Yan-Rong Liu

Risk stratification–directed donor lymphocyte infusion could reduce relapse of standard-risk acute leukemia patients after allogeneic hematopoietic stem cell transplantation

We studied the impact of risk stratification-directed interventions for minimal residual disease (MRD) on relapse and disease-free survival (DFS) prospectively in 814 subjects with standard-risk acute leukemia receiving allotransplantation in first or second complete remission. A total of 709 subjects were MRD(-) after transplantation (Group A); 105 subjects were MRD(+), 49 received low-dose IL-2 (Group B), and 56 received modified donor lymphocyte infusion (DLI) with or without low-dose IL-2 (Group C). Posttransplantation immune suppression for GVHD was also modified based on MRD state. The cumulative risk of relapse was significantly less and DFS was significantly better in subjects in Group C than in subjects in Group B (P = .001 and P = .002, respectively), but was not different from subjects in Group A (P = .269 and P = .688, respectively). Multivariate analyses confirmed that MRD state and modified DLI were significantly correlated with relapse (P = .000, odds ratio [OR] = 0.255 and P = .000, OR = 0.269) and DFS (P = .001, OR = 0.511 and P = .006, OR = 0.436, respectively). These data suggest that risk stratification-directed interventions with modified DLI in patients with standard-risk acute leukemia who are MRD(+) after transplantation may improve transplantation outcomes.

MRD-directed risk stratification treatment may improve outcomes of t(8;21) AML in the first complete remission: results from the AML05 multicenter trial

We aimed to improve the outcome of t(8;21) acute myeloid leukemia (AML) in the first complete remission (CR1) by applying risk-directed therapy based on minimal residual disease (MRD) determined by RUNX1/RUNX1T1 transcript levels. Risk-directed therapy included recommending allogeneic hematopoietic stem cell transplantation (allo-HSCT) for high-risk patients and chemotherapy/autologous-HSCT (auto-HSCT) for low-risk patients. Among 116 eligible patients, MRD status after the second consolidation rather than induction or first consolidation could discriminate high-risk relapse patients (P = .001). Allo-HSCT could reduce relapse and improve survival compared with chemotherapy for high-risk patients (cumulative incidence of relapse [CIR]: 22.1% vs 78.9%, P < .0001; disease-free survival [DFS]: 61.7% vs 19.6%, P = .001), whereas chemotherapy/auto-HSCT achieved a low relapse rate (5.3%) and high DFS (94.7%) for low-risk patients. Multivariate analysis revealed that MRD status and treatment choice were independent prognostic factors for relapse, DFS, and OS. We concluded that MRD status after the second consolidation may be the best timing for treatment choice. MRD-directed risk stratification treatment may improve the outcome of t(8;21) AML in CR1. This trial was registered at http://www.chictr.org as #ChiCTR-OCH-12002406.

Monitoring MRD with flow cytometry: an effective method to predict relapse for ALL patients after allogeneic hematopoietic stem cell transplantation

This study evaluated the prognostic value of minimal residual disease (MRD) monitoring by four-color flow cytometry (FCM) in patients with acute lymphoblastic leukemia (ALL) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). MRD was examined with four-color FCM at different time points in 139 patients (including pediatric and adult patients) with ALL after allo-HSCT. Real-time quantitative polymerase chain reaction (RQ-PCR) was applied to evaluate the MRD of Philadelphia chromosome-positive ALL (Ph+ ALL) patients. Patients who were FCM-positive (FCM+) after transplantation had a lower event-free survival (EFS) of 0.54 and a higher cumulative incidence of relapse (CIR) of 0.54 compared to an EFS of 0.80 and a CIR of 0.08 in FCM-negative (FCM−) patients (EFS, p < 0.001; CIR, p < 0.001). Similar results were obtained in high-risk patients and Ph+ ALL patients. Moreover, a FCM+ status after the second month post-HSCT (defined as MRD positive) proved to be a predictor of leukemia relapse. Multivariate analysis for EFS, OS and CIR showed that MRD status after transplantation was an independent prognostic factor (p < 0.001, p = 0.013, and p < 0.001, respectively). A good correlation was found between the MRD results of FCM and RQ-PCR (n = 126 pairs, Spearman r = 0.8139, p < 0.001). MRD monitoring by four-color FCM post-transplantation is an important tool for relapse prediction in ALL patients. Prompt and appropriate pre-emptive anti-leukemia treatment could be considered based on the status of MRD after HSCT.

