Poor graft function (PGF), including early and late PGF, is a serious complication following allotransplant. We recently reported that bone marrow microenvironment abnormalities may occur in cases of late PGF. Whether these abnormalities occur in early PGF remains unknown. To answer this question, we performed a nested case–control study comparing cellular elements of the bone marrow microenvironment in 10 subjects with early PGF, 30 subjects with late PGF and 40 subjects without PGF. Bone marrow endosteal cells, perivascular cells and endothelial cells were analyzed by flow cytometry and by hematoxylin–eosin and immunohistochemical staining in situ. Subjects with early and late PGF had similar abnormalities in these cell types compared with transplant recipients without PGF. However, none of the aforementioned elements of the bone marrow microenvironment were significantly different between early and late PGF patients. Our data suggest that similar abnormalities in the bone marrow microenvironment may occur in early and late PGF post allotransplant. Cellular approaches, such as the administration of mesenchymal stem cells, promise to be beneficial therapeutic strategies in patients with early or late PGF.
Poor graft function (PGF), including early and late PGF, remains a life-threatening complication and is associated with serious infections or hemorrhagic complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT).1, 2, 3, 4, 5 Especially with the increasing use of HLA-mismatched/haploidentical allo-HSCT (HBMT) in the past 10 years, PGF has become a growing obstacle contributing to high morbidity and mortality after transplantation.6, 7 Sun et al.4 recently reported that subjects with early PGF, with an incidence of ~5.6% in HBMT, have significantly poorer overall survival at 2 years than subjects with good graft function (GGF; 34.6 vs 82.7%, P<0.001). Moreover, Chang et al.5 have identified early PGF as an independent factor for inferior survival after unmanipulated HBMT in multivariate analysis. Nevertheless, the available treatment options for PGF are limited and include the administration of hematopoietic growth factors, a second allo-HSCT, or a CD34+-selected stem cell boost, among others.1, 8, 9, 10 Therefore, obtaining a better understanding of the pathophysiology of PGF promises to guide more effective therapeutic approaches and eventually improve survival.
Rapid and effective hematopoietic recovery has a central role in successful allo-HSCT. Considerable data from murine studies have revealed that effective hematopoiesis depends on the specific bone marrow microenvironment, known as a ‘niche’, where hematopoietic stem cells (HSCs) reside.11, 12, 13, 14 Bone marrow endothelial cells, perivascular cells and endosteal cells have been validated as the key cells that support HSCs in the murine bone marrow microenvironment.11, 12, 13, 14 We recently reported abnormalities of the bone marrow microenvironment in transplant recipients with late PGF2 and prolonged isolated thrombocytopenia.15 Our previous data suggest that an impaired bone marrow microenvironment may contribute to the occurrence of different degrees of delayed hematopoietic reconstitution after allo-HSCT. Thus, we speculate that the occurrence of early PGF may also be associated with a defective bone marrow microenvironment.
Here, we performed a nested case–control study of subjects with early and late PGF to determine whether similar abnormalities of the bone marrow microenvironment occurred at both kinds of PGF. We compared both PGF cohorts with allotransplant recipients with normal bone marrow function.
Materials and methods
We designed a prospective, nested case–control study that included 10 subjects with early PGF who were identified from a cohort receiving allotransplants for hematologic neoplasms between 1 April 2012 and 31 March 2014 at Peking University Institute of Hematology. For each case, three matched controls with late PGF (n=30) and four matched controls with GGF (n=40) were randomly selected. Matching was based on age (±1 year), diagnosis, pre-allotransplant cycles of chemotherapy (±1 cycle) and disease status at HSCT (risk-set sampling).16 The characteristics of the cohorts are summarized in Table 1. The study was approved by the Ethics Committee of Peking University People’s Hospital, and written informed consent was obtained from all the subjects in accordance with the Declaration of Helsinki.
