Xiao-Su Zhao

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.

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.

Copy Number Variations of EphA3 Are Associated With Multiple Types of Hematologic Malignancies

BACKGROUND EphA3 is a component of the Eph receptor family, the largest subgroup of the receptor tyrosine kinase (RTK) family. A recent array-based study implicated the presence of copy-number variations (CNVs) of EphA3 in the genomes of acute myelogenous leukemia. CNVs are present in the general population at varying degrees, and have been found to associate with various types of diseases including hematologic malignancies. However, most of the current studies focused on the genome-wide screening of CNVs, and the functional impact of such regions needs to be extensively investigated in large number of clinical samples. PATIENTS AND METHODS In our study, we collected 617 bone marrow samples from multiple types of hematologic malignancies as well as healthy controls. DNA copy numbers and mRNA levels of EphA3 in these samples were examined. RESULTS We found significant association between the CNVs of EphA3 and these hematologic malignancies including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), and myelodysplastic syndrome (MDS). We also observed a positive correlation between the relative mRNA level and gene dosage of EphA3. CONCLUSION The CNVs of EphA3 were associated with multiple types of hematologic malignancies including ALL, AML, CLL, CML, MM, and MDS.

Monocytic and promyelocytic myeloid-derived suppressor cells may contribute to G-CSF-induced immune tolerance in haplo-identical allogeneic hematopoietic stem cell transplantation

We investigated the effects of granulocyte colony‐stimulating factor (G‐CSF) on monocytic (M), promyelocytic (P), and granulocytic (G) myeloid‐derived suppressor cells (MDSCs) both in bone marrow and peripheral blood of 20 healthy donors and the association of MDSCs subgroups with acute and chronic graft‐versus‐host disease (aGvHD/cGvHD) in 62 patients who underwent haplo‐identical allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Patients who received a higher absolute counts of M‐MDSCs or P‐MDSCs exhibited lower incidence of grade II–IV aGvHD (P = 0.001; P = 0.031) and extensive cGvHD (P = 0.011; P = 0.021). In the multivariate analysis, absolute counts of MDSCs in allografts emerged as independent factors that reduced the occurrence of grade II–IV aGvHD (M‐MDSCs: HR = 0.087, 95% CI = 0.020–0.381, P = 0.001; P‐MDSCs: HR = 0.357, 95% CI = 0.139–0.922, P = 0.033) and extensive cGvHD (M‐MDSCs: HR = 0.196, 95% CI = 0.043–0.894, P = 0.035; P‐MDSCs: HR = 0.257, 95% CI = 0.070–0.942, P = 0.04). Delayed M‐MDSC reconstitution was associated with aGvHD onset. The 3‐year cumulative incidence of transplant related mortality and relapse, 3‐year probability of disease‐free survival, and overall survival did not differ significantly between these subgroups. Our results suggested that G‐CSF‐induced immune tolerance may be mediated by M/P‐MDSCs in allo‐HSCT. Am. J. Hematol. 90:E9–E16, 2015.

Recipient expression of ligands for donor inhibitory KIRs enhances NK‐cell function to control leukemic relapse after haploidentical transplantation

Natural killer (NK) cells that express self‐HLA‐specific receptors (where HLA is human leukocyte antigen) are “licensed” and more readily activated than unlicensed cells; therefore, NK‐cell licensing could influence the antileukemia effects of NK cells following haploidentical stem cell transplantation (haplo‐SCT). In this study, we compared the functionality of reconstituting NK cells, based on CD107α expression and interferon‐γsecretion, in a cohort of 29 patients that expressed (n = 8) or lacked (n = 21) class I human leukocyte antigens for donor inhibitory killer cell immunoglobulin‐like receptors (KIRs) following T‐cell‐replete haplo‐SCT. We also addressed whether recipient expression of class I ligands for donor inhibitory KIRs could predict relapse occurrence in another cohort of 188 patients. A longitudinal analysis indicated that patients presenting class I for all donor inhibitory KIRs showed more capable functional NK effector cells when tested against class I negative K562 cells and primary leukemic cells within 3 months of transplantation. The lowest 7‐year relapse incidence was observed when donor KIRs were ligated by recipient class I (n = 60) compared with donor–host partnerships where donor KIR+ cells were ligated by donor, but not recipient class I (n = 86, p = 0.026) or KIRs that were ligated by neither donor nor recipient class I (n = 42, p = 0.043). This study suggests that haplo‐SCT recipients presenting class I for donor inhibitory KIRs promote NK‐cell licensing, leading to decreased relapse rates.

