John E. Levine

Graft-versus-host disease

Haemopoietic-cell transplantation (HCT) is an intensive therapy used to treat high-risk haematological malignant disorders and other life-threatening haematological and genetic diseases. The main complication of HCT is graft-versus-host disease (GVHD), an immunological disorder that affects many organ systems, including the gastrointestinal tract, liver, skin, and lungs. The number of patients with this complication continues to grow, and many return home from transplant centres after HCT requiring continued treatment with immunosuppressive drugs that increases their risks for serious infections and other complications. In this Seminar, we review our understanding of the risk factors and causes of GHVD, the cellular and cytokine networks implicated in its pathophysiology, and current strategies to prevent and treat the disease. We also summarise supportive-care measures that are essential for management of this medically fragile population.

A biomarker panel for acute graft-versus-host disease.

No validated biomarkers exist for acute graft-versus-host disease (GVHD). We screened plasma with antibody microarrays for 120 proteins in a discovery set of 42 patients who underwent transplantation that revealed 8 potential biomarkers for diagnostic of GVHD. We then measured by enzyme-linked immunosorbent assay (ELISA) the levels of these biomarkers in samples from 424 patients who underwent transplantation randomly divided into training (n = 282) and validation (n = 142) sets. Logistic regression analysis of these 8 proteins determined a composite biomarker panel of 4 proteins (interleukin-2-receptor-alpha, tumor-necrosis-factor-receptor-1, interleukin-8, and hepatocyte growth factor) that optimally discriminated patients with and without GVHD. The area under the receiver operating characteristic curve distinguishing these 2 groups in the training set was 0.91 (95% confidence interval, 0.87-0.94) and 0.86 (95% confidence interval, 0.79-0.92) in the validation set. In patients with GVHD, Cox regression analysis revealed that the biomarker panel predicted survival independently of GVHD severity. A panel of 4 biomarkers can confirm the diagnosis of GVHD in patients at onset of clinical symptoms of GVHD and provide prognostic information independent of GVHD severity.

Prospective trial of chemotherapy and donor leukocyte infusions for relapse of advanced myeloid malignancies after allogeneic stem-cell transplantation

PURPOSE Patients with advanced myeloid malignancies who experience relapse after allogeneic bone marrow transplantation (BMT) have a poor prognosis. Long-term survival after chemotherapy alone, second myeloablative transplant, or donor leukocyte infusions (DLIs) alone is unusual. DLIs may have minimal effectiveness in advanced disease because adequate cellular responses are not able to develop in the presence of bulky, fast-growing disease. A chemotherapy strategy was used to debulk disease before administration of granulocyte colony-stimulating factor (G-CSF)-primed DLIs. PATIENTS AND METHODS Sixty-five patients experiencing hematologic relapse of myeloid malignancy after HLA-matched sibling BMT were prospectively treated with cytarabine-based chemotherapy, then G-CSF-primed DLIs. No prophylactic immunosuppression was provided. RESULTS Twenty-seven of 57 assessable patients experienced a complete response. Graft-versus-host disease (GVHD) was observed in 56% of the patients. Treatment-related mortality was 23%. Overall survival at 2 years for the entire cohort was 19%. Patients with a complete response were more likely to survive, with 1- and 2-year survival rates of 51% and 41%, respectively, with a median follow-up of more than 2 years. The 1-year survival for nonresponders was 5%. A posttransplant remission lasting more than 6 months before relapse was associated with a higher likelihood of response. GVHD was not required for durable remission. CONCLUSION Salvage treatment with chemotherapy before DLI can help some patients with advanced myeloid relapse and is not dependent on GVHD. Patients with short remissions after BMT are unlikely to benefit from this approach, and the approach is associated with significant treatment-related mortality. Modifications of this approach or entirely different approaches will be required for most patients with this difficult clinical problem.

Donor leukocyte infusions in acute lymphocytic leukemia.

