Luxi Song

Effect of low-dose cytarabine, homoharringtonine and granulocyte colony-stimulating factor priming regimen on patients with advanced myelodysplastic syndrome or acute myeloid leukemia transformed from myelodysplastic syndrome.

A total of 32 patients (25 with advanced MDS and 7 with t-AML) were enrolled in this study to evaluate the efficacy and toxicity of the low-dose cytarabine and homoharringtonine in combination with granulocyte colony-stimulating factor (G-CSF) (CHG protocol) in patients with advanced myelodysplastic syndromes (MDS) or MDS-transformed acute myeloid leukemia (t-AML). All the patients were administered the CHG regimen comprising low-dose cytarabine (25 mg/day, intravenous continuous infusion, days 1–14), homoharringtonine (1 mg/day, intravenous continuous infusion, days 1–14), and G-CSF (300 µg/day, subcutaneous injection, days 0–14, interrupted when the peripheral white blood cell count reached >20 × 109/L). The overall response rate was 71.9% after the administration of one course of the CHG regimen. Of the 32 patients, 15 (46.9%) achieved complete remission (CR) and 8 (25%) achieved partial remission (PR). This regimen was followed by a post-remission therapy that included conventional chemotherapy, when CR was achieved. Of the patients with CR who just received post-remission regimens as homoharringtonine and cytarabine (HA) and daunorubicin and cytarabine (DA) 6 relapsed rapidly and just had a mean 6.1 months of CR. Otherwise, the other 8 out of 14 patients with CR alternatively received subsequent chemotherapy, which combined mitoxantrone, idarubicin, pirarubicin, or aclarubicin with cytarabine. The mean CR duration of the 8 patients had reached 10.6 months, and 5 of the 8 still kept a continuous CR. The median overall survival (OS) was 18.2 months. There were no statistically significant differences for CR, PR, and OS when the patients were grouped by age, blasts in bone marrow, and karyotypes, respectively. No treatment-related deaths were observed. Myelosuppression was mild to moderate, and no severe non-hematological toxicity was observed. Thus, a CHG priming regimen as an induction therapy was well tolerated and effective in patients with advanced MDS or t-AML. Stronger and alternative subsequent chemotherapy is necessary for patients with CR to maintain longer CR and better OS.

Whole-exome and targeted sequencing identify ROBO1 and ROBO2 mutations as progression-related drivers in myelodysplastic syndromes

The progressive mechanism underlying myelodysplastic syndrome remains unknown. Here we identify ROBO1 and ROBO2 as novel progression-related somatic mutations using whole-exome and targeted sequencing in 6 of 16 (37.5%) paired MDS patients with disease progression. Further deep sequencing detects 20 (10.4%) patients with ROBO mutations in a cohort of 193 MDS patients. In addition, copy number loss and loss of heterogeneity (LOH) of ROBO1 and ROBO2 are frequently observed in patients with progression or carrying ROBO mutations. In in vitro experiments, overexpression of ROBO1 or ROBO2 produces anti-proliferative and pro-apoptotic effects in leukaemia cells. However, this effect was lost in ROBO mutants and ROBO-SLIT2 signalling is impaired. Multivariate analysis shows that ROBO mutations are independent factors for predicting poor survival. These findings demonstrate a novel contribution of ROBO mutations to the pathogenesis of MDS and highlight a key role for ROBO-SLIT2 signalling in MDS disease progression.

Bone Marrow Mesenchymal Stem Cells in Myelodysplastic Syndromes: Cytogenetic Characterization

Aim: This study compared genetic aberrations in hematopoietic cells (HCs) and mesenchymal stem cells of myelodysplastic syndrome (MDS-MSCs) patients. Methods: We obtained chromosomes with aberrations from 22 patients with MDS and chromosomes from 7 healthy individuals. Chromosomal aberrations in both HCs and MSCs were identified using G-banding. We then performed DNA content analysis of the HCs and MSCs. Results: Cytogenetic aberrations were detected in HCs from 13 of the 22 MDS patients (59%). Chromosomal aberrations in MSCs were detected in 15 of the 22 MDS patients (68%). No chromosomal abnormalities were identified in MSCs of the 7 healthy volunteers. We demonstrate herein that MSCs have distinct genetic abnormalities compared to HCs from the same individual. We observed a random loss of chromosomal material in significant proportions of MSCs. A high proportion of random loss may be a marker of chromosomal instability of MDS-MSCs. However, two case results showed that HCs and MSCs have different altered structural changes. Conclusion: Our results suggest enhanced genetic susceptibility of these cells in MDS patients. Our data indicates that the genetic alterations in MSCs may constitute a particular biological mechanism of MDS pathogenesis.

Interleukin-17-producing CD4+ T lymphocytes are increased in patients with primary immune thrombocytopenia.

