Acute Cutaneous Graft-versus-host Disease Research Articles

Diverse macrophage populations contribute to distinct manifestations of human cutaneous graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major life-threatening complication of allogeneic haematopoietic stem cell transplantation (HSCT), limiting the broad application of HSCT for haematological malignancies. Cutaneous GvHD is described as a post-transplant inflammatory reaction by skin-infiltrating donor T cells and remaining recipient tissue-resident memory T cells. Despite the major influence of lymphocytes on GvHD pathogenesis, the complex role of mononuclear phagocytes (MNPs) in tissues affected by GvHD is increasingly appreciated. To characterize the identity, origin and functions of MNPs in patients with acute cutaneous GvHD. Using single-cell RNA sequencing and multiplex tissue immunofluorescence, we identified an increased abundance of MNPs in skin and blood from 36 patients with acute cutaneous GvHD. In cases of sex-mismatched transplantation, we used expression of X-linked genes to detect rapid tissue adaptation of newly recruited donor MNPs resulting in similar transcriptional states of host- and donor-derived macrophages within GvHD skin lesions. We showed that cutaneous GvHD lesions harbour expanded CD163+ tissue-resident macrophage populations with anti-inflammatory and tissue-remodelling properties including interleukin-10 cytokine production. Cell-cell interaction analyses revealed putative signalling to strengthen regulatory T-cell responses. Notably, macrophage polarization in chronic cutaneous GvHD types was proinflammatory and drastically differed from acute GvHD, supporting the notion of distinct cellular players in different clinical GvHD subtypes. Overall, our data reveal a surprisingly dynamic role of MNPs after HSCT. Specific and time-resolved targeting to repolarize this cell subset may present a promising therapeutic strategy in combatting GvHD skin inflammation.

Risk factors and clinical characteristics of acute and chronic cutaneous graft-versus-host disease in pediatric patients undergoing hematopoietic stem cell transplantation.

Acute and chronic cutaneous graft-versus-host disease (GVHD) are common complications following hematopoietic stem cell transplantation (HSCT) in pediatric patients. In this retrospective study, we explored the risk factors and clinical characteristics of acute and chronic cutaneous GVHD in a case series of children undergoing HSCT at a tertiary referral hospital. We found that 36% of acute cutaneous GVHD was severe and these patients were more likely to have an unrelated donor, and that children with acute cutaneous GVHD who progressed to chronic cutaneous GVHD had a higher proportion of malignant diseases, total body irradiation, and bronchiolitis obliterans compared to those who did not progress to chronic cutaneous GVHD. Our study highlights the importance of identifying and monitoring these high-risk patients to improve the clinical management and outcomes of cutaneous GVHD in pediatric HSCT recipients.

Donor monocyte-derived macrophages promote human acute graft-versus-host disease.

Myelopoiesis is invariably present and contributes to pathology in animal models of graft-versus-host disease (GVHD). In humans, a rich inflammatory infiltrate bearing macrophage markers has also been described in histological studies. In order to determine the origin, functional properties, and role in pathogenesis of these cells, we isolated single-cell suspensions from acute cutaneous GVHD and subjected them to genotype, transcriptome, and in vitro functional analysis. A donor-derived population of CD11c+CD14+ cells was the dominant population of all leukocytes in GVHD. Surface phenotype and NanoString gene expression profiling indicated the closest steady-state counterpart of these cells to be monocyte-derived macrophages. In GVHD, however, there was upregulation of monocyte antigens SIRPα and S100A8/9 transcripts associated with leukocyte trafficking, pattern recognition, antigen presentation, and costimulation. Isolated GVHD macrophages stimulated greater proliferation and activation of allogeneic T cells and secreted higher levels of inflammatory cytokines than their steady-state counterparts. In HLA-matched mixed leukocyte reactions, we also observed differentiation of activated macrophages with a similar phenotype. These exhibited cytopathicity to a keratinocyte cell line and mediated pathological damage to skin explants independently of T cells. Together, these results define the origin, functional properties, and potential pathogenic roles of human GVHD macrophages.

Dermoscopy of Cutaneous Graft-Versus-Host-Disease in Patients After Allogeneic Hematopoietic Stem Cell Transplantation.