CXCR4 is a good survival prognostic indicator in multiple myeloma patients

SDF-1α and its receptor CXCR4 are involved in multiple myeloma (MM) by attracting and activating plasma cells in the bone marrow. CXCR4 expression in MM cells is inversely correlated with disease activity. The aim of this study was to evaluate CXCR4 as a prognostic tool in MM, as well as other markers of disease, such as chromosomal aberrancies. Purpose was to investigate the expression levels of SDF-1α before and after bortezomib and thalidomide treatment. From February 2006 to April 2012, CXCR4 expression was prospectively assessed in bone marrow samples from a large population of patients (n=227) using flow cytometry. Clinical characteristics were collected and chromosomal aberrancies were assessed in 144 patients. SDF-1α levels were determined using ELISA in peripheral blood samples from 40 patients before and after chemotherapy. Our results show that CXCR4 was present in 43.2% (98/227) of newly diagnosed MM patients and that CXCR4 expression was significantly correlated with CD117 (P<0.05). CXCR4-positive MM patients had a significantly longer estimated survival time than CXCR4-negative patients (median of 48 vs. 42 months, P<0.05). Multivariate survival analyses identified that the +1q21/CXCR4- phenotype is an independent survival predictor, along with the International Staging System (ISS) stage. No significant difference was observed in expression levels of SDF-1α before and after bortezomib/thalidomide treatment. In conclusion, +1q21/CXCR4- could be an independent survival prognosis predictor in MM patients. Expression levels of SDF-1α before and after bortezomib/thalidomide treatment are not different, although they are higher than in controls.

Higher frequency of regulatory T cells in granulocyte colony-stimulating factor (G-CSF)-primed bone marrow grafts compared with G-CSF-primed peripheral blood grafts

BackgroundRegulatory T cells (Treg) in allografts are important for the prevention of graft-versus-host disease (GVHD) post-transplantation. The aim of this study was to compare the contents of Tregs and effector T cells in granulocyte colony-stimulating factor (G-CSF)-primed bone marrow grafts (G-BM) and peripheral blood grafts (G-PB).MethodG-BM and G-PB were obtained from 20 allogeneic donors. T-cell subgroups, including conventional T cells and different types of Treg cells, as well as the percentage of Ki67 expression on CD4+CD25highFoxp3+ Treg cells, were analyzed using flow cytometry. The levels of interferon-γ (IFN-γ) and interleukin-17 (IL-17) secreted by T cells stimulated with PMA and ionomycin were also determined by flow cytometry.ResultsThe percentage of CD4+CD25highCD127-/dimCD62L+ Treg cells was significantly higher in the G-BM group, with higher proportions of CD45RA+ naïve Treg cells and higher expression of CD69 on Treg cells in G-BM (P < 0.05). The percentage of Ki67 expression in CD4+CD25highFoxp3+ Treg cells in G-BM was significantly higher than that on G-PB. The suppressive functions of Treg cells in inhibiting T-cell activation were comparable between G-BM and G-PB. The proportions of CD4+CD25−CD69+ Treg subsets as well as Th1 cells in G-BM were also significantly higher than those in G-PB (P < 0.001). The proportions of conventional T cells and Th17 effector cells were comparable in G-BM compared with those in G-PB. Thus, the ratio of conventional T cells and CD4+CD25highCD127-/dim regulatory T cells were lower in G-BM than that in G-PB (P = 0.014).ConclusionIn addition to the much higher T-cell counts in G-PB grafts that may contribute to more severe GVHD, the higher frequency of Treg cells and lower ratio of conventional T cells to Treg cells in G-BM compared with G-PB grafts might reduce GVHD post-transplantation in G-BM compared with G-PB transplantation.

