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1 Biol Blood Marrow Transplant 19 (2013) 767e776 Current Practice in Diagnosis and Treatment of Acute Graft-versus-Host Disease: Results from a Survey among German-Austrian-Swiss Hematopoietic Stem Cell Transplant Centers American Society for Blood ASBMT and Marrow Transplantation Daniel Wolff 1, *, Francis Ayuk 2, Ahmet Elmaagacli 3, Hartmut Bertz 4, Anita Lawitschka 5, Michael Schleuning 6, Ralf-Georg Meyer 7, Armin Gerbitz 8, Inken Hilgendorf 9, Gerhard C. Hildebrandt 10, Matthias Edinger 1, Stephan Klein 11, Jörg Halter 12, Sabine Mousset 13, Ernst Holler 1, Hildegard T. Greinix 14 1 Department of Hematology and Clinical Oncology, University of Regensburg, Germany 2 Interdisciplinary Clinic for Stem Cell Transplantation, University Cancer Center Hamburg, Germany 3 Department of Bone Marrow Transplantation, University Hospital Essen, Germany 4 Department of Hematology and Oncology, Albert Ludwigs-University Medical Center, Freiburg, Germany 5 St. Anna Children s Hospital, Vienna, Austria 6 Center of Blood Stem Cell and Marrow Transplantation, DKD, Wiesbaden, Germany 7 Department of Medicine III, University Medical Center, Mainz, Germany 8 Department of Medicine V, University of Erlangen, Germany 9 Department of Hematology, Oncology, Palliative Care, University Medical Center Rostock, Germany 10 Division of Hematology and Oncology, LSUHSC-Shreveport, Shreveport, Louisiana, USA 11 Department of Hematology and Oncology, University Hospital Mannheim, Germany 12 Department of Hematology, University Hospital Basel, Switzerland 13 Department of Internal Medicine II, University of Frankfurt, Germany 14 Department of Internal Medicine I, Medical University of Vienna, Austria Article history: Received 1 November 2012 Accepted 23 January 2013 Key Words: Allogeneic hematopoietic stem cell transplantation Acute graft-versus-host disease Immunosuppressive therapy abstract To assess current clinical practice in diagnosis and treatment of acute graft-versus-host disease (agvhd), we performed a survey among German, Austrian, and Swiss allogeneic hematopoietic stem cell transplantation (allo-hsct) centers. Thirty-four of 72 contacted centers (47%) completed both the diagnostic and therapeutic sections of the survey, representing 65% of allo-hsct activity within the participating countries in Three pediatric centers answered as requested only the diagnostic part of the survey. In the presence of diarrhea and decreased oral intake after engraftment, only 4 centers (12%) do not perform any endoscopy before the start of immunosuppressive treatment. In case of a skin rash with the differential diagnosis of drug reaction, only 12 centers (35%) perform a skin biopsy up front, whereas 19 do so after failure of systemic steroids. In the presence of rapidly increasing cholestasis occurring without any other signs of agvhd, 11 centers (32%) perform a liver biopsy up front and 14 only after failure of steroid treatment, whereas 9 centers do not perform a liver biopsy at all. Twenty centers (59%) use a percutaneous approach, 12 a transvenous approach, and 1 mini-laparoscopy for liver biopsies. First-line treatment of cutaneous agvhd stage 1 consists of topical treatment alone in 17 of 31 responding centers (61%), whereas isolated cutaneous agvhd stage III is treated with systemic steroids (prednisolone below 0.5 mg/kg/day n ¼ 2, 0.5 to 1.0 mg/kg/day n ¼ 10, above 1.0 to 2.5 mg/kg/day n ¼ 19) without or with topical agents (steroids n ¼ 10; calcineurin inhibitors n ¼ 3). In gastrointestinal manifestations of agvhd, 9 centers (29%) add topical to systemic steroids, and 3 consider topical steroids as the only treatment for mild gastrointestinal and cutaneous agvhd. The choice of agent for second-line treatment as well as the sequence of administration are extremely heterogeneous, most likely due to a lack of convincing data published. Most frequently used are mycophenolate mofetil (n ¼ 14) and extracorporeal photopheresis (n ¼ 10). Our survey also demonstrates that clinicians chose salvage therapies for steroid-refractory agvhd based on their centers own clinical experience. Ó 2013 American Society for Blood and Marrow Transplantation. INTRODUCTION Acute graft-versus-host disease (agvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct) remains the leading cause for early morbidity and mortality. Historically, agvhd has been diagnosed clinically, and