IntroductionProgress in the transplant procedure has resulted in a higher proportion of patients with long-term survival after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Cutaneous graft-versus-host disease (GvHD) occurs often among patients who have undergone allo-HSCT. Routine diagnosis of skin and mucosal lesions is based primarily on clinical evaluation and histopathologic confirmation of skin biopsies. However, biopsy is an invasive method and histopathologic analysis is time-consuming, often accompanied by a lack of clinical correlation. There is therefore an urgent need for non-invasive, reproducible in vivo imaging methods that could be used in patients with cutaneous GvHD—both in the setting of initial diagnosis and during follow-up.The aim of the study reported here was to determine the role of dermoscopic monitoring of skin lesions in allo-HSCT recipients with consecutive histopathologic support as a non-invasive, alternative method to diagnose GvHD.MethodsTwenty patients were examined by dermoscopy upon the manifestation of skin changes in the course of GvHD. Consecutive skin biopsies for histopathologic analysis were obtained from the suspected skin locations determined during dermoscopy.ResultsGraft-versus-host disease was confirmed by histopathology in 19 of the 20 allo-HSCT recipients. Four patients developed symptoms of acute cutaneous GvHD (grade 1, n = 2; grade 2, n = 1; grade 3, n = 1), and 15 patients developed chronic cutaneous GvHD. The most frequent dermoscopic signs (irrespective of whether GvHD was chronic or acute) were vessels and scaling (both n = 14, 73.7%). Hyperpigmentation and white patchy areas were present in eight patients (42.1%). Fair to moderate levels of agreement were found between presence of melanophages in the skin sample and dermoscopic granularity (Cohen’s Kappa [κ] = 0.39), scaling (κ = − 0.3) and vessels (κ = − 0.42). The finding of white patchy areas was inversely associated with lymphocytic infiltration (κ = − 0.55).ConclusionThe results of this study suggest that dermoscopy may be a useful tool for diagnosing cutaneous GvHD in allo-HSCT recipients. Combining the clinical picture with dermoscopic features may bring us closer to a faster and easier diagnosis of GvHD.

Cutaneous graft-versus-host disease incidence is similar in haploidentical and matched unrelated hematopoietic transplant recipients: A retrospective cohort study

Cutaneous graft-versus-host disease (GVHD) is common after hematopoietic cell transplants. Haploidentical transplants (Haplo) have historically higher rates of GVHD with overall outcomes improved with the use of posttransplant cyclophosphamide. Specific cutaneous outcomes have not been explored in haploidentical versus matched unrelated donor (MUD) transplants. We sought to examine the incidence of GVHD in MUD and Haplo transplants. This is a retrospective cohort study of patients' records that received MUD or Haplo transplants between 2010 and 2015 with determination of GVHD severity and features by one investigator. The Haplo cohort included more minorities (22.7% vs 6.8%; P<.001). The incidence of acute cutaneous GVHD was similar (Haplo 47.7% [95% confidence interval {CI} 37.0-58.6%] vs MUD 42.6% [95% CI 37.9-47.3%]; P=.41). Chronic GVHD was also similar (Haplo 17.1% [95% CI 9.9-26.6%] vs MUD 12.8% [95% CI 9.9-16.3%]; P=.31). The Haplo group had lower rates of sclerosis (13.3% [95% CI 1.7-4.05%] vs 50.9% [95% CI 37.3-64.4%]; P=.0095). Other secondary outcomes showed no difference. Severity of GVHD was determined retrospectively and not all patients were seen by a dermatologist. No difference was observed between rates or severity of acute or chronic GVHD. Sclerosis was less common in the Haplo group.

Cutaneous Graft-Versus-Host Disease: Diagnosis and Treatment.

Graft-versus-host disease (GVHD) is an immunological reaction and a frequent complication following allogeneic hematopoietic stem cell transplantation. It is associated with high mortality rates and may have a significant negative impact on the patient’s quality of life, particularly in the chronic-stage setting. Many different organs can be involved, which leads to a wide range of clinical manifestations. In this context, dermatologists play a key role by diagnosing and treating GVHD from the outset since cutaneous features are not just the most common but are also usually the presenting sign. Several skin-direct therapies are available and may be indicated as monotherapy or adjuvant treatment in order to allow faster tapering and withdrawal of systemic immunosuppression. Treatment of steroid-refractory patients remains a challenge and, to date, no consensus has been reached for one single agent in second-line therapy. This article aims to review skin involvement as well as provide and update discussion on therapeutic options for both acute and chronic cutaneous GVHD.