Clinical definitions and evaluation
GGF2, 15 was defined as a persistent successful engraftment (neutrophils >0.5 × 10E+9/L for 3 consecutive days, platelets >20 × 10E+9/L for 7 consecutive days without platelet transfusion and hemoglobin concentration >70 g/L without RBC transfusion) >28 days post transplant. PGF (both early and late)2 was defined as the presence of the following two to three cytopenic counts: (1) neutrophils ⩽0.5 × 10E+9/L, (2) platelets ⩽20 × 10E+9/L and/or (3) hemoglobin concentration ⩽70 g/L for ⩾3 consecutive days beyond day +28 post transplant or in accordance with platelet and/or RBC transfusion requirements. Hypoplastic–aplastic bone marrow with complete donor chimerism also had to be present (see below). Subjects with hematologic evidence of relapse were excluded. Early (primary) PGF was defined as a slow or incomplete recovery of bone marrow function ⩾28 days post transplant. Late (secondary) PGF was defined as a loss of bone marrow function after achieving normal bone marrow function and without relapse.
Chimerism analyses were performed by DNA fingerprinting for short tandem repeats in blood samples and/or by chromosome fluorescent in situ hybridization of bone marrow samples. Complete donor chimerism was defined as no recipient hematopoietic or lymphoid cells detected; the sensitivity of these methods enables the detection of as low as 0.1% of recipient signals.17 Diagnoses were categorized as standard or high risk. Standard risk was defined as first or second CR (CR1 or CR2) of acute leukemia or myelodysplastic syndrome. All the other subjects were classified as high risk. Hematologic relapse was defined by the reappearance of blasts in the blood or bone marrow (>5%) or in any extra-medullary site after CR based on histological criteria. GvHD was scored as acute or chronic as previously described.
Donor selection, HLA-typing, graft harvesting, conditioning therapy and GvHD prophylaxis were done as reported.2, 17, 18, 19 The patients were screened pre-transplant for CMV infection by serology. Weekly real-time quantitative PCR was used to detect CMV reactivation in blood samples. CMV infections were treated with ganciclovir or foscarnet as described.20 After allo-HSCT, rhG-CSF (5 μg/kg/day) was administered to recipients of HLA-mismatched related transplants from day +6 until neutrophils were >0.5 × 10E+9/L for 3 consecutive days. rhG-CSF was not administered to recipients of HLA-identical sibling transplants, except in cases where neutrophils were <0.5 × 10E+9/L until day +21. The subjects received RBCs if their hemoglobin concentrations were ⩽70 g/L, or following platelet transfusion if their platelets were ⩽20 × 10E+9/L.
The gating strategies used for perivascular cells and bone marrow endothelial cells were previously reported2, 15 and are shown in Figure 1. An aliquot of 5 ml of fresh bone marrow was stained with mouse anti-human CD146-PE/CD34-PECy7/CD45-V500/vascular endothelial growth factor receptor 2 (VEGFR2) Alexa fluor 647-conjugated monoclonal antibodies (Becton Dickinson, San Jose, CA, USA). Multi-parameter flow cytometric analyses were performed using a BD LSRFortessa (Becton Dickinson), and 2 000 000 events were routinely collected. Aliquots of unstained samples were used as negative controls. The data were analyzed using BD LSRFortessa software (Becton Dickinson).
Bone marrow trephine biopsies were stained with hematoxylin–eosin. Immune histochemistry staining was performed using rabbit anti-human osteopontin, CD34 and CD146 primary antibodies (Abcam, Cambridge, MA, USA) as previously described.2, 15 The slides were read blindly by two observers. Each section was analyzed using light microscopy (Axiovert 200; Carl Zeiss, Jena, Germany). Bone marrow cellularity was categorized into three groups.2, 15 Endosteal cells were scored as numbers of osteopontin-positive cells on the line of trabecular bone per high-power field. Numbers of CD146-positive perivascular cells per microvessel were enumerated. Bone marrow microvessel density was enumerated using a CD34-reactive monoclonal Ab. The median numbers of vessels per trabecular bone was quantified.
Statistical analyses were performed using one-way analysis of variance to compare the three groups. Subject variables were compared using the χ2 test for categorical variables and the Mann–Whitney U-test for continuous variables. Analyses were performed using SPSS 16.0 (IBM, Armonk, NY, USA) and GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA) software packages, and P-values <0.05 were considered statistically significant.