Donor leukocyte infusion (DLI) has well-documented activity in CML but the role of DLI in other diseases is less well defined. To evaluate the strategy in acute lymphocytic leukemia (ALL) we evaluated 44 ALL patients from 27 centers who were treated with DLI. Patients with persistent or recurrent disease received DLI from the original marrow donor (30 matched related, four mismatched family, and 10 matched unrelated). Chemotherapy was given before DLI to 28 patients. Of 15 patients who received no pre-DLI chemotherapy, two achieved complete remissions, lasting 1112 and 764+ days. In four patients who received DLI as consolidation of remission induced by chemotherapy or immunosuppression-withdrawal, duration of remission post DLI was 65, 99, 195 and 672+ days. Of 25 patients who received DLI in the nadir after chemotherapy, 13 survived ⩾30 days post DLI but did not achieve remission, seven died within less than 30 days post DLI, and five entered remissions that lasted 42, 68, 83, 90, 193 days. Seven patients who did not respond to the initial DLI received a second DLI; none of these patients attained durable remission. Eighteen of 37 evaluable patients developed acute GVHD and five of 20 evaluable patients developed chronic GVHD. Overall actuarial survival is 13% at 3 years. In conclusion, DLI has limited benefit in ALL. New approaches are needed in this group of patients. Bone Marrow Transplantation (2000) 26, 511–516.

ST2 as a Marker for Risk of Therapy-Resistant Graft-versus-Host Disease and Death

BACKGROUND No plasma biomarkers are associated with the response of acute graft-versus-host disease (GVHD) to therapy after allogeneic hematopoietic stem-cell transplantation. METHODS We compared 12 biomarkers in plasma obtained a median of 16 days after therapy initiation from 10 patients with a complete response by day 28 after therapy initiation and in plasma obtained from 10 patients with progressive GVHD during therapy. The lead biomarker, suppression of tumorigenicity 2 (ST2), was measured at the beginning of treatment for GVHD in plasma from 381 patients and during the first month after transplantation in three independent sets totaling 673 patients to determine the association of this biomarker with treatment-resistant GVHD and 6-month mortality after treatment or transplantation. RESULTS Of the 12 markers, ST2 had the most significant association with resistance to GVHD therapy and subsequent death without relapse. As compared with patients with low ST2 values at therapy initiation, patients with high ST2 values were 2.3 times as likely to have treatment-resistant GVHD (95% confidence interval [CI], 1.5 to 3.6) and 3.7 times as likely to die within 6 months after therapy (95% CI, 2.3 to 5.9). Patients with low ST2 values had lower mortality without relapse than patients with high ST2 values, regardless of the GVHD grade (11% vs. 31% among patients with grade I or II GVHD and 14% vs. 67% among patients with grade III or IV GVHD, P<0.001 for both comparisons). Plasma ST2 values at day 14 after transplantation were associated with 6-month mortality without relapse, regardless of the intensity of the conditioning regimen. CONCLUSIONS ST2 levels measured at the initiation of therapy for GVHD and during the first month after transplantation improved risk stratification for treatment-resistant GVHD and death without relapse after transplantation. (Funded by the National Institutes of Health.)

Donor leukocyte infusions for multiple myeloma

Donor leukocyte infusion (dli) has well-documented activity in cml, but the role of dli in other diseases is less well defined. to evaluate the strategy in multiple myeloma (mm) we evaluated 25 mm patients from 15 centers who were treated with dli. patients with persistent or recurrent disease after allogeneic bmt received dli from the original marrow donor (23 matched related, one mismatched family, and one matched unrelated). chemotherapy was given before dli in three patients. two of 22 patients responded completely to dli alone and three patients responded to the combination of dli and chemotherapy. nine patients who had not had sufficient disease control after dli were given additional dlis; five of these patients had either complete (two) or partial (three) responses. thirteen of 25 evaluable patients developed acute gvhd and 11 of 21 evaluable patients developed chronic gvhd; all responders developed gvhd. no patients developed post-dli pancytopenia. four patients had responses which lasted >1 year after DLI, three patients had responses which lasted <1 year, and three patients had ongoing responses but with follow-up <1 year. In conclusion, DLI has anti-myeloma activity but the strategy is limited by no response or short duration of response in a significant percentage of patients and by significant GVHD in the majority of the responders. Bone Marrow Transplantation (2000) 26, 1179–1184.

Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease.

Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 μmol/L to 11.8 ± 2.8 μmol/L in all post-transplant samples and to 3.5 ± 3 μmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT.

Etanercept, mycophenolate, denileukin, or pentostatin plus corticosteroids for acute graft-versus-host disease: a randomized phase 2 trial from the Blood and Marrow Transplant Clinical Trials Network

Acute graft-versus-host disease (aGVHD) is the primary limitation of allogeneic hematopoietic cell transplantation. Corticosteroids remain the standard initial therapy, yet only 25% to 41% of patients completely respond. This randomized, 4-arm, phase 2 trial was designed to identify the most promising agent(s) for initial therapy for aGVHD. Patients were randomized to receive methylprednisolone 2 mg/kg per day plus etanercept, mycophenolate mofetil (MMF), denileukin diftitox (denileukin), or pentostatin. Patients (n = 180) were randomized; their median age was 50 years (range, 7.5-70 years). Myeloablative conditioning represented 66% of transplants. Grafts were peripheral blood (61%), bone marrow (25%), or umbilical cord blood (14%); 53% were from unrelated donors. Patients who received MMF for prophylaxis (24%) were randomized to a non-MMF arm. At randomization, aGVHD was grade I to II (68%), III to IV (32%), and (53%) had visceral organ involvement. Day 28 complete response rates were etanercept 26%, MMF 60%, denileukin 53%, and pentostatin 38%. Corresponding 9-month overall survival was 47%, 64%, 49%, and 47%, respectively. Cumulative incidences of severe infections were as follows: etanercept 48%, MMF 44%, denileukin 62%, and pentostatin 57%. Efficacy and toxicity data suggest the use of MMF plus corticosteroids is the most promising regimen to compare against corticosteroids alone in a definitive phase 3 trial. This study is registered at http://www.clinicaltrials.gov as NCT00224874.

Regenerating islet-derived 3-alpha is a biomarker of gastrointestinal graft-versus-host disease

There are no plasma biomarkers specific for GVHD of the gastrointestinal (GI) tract, the GVHD target organ most associated with nonrelapse mortality (NRM) following hematopoietic cell transplantation (HCT). Using an unbiased, large-scale, quantitative proteomic discovery approach to identify candidate biomarkers that were increased in plasma from HCT patients with GI GVHD, 74 proteins were increased at least 2-fold; 5 were of GI origin. We validated the lead candidate, REG3α, by ELISA in samples from 1014 HCT patients from 3 transplantation centers. Plasma REG3α concentrations were 3-fold higher in patients at GI GVHD onset than in all other patients and correlated most closely with lower GI GVHD. REG3α concentrations at GVHD onset predicted response to therapy at 4 weeks, 1-year NRM, and 1-year survival (P ≤ .001). In a multivariate analysis, advanced clinical stage, severe histologic damage, and high REG3α concentrations at GVHD diagnosis independently predicted 1-year NRM, which progressively increased with higher numbers of onset risk factors present: 25% for patients with 0 risk factors to 86% with 3 risk factors present (P < .001). REG3α is a plasma biomarker of GI GVHD that can be combined with clinical stage and histologic grade to improve risk stratification of patients.

Anti-CD20 Chimeric Monoclonal Antibody Treatment of Refractory Immune-Mediated Thrombocytopenia in a Patient with Chronic Graft-versus-Host Disease