Long-Term Survival and Absence of Late Relapses in Patients Who Underwent Allogeneic Stem Cell Transplantation for Advanced Cutaneous T-Cell Lymphoma

Long-Term Survival and Absence of Late Relapses in Patients Who Underwent Allogeneic Stem Cell Transplantation for Advanced Cutaneous T-Cell Lymphoma

Clinical research on unrelated cord blood transplantation treatment for mucopolysaccharidosis I

Objective To explore the efficacy of unrelated cord blood transplantation treatment of mucopolysaccharidosis Ⅰ(MPS Ⅰ). Methods A 4-year-and-2-month-old boy with MPS Ⅰ who received treatment of human leucocyte antigen-mismatched unrelated cord blood stem cell transplantation after diagnosis was identified.The pre-treatment regimen was Busulfan+ Cyclophosphamide+ Fludarabine(Bu/Cy4+ Flud). Bu with the dosage of 1.2 mg/kg, once every 6 hours, 4 days; Cy with the dosage of 50 mg/(kg·d) for 4 days and Flud with the dosage of 30 mg/(m2·d) lasted for 4 days, respectively.The day that the graft was transplanted was defined as 0 day, days before transplantation as negative days, days after transplantation as positive days.After pre-treatment, 4.60×107/kg of cord blood nucleated cells and 3.05×105/kg CD34 positive cells were transplanted into the child.The combination of Antihuman thymocyte globulin, Cyclosporin A and Mycophenolate mofetil was administrated for prophylaxis of graft versus host disease(GVHD). After transplantation, the patient was given granulocyte colony stimulating factor to promote reconstitution of hematopoiesis. Results The myeloid and platelet engraftment time was respectively 15 days and 24 days after transplantation.Short tandem repeat (STR) DNA fingerprinting showed a full donor chimerism on day 21 after transplantation, and the full donor chimerism was stable afterwards.The peripheral-blood α-L-iduronidase(IDUA) activity returned to the normal value, and the IDUA gene sequencing did not demonstrate any mutation in 83 days after transplantation.On day 12 after transplantation, pulmonary infection with pulmonary hypertension occurred.Grade-Ⅱ acute intestinal GVHD occurred on day 15, Grade-Ⅱ acute cutaneous GVHD on day 51, and chronic GVHD (cutaneous, localized) on day 180.Otherwise, the patient complicated with hemorrhagic cystitis on day 35.These complications was cured favourably.In an 18-month-follow-up, the height of the boy increased by 3 cm, and his body weight had increased by 2.4 kg.His corneas regained clear, and his hepatosplenomegaly disappeared.The glycosaminoglycan of urine was negative.The neurocognitive performance of the boy had a little improvement.The abnormalities of fingers and other skeletons had no marked change. Conclusions Unrelated cord blood transplantation for MPS Ⅰ have definited effect.It is the first case report in China on treatment of MPS Ⅰ by unrelated cord blood transplantation.The researchers have accumulated some preliminary experience for future treatment of MPS Ⅰ by unrelated cord blood transplantation. Key words: Hematopoietic stem cell transplantation; Cord blood; Mucopolysaccharidosis

Épidermolyse bulleuse acquise et réaction du greffon contre l’hôte

Epidermolysis bullosa acquisita (EBA) is a rare acquired blistering disorder caused by production of auto-antibody directed against type-VII collagen. Autoimmune disorders can occur after allogenic bone marrow transplantation as manifestations associated with chronic graft-versus-host disease (GVHD). To date, there have been 10 cases reported in the literature concerning autoimmune blistering diseases following allogenic stem-cell transplants. Herein we describe a new case involving EBA. A 46-year-old woman developed EBA 4 years after allogenic cord blood transplantation for non-Hodgkin T-cell lymphoma complicated by acute digestive and cutaneous GVHD. At physical examination, she had some cutaneous blisters on the abdomen, arms and face, as well as numerous erosions in the buccal cavity. Direct immunofluorescence microscopy revealed linear IgG and C3 deposits along the dermal-epidermal basement membrane zone. Indirect immunofluorescence showed weak IgG G4 anti-basement membrane zone antibodies, which reacted with the dermal side of 1M NaCl-split skin; the autoantibodies were directed against type-VII collagen. This second case of EBA was evocative of a GVHD blistering disease. After the therapeutic failure of dapsone and of combined mycophenolate-prednisone, treatment with rituximab proved effective. EBA may form part of the autoimmune signs associated with chronic GVH. The destruction of basement membrane and of epidermal basal cells that occurs in GVH could give rise to autoimmune bullous disease. However, in our patient, in whom manifestation of chronic GVH was restricted to the lungs, it is difficult to rule out the fortuitous onset of EBA, which presented at a sizeable interval after acute GVH.

