020) . In multivariable analyses, age <0 years (OR=4.302, 95% CI 1.245-14.820, P=0.021) , RBC transfusion dependence (OR=3.774, 95% CI 1.400-10.177, P=0.009) , and U2AF1 mutation (OR=3.414, 95% CI 1.168-9.978, P=0.025) were significantly correlated with response. Variables that independently predicted the response were combined to generate the predictive model. According to the model, the overall response rates of patients with 0, 1, 2, and 3 risk factors were 65%, 30%-35%, 10%-15%, and 3%, respectively. Conclusion: CsA combined with danazol with or without thalidomide regimen could improve cytopenia symptoms in patients with MDS with low-percentage bone marrow blasts. At age <60 years, no transfusion dependence of RBC and wild-type U2AF1 mutation is a favorable prognostic factor.

[Evaluation of the prognostic value of MIPSS70-plus in Chinese patients with primary myelofibrosis].

Objective: To evaluate the prognostic value of MIPSS70-plus in Chinese patients with primary myelofibrosis (PMF) . Methods: A total of 113 Chinese patients with PMF were retrospectively analyzed. The Kaplan-Meier method, Log-rank test, and Cox proportional hazard regression model were performed to evaluate the prognostic factors. The likelihood ratio test was used to evaluate the predictive power between MIPSS70-plus and DIPSS systems. Results: The median age of the Chinese patients was 55 (range: 20-70) years, including 71 males and 42 females. According to the standard of MIPSS70-plus system, 99 patients (79.6% ) had a favorable karyotype and 23 patients (20.4% ) had an unfavorable karyotype. JAK2V617F in 55.8% (n=63) , CALR exon9 in 17.7% (including 15 CALR type 1 and 5 CALR type 2, n=20) , MPLW515 in 4.4% (n=5) , and triple negative (no detectable JAK2, MPL, and CALR mutations) in 22.1% of patients in our cohort were found by target-specific next-generation sequencing approach. At least one high-molecular risk mutations were presented in 45.1% (n=51) of patients, with ASXL1 in 38.9% (n=44) , SRSF2 in 7.1% (n=8) , IDH1/2 in 4.4% (n=5) , and EZH2 in 3.5% (n=4) of patients. A total of 28 patients (26.7% ) were in low risk, 20 (19.0% ) in intermediate risk, 41 (39.0% ) in high risk, and 16 (15.3% ) in very-high risk categories, which were delineated for the MIPSS70-plus model. A 2-year OS was 100% in low risk, 89.7% (95% CI 76.2% -100.0% ) in intermediate risk, 64.8% (95% CI 47.0% -82.6% ) in high risk, and 35.0% (95% CI 10.3% -59.7% ) in very-high risk categories, which had a significant difference (P<0.001) . A significantly higher predictive power for survival of the MIPSS70-plus group was observed compared with the DIPSS group (P=0.001, -2 log-likelihood ratios of 86.355 vs 95.990 for the MIPSS70-plus and DIPSS systems, respectively) . Conclusion: The MIPSS70-plus had significantly higher predictive power than the DIPSS.

[Overall survival and prognosis of patients with polycythemia vera: an analysis based on 906 patients from a single center].

