Pharmacogenetics of steroid‐responsive acute graft‐versus‐host disease

Glucocorticoids are central to effective therapy of acute graft‐versus‐host disease (GVHD). However, only about half of the patients respond to steroids in initial therapy. Based on postulated mechanisms for anti‐inflammatory effectiveness, we explored genetic variations in glucocorticoid receptor, co‐chaperone proteins, membrane transporters, inflammatory mediators, and variants in the T‐cell receptor complex in hematopoietic cell transplant recipients with acute GVHD requiring treatment with steroids and their donors toward response at day 28 after initiation of therapy. A total of 300 recipient and donor samples were analyzed. Twenty‐three SNPs in 17 genes affecting glucocorticoid pathways were included in the analysis. In multiple regression analysis, donor SNP rs3192177 in the ZAP70 gene (O.R. 2.8, 95% CI: 1.3‐6.0, P=.008) and donor SNP rs34471628 in the DUSPI gene (O.R. 0.3, 95% CI: 0.1‐1.0, P=.048) were significantly associated with complete or partial response. However, after adjustment for multiple testing, these SNPs did not remain statistically significant. Our results, on this small, exploratory, hypothesis generating analysis suggest that common genetic variation in glucocorticoid pathways may help identify subjects with differential response to glucocorticoids. This needs further assessment in larger datasets and if validated could help identify subjects for alternative treatments and design targeted treatments to overcome steroid resistance.     

Modified donor lymphocyte infusion‐associated acute graft‐versus‐host disease after haploidentical T‐cell‐replete hematopoietic stem cell transplantation: incidence and risk factors

We performed a study to investigate the profile of donor lymphocyte infusion (DLI)‐associated acute graft‐versus‐host disease (GVHD) in haploidentical T‐cell‐replete hematopoietic stem cell transplantation (HSCT). A total of 124 patients receiving modified DLI after haploidentical T‐cell‐replete HSCT were enrolled. The cumulative incidence of DLI‐associated acute GVHD was 53.2% for grades II–IV and 28.4% for grades III–IV. The duration of GVHD prophylaxis after DLI was the only risk factor for DLI‐associated grades III–IV acute GVHD (p < 0.05). The cumulative incidence of grades III–IV acute GVHD in patients with prophylaxis more than six, four to six, two to four, and <2 wk were 9.3%, 14.4%, 31.6%, and 49.5%, respectively (p = 0.018). Furthermore, DLI‐associated grades III–IV acute GVHD was the only risk factor for overall survival (p = 0.038, OR   = 2.869) and transplant‐related mortality (p = 0.018, OR = 3.296) but not a risk factor for relapse after DLI (p = 0.840). This study confirms for the first time that the duration of GVHD prophylaxis after DLI is the only risk factor for the development of grades III–IV acute GVHD. Donor lymphocyte infusion with prophylaxis more than six wk was associated with a lower incidence of grades III–IV acute GVHD.     

Serum microRNA155 is increased in patients with acute graft‐versus‐host disease

To explore the clinical relevance of three lymphocyte‐related serum microRNAs (miR‐155, miR‐214, and miR‐326) to the pathogenesis of graft‐versus‐host disease (GVHD), 64 subjects who received allogeneic peripheral blood stem cell transplantation (allo‐PBSCT) were recruited in this study, of whom 19 subjects did not develop GVHD, 25 subjects were diagnosed with acute GVHD (aGVHD), and 20 subjects were diagnosed with chronic GVHD (cGVHD). Serum miRNAs were determined by real‐time RT‐PCR. Expression level of miRNAs and the expression signatures of miRNAs as a panel were analyzed among the three groups. The expression level of miR‐214 and miR‐326 showed no significant difference between GVHD and non‐GVHD groups. However, miR‐155 was significantly up‐regulated in GVHD patients. There was a correlation between the level of miR‐155 and the severity of aGVHD. Moreover, serum IFN‐gamma, IL‐17, and IL‐9 levels were higher in aGVHD patients with high miR‐155. In conclusion, the expression level of lymphocyte‐related miR‐155 in serum was significantly increased in aGVHD patients. The miR‐155 may be considered as a potential targeted therapy for aGVHD patients.     