Diverse T Cell Responses Lead To The Different Manifestations Of Cutaneous Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HCT). GVHD is classified into an acute (aGVHD) and a chronic form (cGVHD) based on NIH-defined, clinical criteria. In skin, aGVHD presents as a maculopapular rash, while cGVHD shows at the beginning the signs of lichenoid tissue inflammation (chronic inflammatory GVHD, ciGVHD) and, later, exhibits scleroderma-like features (chronic sclerotic GVHD, csGVHD). Mechanisms involved in the initiation and propagation of the different forms of GVHD are a main focus of HCT research, but remain controversially discussed. Acute GVHD has long been thought to mainly involve a type 1 T cell response, but more recent data have shown an involvement of Th2, Th17 and Treg cells. These T cell subsets, and particularly an altered regulatory T cell homeostasis, have also been reported to play a role in chronic GVHD, the pathogenesis of which is much less understood. Taken together, it is unclear which exact mechanisms are operative in the T cell immunopathogenesis of cutaneous GVHD. In this study, we searched for cellular and molecular patterns that would allow us to distinguish between the different forms of cutaneous GVHD and to draw conclusions about the pathomechanisms involved. To address this issue, we collected lesional skin biopsies from patients diagnosed with aGVHD (n=25), ciGVHD (n=17) and csGVHD (n=7). Clinical grading of acute and chronic GVHD was performed according to the modified Glucksberg criteria and the NIH consensus criteria, respectively. 14 HCT recipients underwent serial skin biopsies at different time points prior/after HCT (prior/after conditioning, day 20-30 post-HCT, day 100-120 post-HCT). Skin biopsies obtained were subjected to phenotypic and functional analyses. The cellular infiltrate was assessed by immunofluorescence stainings; interleukins and chemokines were measured by real-time RT-PCR. Cytokine secretion profiles of stimulated T cells from collagenase-digested GVHD skin biopsies were analyzed by intracellular flow cytometry. While CD4+ and CD8+ T cells dominated the inflammatory infiltrate in both acute and chronic GVHD skin lesions, the analysis of the quality of the T cell-mediated immune response revealed striking differences. In acute cutaneous GVHD, we found a “Th2 signature” as evidenced by highly increased transcripts of Th2 cytokines (IL-4, IL-5 and IL-13) and chemokines (CCL17, CCL20). Surprisingly, we also observed a strong up-regulation of IL-22, but not IL-17 mRNA, which was paralleled by an increase of IL-22-single-producing CD4+ T cells. IL-17/IL-22 double-positive T cells were almost absent. In sharp contrast to aGVHD, the immune response occurring in ciGVHD showed a mixed Th1/Th17 signature. This was evidenced by an up-regulation of Th1/Th17 cytokine (IFN-γ, IL-12/IL-23p40 and IL23p19) and chemokine transcripts (CCL5, CCR5, CXCL9, CXCL10). In accordance with this finding, increased numbers of IFN-ү- and IL-17-single-producing CD8+ T cells infiltrated ciGVHD skin lesions.Chronic sclerotic GVHD exhibited distinct features, including an abundance of mast cells and a down-regulation of T cell cytokines and chemokines relative to normal skin. In an attempt to identify a factor predicting the occurrence of acute cutaneous GVHD, we analyzed the cellular and molecular signatures in skin of HCT recipients before GVHD onset. In accordance with the finding of a Th2 response occurring in aGVHD, levels of TSLP, a keratinocyte-derived cytokine skewing the immune response towards a Th2 direction, were increased at day 20 after HCT in non-lesional skin of patients who would later develop aGVHD. In conclusion, the present study provides new insights into the diverse pathomechanisms operative in the acute and chronic form of cutaneous GVHD, respectively. Our findings allow to more accurately distinguish aGVHD from ciGVHD based on different cellular and molecular patterns. The analysis of TSLP expression in skin (before disease onset) could be helpful in identifying HCT recipients at risk for developing acute cutaneous GVHD. Disclosures: No relevant conflicts of interest to declare.

CD30-Targeted Therapy with Brentuximab Vedotin and DLI in a Patient with T-Cell Posttransplantation Lymphoma

CD30-Targeted Therapy with Brentuximab Vedotin and DLI in a Patient with T-Cell Posttransplantation Lymphoma

Steroid refractory psoriasiform cutaneous graft versus host disease successfully treated by extracorporeal photopheresis: A case report

Graft-versus-host disease (GVHD) is a frequent complication occurring after allogeneic hematopoietic stem cell transplantation. It can be classified as acute and chronic GVHD based on the time of onset following transplantation and clinical presentation. Cutaneous involvement is the most common feature of acute GVHD, with maculopapular exanthema and perifollicular papular lesions. Steroid refractory GVHD is associated with a significant morbidity and mortality. We present a very rare case with acute cutaneous GVHD mimicking psoriasis vulgaris occurring after allogeneic peripheral blood stem cell transplantation for chronic lymphocytic leukemia. The patient’s rash resembled psoriasis vulgaris and showed histologic features of both psoriasis and acute GVHD. Despite concomitant cyclosporine A and methylprednisolone therapy no response was obtained. Therefore, we administered extracorporeal photopheresis and achieved the desired therapeutic effect.