Objective: To explore predictors of overall survival (OS) in Chinese patients with polycythemia vera (PV) . Methods: A total of 906 consecutive newly diagnosed patients with PV seen at the Blood Diseases Hospital, Chinese Academy of Medical Sciences, from June 2007 to February 2020 were included, and their data were collected. PV was diagnosed according to 2016 World Health Organization (WHO) diagnostic definitions. OS and prognostic factors were retrospectively analyzed. Results: Among the 906 patients, 439 were male (48.5%) and 467 were female (51.5%) . The median age was 57 years (range: 18-91 years) . 31.6% (276/874) of the patients had a thrombosis history at diagnosis, and 4.6% (25/541) of the patients had abnormal cytogenetics. The median follow-up was 54 months (95% confidence interval [CI] 8-130 months) . The 5- and 10-year cumulative deaths were 5.8% (95% CI 4.8%-6.7%) and 11.1% (95% CI 9.3%-12.9%) , respectively. Univariate analysis showed that age ≥60 years, thrombosis history, white blood cells (WBC) ≥15×10(9)/L, platelet (PLT) ≥450×10(9)/L, and platelet distribution width (PDW) ≥15 fl significantly correlated with worse OS, and palpable spleen correlated with better OS. Multivariate analysis showed that age ≥60 years (HR=4.3, 95% CI 2.1-9.2, P<0.001) and PDW ≥15 fl (HR=2.1, 95% CI 1.1-4.0, P=0.023) were independent prognostic factors for worse OS. The 5-year cumulative death for patients with PDW ≥15 fl or PDW<15 fl was 8.6% (95% CI 5.9%-11.3%) or 4.4% (95% CI 3.4%-5.4%) , respectively. The 5-year cumulative death for patients defined as low-, intermediate-, and high-risk patients by international working group score system for PV (IWG-PV) were 0.8% (95 CI 0.2%-1.4%) , 4.0% (95% CI 2.7%-5.3%) , and 12% (95% CI 9.6%-14.4%) , respectively, with a significant difference among the three cohorts (P<0.05) . PDW ≥ 15 fl significantly affected OS for intermediate- and high-risk patients (HR=2.3, 95% CI 1.2-4.2, P=0.009) defined by IWG-PV score system, but not for low-risk patients (HR=3.1, 95% CI 0.2-52.0, P=0.405) . Conclusions: Age ≥60 years and PDW ≥15 fl were independent prognostic factors for worse OS in PV. IWG-PV score system effectively predicted OS for Chinese patients with PV.

[Pathological characteristics of megakaryocytes in myeloproliferative neoplasms and their correlation with driver gene mutations].

Objective: To investigate the pathological characteristics of megakaryocytes in myeloproliferative neoplasms(MPN)and their correlations with driver gene mutations. Methods: Trephine specimens administered for 160 patients with MPN from February 2012 to October 2017 were reevaluated according to the World Health Organization(WHO)'s(2016)diagnostic criteria. Results: This cohort of patients included 72(45.0%)men, with the median age of 59(range, 13-87)years, comprising 39 with polycythemia vera(PV), 33 with essential thrombocythemia(ET), 37 with prefibrotic/early-primary myelofibrosis(pre-PMF), 37 with overt PMF, 1 with post-ET MF, 2 with post-PV MF, and 11 with MPN-unclassifiable(MPN-U)after the re-diagnosis. With PV, ET, pre-PMF, and overt PMF changes, proportions of dense clusters, hypolobulated nuclei, and naked nuclei of megakaryocytes gradually increased, whereas erythropoiesis gradually decreased. Proportions of reticulin, collagen, and osteosclerosis grades of ≥1 also increased. Dense clusters, hypolobulated nuclei, and naked nuclei of megakaryocytes were negatively correlated with erythropoiesis and positively correlated with granulopoiesis and fibrosis. In patients with pre- and overt PMF, dense clusters and naked nuclei of megakaryocytes were positively correlated with fibrosis. Patients with JAK2V617F MPN had significantly increased erythropoiesis(P=0.022). Patients with CALR-mutated MPN were characterized by increased loose and dense clusters; paratrabecular distribution and naked nuclei of megakaryocytes(P=0.055, P=0.002, P=0.018, P=0.008); and increased reticulin, collagen, and osteosclerosis(P=0.003, P<0.001, P=0.001). In patients with pre- and overt PMF, patients with JAK2V617F had increased cellularity(P=0.037). CALR-mutated patients had increased dense clusters and giant sizes of megakaryocytes, collagen, and osteosclerosis(P=0.055, P=0.059, P=0.011, P=0.046). Conclusion: Megakaryocytes showed abnormal MPN morphology and distribution, which were related to fibrosis. CALR mutation was probably associated with abnormal morphology and distribution of megakaryocytes and fibrosis.

[Efficacy and safety of generic azacitidine in Chinese patients with higher-risk myelodysplastic syndromes: a multicenter, prospective, single-arm study].