Limited benefit of pentostatin salvage therapy for steroid‐refractory grade III‐IV acute graft‐versus‐host disease

Corticosteroid‐refractory (SR) acute graft‐versus‐host disease (aGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Multiple agents have been evaluated in this setting, but the benefit of pentostatin has not been described well. We report a single‐center experience of pentostatin salvage therapy for SR aGVHD. Fifteen patients received pentostatin for SR aGVHD from March 2005 till March 2010 after failure to respond to methylprednisolone ≥2 mg/kg/d for at least seven d. All patients had grade III‐IV aGVHD prior to pentostatin therapy. Thirteen (87%), 10 (67%), and six (40%) patients had gastrointestinal (GI), skin, and liver involvement of aGVHD, respectively. Pentostatin was given at a median of 33 d after steroid therapy initiation. The dose of pentostatin was 1.4 mg/m² daily for three d, repeated every two wk. Eight (53%) patients also received additional therapies. Complete responses were noted in two patients (both in skin). However, one patient relapsed and did not respond to additional salvage treatment. Partial responses were seen in three patients. Fourteen died of GVHD‐related causes. This study suggested that pentostatin is of limited benefit in the treatment for SR grade III‐IV aGVHD.     

Multidonor bone marrow transplantation improves donor engraftment and increases the graft versus tumor effect while decreasing graft‐versus‐host disease

In partially matched donor transplantation, mandatory T‐cell depletion (TCD) increases the risks of rejection/graft failure, relapse, and post‐transplant infections. A multi‐donor approach was offered to resolve some of these drawbacks. This hypothesis was previously tested in a TCD fully mismatched murine model. However, the effect of multi‐donor transplantation (MDT) on graft‐versus‐host disease (GVHD) and graft versus tumor (GVT) effect were never tested. To assess the safety and efficacy of MDT, we used it in non‐TCD transplantation and murine breast carcinoma model. We found that when transplanting non‐TCD MDT composed by C57Bl/6 and C3H cells into BALB/c, a consistent trichimerism is established, dominated by C57Bl/6 cells. Following MDT the study animals experienced reduced GVHD compare with those transplanted from C57Bl/6 alone, while the GVT effect was superior. We conclude that MDT may serve as a technique that suppresses GVHD while maintaining the GVT effect.     

Ultrasonographic evaluation of gastrointestinal graft‐versus‐host disease after hematopoietic stem cell transplantation

Gastrointestinal graft‐versus‐host disease (GI‐GVHD) is a major and life‐threatening complication of hematopoietic stem cell transplantation (HSCT). This study evaluated the efficacy of ultrasonography (US) for assessing and monitoring GI‐GVHD. GI tract was evaluated by US in 81 patients. US findings were positive in 43 patients, including 11 false positive, and negative in 38 patients. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of US for the diagnosis of GI‐GVHD were 100%, 78%, 74%, 100%, and 86%, respectively. Diffuse wall thickening of the ileum was the most frequent finding in patients with GI‐GVHD. Severity of GI‐GVHD was correlated with the thickness of internal low echoic layer of the wall, the echogenicity of mesenteric fat tissue, and the intensity of Doppler signaling. We classified US findings of GI‐GVHD into four US grades. There was a significant correlation between clinical stage of GI‐GVHD and the US grade. These ultrasonographic abnormalities were improved with clinical improvement of GI‐GVHD upon treatment. Thus, US is an effective and efficient non‐invasive means of identifying the extent and severity of GI‐GVHD and monitoring response to treatment.     

Successful treatment for graft‐versus‐host disease after pancreas transplantation

Graft‐versus‐host disease (GVHD) after pancreas transplantation is a rare but serious complication: All previously reported cases were fatal. We herein report three cases of GVHD after pancreas transplantation with favorable outcomes. Patients with a history of kidney (and pancreas) transplantation subsequently received a pancreas (and kidney) transplantation (i.e., pancreas retransplantation or pancreas after kidney transplantation) and developed acute GVHD. All of them responded to increased immunosuppression (e.g., steroid bolus, antithymocyte globulin) and retained normal graft function. Because the clinical manifestations are non‐specific, vigilance is necessary to make an accurate diagnosis. We underscored the importance of a biopsy of involved organs and the clinicopathologic correlation in the early diagnosis of GVHD. Augmented immunosuppression to prevent progression from a self‐limited disease to life‐threatening pancytopenia or sepsis may be most critical to improve outcome.     