Cutaneous Graft versus Host Disease in Pediatric Multivisceral Transplantation

Multivisceral transplantation (MvTx) is the concurrent transplantation of the stomach, pancreaticoduodenal complex, and intestine, with or without the liver. Its use is increasing worldwide as it has been considered as a therapy for patients with functional disturbance of several organs. Graft-versus-host disease (GvHD) has been a relevant clinical problem in MvTx ever since the procedure was first performed, but little has been reported about its specific cutaneous features. Our study included all pediatric patients with clinical and histopathologic evidence of cutaneous GvHD who received MvTx between October 1999 and December 2010 in University Hospital La Paz. Seventeen children underwent MvTx at our center during this period of time. Five patients developed cutaneous GvHD (29.4%). The median onset was 45.2 days after transplantation. Acute cutaneous GvHD, consisting of symmetrical maculopapular exanthema with prominent acral erythema and accentuated lesions on the face and pinnae, was clinically suspected and pathologically confirmed in four patients (80%). Three children (60%) experienced disease progression to a formation and a positive Nikolsky sign. Only one girl (20%) showed lichenoid GvHD. The first therapeutic approach was steroids and tacrolimus adjustment; many other drugs were used in refractory cases. Three of the five patients (60%) died with concomitant GvHD, the immediate cause of death being another comorbid disease. Knowledge of the features of cutaneous GvHD in MvTx allows clinicians early recognition and prompt therapeutic intervention that may prevent progression to higher-grade disease and improve outcomes for these patients.

TH17 Cells Are Critical for Skin-Specific Pathological Injury in Acute Graft-Versus-Host Disease

Interleukin-17 (IL-17), which is important for host defens, has been implicated in autoimmune and chronic inflammatory diseases. As knockout mice lack IL-17 expression in δγT, NKT-like cells, studies investigating the association between TH17 cells and cutaneous graft-versus-host disease (GVHD) in animal models have reported conflicting results. To determine the role of TH17 cells in cutaneous GVHD, we developed an acute GVHD model using C57BL/6(H-2b) donors to BABL/c (H-2d) recipients. Blood samples and skin were examined for inflammation and infiltrating cells using histology and fluorescence-activated cell sorter (FACS) on days 6 and 15 after bone marrow transplantation. We found donor T cells to mediate severe cutaneous inflammation, which was ameliorater by administration of halofuginone (HF) to the recipients. Mechanistically, we demonstrate the severe tissue damage during this disorder to be associated with the production of IL-17 and the expansion of IL-17-producing CD4+ cells. Specific inhibition of TH17 differentiation and function by HF reduced disease severity. Thus, TH17 cells are sufficient to induce acute cutaneous GVHD.

In Vivo Reflectance Confocal Microscopy in the Early Diagnosis of Acute Cutaneous Graft-Versus-Host Disease: A Pilot Study

Graft-versus-host disease (GVHD) is a common and potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation. The diagnosis of cutaneous GVHD can be challenging, as it is based on clinical presentation, which often simulates other processes. Early recognition and treatment are essential to reduce the associated morbidity and mortality. Reflectance confocal microscopy (RCM) is a high-resolution, noninvasive technique that may facilitate a timely diagnosis and prompt institution of treatment. We sought to describe the features of acute cutaneous GVHD under RCM and to correlate these with the histopathological findings. Patients were recruited prospectively to undergo clinical and RCM examination. A total of 19 patients were enrolled, five of whom developed acute cutaneous GVHD following an allogeneic hematopoietic stem cell transplant. RCM images from these five cases were examined and the key confocal features were compared to histopathological findings. On RCM examination, features of acute cutaneous GVHD, such as apoptotic keratinocytes, inflammatory cell infiltration and interface dermatitis, were identified and were shown to correlate well with histopathological findings. The open-label, unblinded nature of this study allows for potential observer bias and the small sample size may not reflect the findings in a larger population. The sensitivity and specificity of RCM in this population remain to be determined. The findings support further examination of the use of RCM in the rapid, bedside diagnosis of acute cutaneous GVHD.