Objective: To evaluate the efficacy, safety, and pharmacokinetics of the generic azacitidine in Chinese patients with higher-risk myelodysplastic syndromes(MDS). Methods: Between October 2013 and 2016, 72 patients were eligible for enrollment at 9 sites from China received generic subcutaneous azacitidine 75 mg·m(-2)·d(-1) for 7 days per 28-day cycle, for ≥6 cycles. Pharmacokinetic blood samples were collected on day 1 of a single-dose. Results: For each patient at cycle 6 or at the time of study discontinuation, whichever came first, the overall response rate, which included complete remission (CR)and partial remission(PR), was 6.9%(5/72), the rate of patients who had the best effect with CR or PR during the treatment was 12.5%(9/72). Patients who were dependent on red-blood-cell transfusions and platelet transfusions at baseline became transfusion independent were 46.3%(19/41)and 41.2% (7/17), respectively. The median time of treatment was 6 cycles, and the median OS was 16.1 months (95%CI 10.9-20.6 months). For 36 patients(50%)received treatment at ≥6 cycles, and the median OS was 22.3 months(95%CI 16.1- not evaluative). Most common grade Ⅲ-Ⅳ hematologic treatment-emergent adverse events were neutropenia(55%), leukopenia(47%), and thrombocytopenia(61%). Pharmacokinetic profiles were similar for generic and original azacitidine in Chinese patients. Conclusion: Generic azacitidine treatment was favorable and safe and can be used as a standard treatment for patients with higher-risk MDS.

[Molecular features and prognostic value of RAS mutations in patients with myelodysplastic syndromes].

Objective: To explore the molecular features and prognostic value of RAS mutations in patients with myelodysplastic syndromes (MDS) . Methods: 112-gene targeted sequencing was conducted to detect RAS mutations in 776 patients with newly diagnosed primary MDS from December 2011 to December 2018. The mutual exclusivity and co-occurrence in gene mutations and clonal architecture were explored. Moreover, the prognostic significance of RAS mutations in MDS was analyzed. Results: RAS gene mutations were found in 52 (6.7% ) cases, 38 (4.9% ) of whom harbored NRAS mutation, 18 (2.3% ) KRAS mutation, and 4 (0.5% ) both NRAS and KRAS mutations. All the NRAS mutations and 65% of the KRAS mutations were located in codons 12, 13, and 61. PTPN11, FLT3, U2AF1, RUNX1, WT1, ETV6, and NPM1 mutations were enriched in patients with RAS mutations (Q<0.05) . Around 80% of RAS mutations represented subclonal lesions in patients who harbored at least two different mutations. Patients with RAS mutations were more frequently diagnosed with MDS with excess blast (MDS-EB) (82.7% vs. 35.2% , P<0.001) and had higher levels of white blood cell count (4.33×10(9)/L vs. 2.71×10(9)/L, P<0.001) , neutrophil absolute count (2.13×10(9)/L vs. 1.12×10(9)/L, P<0.001) , and bone marrow blast percentage (7% vs. 2% , P<0.001) but lower levels of platelet count (48×10(9)/L vs. 62×10(9)/L, P=0.048) . RAS mutations were correlated with higher-risk categories in the Revised International Prognostic Scoring System (IPSS-R) (71.1% vs. 37.9% , P<0.001) . The median overall survival of patients with NRAS mutations was shorter than the others (P=0.011) , while the significance was lost in the multivariable model. Conclusion: RAS gene mutations always occurred in the late-stage MDS and co-occurred with other signal transduction- and transcription factor-related gene mutations. PTPN11, a RAS pathway-related gene, is an independent poor prognostic factor in MDS patients.

[Features and clinical significance of gene mutations in patients with myelodysplastic syndromes with ring sideroblasts].