Association of an Impaired Bone Marrow Microenvironment with Secondary Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

Poor graft function (PGF) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Whether abnormalities of the bone marrow (BM) microenvironment are involved in the pathogenesis of PGF is unclear. In the present prospective nested case-control study, 19 patients with secondary PGF, 38 matched patients with good graft function (GGF) after allo-HSCT, and 15 healthy donors (HDs) were enrolled. The cellular elements of the BM microenvironment, including endosteal cells, perivascular cells, and vascular cells, were analyzed by flow cytometry as well as hematoxylin and eosin and immunohistochemical staining in situ. The median time to occurrence of secondary PGF was 90 days post-transplantation (range, 58 to 264 days). The patients with PGF showed markedly hypocellular marrow (10% versus 45% versus 45%; P < .0001) with scattered hematopoietic cells and significantly lower CD34(+) cells (0.07% versus 0.26% versus 0.26%; P < .0001), endosteal cells (4 per high-power field [hpf] versus 16 per hpf versus 20 per hpf; P < .001), perivascular cells (0.008% versus 0.10% versus 0.12%; P < .0001), and endothelial progenitor cells (0.008% versus 0.16% versus 0.18%; P < .0001) compared with GGF allo-HSCT recipients and HDs, respectively. Multivariate analyses revealed that endothelial progenitor cells (odds ratio, 150.72; P = .001) and the underlying disease (odds ratio, 18.52; P = .007) were independent risk factors for secondary PGF. Our results suggest that the impaired BM microenvironment may contribute to the occurrence of secondary PGF post-HSCT.

Imatinib mesylate versus allogeneic hematopoietic stem cell transplantation for patients with chronic myelogenous leukemia in the accelerated phase

The relative merits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and imatinib for chronic myelogenous leukemia in the accelerated phase (AP-CML) have not previously been evaluated. This cohort study was designed to compare the outcomes of imatinib (n = 87) versus allo-HSCT (n = 45) for AP-CML. A multivariate analysis of the total population revealed that a CML duration ≥ 12 months, hemoglobin < 100 g/L, and peripheral blood blasts ≥ 5% were independent adverse prognostic factors for both overall survival (OS) and progression-free survival (PFS). Both treatments resulted in similar survival in low-risk (no factor) patients, with 6-year event-free survival (EFS), OS, and PFS rates of more than 80.0%. Intermediate-risk (any factor) patients showed no difference in EFS and OS, but 6-year PFS rates were 55.7% versus 92.9% (P = .047) with imatinib versus allo-HSCT, respectively. Among high-risk (at least 2 factors) patients, imatinib was by far inferior to allo-HSCT, with 5-year EFS, OS, and PFS rates of 9.3% versus 66.7% (P = .034), 17.7% versus 100% (P = .008), and 18.8% versus 100% (P = .006), respectively. We conclude that allo-HSCT confers significant survival advantages for high- and intermediate-risk patients with AP-CML compared with imatinib treatment; however, the outcomes of the 2 therapies are equally good in low-risk patients. All trials were registered with the Chinese Clinical Trial Registry (www.chictr.org) as CHiCTR-TNC-10000955.