Clinical differentiation of acute cutaneous graft-versus-host disease from drug hypersensitivity reactions

The early diagnosis of acute cutaneous graft-versus-host disease (GVHD) is challenging as there are no specific features. Drug hypersensitivity reaction (DHR) is the most frequently encountered condition to differentiate from GVHD. We sought to investigate the clinical characteristics of acute cutaneous GVHD in comparison with DHR. A retrospective chart review was conducted for allogeneic hemopoietic-cell transplantation recipients with maculopapular rashes who had undergone hemopoietic-cell transplantation less than 100 days earlier. We analyzed the clinical characteristics of 22 patients with acute cutaneous GVHD in comparison with 17 patients with DHR. Facial involvement was more frequent in the GVHD group (59%) than in the DHR group (24%). The difference was more significant when both the face and palms/soles were involved (36% in the GVHD group, no patients in the DHR group). Diarrhea was significantly more common in the GVHD group (73%) than in the DHR group (12%). The presence of both diarrhea and hyperbilirubinemia occurred only in the GVHD group (41%). In the GVHD group, the patients developed diarrhea and hyperbilirubinemia gradually with most patients exhibiting diarrhea and hyperbilirubinemia after 2 days. A retrospective design, small number of patients, and single-center design are limitations. Facial involvement, and moreover involvement of the face and palms/soles, suggests the diagnosis of GVHD versus DHR. The presence of diarrhea, and diarrhea accompanied by hyperbilirubinemia is thought to favor the diagnosis of GVHD. Rashes with more than 2 to 3 days of duration and not accompanied by diarrhea and hyperbilirubinemia are less likely to be GVHD.

Organ-Specific Protection from CD8+ T Cell-Mediated Acute Skin GVHD by Bortezomib Administration Correlates with Decreased IL-6.

AbstractAbstract 3735We have previously demonstrated that while early administration of bortezomib after murine allogeneic bone marrow transplantation (allo-BMT) can prevent acute graft versus host disease (GVHD), delayed administration of bortezomib results in increased GVHD-dependent gastrointestinal toxicity (PNAS 2004, Blood 2005). This was primarily mediated by donor CD4+ T cells in fully MHC-mismatched allo-BMT (Blood 2008). In the current study, we assessed continuous bortezomib administration on CD8+ T cell-mediated acute GVHD. Using an MHC-matched, mHAg-mismatched model (C3.SW donor cells into lethally irradiated C57BL/6 recipients) in which acute GVHD is primarily induced by CD8+ T cells, we confirmed that significant skin and liver GVHD was observed after CD8+ T cell transfer. Histological evaluation of acute liver GVHD displayed severe lymphocyte infiltrates and severe subacute bile duct hepatitis consistent with hepatic GVHD. Severe acute skin GVHD was noted by persistent fur loss and skin ulcerations. Cutaneous histopathological assessment exhibited severe inflammatory infiltrates, epidermal hypertrophy, loss of hair follicles, satellite cell necrosis and dermal fibrosis. Notably, after allo-BMT, 6 weeks or thereafter, a significant increase in serum IL-6 levels was observed in the mice exhibiting marked skin damage, suggesting organ-specific correlation of higher serum IL-6 expression with acute cutaneous GVHD associated injury. We next investigated the effects of continuous treatment with bortezomib in this GVHD model. Surprisingly, we found that continuous bortezomib administration at the conventional dose of 0.75–1 mg/kg twice per week resulted in fractional GVHD- dependent lethal toxicity, exacerbating lymphocyte infiltration in liver, gut and lung as evidenced by pathological examination. However, skin GVHD pathology was markedly improved. Due to the limitations of conventional doses of bortezomib for treatment of acute GVHD, we then performed a low-dose bortezomib treatment at 0.125 mg/kg, twice per week. We found that mice receiving low-dose bortezomib were protected from lethal toxicity and displayed significant amelioration of skin GVHD pathology. Significant decreases in serum IL-6 levels were also consistently observed consistent with the amelioration of skin GVHD. Interestingly, while skin GVHD was markedly improved, there was no difference in liver, gut and lung pathology compared to control GVHD recipients indicating an organ-specific protection. These results demonstrate that continuous bortezomib can be applied in CD8+ T cell-mediated acute GVHD but necessitates lower dosing and organ-specific protection is observed. Disclosures:No relevant conflicts of interest to declare.