Cytomegalovirus is a potential risk factor for late‐onset hemorrhagic cystitis following allogeneic hematopoietic stem cell transplantation

Late‐onset hemorrhagic cystitis (LOHC) is a common complication following allogeneic hematopoietic stem cell transplantation (allo‐HSCT) and is primarily associated with viral infection. We prospectively quantified cytomegalovirus (CMV), BK virus (BKV), and adenovirus in urine and plasma using Q‐RT‐PCR in 50 consecutive patients to define the relationship between virus and LOHC. Of the 50 patients, 21 developed LOHC at a median of 29 days (range 4–64 days), with a cumulative incidence of 42% (±7.1%). The cumulative incidence of LOHC on day 100 in patients with and without CMV viremia (prior to or at the onset of LOHC) were 56.3% (±8.9%) and 16.7% (±9.1%) (P = 0.018), respectively, and it was 59.3% (±9.8%) and 21.7% (±8.8%) in patients with and without CMV viruria (prior to or at the onset of LOHC) (P = 0.021), respectively. The cumulative incidence of LOHC was also higher in patients with a plasma BKV load increased ≥3 log10 or with a urine BKV load increased ≥4 log10 than those without the increase (P < 0.001). Only one patient with LOHC was tested positive for ADV. Both the univariate and multivariate analyses showed that CMV viremia (HR = 3.461, 95% CI: 1.005–11.922, P = 0.049) and a plasma BKV load that was increased ≥3 log10 (HR = 10.705, 95%CI: 2.469–46.420, P = 0.002) were independent risk factors for the development of LOHC. We conclude that both CMV viremia and an increase of plasma BKV are independent risk factors for LOHC. And the role of CMV viremia was firstly demonstrated. Am. J. Hematol. 89:55–61, 2014.

Early lymphocyte recovery predicts superior overall survival after unmanipulated haploidentical blood and marrow transplant for myelodysplastic syndrome and acute myeloid leukemia evolving from myelodysplastic syndrome

Abstract We investigated whether early lymphocyte recovery, after unmanipulated, haploidentical, blood and marrow transplant (HBMT), affected clinical outcomes in 78 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia evolving from MDS. Lymphocyte recovery was based on the absolute lymphocyte count on day 30 (ALC-30). Patients with high ALC-30 (≥ 300 cells/μL) had lower relapse rates (13.8% vs. 35.5%, p = 0.049) and lower incidence of bacterial infections (3.4% vs. 25.8%, p = 0.015) than those with low ALC-30 values. Multivariate analysis showed that a high ALC-30 was associated with improved overall survival (OS, hazard ratio [HR]: 0.099, 95% confidence interval [CI]: 0.029–0.337; p < 0.0001), improved leukemia-free survival (HR: 0.245, 95% CI: 0.112–0.539; p < 0.0001), lower relapse rate (HR: 0.096, 95% CI: 0.011–0.827; p = 0.033) and lower transplant-related mortality (TRM, HR: 0.073, 95% CI: 0.016–0.324; p = 0.001). Combinations of three mismatches in the human leukocyte antigen loci were associated with a higher TRM (HR: 5.026, 95% CI: 1.392–18.173; p = 0.014). Our results suggest that the ALC-30 can predict a favorable OS after unmanipulated HBMT.

Improved clinical outcomes of rhG-CSF-mobilized blood and marrow haploidentical transplantation compared to propensity score-matched rhG-CSF-primed peripheral blood stem cell haploidentical transplantation: a multicenter study.

The effects of haploidentical rhG-CSF-mobilized blood and marrow transplantation (HBMT) on hematological malignances are well established. Previous prospective single-center studies have demonstrated better survival after HBMT versus haploidentical rhG-CSF-mobilized peripheral blood stem cell transplantation (HPBSCT) for acute leukemia (AL) not in remission (NR) or in more than the second complete remission (>CR2). To test the hypothesis that HBMT is still superior to HPBSCT for patients with AL, multiple myeloma (MM), or non-Hodgkin lymphoma (NHL) in CR1/CR2 and for patients with chronic myeloid leukemia in the first and second chronic phase lacking a matched donor, we designed a propensity score method-based multicenter study. Hematopoietic recovery, acute graft-versus-host disease (aGVHD), and chronic GVHD were comparable between the HBMT group (n=168) and the HPBSCT group (n=42). No significant differences were found in non-relapse mortality rate (20.17%±3.58% and 27.24%±7.16%, P=0.18) or relapse rate (19.96%±3.72% and 28.49%±8.25%, P=0.32) between the HBMT group and the HPBSCT group. HBMT recipients had better overall survival (65.0%±4.2% and 54.2%±8.3%, P=0.037) and disease-free survival (59.9%±4.6% and 44.3%±8.7%, P=0.051). Multivariate analysis showed that HPBSCT was associated with poorer DFS (HR (95%CI), 1.639 (0.995–2.699), P=0.052). Our comparisons showed that HBMT was superior to HPBSCT as a post-remission treatment for patients lacking an identical donor.