A mathematical model provides new insights into solid organ transplant‐associated acute graft‐versus‐host disease

Solid organ transplantation‐associated acute graft‐versus‐host disease (SOT‐aGvHD) is a rare but highly fatal condition. Our poor understanding of this entity in addition to its rarity has hampered treatment progress and most patients succumb to the disease. A mechanistic mathematical model is developed to replicate and explain the complex pathogenesis of SOT‐aGvHD. The model captures a number of important features of SOT‐aGvHD including (i) the occurrence of stable and persistent mixed chimerism in some, but not all, cases, (ii) fluctuation in chimerism in some persistently mixed chimeric cases, (iii) rare occurrence of full donor chimerism, and (iv) beneficial effect of escalating immunosuppression in some cases of SOT‐aGvHD and detrimental effect in others. In addition, the model predicts the conditions under which escalation or de‐escalation of immunosuppression would be the preferred treatment strategy. In an exceedingly rare condition such as SOT‐aGvHD, where prospective trials are not feasible, mathematical modeling can provide useful insights into pathogenesis and treatment.     

R707, a fully human antibody directed against CC‐chemokine receptor 7, attenuates xenogeneic acute graft‐versus‐host disease

Acute graft‐versus‐host disease (aGVHD) remains a barrier to the success of allogeneic hematopoietic stem cell transplantation (HSCT). Previously, we demonstrated that CC‐chemokine receptor 7 (CCR7) is critical for aGVHD pathogenesis but dispensable for beneficial graft‐versus‐leukemia responses. As a result, we evaluated a fully human anti‐CCR7‐blocking antibody as a new approach to prevent aGVHD in preclinical models. Here we report that antibody R707 is able to block human CCR7 signaling and function in vitro in response to its 2 natural ligands. The antibody was less active against the murine orthologue, however, and failed to substantially limit aGVHD in a standard murine allogeneic HSCT model. Nevertheless, R707 significantly reduced xenogeneic aGVHD induced by human peripheral blood mononuclear cells (PBMCs). R707 limited CD4⁺ and in particular CD8⁺ T cell expansion during the period of antibody administration. These effects were transient, however, and T cell numbers recovered after antibody cessation. R707 did not substantially impair the antitumor potential of the PBMC inoculum as antibody‐treated mice retained their capacity to reject a human acute myeloid leukemia cell line. Collectively, these data indicate for the first time that an antibody directed against CCR7 might represent a viable new approach for aGVHD prevention.     

Mycophenolate mofetil use after unrelated hematopoietic stem cell transplantation for prophylaxis and treatment of graft‐vs.‐host disease in adult patients in Japan

Our previous study of 301 patients who received hematopoietic stem cell transplantation (HSCT) from related donors demonstrated the efficacy of mycophenolate mofetil (MMF) for prophylaxis and treatment of graft‐vs.‐host disease (GVHD). In this study, we investigated the safety and efficacy of MMF in 716 adult patients who received unrelated HSCT. The incidences of Grade II–IV and III–IV acute GVHD in the prophylactic administration group were 38.3% and 14.3%, respectively. These rates were not statistically significant when evaluating the MMF dosage and graft source. The incidences of limited and extensive chronic GVHD were 16.6% and 11.1%, respectively. In the therapeutic administration group, 69.1% of the subjective symptoms for both acute and chronic GVHD improved. With respect to the adverse events, 75 infections and 50 cases of diarrhea were observed, and the frequency of these events increased with increasing MMF dose. The overall survival rate was 36.4% after a median follow‐up period of three yr. This study shows that MMF is safe and effective for the prevention and treatment of GVHD in patients who have received HSCT from unrelated donors.     

Role of Toll‐like receptor 4 signaling in cutaneous chronic graft‐versus‐host disease

Cutaneous damage is one of the characterized manifestations in chronic graft‐versus‐host disease (cGVHD). When local effective immunity in the skin is altered to a dysimmune reaction, cutaneous injuries occur. Toll‐like receptor 4 signaling is regarded as a central mediator of inflammation and organ injury. In this study, we found that TLR4 mRNA in peripheral blood from patients with cutaneous cGVHD was markedly increased compared with that from non‐GVHD patients and healthy controls. In addition, NF‐κB expression, TLR4 downstream signaling, and TLR4‐mediated cytokines, including IL‐6 and ICAM‐1, were upregulated. Moreover, ICAM‐1 was widely distributed in skin biopsies from patients with cutaneous cGVHD. We also found that LPS induced TLR4‐mediated NF‐κB activation and IL‐6 and ICAM‐1 secretion in human fibroblasts in vitro. Thus, TLR4, NF‐κB, IL‐6, and ICAM‐1 contribute to the inflammatory response that occurs in cutaneous cGVHD, indicating the TLR4 pathway may be a novel target for cutaneous cGVHD therapy.     