Chronic graft-versus-host disease occurs in approximately 50% of long-term survivors of transplantation with marrow from HLA-identical donors (1); the risk for this disease increases with use of peripheral blood stem cells (2). Chronic graft-versus-host disease shares many of the clinical manifestations of autoimmune collagen vascular diseases, including oral ulceration, lichen planus, xerostomia, keratoconjunctivitis sicca, polyserositis, esophagitis and esophageal stricture, vaginal ulceration and stricture, intrahepatic obstructive liver disease, obstructive pulmonary disease, scleroderma, morphea, fasciitis, and myositis (3). Cytopenias, particularly thrombocytopenia, are a common feature of both chronic graft-versus-host disease and collagen vascular disease, and thrombocytopenia in chronic graft-versus-host disease is associated with poorer survival (4, 5). Antiplatelet antibodies are frequently detected in patients with thrombocytopenia associated with chronic graft-versus-host disease (6). Most patients with chronic graft-versus-host disease have evidence of B-cell dysregulation, with a high prevalence of autoantibodies to several cell surface and intracellular antigens (7). The role of these autoantibodies in the pathogenesis of chronic graft-versus-host disease is unclear. Autoimmune thrombocytopenia in chronic graft-versus-host disease may represent an instance of B-cell dysregulation leading to clinical disease. Rituximab is a humanized murine monoclonal antibody commonly used to treat B-cell lymphomas (8). This antibody is highly effective for in vivo depletion of B cells. Circulating B cells become undetectable after a single 375-mg/m2 infusion of rituximab; recovery of B cells begins at 6 to 9 months after treatment, and counts normalize by 9 to 12 months (9). Because of these biological properties of rituximab and the association of antiplatelet autoantibody with thrombocytopenia in patients with chronic graft-versus-host disease (6), we hypothesized that rituximab might have clinically significant activity in the treatment of refractory immune-mediated thrombocytopenia. We describe a patient with chronic graft-versus-host disease who developed severe refractory thrombocytopenia that responded to anti-CD20 chimeric antibody therapy. The rationale for this treatment was to eliminate B cells producing autoantibodies and thereby reverse the thrombocytopenia. Case Report A 32-year-old woman, gravida 3 para 1, presented with blurred vision and was found to have retinal hemorrhages. Complete blood count showed a leukocyte count of 451 109 cells/L with 2% basophils. A diagnosis of chronic myelogenous leukemia was confirmed by the presence of the Philadelphia chromosome in the marrow sample. The patient began receiving hydroxyurea to reduce her leukocyte count and was referred for stem-cell transplantation. She received 16 doses of busulphan (1 mg/kg of body weight every 6 hours) followed by cyclophosphamide (60 mg/kg daily for 2 days) in preparation for transplantation. Filgrastim-mobilized peripheral blood stem cells were harvested from an HLA-matched brother and were infused into the patient 3 months after diagnosis. The patients blood group was A+, and she was seropositive for cytomegalovirus; the donors blood group was O+, and he was seronegative for cytomegalovirus. Prophylaxis against graft-versus-host disease consisted of tacrolimus and methotrexate (10). The patient had prompt hematologic reconstitution, with an absolute neutrophil count of 0.5 109 cells/L on day 12 after transplantation and a platelet count greater than 100 109 cells/L on day 16 after transplantation. Therapy with ganciclovir, 5 mg/kg twice weekly, was started on day 16 as prophylaxis against cytomegalovirus infection. On day 28 after transplantation, the patient developed a maculopapular rash of the upper torso and diarrhea. Skin biopsy confirmed acute graft-versus-host disease, and she began receiving methylprednisolone, 1 mg/kg daily. The ganciclovir dose was increased prophylactically to 5 mg/kg 5 times per week. Bone marrow aspirate on day 100 after transplantation showed a normal male karyotype, and full donor chimerism was confirmed by polymerase chain reaction of microsatellite markers. The patient responded well to steroid therapy for the skin and gastrointestinal symptoms of graft-versus-host disease, but symptoms of xerophthalmia and xerostomia developed and steroid therapy was continued through day 127. On day 142, she underwent punctal occlusion of both eyes for severe xerophthalmia. Other manifestations of chronic graft-versus-host disease were progressive xerostomia with lichenoid changes of the oral mucosa. During the patients course of therapy with steroids and the increased dose of ganciclovir, her platelet count remained greater than 100 109 cells/L. On day 211, a complete blood count showed an absolute neutrophil count of 2.7 109 cells/L, hemoglobin value of 131 g/L, and platelet count of 178 109 