Administration of imatinib after allogeneic hematopoietic stem cell transplantation may improve disease-free survival for patients with Philadelphia chromosome-positive acute lymphobla stic leukemia

BackgroundMaintenance therapy with imatinib during the post-transplant period has been used for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL); however, its efficacy has not been demonstrated. A study was designed to investigate the safety of imatinib and its efficacy in preventing hematological relapse and improving disease-free survival (DFS) when administered after allogeneic hematopoietic stem cell transplantation (allo-HCT).MethodsPatients with Ph + ALL that received allo-HCT were enrolled in the study. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to detect BCR-ABL transcript levels. Imatinib therapy was initiated if patient neutrophil counts were > 1.0 × 109/L and platelet counts were > 50.0 × 109/L, or if they displayed either elevated BCR-ABL transcript levels in two consecutive tests, or a BCR-ABL transcript level ≥ 10-2 after initial engraftment. Patients receiving imatinib after relapse were assigned to the non-imatinib group. The imatinib treatment was scheduled for 3–12 months, until BCR-ABL transcript levels were negative at least for three consecutive tests or complete molecular remission was sustained for at least 3 months.ResultsA total of 82 patients were enrolled. Sixty-two patients initiated imatinib therapy post-HCT. Imatinib therapy was initiated at a median time of 70 days post-HCT. Grade 3–4 adverse events (AEs) occurred in 17.7% of patients. Ten patients (16.1%) terminated imatinib therapy owing to AEs. Among the patients in imatinib and non-imatinib groups, the estimated 5-year relapse rate was 10.2% and 33.1% (p = 0.016), and the 5-year probability of DFS was 81.5% and 33.5% (p = 0.000) with the median follow-up of 31 months (range, 2.5-76 months) and 24.5 months (range, 4–72 months), respectively. Multivariate analysis identified imatinib maintenance therapy post-HCT as an independent prognostic factor for DFS (p = 0.000, hazard ratio [HR] =4.8) and OS (p = 0.000, HR = 6.2).ConclusionsThese results indicate that relapse rate can be reduced and DFS may be improved in Ph + ALL patients with imatinib maintenance therapy after HCT. BCR-ABL monitoring by qRT-PCR can guide maintenance therapy with imatinib including initiation time and treatment duration after allo-HCT.

Expression patterns of WT1 and PRAME in acute myeloid leukemia patients and their usefulness for monitoring minimal residual disease.

Both WT1 and PRAME are highly expressed in acute myeloid leukemia (AML) patients. To assess the efficacy of their simultaneous detection for the purpose of monitoring minimal residual disease (MRD), we used real-time quantitative RT-PCR to quantify both WT1 and PRAME transcript levels in the bone marrow of 204 newly diagnosed AML patients, and 21 patients were serially monitored for a median of 11 months. The 22 normal bone marrow samples which served as controls collectively expressed low levels of WT1 and PRAME. An increase of >1-log over the upper limit of normal bone marrow was defined as positive. The positive rates of WT1 and PRAME for all patients were 79.2% and 55.4%, respectively. For the 108 patients lacking a specific fusion gene, the positive rate of WT1 was significantly higher than that of PRAME (76.9% vs. 35.2%, P<0.001). PRAME was positive in 9/25 WT1 (-) patients and the log increase of PRAME was higher than that of WT1 in 12/83 WT1 (+) patients. Dynamic patterns of WT1 and PRAME during follow-up showed that a consistent elevation or a rise over time to exceed the normal range predicted clinical relapse. The exception was that one patients WT1 significantly decreased at relapse compared to diagnosis. Therefore, a simultaneous detection of WT1 and PRAME might not only provide AML patients with either a positive or a more sensitive molecular marker for MRD monitoring. Moreover, it might also avoid false negativity in the case of expression alteration.

Combined use of WT1 and flow cytometry monitoring can promote sensitivity of predicting relapse after allogeneic HSCT without affecting specificity.

Either WT1 or leukemia-associated aberrant immune phenotypes (LAIPs) was one of the minimal residual disease (MRD) parameters used to predict leukemia relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We first evaluated the clinical value of various positive MRD standards for accurately indicating relapse based on WT1 and FCM data in adult patients with acute leukemia (AL). In total, 824 AL patients treated with allo-HSCT were enrolled in this study. We compared the sensitivity and specificity of diverse, multiple-criteria MRD prognostic standards based on WT1 and FCM assays. Higher sensitivity was achieved without a loss of specificity when MRDco+, which was defined as two consecutive WT10.6+ or FCM+ or both WT10.6+ and FCM+ in the same sample within a year posttransplantation, was used as the positive MRD standard. Similar results were observed, even in 484 patients who had both abnormal WT1 and LAIPs values before transplant. A multivariate analysis showed that MRDco+ was an independent risk factor for leukemia relapse after transplant in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). The combined use of FCM and WT1 monitoring could distinguish between patients with low and high risks of relapse. Various positive MRD standards were useful for guiding intervention.