the extent of clinical symptoms defines its organ and overall severity [1]. Financial disclosure: See Acknowledgments on page 774. * Correspondence and reprint requests: Daniel Wolff, MD, Department of Hematology and Oncology, University of Regensburg, F.J. Strauss Allee 11, Regensburg, Germany. address: daniel.wolff@ukr.de (D. Wolff) /$ e see front matter Ó 2013 American Society for Blood and Marrow Transplantation. However, none of these is specific for agvhd, and relevant differential diagnoses such as allergies, infections, or toxicities may result in identical or very similar clinical symptoms. Moreover, retrospective analyses identified a significant risk for falsely positive diagnosis of cutaneous or liver GVHD if the diagnosis was only based on clinical symptoms [2-4]. Despite the obvious importance of histological confirmation of agvhd, and in contrast to solid organ transplantation, in which histological confirmation of rejection is routine clinical practice, common guidelines for diagnostic standards in allo-hsct recipients are missing, mainly due to the lack of prospective trials evaluating the impact of histology on

2 768 D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e776 treatment and prognosis. The same holds true for treatment of agvhd. Although first-line treatment consists of steroids with doses of prednisolone/methylprednisolone ranging from 1 to 2.5 mg/kg body weight based on prospective randomized trials, decisions on second-line therapy are derived solely from uncontrolled phase II trials or retrospective analyses. Consequently, no accepted treatment algorithm for second-line treatment of steroid-refractory agvhd exists [5]. Therefore, the German-Austrian-Swiss Working Group on GVHD performed a survey on current clinical practice sent to 72 allo-hsct centers within Germany, Austria, and Switzerland to document the current clinical routine. METHODS A paper-based survey was distributed electronically to 72 allo-hsct centers within Germany, Austria, and Switzerland and was returned, digitalized, and evaluated anonymously. In parallel, the number of allo-hsct performed in 2011 per participating transplant center was obtained from the German, Austrian, and Swiss Registries for stem cell transplantation to document the transplant activities of responding centers. The survey contained 9 questions regarding the issue of histopathological confirmation of suspected agvhd describing predefined clinical scenarios and requesting the description of a center s diagnostic approach. Two questions on diagnostic challenges related to a case scenario with clinical symptoms of lower gastrointestinal (GI) agvhd but negative histopathology on initial endoscopic biopsy. In addition, 9 questions focused on specific situations of first-line treatment of agvhd after T cellereplete transplantations, including 1 question reflecting the situation of overlap syndrome with predominant symptoms of acute GI involvement followed by 4 questions on second-line treatment. Of 72 allo-hsct centers, 29 German, 3 Austrian, and 2 Swiss centers responded to the survey, representing 65% of allo-hsct activity within the participating countries in Three pediatric centers responded only to the diagnostic part of the survey as decided within the Pediatric Working Party of the European Group for Blood and Marrow Transplantation (EBMT). RESULTS Diagnosis of GI GVHD The first question focused on the diagnostic approach in case of isolated clinical symptoms of GI GVHD consisting of decreased oral intake and diarrhea on day 24 after allo-hsct, mild edema of the colon descendens in ultrasound, and negative microbiology results of stool specimens. Twentyone of 34 centers (62%) would perform a colonoscopy before initiation of treatment, including 8 centers performing additional gastroscopy. Two centers would perform a gastroscopy alone and 4 a sigmoidoscopy alone. Three additional centers would combine gastroscopy and sigmoidoscopy. Three centers would only perform endoscopy if first-line treatment of agvhd failed, and only 1 center would not use endoscopy at all. The second question related to the approach in case of normal histology obtained from the descending colon of the above-mentioned patient who showed significant regression of symptoms of GVHD under empirical corticosteroid treatment. Twenty centers (59%) stated that they would assume the histological result was false negative and would