Objective: To explore the features and clinical significance of gene mutations in patients with myelodysplastic syndromes with ring sideroblasts (MDS-RS) . Methods: A total of 255 newly diagnosed primary MDS-RS patients were retrospectively reviewed from our center from January2001 to June 2019. SF3B1 gene mutations were detected by Sanger sequencing in 129 patients, and next generation sequencing (NGS) was performed in the other 126 patients using a set of selected 112-genes. Results: A total of 193 (75.7%) patients presented with SF3B1 mutation, predominantly mutant at amino acid position 700 (K700E) (n=147, 76.2%) . Non-SF3B1 gene mutations were TET2 (16.7%) , ASXL1 (14.3%) , U2AF1 (11.1%) , TP53 (7.9%) , SETBP1 (6.3%) , and RUNX1 (6.3%) . RS 5%-<15% patients had a higher SETBP1 mutation frequency than RS≥15% patients (21.4% vs 4.5%, P=0.044) . Mutation frequencies of other genes were similar in both groups (all P>0.05) . SF3B1 variant allele frequencies (VAF) had positive correlation with marrow RS percentage but without statistical significance in RS 5%-<15% group (P=0.078, r=0.486) . SF3B1 mutant patients presented with higher marrow RS percentage compared with wild-type patients[40.0% (15.0%-80.0%) vs 25.5% (15.0%-82.0%) , P<0.001], and SF3B1 VAF positively correlated with RS percentage (P=0.009, rs=0.261) in RS≥15% group. Age, ANC, PLT, mean RBC corpuscular volume, RS percentage, IPSS-R cytogenetics, and IPSS-R risk score were significantly different between patients with SF3B1 mutations and wild-type SF3B1 (all P<0.05) . Multivariable survival analyses adjusted by age and IPSS-R cytogenetics revealed that SF3B1 mutation was an independent favorable prognostic factor (HR=0.265, 95% CI 0.077-0.917, P=0.036) , and TP53 mutation was an adverse variable independent of SF3B1 mutation (HR=6.272, 95% CI 1.725-22.809, P=0.005) . According to the mutant status of SF3B1 and TP53, MDS-RS patients were categorized into 4 groups, namely, with SF3B1 and TP53 mutation, with wild-type SF3B1 and TP53, with wild-type SF3B1 but TP53 mutation, and with SF3B1 mutation but wild-type TP53. There was a significant difference for OS among these 4 groups (P<0.001) . The former 3 groups showed no significant difference in OS in multiple comparisons. However, the SF3B1 mutation but wild-type TP53 group had a better OS than wild-type SF3B1 but TP53 mutation group and wild-type SF3B1 and TP53 group, whereas a similar OS compared with SF3B1 and TP53 mutation group. Conclusion: SF3B1 mutations were prevalent in MDS-RS patients with the most common mutation at amino acid position 700 (K700E) . SF3B1 mutation was an independent favorable prognostic variable, whereas TP53 mutation was an independent adverse variable. SF3B1 mutation could coordinate with TP53 mutation for more sophisticated prognosis stratification in MDS-RS patients.

Phylogenetic diversity is a better predictor of wetland community resistance to Alternanthera philoxeroides invasion than species richness.

Highly biodiversity communities have been shown to better resist plant invasions through complementarity effects. Species richness (SR) is a widely used biodiversity metric but lacks explanatory power when there are only a few species. Communities with low SR can have a wide variety of phylogenetic diversities (PD), which might allow for a better prediction of invasibility. We assessed the effect of diversity reduction of a wetland community assemblage typical of the Beijing area on biotic resistance to invasion of the exotic weed Alternanthera philoxeroides and compared the reduction in SR and PD in predicting community invasibility. The eight studied resident species performed similarly when grown alone and when grown in eight-species communities together with the invasive A. philoxeroides. Variation partitioning showed that PD contributed more to variation in both A. philoxeroides traits and community indicators than SR. All A. philoxeroides traits and community indicators, except for evenness index, showed a linear relationship with PD. However, only stem length of A. philoxeroides differed between the one- and two-species treatments, and the diversity index of the communities differed between the one- and two-species treatments and between the one- and four-species treatments. Our results showed that in natural or semi-natural wetlands with relatively low SR, PD may be a better predictor of invasibility than SR. When designing management strategies for mitigating A. philoxeroides invasion, deliberately raising PD is expected to be more efficient than simply increasing species number.

[Mean corpuscular volume ≤100 fl was an independent prognostic factor in patients with myelodysplastic syndrome and bone marrow blast<5 percent].