High frequency of CD4+ CD25- CD69+ T cells is correlated with a low risk of acute graft-versus-host disease in allotransplants

Regulatory T cells (Tregs) maintain transplantation tolerance and suppress graft-versus-host disease (GvHD) in humans. We monitored 17 subjects with acute GvHD to determine whether Treg frequency correlates with acute GvHD. We found the percent of CD4(+) CD25(-) CD69(+) Tregs decreases when acute GvHD develops and increases after acute GvHD is controlled. We next sequentially studied 50 subjects receiving conventional allotransplants. We show a high frequency and increased numbers of CD4(+) CD25(-) CD69(+) Tregs are associated with a reduced risk of acute GvHD. We also show that CD4(+) CD25(-) CD69(+) Treg numbers increase substantially early after allografts and that a low percent of CD4(+) CD25(-) CD69(+) Tregs is associated with an increased risk of acute GvHD. Reconstitution of Tregs early post-transplant is associated with less acute GvHD. These data imply that CD4(+) CD25(-) CD69(+) Tregs are a novel subset of regulatory T cells that may protect against acute GvHD after allotransplants.     

A gut microbiota score predicting acute graft‐versus‐host disease following myeloablative allogeneic hematopoietic stem cell transplantation

Although studies have reported that intestinal microbiota are associated with acute graft‐versus‐host disease (aGVHD), they lacked a satisfactory method for predicting aGVHD. We collected stool and blood samples at day 15 posttransplant from 150 patients from two centers who underwent myeloablative conditioning allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Stool microbiota were detected by 16S ribosomal RNA gene sequencing; inflammatory factors and T lymphocytes were detected by multiplex immunoassays and flow cytometry, respectively. A gut microbiota score (GMS) from a LASSO (least absolute shrinkage and selection operator) model was developed and validated to predict aGVHD. In the discovery cohort, the GMS could predict II‐IV aGVHD (area under the receiver operating characteristic [ROC] curve [AUC] = 0.904, P < .0001). Furthermore, the validation model was consistent with the discovery set (AUC = 0.887, P < .0001). Regulatory T/T‐helper 17 (Treg/Th17) cells ratio in the low GMS subgroup was higher compared with the high GMS (P = .012), and the validation set is consistent with the discovery set (P = .003). In addition, high cytokine levels were associated with high GMS. In conclusion, the GMS at neutrophil engraftment could predict aGVHD, and it was a potential and novel method. The GMS was associated with the inflammatory factor and Treg/Th17 balance.     

The use of ruxolitinib for acute graft‐versus‐host disease developing after solid organ transplantation

Development of graft‐versus‐host disease (GvHD) is a rare complication after transfusions or solid organ transplantation. Patients typically present with a skin rash, diarrhea, liver failure, and bone marrow aplasia. A diagnosis of transfusion/transplantation associated‐GvHD is made based on the clinical and histologic evidence, yet it is often delayed due to the nonspecific symptoms attributed to the patient's underlying illness. Several therapeutic approaches are being used including both increasing and withdrawing immunosuppression, and the use of cellular therapies. Unfortunately, the success rate of these approaches is low and the mortality of this complication is very high. New approaches are needed. We report on three cases of GvHD developing after solid organ transplantation treated with ruxolitinib.     

Low‐dose methotrexate may preserve a stronger antileukemic effect than that of cyclosporine after modified donor lymphocyte infusion in unmanipulated haploidentical HSCT

To compare the impacts of low‐dose methotrexate (MTX) with cyclosporine (CSA) on graft‐versus‐host disease (GVHD) and graft‐versus‐leukemia (GVL) effect after haploidentical modified donor lymphocyte infusion (DLI). Fifty‐five consecutive patients who had relapsed acute leukemia after haploidentical hematopoietic stem cell transplantation (HSCT) and received modified DLI were retrospectively studied. Forty‐one patients received CSA and 14 received low‐dose MTX after DLI to prevent DLI‐associated GVHD. The incidence of acute GVHD and grade 2–4 acute GVHD in MTX group showed a trend toward being higher than in CSA group (61.0% vs. 37.3%, p = 0.198 and 61.0% vs. 35.5%, p = 0.155). However, no significant difference in the incidence of grade 3–4 acute GVHD between two groups (p = 0.982) was observed. Moreover, compared with CSA, patients treated with MTX had lower re‐relapse rate (38.1% vs. 80.8%, p = 0.029), better disease‐free survival (DFS) (51.9% vs. 15.6%, p = 0.06), and higher absolute lymphocyte counts at 30, 45, 60, and 90 d after modified DLI (p < 0.05). This study suggested that after haploidentical modified DLI, low‐dose MTX is at least as effective as CSA in the prevention of DLI‐associated GVHD and probably allowed stronger GVL effect than CSA. This phenomenon was probably due to a direct antitumor effect and a better reconstitution of lymphocytes after modified DLI induced by low‐dose MTX.     