continue GVHD treatment. Twelve centers (35%) would assume GVHD was present but would speed up taper of steroids, and 2 centers would rapidly reduce steroid dose. The third question dealt with the approach in case the above-mentioned patient had negative histology of the sigmoid but continued to have substantial amounts of diarrhea despite therapy with methylprednisolone at 2.5 mg/kg/ day for 10 days. In addition, ultrasound exam revealed persistent mild to moderate edema of the bowel wall of the descending colon and ileum. Three centers responded that they would not perform endoscopy but would assume steroid-refractory agvhd. Fifteen centers would repeat the colonoscopy and 11 centers, colonoscopy and gastroscopy. One center would solely perform a Fluorodeoxyglucosepositron emission tomographyecomputed tomography, 1 a sigmoidoscopy, and 1 would combine gastroscopy and sigmoidoscopy, whereas 2 centers would only perform gastroscopy, respectively. One of the centers combining gastroscopy and colonoscopy would add magnetic resonance tomography enterography. Next, we asked whether endoscopic exams would be performed more frequently if sensitivity of histopathological evaluation is increased to 75% and specificity to 90%. Thirty centers (88%) agreed to this approach. Diagnosis of Cutaneous GVHD The fifth question related to a cutaneous rash involving more than 50% of the body surface on day 14 (2 days after hematopoietic engraftment) without liver or GI abnormalities and no pruritus or other impairment of the patient s general condition on day 14 (2 days after hematopoietic engraftment). Of note, the skin rash appeared after starting therapy with piperacillin/tazobactam. Twelve centers stated they would change the antimicrobial therapy, perform a skin biopsy, and start systemic corticosteroid treatment thereafter. Sixteen centers would change the antimicrobial agent, apply topical steroids, and only perform a skin biopsy in case of lack of improvement. Three centers would start systemic corticosteroids and only perform a skin biopsy in case of persistence of the rash. Three centers would not consider a skin biopsy at all. The sixth question asked whether skin biopsies in isolated cutaneous agvhd would be performed more frequently if sensitivity of histopathological evaluation is increased to 75% and specificity to 90%. Twenty-three centers (68%) agreed to this approach. Diagnosis of Liver GVHD The seventh question of the survey related to a scenario on day 25 (12 days after hematopoietic engraftment) with rapidly progressive cholestasis and suspected agvhd of the liver stage 3, normal ultrasound exam of the liver, negative microbiology results, and lack of prior liver toxicity in the patient s history. Four centers stated they would perform a liver biopsy followed immediately by administration of systemic corticosteroids and ursodeoxycholic acid (UDCA). Two centers would perform a liver biopsy and administer UDCA alone, and 1 center would perform a liver biopsy followed by steroids alone. Four centers would just perform a liver biopsy and wait for the result before starting corticosteroid therapy. Fourteen centers would perform a liver biopsy if first-line treatment with steroids failed, and 9 centers would not consider a liver biopsy in this situation. The eighth question dealt with the technique applied for performing liver biopsies. Twelve centers would use a transjugular and 20 a transcutaneous approach, and 1 center would consider both approaches equally. One center would perform a mini-laparoscopy. The ninth question asked whether liver biopsies would be performed more frequently if sensitivity of histopathological evaluation is increased to 75% and specificity to 90%. Twentysix centers (76%) agreed. First-Line Treatment of agvhd Due to a concomitant survey of the EBMT on treatment of agvhd in children, 3 pediatric centers did not answer the