The median time to PGF occurrence in patients with early and late PGF was 30 days (range, 28–30 days) vs 90 days (range, 60–210 days) post transplant (P<0.0001). Complete donor chimerism was confirmed by PCR-based DNA fingerprinting of short tandem repeats in bone marrow cells at the time of PGF. As shown in Table 1, none of the demographic or clinical characteristics of the assessed individuals, including recipient age, gender, underlying disease, disease status pre-transplantation, median time from diagnosis to transplantation, source of stem cells, transplanted total nucleated cell dose, CD34+ cell dose, donor age, donor HLA match, sex/ABO mismatch, pre-HSCT cycles of chemotherapy, conditioning, history of GvHD or CMV, and anti CMV therapy with ganciclovir, showed significant differences between subjects with early and late PGF (P>0.05).
Subjects with early and late PGF had similar degrees of bone marrow hypocellularity compared with those of the GGF cohort (10% and 10% vs 45%, P<0.0001; Figure 2). The median WBCs (1.34 × 10E+9/L vs 1.62 × 10E+9/L, P=0.16), neutrophils (0.34 × 10E+9/L vs 0.35 × 10E+9/L, P=0.99) and platelets (12 × 10E+9/L vs 18 × 10E+9/L, P=0.15) were similar in subjects with early and late PGF. Hemoglobin concentration (58 g/L vs 68 g/L, P=0.001) was significantly lower in the early PGF cohort.
Subjects with early and late PGF had few CD34+ cells compared with subjects with GGF (0.05 and 0.07% vs 0.26%; P<0.0001; Figure 3).
The numbers of CD45−CD34−CD146+ perivascular cells (0.008 vs 0.01 vs 0.10%, P<0.0001) and CD45−CD34+VEGFR2+ bone marrow endothelial cells (0.007 and 0.008 vs 0.16%; P<0.0001; Figures 1 and 3) were dramatically decreased in subjects with early and late PGF compared with subjects with GGF. However, the aforementioned elements of the bone marrow microenvironment displayed no significant differences between early and late PGF patients.
In situ histological analysis of the bone marrow trephine biopsies from the recipients was performed to further characterize the human bone marrow microenvironment post transplantation. Trabecular bone was lined by fewer detectable endosteal cells per high-power field in early and late PGF compared with GGF (3 and 4 vs 16; P<0.0001; Figure 4). Consistent with the flow cytometry data, the frequency of CD146-positive perivascular cells per microvessel was decreased in early and late PGF compared with GGF (2 and 2 vs 4; P<0.05). The median numbers of CD34-positive microvessels per trabecular bone was also significantly reduced (2 and 2 vs 5; P<0.05; Figure 5). In contrast, the numbers of endosteal, perivascular and CD34+ vascular cells detected by immunohistochemical staining were not significantly different between the early and late PGF groups.
In this prospective, nested case–control study, we demonstrated for the first time that anatomically and phenotypically defined cellular elements,11, 12, 13, 14 including endosteal, perivascular and vascular cells, of the bone marrow microenvironment are similarly impaired in early and late PGF patients compared with GGF patients. Our data indicate that an impaired bone marrow microenvironment was associated with the occurrence of early and late PGF post allotransplant.
Effective cross-talk between HSCs and the bone marrow microenvironment has an important role in murine hematopoiesis.11, 12, 13, 14 Consistent with our previous study in late PGF patients,2 in the current study, we found that although transplanted numbers of donor CD34+ cells were normal, CD34+ cells and the cellular elements of the bone marrow microenvironment were similarly impaired in early and late PGF patients compared with GGF patients. Several risk factors, including CD34+ cell numbers in the graft, disease state, drug-induced toxicity, host vs graft reaction, GvHD and infections, especially with CMV, have been associated with PGF in previous studies.7, 21 However, the etiology of bone marrow microenvironment impairment and how this impairment drives PGF occurrence in allotransplant patients remains unknown.