A case of congenital bone marrow failure with radio-ulnar synostosis

Bone marrow failure is often associated with skeletal defects, as seen in Fanconi anemia [1], Diamond-Blackfan anemia [2], or thrombocytopenia-absent radius syndrome [3]. Congenital amegakaryocytic thrombocytopenia with radio-ulnar synostosis has been reported to be related with HOXA11 mutation [4]. We present a boy with congenital bone marrow failure with radio-ulnar synostosis, but without HOXA11 mutation. A boy, born to non-consanguineous parents, was delivered via Cesarean section at 31 weeks’ gestation for hydrops fetalis resulting from severe anemia. He had severe anemia (hemoglobin 2.7 g/dl), severe neutropenia (white blood cells 3,220/ll with 1.5% neutrophils), and moderate thrombocytopenia (platelets 89,000/ll) at birth. Neutrophils continued to be nearly absent and platelet counts progressively decreased below 20,000/ll by 2 weeks of age. He had no hepatosplenomegaly, lymphadenopathy, or cafe-au-lait spot, but had hydrocele testicle, sensorineural hearing loss, and overlapping fingers without abnormalities of bone. Supination of his bilateral forearms was restricted and X-rays revealed bilateral proximal radio-ulnar synostosis (Fig. 1). Bone marrow aspiration showed aplasia without dysplasia. Chromosomal analysis revealed a 46, XY, inv(9)(p12q13) karyotype, which is considered to be normal variant without physiologic role. He lacked a family history of hematological or skeletal disorders. No excessive chromosomal breakage was detected by mitomycin C stimulation, and no mutation was found for the Shwachman–Bodian–Diamond syndrome (SBDS) gene. He had repeated bacterial and viral infections after birth, requiring antibiotics and prophylactic antifungal agents. He also needed repeated transfusions of red blood cells and platelets until 8 months of age when he underwent a 5/6 human leukocyte antigen (HLA)-matched unrelated bone marrow transplantation (BMT). Pretransplant conditioning was with fludarabine, cyclophosphamide, and thoracicabdominal irradiation (2 Gy). Prophylaxis for graft-versushost disease (GVHD) was short-term methotrexate and tacrolimus. Engraftment was prompt and grade 1 acute cutaneous GVHD was observed. By 20? days following BMT, he became independent of all transfusions. He has had normal hematological studies for 14 months after BMT. Limb formation is regulated by HOX genes during embryonic development. The most 50 members of the HOXA and HOXD clusters (HOXA9–HOXA13 and HOXD9–HOXD13) are particularly important in limb development [5]. HOX genes are also well known to be associated with hematopoiesis and leukemogenesis [6]. The effects of Hoxa10 and Hoxd11 were found to be on forearm development from studies of mutant mice [7, 8]. Radio-ulnar synostosis with congenital thrombocytopenia has been reported to be associated with HOXA11 mutation [4], which prompted us to examine HOX genes. However, we detected no mutations of HOXA11 and HOXD11. Of the three patients with radio-ulnar synostosis and thrombocytopenia reported by Thompson et al. [9], one H. Yoshida Y. Hashii T. Okuda S. Kusuki E. Sato H. Ohta (&) K. Ozono Department of Pediatrics, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita 565-0871, Japan e-mail: ohta@ped.med.osaka-u.ac.jp

Highlighting TH-1 Rather Than TH-17 Cytokine Network in Acute Cutaneous Gvhd

Graft versus Host disease (GVHD) is a major lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). As skin is one of the major organs involved and the most accessible, it serves as an ideal organ site to further understand the pathogenesis and progression of acute GVHD and could serve as a window into pathophysiology of other organ sites. Cytokine network aberrations are a central feature of the pathogenesis of acute cutaneous GVHD, terminating in the hallmark of premature apoptosis of keratinocytes. The purpose of this study is to delineate the cytokine network aberrations at the molecular and cellular level in affected skin from patients with acute GVHD with the goal of developing novel therapeutic approaches to defined targets with the goal of preventing or reducing acute GVHD. We performed immuno-phenotypic profiling of acute cutaneous GVHD biopsies determining relative numbers of various types of immunocytes including T cells, dendritic cells, and macrophages. Cytokine expression (IFNγ and TNFα) was determined by RT-PCR on RNA extracted from eight different lesional skin and two non-lesional biopsies. We analyzed for TH-1 (IFN γ and TNFα), TH-2 (IL-4) and TH-17 (IL-17A, IL-22) cytokine mRNAs. We observed that TH-1 cytokine mRNAs were detected in 7 out of 8 different lesional skin biopsies but none of two nonlesional skin biopsies. Interestingly TH-2 and TH-17 cytokine mRNAs were not detectable in acute cutaneous GVHD lesions or nonlesional skin biopsies. RNA extracted from a 2-way mixed lymphocyte reaction (MLR) served as a positive control for these RT-PCR results. Given recent data that suggested that Notch-1 regulated TH-1 polarization by transcriptionally controlling the Tbx gene (MinterLM et al 2005), we hypothesized that inhibiting Notch signaling might be of potential therapeutic importance given these results of TH-1 polarization in acute skin GVHD. To block Notch signaling we utilized several pharmacological gamma secretase inhibitors (which may have therapeutic applications), and supplemented this pan-Notch receptor inhibition approach by use of a Notch-1 specific siRNA. Preliminary studies suggest that both approaches reduce IFN γ levels in allogeneic T cell responses in-vitro. Further experiments are in progress to confirm these findings and to determine the molecular mechanism. This study begins to define the cytokine network operative within acute cutaneous GVHD lesions highlighting the finding that TH-1 rather than TH-17 cytokines may be the more important target.