Characteristics of BCR–ABL kinase domain point mutations in Chinese imatinib-resistant chronic myeloid leukemia patients

To explore the characteristics of BCR–ABL kinase domain point mutations in Chinese chronic myeloid leukemia (CML) patients, a cohort of 127 patients with hematologic or cytogenetic resistance to imatinib are screened by direct sequencing. Mutations are found in 74 patients (58%). More patients with hematologic resistance show mutations compared to patients with cytogenetic resistance (70% vs 44%, p = 0.002), and more patients with acquired resistance present mutations compared to patients with primary resistance (68% vs 48%, p = 0.031). Frequencies of mutations were similar in early chronic phase (ECP), late chronic phase (LCP), accelerated phase, and blast phase (BP) patients (56%, 58%, 50%, and 69%, respectively; p > 0.05). Overall, 25 mutants are found in 21 amino acid sites, and four of them (I418V, E450A, E453L, and E455K) are first reported here. All patients with these four mutants either progress to or reenter the BP. The most frequent mutant is M244V, followed by Y253H, F359C/V/I, G250E, E255K, and T315I. Only seven patients (9%) have T315I mutants, and all showed hematologic resistance. Three of them were in the ECP and three in the LCP. Look-back studies show that mutants were detected 0–20 (median 7) months ahead of the appearance of clinical resistance in 15 tested patients with acquired resistance. ABL mutations are common in Chinese imatinib-resistant CML patients and are associated with clinical resistance. Chinese patients also seem to have unique profiles in the types and frequencies of some mutants, the disease phases of patients with T315I mutation, and the frequency of mutations in CP patients.

Two Single-Nucleotide Polymorphisms with Linkage Disequilibrium in the Human Programmed Cell Death 5 Gene 5′ Regulatory Region Affect Promoter Activity and the Susceptibility of Chronic Myelogenous Leukemia in Chinese Population

Purpose: Chronic myelogenous leukemia (CML) is a disease characterized cytogenetically by the presence of the Philadelphia chromosome. Recent studies suggested that altered PDCD5 expression may have significant implications in CML progression. The aim of this study was to identify single-nucleotide polymorphisms (SNP) within the programmed cell death 5 (PDCD5) promoter region and show their functional relevance to PDCD5 expression as well as their genetic susceptibility to CML. Experimental Design: One hundred twenty-nine CML subjects and 211 healthy controls were recruited for identification of SNPs and subsequent genetic analysis. Luciferase reporter assays were carried out to show the functional significance of the SNPs located in the promoter region to PDCD5 expression. Real-time quantitative PCR and Western blot analysis were done to determine the expression differences of PDCD5 in CML patients with different genotypes. Results: Two SNPs were identified within the PDCD5 promoter. They are −27A>G and −11G>A (transcription start site as position 1), respectively. The complete linkage disequilibrium was found between these two polymorphisms. The frequencies of −27G+/−11A+ genotype and −27G/−11A allele were significantly higher in CML patients than in healthy controls (genotype: 26.36% versus 11.85%, χ2=11.75, P < 0.01; allele: 13.57% versus 6.40%, χ2 = 9.48, P < 0.01). Luciferase reporter assays revealed that the promoter with −27G/−11A had significantly lower transcriptional activity and could not be up-regulated after apoptotic stimulations compared with the promoter with −27A/−11G. PDCD5 expression analysis in mononuclear cells derived from CML patients and cell lines with different −27/−11 genotypes showed consistent results with the reporter assays. Conclusions: These data suggest that −27G/−11A is associated with reduced PDCD5 promoter activity and increased susceptibility to CML.