Objective: To explore the prognostic effects of mean corpuscular volume (MCV) in patients with myelodysplastic syndromes (MDS) . Methods: 321 newly diagnosed, untransfused primary MDS patients who administered from December 2009 to December 2017 were enrolled. The association of MCV with prognosis and several clinical features and genetic mutations were analyzed. Results: Patients were divided into MCV≤100 fl (n=148) and MCV>100 fl (n=173) cohorts. Median overall survival of patients with MCV≤100 fl was shorter than their counterparts (27 months vs 72 months, P<0.001) . In subgroup analysis, MCV≤100 fl patients had worse survivals in bone marrow blast <5% cohort (34 months vs not reached, P=0.002) , but not so in ≥5 % cohort (17 months vs 20 months, P=0.078) . MCV≤100 fl was still an independent adverse variable (HR=1.890, 95%CI 1.007-3.548, P=0.048) after adjusting for clinical and laboratory variables and mutation topography in bone marrow blasts<5% cohort. In bone marrow blasts<5% cohort, patients with MCV≤100 fl had higher hemoglobin levels [90 (42-153) g/L vs 78.5 (28-146) g/L, P=0.015].The proportions of Revised International Prognostic Scoring System (IPSS-R) high/very high risks and poor/very poor IPSS-R karyotypes were higher in MCV≤100 fl cohort (28.8% vs 10.8%, P=0.003; 24.7% vs 12.9%, P=0.049) . MCV≤100 fl cohort had more genetic mutations than those with MCV>100 fl though without significance (0.988 vs 0.769, P=0.064) . Mutated SF3B1 was less frequently in MCV≤100 fl cohort (4.7% vs 15.4%, P=0.018) . Conclusion: MCV≤100 fl was an independent adverse variable after adjusting for clinical and laboratory variables and mutation topography in MDS patients with bone marrow blasts<5%.

[Genetic characteristics and prognostic values of RAS mutations in patients with myelofibrosis].

Objective: To explore the genetic characteristics, clinical features, and prognostic values of RAS mutations in patients with myelofibrosis (MF) . Methods: We analyzed 112-gene targeted sequencing data from 226 patients who had a diagnosis of either primary myelofibrosis (PMF) or post-polycythemia vera/post-essential thrombocythemia (post-PV MF and post-ET MF) from December 2011 to December 2019. A retrospective analysis of the genetic characteristics, clinical features, and prognosis of RAS mutations was performed. Results: Among 266 patients diagnosed PMF or post-PV/ET MF, RAS mutations were found in 14 (6.2%) cases, including 9 (4.0%) cases of NRAS mutations, 8 (3.5%) cases of KRAS mutations, and 3 (1.3%) cases of both NRAS and KRAS mutations. All of the NRAS mutations were located in codons 12 and 13. The median VAFs of RAS mutations were significantly lower than those of the driver mutations, confirming that they represent sub-clonal events that are acquired during the disease course. SETBP1, SRSF2, and MPL tended to be clustered with RAS mutations. Patients with RAS mutations had a higher number of additional oncogenic mutations (median, 3.36 vs 1.17, P<0.001) . RAS mutations had a statistically significant association with elevated monocyte cell counts (P=0.003) , lower platelet counts (P=0.026) , higher bone marrow blasts (P=0.022) , splenomegaly (P=0.005) , and very high-risk (VHR) karyotype abnormality percentage (P=0.031) . In univariate analysis, the OS of patients with NRAS mutations were significantly inferior in the entire MF and PMF cohorts (P=0.001, P=0.008) . In a multivariate model, NRAS retained an independent negative prognostic factor in PMF. Conclusion: RAS gene mutations were constantly related to elevated monocyte cell counts, lower platelet counts, higher bone marrow blasts, and VHR karyotype abnormality percentage that usually defined high-risk disease and often occurred as sub-clonal events. NRAS mutation is an independent poor prognostic factor in PMF.

[Fibrosis-driving cells in patients with primary myelofibrosis and myelodysplastic syndromes with myelofibrosis].