The present study was performed to investigate the role of interleukin-17-producing CD4-positive T cells in the pathogenesis of primary immune thrombocytopenia (ITP). Peripheral blood was collected from ITP patients and healthy controls. The immunophenotyping of T lymphocytes and the detection of T helper 1/2/17 (Th1/Th2/Th17) cells were performed by flow cytometry, Th1/Th2/Th17-associated cytokines were determined by cytokines microarray and ELISA. The association between Th17 and T regulatory cells (Tregs) was also investigated. The percentage of Th17 and Th1 cells were markedly increased in ITP patients especially in those with severe ITP compared with normal controls. Th17 cytokines microarray revealed the upregulation of proinflammatory cytokines and downregulation of inflammatory inhibitory cytokines in ITP patients compared with that in the normal controls. Further ELISA analysis verified high levels of Th17-associated proinflammatory cytokines such as interleukin-17A/F, interleukin-6 and interleukin-23 and low levels of inflammatory inhibitory factors including interleukin-10 and transforming growth factor-&bgr; in ITP patients compared with normal controls. We also observed that the ratio of Th17/Treg was significantly higher in severe ITP than that in mild ITP and normal controls and inversely correlated with platelet count. In addition, Tregs from ITP patients could suppress the secretion of interferon-&ggr; by effector CD4-positive T cells, but had no effect on interleukin-17 production in vitro. Th17 cells are increased in ITP patients, and the inversion of Th17/Treg may contribute to the activation of autoimmunity.

Establishment and Validation of an Updated Diagnostic FCM Scoring System Based on Pooled Immunophenotyping in CD34+ Blasts and Its Clinical Significance for Myelodysplastic Syndromes

Abnormal immunophenotypes of hematopoietic cells can be detected by flow cytometry (FCM) to assist the diagnosis of myelodysplastic syndromes (MDS). We previously established a FCM scoring system for the diagnosis of low-grade MDS. In this study, additional valuable antigens were involved in an updated FCM scoring system (u-FCMSS) for all MDS subtypes. The u-FCMSS showed better sensitivity and specificity (89.4% and 96.5%) in distinguishing MDS from non-clonal cytopenia diseases. Validation analysis of u-FCMSS exhibited comparable sensitivity and specificity (86.7% and 93.3%) and high agreement rate (88.9%) of FCM diagnosis with morphological diagnosis at optimal cut-off (score 3). The distribution of FCM scores in different disease stages was also analyzed. The results suggested that early scoring from abnormal expression of mature myeloid/lymphoid antigens and advanced scoring from abnormal expression of stem/progenitor antigens expression constituted the majority of FCM scores of low-grade and high-grade MDS, respectively. High early scoring was generally accompanied by low IPSS-R score and superior survival, whereas high advanced scoring was accompanied by high IPSS-R score and inferior survival. In addition, the low-risk MDS patients with high early scoring and low advanced scoring were revealed as candidates for immunosuppressive therapy, whereas those with high advanced scoring and low early scoring may be more suitable for decitabine treatment. In conclusion, the u-FCMSS is a useful tool for diagnosis, prognosis and treatment selection in MDS. Differences in classes of antigens expressed and in distribution of FCM scores may reflect distinctive stage characteristics of MDS during disease progression.

Cytogenetic response based on revised IPSS cytogenetic risk stratification and minimal residual disease monitoring by FISH in MDS patients treated with low-dose decitabine

The features of cytogenetic response have been not well described in myelodysplastic syndrome (MDS) patients receiving low-dose decitabine treatment. In this study, we observed and analyzed the response characteristics based on the revised IPSS (IPSS-R) cytogenetic risk stratification in eighty-seven MDS patients who received low-dose decitabine treatment (15-20 mg/M(2)/d×5/per course). Twenty-seven of 44 patients (61.3%) with abnormal karyotypes achieved a cytogenetic response, including 18 cases with complete cytogenetic response (cCR). The patients carrying poor or very poor karyotypes achieved better clinical and cytogenetic response than those with intermediate or good karyotypes. Among the patients with poor or very poor karyotypes, those carrying chromosome 7 aberrance showed a better treatment response than the other patients. Four patients (22.2%) of the patients who achieved clinical CR presented with a cytogenetic PR (partial response) or NR (no response). Over 5% of the clonal cells determined by FISH analysis were in the two patients who presented cytogenetic CR. Longer median OS (24 months) were observed in the patients who achieved a cytogenetic response than in those who did not (12 months) (P=0.007). The patients with poor or very poor karyotypes could achieve survival comparable to that of the patients with good or very good karyotypes after decitabine treatment (18 vs. 20 months, P=0.365). IPSS-R cytogenetic risk stratification could be used to predict the clinical and cytogenetic response to decitabine treatment in MDS patients, and the predicting effect may be related to chromosome 7 involvement. Analysis with FISH and G-banding should be available in determining the minimal residual disease in MDS patients after treatment.