Adhesion Molecule Blockade with the Humanized Anti-LFA-1 Monoclonal Antibody Efalizumab Improves Steroid Refractory Acute and Chronic Graft-Versus-Host Disease of the Skin

The integrin LFA-1 is important for naïve T-cell homing to lymph nodes and bronchial associated lymphoid tissue (BALT), and appears to play a major role in the trafficking of effector T-cells to cutaneous and possibly hepatic sites of inflammation. Efalizumab, a humanized monoclonal antibody directed against the CD11a subunit of LFA-1, is currently approved for the management of moderate to severe plaque psoriasis. Given the pathophysiologic similarities between psoriasis and cutaneous graft-versus-host disease (GVHD), we launched a small exploratory study examining the use of efalizumab for steroid refractory GVHD of the skin.Methods: Patients with evidence of an inflammatory, erythematous rash and a skin biopsy consistent with cutaneous GVHD who had failed to improve after at least 4 weeks of methylprednisilone at 0.5mg/kg were eligible for enrollment. Patients were scheduled to receive weekly subcutaneous efalizumab at 1mg/kg for a total of 8 weeks. Using digital photography and “ImageJ” software obtained from The National Institutes of Health, each patient's rash was quantified as a percentage of their total body surface area (BSA) at baseline and after completing therapy. A complete response (CR) was defined as the total resolution of skin disease, and a partial response was defined as ≥ 50% reduction in the proportion of BSA involved by rash. Skin biopsies were taken before and after therapy to study baseline pathology and to assess for histological improvement.Results: Two patients were treated at our institution. Patient #1 presented with longstanding chronic GVHD of the skin, and received 8 doses of study medication according to schedule. After therapy, she demonstrated a PR, with a reduction in cutaneous involvement from 15% to 6%. The efalizumab appeared to be well tolerated, with the exception of grade II nausea. Patient #2 exhibited grade IV acute GVHD of the skin that developed shortly after an allogeneic transplant for refractory Hodgkin's lymphoma following an autologous transplant two years earlier. The patient received 6/8 scheduled efalizumab doses, but was subsequently taken off study after developing transient coagulase negative staphylococcal bacteremia. In spite of his abbreviated therapeutic course and advanced disease, he attained a CR. Approximately 6 weeks after his final efalizumab dose, the patient developed parainfluenza pneumonia that ultimately proved fatal. Both subjects showed histological improvement following efalizumab therapy, with a reduction in dermal T-cell infiltration (see table). Neither demonstrated elevated levels of IL-17 in the skin by RT-PCR at any time point.Conclusions: Efalizumab appears to show signs of activity against acute and chronic cutaneous GVHD. While the late infectious complications that developed in patient #2 are concerning, the current sample size is too small to draw firm conclusions regarding the toxicity profile of this agent in the bone marrow transplant (BMT) setting, particularly given the heavily pretreated status of both subjects. Our digital photography technique proved to be a feasible method for assessing response to therapy, and would allow for central review by a blinded operator. Further studies to better characterize the efficacy and toxicity of efalizumab in the BMT setting appear warranted.Patient #1Patient #2Day 0Week 8Day 0Week 8Epidermal thickness (um)312.493.861.936.1CD3 Epidermis364500CD3 Dermis18538180CD8 Epidermis382000CD8 Dermis5622167CD11a Epidermis20200CD11a Dermis742990Cell counts are in #cells/1.2mm surface epidermal length at 10x magnification