Long‐term outcomes of antithymocyte globulin in patients with hematological malignancies undergoing myeloablative allogeneic hematopoietic cell transplantation: a systematic review and meta‐analysis

Antithymocyte globulin (ATG) has shown efficacy in preventing acute GVHD (aGVHD) in allogeneic hematopoietic cell transplantation (allo‐HCT), but its efficacy in chronic GVHD (cGVHD) and long‐term outcomes remains controversial. We conducted a systematic review and meta‐analysis to evaluate potential benefit and risk of prophylactic ATG use in myeloablative HCT. We searched Pubmed, EMBASE, Cochrane databases, and included 10 trials (two RCTs and eight retrospective) comparing ATG use vs. control with a total of 1859 patients. The median follow‐ups were over two yr. Outcomes assessed included overall cGVHD, extensive cGVHD, overall survival (OS), disease‐free survival, relapse, and causes of death. Our results showed ATG significantly decreased overall cGVHD (RR = 0.59; 95% CI: 0.53–0.66, p < 0.00001), extensive cGVHD (RR = 0.34; 95% CI: 0.25–0.47, p < 0.00001). Pooled results also showed ATG use was associated with a marginal increased risk of relapse (RR = 1.28; 95% CI: 1.01–1.63, p = 0.04), and a non‐inferior OS (HR = 0.86; 95% CI: 0.74–1.01, p = 0.06). We conclude prophylactic use of ATG exerts a favorable effect in reducing cGVHD without survival impairment in a long term, although a higher relapse rate is a major threat.     

The lack of memory B cells including T cell independent IgM+ IgD+ memory B cells in chronic graft‐versus host disease is associated with susceptibility to infection

The chronic graft‐versus host disease (cGVHD) is associated with a perturbed B cell homeostasis and an increased infection rate. Aiming to determine the impact of lymphocyte subsets on cGVHD, blood samples from 98 patients at least 100 days following allogeneic haematopoietic stem cell transplantation (median 1066 days) were analyzed, serum levels of immunoglobulins measured and the incidence of severe infections retrospectively documented. Absolute CD19⁺ B cell counts, including counts of immature (CD10⁺ CD38⁺⁺ CD20⁺ IgM⁺⁺) and transitional (CD10^? CD38⁺⁺ CD20⁺ IgM⁺⁺) as well as class switched memory (CD19⁺ CD27⁺ IgM^? IgD^?) B cells in patients with active cGVHD (n = 52) were significantly decreased as compared to those with inactive (n = 18) or without cGVHD (n = 28). In addition, nonclass switched IgM⁺ memory B cells (CD19⁺ CD27⁺ IgM⁺ IgD⁺) were absent in patients with cGVHD, but not in patients with inactive (0.4 × 10⁶/l) or without (1.7 × 10⁶/l) cGVHD (both P < 0.001). In line with these results we found significantly decreased lgG levels in patients with cGVHD, which was associated with a significantly higher rate of severe infections in cGVHD patients. Our data underline the close association of diminished B cell counts with cGVHD and the onset of severe infections. The lack of IgM⁺ memory B cells in patients with cGVHD may indicate functional asplenia.     

Graft‐versus‐host disease after orthotopic liver transplantation: multivariate analysis of risk factors

Graft‐versus‐host disease (GVHD) is a rare, fatal complication following orthotopic liver transplantation (OLT). To date, several risk factors have been proposed, but reports on these factors have been inconclusive. This is a retrospective, case–control study of prospectively collected data from 2775 OLTs performed at our institution. Eight cases of GVHD after OLT were diagnosed on the basis of the patient's clinical characteristics, and the findings were confirmed with skin and colonic biopsies. Each case was matched to three controls based on the diagnosis of liver disease, recipient's age, and blood group. Univariate and multivariate analyses were performed to identify risk factors associated with the development of GVHD after OLT. The univariate and multivariate analyses identified two main risk factors associated with development of GVHD in OLT recipients, a difference between recipient and donor age of >20 yr, and any human leukocyte antigen class I matches. Taking these two risk factors into consideration while matching prospective donors and recipients may reduce further incidence of GVHD in OLT patients. However, further studies are recommended to validate these findings.