Evidence from in vitro and murine studies indicate several risk factors, including GvHD and CMV infection, that can damage the bone marrow microenvironment, resulting in PGF.22, 23 Consistent with this idea, Shono et al.22 reported that impaired hematopoiesis was not caused by direct damage to CD34+ cells in GvHD but was instead because of GvHD-targeted impaired osteoblasts in the bone marrow endosteal niche, which consequently led to hematopoietic dysfunction after murine MHC-mismatched transplant. As reported by Yao et al.,23 the vascular niche is a target of GvHD in the MHC-haploidentical-matched murine transplant model. Moreover, the CD34+ and bone marrow stromal cell compartments can be reduced in the presence of CMV infection.24 On the basis of our preliminary data and on previous reports,22, 23, 24 it is conceivable that some post-HSCT events, such as GvHD, CMV reactivation and some myelotoxic drugs, can induce bone marrow microenvironment impairment, which may subsequently lead to defective HSCs and the occurrence of PGF. Nevertheless, definitive proof of our hypothesis regarding the causal relationship between donor CD34+ cells and recipient bone marrow microenvironment in PGF will either require further functional evaluation of donor CD34+ cells in the context of xenotransplantation or the demonstration that a second transplant from the same or a different donor would also develop PGF in the future.
Owing to the lack of mechanistic studies, the optimal therapeutic approaches for PGF have not been well established. The stimulation of engrafted HSCs (for example, the use of hematopoietic growth factors such as rhG-CSF and TPO) represents a temporary approach for alleviating PGF. The prognosis of a second allo-HSCT is dismal even with conditioning. The high frequencies of treatment-related mortality, opportunistic infections and GvHD pose a significant obstacle to a second allo-HSCT. More recently, Askaa et al.,8 Klyuchnikov et al.9 and Stasia et al.10 reported promising results in treating PGF patients with a CD34+-selected stem cell boost without further conditioning. However, it should be noted that most patients who received the boosted CD34+ cell infusion had late PGF. Therefore, obtaining better understanding of the mechanisms of early and late PGF will enhance the effectiveness of choosing a therapeutic approach based on pathophysiology in the future, rather than choosing on the basis of etiology, as is done currently.
Mesenchymal stem cells (MSCs), a type of multipotent adult stem cell that can be isolated from bone marrow, cord blood and adipose tissue, have the capacity to repair the bone marrow microenvironment, support hematopoiesis and regulate immunologic responses.3, 25, 26 Clinical applications of MSCs are evolving rapidly, with the goal of promoting hematopoietic recovery after transplantation.3, 26 If PGF is diagnosed, and infection, GvHD or a myelotoxic agent is not responsible for insufficient bone marrow function, the administration of MSCs is an effective option for improving graft function. The positive effects produced by MSCs on hematopoietic engraftment have been demonstrated in PGF after allo-HSCT.3, 26
In conclusion, this prospective, nested case–control study provides evidence that bone marrow endothelial cells, perivascular cells and endosteal cells are similarly reduced in allotransplant patients with early and late PGF. On the basis of their similar pathophysiologies, cellular approaches, such as the administration of MSCs,3, 26 may facilitate hematopoietic recovery and eventually improve the outcomes of patients with early or late PGF.
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Professor Robert Peter Gale (Imperial College London) kindly reviewed the manuscript. This work was supported by the National Natural Science Foundation of China (grant nos 81370638, 81570127, 81530046 and 81230013), the Beijing Municipal Science and Technology Program (grant nos Z151100004015164, Z151100001615020, Z141100000214011), the National High Technology Research Development Program of China (grant no. SS2013AA020104). We thank Professor Qing Ge for helpful discussions and suggestions. The language editing service American Journal Experts (www.americanjournalexperts.com) provided English editorial assistance to the authors during the preparation of this manuscript. We thank all the core facilities at Peking University Institute of Hematology for the sample collection.
X-JH designed the study and supervised the analyses and manuscript preparation. YK performed the research, analyzed and interpreted the data, performed statistical analysis and wrote the manuscript. All the other authors participated in the collection of patients’ data. All the authors agreed to submit the final manuscript.
The authors declare no conflict of interest.
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