Objective: To compare fibrosis-driving cells in patients with primary myelofibrosis (PMF) and patients with myelodysplastic syndromes (MDS) with myelofibrosis (MF) (MDS-MF) . Methods: Bone marrow biopsy sections of patients with newly diagnosed PMF and MDS (10 each randomly selected for MF-0/1, MF-2, and MF-3) were stained with specific immunofluorescence antibodies to label Gli1, LeptinR, alpha smooth muscle actin (α-SMA) , CD45, and ProcollagenⅠ. Images captured by confocal microscopy were analyzed by Fiji-ImageJ to calculate the cell counts of Gli1(+), LeptinR(+) cells, and fibrosis-driving cells including α-SMA(+), α-SMA(+)/Gli1(+), α-SMA(+)/LeptinR(+), and ProcollagenⅠ(+)/CD45(+) cells. Results: Patients with PMF and MDS with MF-2/3 had higher LeptinR(+), α-SMA(+), α-SMA(+)/Gli1(+), and Procollagen Ⅰ(+)/CD45(+) cell counts compared with those with MF-0/1 (all P values<0.05) . However, patients with PMF with MF-2/3 presented with higher Gli1(+) and α-SMA(+)/LeptinR(+) cell counts than those with MF-0/1 (P=0.001 and 0.006) , whereas these cells were similar between patients with MDS with MF-0/1 and MF-2/3 (P=0.169 and 0.067) . In patients with MF-0/1, all fibrosis-driving cells did not differ between PMF and MDS (all P>0.05) . However, in patients with MF-2/3, Procollagen Ⅰ(+)/CD45(+) cell counts were higher in patients with PMF compared with those with MDS (P=0.007) , while other fibrosis-driving cell counts were similar between these two groups (all P>0.05) . MF grade and fibrosis-driving cell counts were not correlated with overall survival in patients with either PMF or MDS. Conclusion: α-SMA(+) cells in patients with PMF originated from both Gli1(+) and LeptinR(+) cells, whereas α-SMA(+) cells in patients with MDS-MF only originated from Gli1(+) cells; patients with PMF had higher ProcollagenⅠ(+)/CD45(+) cell counts than those with MDS-MF.

[Analysis of clinical characteristics, treatment response rate and survival of 77 myelodysplastic syndrome patients with del (5q) syndrome].

Objective: To observe the clinical characteristics, treatment responses and prognosis of patients with myelodysplastic syndrome (MDS) -del (5q) syndrome who met WHO (2016) diagnostic typing criteria. Methods: A total of 77 patients with del (5q) syndrome, according to WHO (2016) classification, were retrospectively analyzed between January 2008 and April 2018 in the Blood Diseases Hospital, Chinese Academy of Medical Sciences. Clinical characteristics, lenalidomide (LEN) efficacy and survivals were compared between the patients with del (5q) alone and those with one additional cytogenetic abnormality (ACA) with the exception of monosomy 7 or del (7q) . Treatment response and overall survival (OS) were compared between patients who were treated with LEN and traditional non-LEN drugs. Results: Of 77 patients, 64 were isolated del (5q) and 13 were del (5q) with ACA. There were significant differences of the median age and percentage of patients who had small megakaryocytes in bone marrow smear by immunohistochemistry (CD41) between the patients with isolated del (5q) and the patients with del (5q) + ACA[58 (29-64) years old vs 63 (31-82) years old, z=2.164, P=0.030; and 91.7%vs 60.0%, P=0.046, respectively]. The overall hematological response rate (78.9%vs 80.0%) , complete hematological remission (CR) rate (57.9% vs 60.0%) , cytogenetic response (CyR) rate[69.2% (9/13) vs 66.7% (4/6) ] and complete cytogenetic response (CCyR) rate [61.5% (8/13) vs 33.3% (2/6) ] of LEN were similar between the patients with isolated del (5q) (n=19) and with del (5q) + ACA (n=10) , as well as the median Overall survival (OS) between these two groups of patients (62 months vs 78 months, P=0.388) . The hematological response rate (79.3% vs 36.0%) , CR rate (58.6% vs 8.0%) , CyR rate [68.4% (13/19) vs 11.1% (1/9) ] and CCyR rate [52.6% (10/19) vs 0 (0/9) ] were higher among patients treated with LEN (n=29) than those treated with non-LEN therapy (n=25) . There was no statistically significant difference in OS between the patients with LEN or non-LEN therapy (78 months vs 62 months, P=0.297) . Conclusion: Comparing del (5q) syndrome patients with isolated del (5q) or with del (5q) + ACA, two groups of patients had similar clinical characteristics, median OS and LEN efficacy. LEN showed better treatment response than traditional drugs in patients with del (5q) syndrome.