cells/L. On day 230, the patients platelet count decreased to 88 109/cells/L. On day 238, it decreased further to 28 109 cells/L. No microangiopathic changes were seen on peripheral smear. Bone marrow aspiration and biopsy showed normal trilineage hematopoietic maturation with an adequate number of megakaryocytes. Flow cytometry showed the presence of platelet-associated IgG on washed, formalin-fixed platelets. Intravenous immunoglobulin, 500 mg/kg, was administered daily for 3 days; the platelet count increased to 159 109 cells/L, but for only 2 weeks. The patient then received 5 doses of intravenous immunoglobulins with methylprednisolone, 64 mg/d. On day 278, therapy with mycophenolate was started to provide additional immunosuppression. The combination of intravenous immunoglobulins, steroids, and mycophenolate resulted in an increase in platelet count to 114 109 cells/L by day 299. On day 308, the patients platelet count decreased to 51 109 cells/L. A dose of anti-D antibody (50 g/kg) was given, and the platelet count increased to 127 109 cells/L. However, the patient experienced significant hemolysis; her hemoglobin value decreased from 131 g/L to 78 g/L, an expected side effect related to the destruction of Rho (D)positive red cells. On day 337, she underwent laparoscopic splenectomy; an accessory spleen was also removed. By day 341, the platelet count increased to 139 109 cells/L but decreased to 2 109 cells/L 1 week later. Intravenous vincristine, 2 mg, was administered in four weekly doses starting on day 356, but it did not produce a response. The patient subsequently received intravenous cyclophosphamide, 1.5 g/m2, on day 384, again without response. On day 404, the patient received the first of four weekly doses of anti-CD20 antibody (rituximab [Rituxan, Genentech/IDEC, South San Francisco, California]). Peripheral blood flow cytometry on day 377 showed a CD19+ cell count (B cells) of 0.116 109 cells/L; by day 474, after rituximab therapy, CD19+ cells were absent. A gradual but sustained increase in the platelet count was noted after 2 doses of rituximab. The dose of mycophenolate mofetil was tapered and therapy was discontinued 3 weeks after initiation of rituximab therapy. At the time of this report, the patient has no clinically significant signs or symptoms of chronic graft-versus-host disease despite continued tapering of the tacrolimus dose and discontinuation of this therapy on day 667. The platelet-associated antibody assay remained weakly positive at day 485 and became negative on day 532. The patient has now been followed for 11 months since initiation of rituximab therapy, and the platelet count has not decreased (Table and Figure). Table. Chronological Clinical Course of the Patient Figure. Clinical course of a patient with chronic graft-versus-host disease in whom severe refractory immune-mediated thrombocytopenia responded to treatment with anti-CD20 monoclonal antibody. Discussion The reversal of our patients immune-mediated thrombocytopenia was especially noteworthy because of the lack of a sustained response to intensive immunosuppression, including treatment with steroids, mycophenolate, tacrolimus, vincristine, cyclophosphamide, intravenous immunoglobulins, anti-D antibody, and splenectomy. The improvement of thrombocytopenia was also associated with decreased levels of platelet-associated antibody and lack of progression of chronic graft-versus-host disease despite the discontinuation of immunosuppression. It is possible that cyclophosphamide therapy contributed in part to the platelet response. Reiner and colleagues (11) reported a series of 20 cases of refractory idiopathic thrombocytopenia purpura treated with pulse cyclophosphamide (1 to 1.5 g/m2), with complete remission in 65% of patients and partial remission in 20%. However, most patients required multiple pulses (mean, 2; maximum, 4), and the mean time to response was 7 weeks. Considering the intensity of the immunosuppressive therapy that our patient received and the lack of platelet response at the time of neutrophil recovery, it is unlikely that cyclophosphamide contributed substantially. Studies of B cells in patients with chronic graft-versus-host disease have not convincingly explained the clinical abnormalities, including reduced B-cell count, decreased Ig production, and increased spontaneous Ig production (12), seen in this condition. Clonal dysregulation of B cells in chronic graft-versus-host disease has been suggested by the presence of monoclonal gammopathy (13, 14) and elevated serum IgG and IgM levels (15). Antibody production is a prominent feature of many animal models of chronic graft-versus-host disease (16, 17). Mechanistic studies showed that secretion of Th2 cytokines, such as interleukin-4 and interleukin-5, by donor CD4+ cells are critical to B-cell activation and autoantibody production. Most of the autoantibodies are of the IgG subclass (16), and abnormal cross-linking of IgG receptors on t