Iron overload promotes erythroid apoptosis through regulating HIF-1a/ROS signaling pathway in patients with myelodysplastic syndrome

Erythroid apoptosis increases significantly in myelodysplastic syndrome (MDS) patients with iron overload, but the underlying mechanism is not fully clear. In this study, we aim to explore the effect of HIF-1a/ROS on erythroid apoptosis in MDS patients with iron overload. We found that iron overload injured cellular functions through up-regulating ROS levels in MDS/AML cells, including inhibited cell viability, increased cell apoptosis and blocked cell cycle at G0/G1 phase. Interestingly, overexpression of hypoxia inducible factor-1a (HIF-1a), which was under-expressed in iron overload models, reduced ROS levels and attenuated cell damage caused by iron overload in MDS/AML cells. And gene knockdown of HIF-1a got the similar results as iron overload in MDS/AML cells. Furthermore, iron overload caused high erythroid apoptosis was closely related with ROS in MDS patients. Importantly, the HIF-1a protein levels of erythrocytes elevated obviously after incubation with desferrioxamine (DFO) from MDS patients with iron overload, accompanied by ROS levels inhibited and erythroid apoptosis reduced. Taken together, our findings determine that the HIF-1a/ROS signaling pathway plays a key role in promoting erythroid apoptosis in MDS patients with iron overload.

Increased PD-1/STAT1 ratio may account for the survival benefit in decitabine therapy for lower risk myelodysplastic syndrome

Abstract Decitabine is an effective therapy for patients with lower risk myelodysplastic syndrome (MDS). However, the mechanisms of decitabine’s therapeutic effect are not well established. Forty-four lower risk MDS patients received decitabine therapy. 59.1% patients achieved treatment response, and 53.8% patients who were RBC/platelet-dependent cast off the transfusion burden. The median overall survival (OS) was 19.0 months after decitabine treatment. Moreover, polarization toward type 1 in the CD8 + subset was enhanced, and a significantly increased expression of the PD-1, PD-L1, and PD-1/STAT1 ratio was observed in these lower risk MDS. The patients with amplification of PD-1/STAT1 ratio (2–4) achieved longer OS. Thus, our results suggest that the effect mechanism of decitabine toward lower risk MDS may be the moderate increase of PD-1/STAT1, which contributes to hematopoietic improvement. These findings suggest that a different PD-1-related strategy from those used to treat higher risk patients could be used for lower risk MDS patients.

TP53 mutations predict decitabine-induced complete responses in patients with myelodysplastic syndromes.

To identify the molecular signatures that predict responses to decitabine (DAC), we examined baseline gene mutations (28 target genes) in 109 myelodysplastic syndrome (MDS) patients at diagnosis. We determined that TP53 mutations predicted complete response (CR), as 10 of 15 patients (66·7%) who possessed TP53 mutations achieved a CR. Univariate and multivariate analyses showed that TP53 mutations are the only molecular signatures predictive of a CR to DAC in MDS. Among the ten patients with TP53 mutations who achieved a CR, nine presented with complex karyotypes due to abnormalities involving chromosome 5 and/or chromosome 7, and eight possessed monosomies. Although TP53 mutations were associated with a higher frequency of CRs, they were not associated with improved survival. Poor outcomes were attributed to early relapses and transformation to acute myeloid leukaemia after CR. Post‐DAC therapy patient gene mutation profiles showed that most CR patients exhibited fewer gene mutations after achieving a CR. It seems that suppression of these gene mutations was facilitated by DAC, resulting in a CR. In summary, TP53 mutations might predict decitabine‐induced complete responses in patients with MDS. DAC‐induced responses may result from partial suppression of malignant clones containing mutated TP53 genes.

Exploration of the role of gene mutations in myelodysplastic syndromes through a sequencing design involving a small number of target genes

Novel sequencing designs are necessary to supplement the recognized knowledge of myelodysplastic syndrome (MDS)-related genomic alterations. In this study, we sequenced 28 target genes in 320 Chinese MDS patients but obtained 77.2% of recall factors and 82.8% of genetic abnormalities (including karyotype abnormalities). In addition to known relationships among mutations, some specific chromosomal abnormalities were found to link to specific gene mutations. Trisomy 8 tended to be linked to U2AF1 and ZRSR2 mutations, and 20q- exhibited higher SRSF2/WT1 and U2AF1 mutation frequency. Chromosome 7 involvement accounted for up to 50% of RUNX1 mutations and 37.5% of SETBP1 mutations. Patients carrying a complex karyotype were prone to present TP53 mutations (36.1%). However, individuals with normal karyotypes rarely possessed mutations in the TP53, RUNX1 and U2AF1. Moreover, DNMT3A, TP53, SRSF2, STAG2, ROBO1/2 and WT1 predicted poor survival and high AML transformation. By integrating these predictors into international prognostic scoring system (IPSS) or revised IPSS, we built a set of mutation-based prognostic risk models. These models could layer different degrees of risk in patients more satisfactorily. In summary, this sequencing design was able to detect a number of gene mutations and could be used to stratify patients with varied prognostic risk.