3 D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e Figure 1. (A) Treatment of isolated cutaneous agvhd grade I. (B) Treatment of isolated cutaneous agvhd stage 3. questions on treatment, resulting in 31 responding centers. First, we asked about the therapeutic approach in isolated cutaneous agvhd stage I (<25% body surface area) without general symptoms and low risk for relapse of original disease occurring on day 20 after allo-hsct from a human leukocyte antigen (HLA)-identical sibling donor. Results are shown in Figure 1A. In case of use of systemic steroids, doses of prednisolone/methylprednisolone not above 1 mg/kg/day were administered. The second question related to a scenario of agvhd of the skin stage 3 with involvement of 75% of body surface area on day 40 after withdrawal of mycophenolate mofetil (MMF) but during continuous GVHD prophylaxis with cyclosporine (CsA). Results are shown in Figure 1B. Topical steroids were administered in addition to prednisolone at 0.5 mg/kg/day (n ¼ 1), 0.5 to 1 mg/kg/day (n ¼ 3), or 1 to 2.5 mg/kg/day (n ¼ 4), respectively. Topical calcineurin inhibitors (CNIs) with (n ¼ 2) or without topical steroids (n ¼ 1) were used in addition to systemic steroids by some allo-hsct centers. Next, we presented a clinical situation of agvhd grade III (skin stage 2, liver stage 3, no GI involvement) starting on day 22 under GVHD prophylaxis with CsA and methotrexate. Fourteen centers stated they would perform a skin and 2 a liver biopsy. Responses to therapeutic strategies are shown in Figure 2A. Eighteen centers (58%) would use UDCA in addition. Three centers applying steroid doses between 1 and 2.5 mg/ kg/day would consider higher doses in selected patients. The fourth question focused on upper GI GVHD describing a patient with nausea and localized erythema of the skin starting on day 26 after allo-hsct from an HLA-identical unrelated donor. Eight centers would perform a skin biopsy and 20 a gastroduodenoscopy before start of therapy. Responses to therapeutic strategies are shown in Figure 2B. Nine centers would apply additional topical steroids. The fifth question dealt with therapeutic procedures concerning a patient with agvhd grade III with localized Figure 2. (A) Treatment of agvhd grade III involving skin and liver. (B) Treatment of agvhd grade II with localized skin involvement and nausea. (C) Treatment of agvhd grade III with localized skin involvement and severe diarrhea. skin involvement and severe diarrhea. Four centers would perform a skin biopsy and colonoscopy, 2 centers would combine a skin biopsy with a sigmoidoscopy, and 1 additional center would perform a skin biopsy, sigmoidoscopy, and gastroscopy. Eight centers would only perform colonoscopy. Two centers would combine colonoscopy with gastroscopy. Seven centers would only perform sigmoidoscopy. Three centers would only perform an abdominal ultrasound. Sixteen centers would combine abdominal ultrasound with endoscopy. Four centers did not mention any diagnostic approach. Responses to therapeutic strategies are shown in Figure 2C. The sixth question described a patient with rapidly progressing agvhd overall grade IV consisting of skin GVHD stage 4, severe diarrhea (stage 3), and liver GVHD stage 3 on day 14 after allo-hsct from an HLA-mismatched unrelated donor who had received CsA and MMF for GVHD prophylaxis. Before the start of therapy, 2 centers would only perform a skin biopsy, 5 would combine skin biopsy with colonoscopy, and 7 would combine skin biopsy with sigmoidoscopy. Four centers would solely perform a colonoscopy, whereas 2 centers would solely perform a sigmoidoscopy. Eight centers did not mention any diagnostic approach. Three centers would do abdominal ultrasound without any

4 770 D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e776 further diagnostic approach. Fifteen centers would perform abdominal ultrasound in addition to endoscopy or/and skin biopsy. Responses to therapeutic strategies are shown in Figure 3A. Twelve centers would apply steroids alone at a dose between 1 and 2.5 mg/kg/day of prednisolone, with 3 centers using topical steroids in addition. Others stated to combine systemic steroids with either infliximab (n ¼ 1), pentostatin (n ¼ 1), etanercept (n ¼ 2), or extracorporeal photopheresis (ECP) (n ¼ 1). Four centers would only administer high-dose steroids above 2.5 mg/kg/day prednisolone. Others would use combinations of high-dose steroids with alemtuzumab at 10 mg (n ¼ 2), mesenchymal stromal cells (n ¼ 1), etanercept (n ¼ 6), ECP (n ¼ 1), or basiliximab (n ¼ 1). Five centers would add topical steroids to high-dose steroids. One center did answer the diagnostic part only. Next, we dealt with late-onset agvhd overall grade III (lower GI stage 2, skin stage 3) occurring on day 160 after allo-hsct and 20 days after complete withdrawal of GVHD prophylaxis. Three centers stated they would only perform a skin biopsy, others would combine a skin biopsy with colonoscopy (n ¼ 4) or sigmoidoscopy (n ¼ 5), 4 centers would perform only a sigmoidoscopy, 6 only a colonoscopy, whereas 9 centers would not perform histological confirmation at all. Responses to therapeutic strategies are shown in Figure 3B. Eight centers would apply additional enteral budesonide. With regard to the applied corticosteroid, 11 centers (35.5%) would use either prednisolone or methylprednisolone, whereas 11 would administer only prednisolone and 9 (29%) only methylprednisolone. Four centers would apply the steroid dose once in the morning, 15 in the morning and at night, 3 in the morning and at noon, and 5 either in the morning or in the morning and at noon depending on the severity of GVHD. Four split the steroid dose into 3 doses. The eighth question focused on treatment of National Institutes of Healthedefined overlap syndrome occurring on day 160 after allo-hsct and 30 days after withdrawal of GVHD prophylaxis consisting of lichenoid changes of the oral mucosa, sicca symptoms of the eyes, diffuse erythema of the skin (60% of body surface area), and diarrhea at 1 liter per day. Ultrasound showed wall edema of the descending colon with a wall thickness of 8 mm. Before treatment, 1 center would perform only a skin biopsy, 4 would combine skin biopsy and colonoscopy, 3 skin biopsy and sigmoidoscopy, 4 a sigmoidoscopy alone, and 2 would combine sigmoidoscopy and gastroscopy. Five centers would perform only a colonoscopy. Three would combine colonoscopy and gastroscopy. Gastroscopy alone would be performed by 2 centers. Seven centers would not seek histological confirmation. Responses to therapeutic strategies are shown in Figure 3C. Seventeen centers would apply additional topical steroids. The last question focused on the general use of immunosuppressive agents for first-line treatment of agvhd. Results are listed in Table 1. One center each mentioned occasional use of antithymocyte globulin (ATG), mesenchymal stromal cells, and tacrolimus. Second-Line Treatment First, we focused on the time point of initiation of secondline treatment describing a patient with low risk of relapse of original disease with agvhd grade III (skin stage 1, liver stage 2, and GI stage 3) whose cutaneous and hepatic manifestations responded to methylprednisolone at 2 mg/kg/day, Figure 3. (A) Treatment of multiorgan agvhd grade IV. (B) Treatment of lateonset agvhd grade III involving the skin and gut. (C) Treatment of overlap syndrome including GI involvement. enteral budesonide, MMF, and CsA but who continued to suffer from diarrhea. GI GVHD had been confirmed histologically at the time of diagnosis. Four centers would repeat endoscopy. Five centers would start second-line treatment between 3 and 4 days after initiation of steroids, 18 between 5 and 8 days, 7 between 9 and 14 days, and only 1 center would wait 14 days before initiation of second-line treatment. The second question focused on the sequence of treatment options applied in a patient with steroid-refractory agvhd who had developed histologically proven GI involvement stage 3 on day 25 while being on GVHD prophylaxis with CsA and methotrexate and who did not respond to budesonide and prednisolone at 2.5 mg/kg/day. Responses to therapeutic strategies are shown in Figure 4.Of note, 8 centers mentioned 2 second-line options. In addition, the centers were asked about treatment options considered for second-line therapy of agvhd (Table 2) and after failure of second-line treatment (Table 3) regardless of a patient s clinical features. One center mentioned application of steroids into the arteria mesenterica. DISCUSSION Although GVHD is the leading cause of morbidity and mortality after allo-hsct, standards for diagnosis and treatment remain to be established. This is reflected by the

5 D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e Table 1 Applied Treatment Options for First-Line Therapy of agvhd Treatment Option Never Used Considered in General Rarely Used Frequently Used Always Used Not Mentioned but Not Applied yet Budesonid/Beclomethason MMF Steroid-pulse ECP Etanercept Sirolimus Everolimus Alemtuzumab Rituximab Values are percents. diversity of diagnostic and therapeutic approaches documented within our survey. Interestingly, attempted histological confirmation of GI manifestations of agvhd is part of the clinical routine in most responding allo-hsct centers, confirming previous reports [6,7]; however, the optimal approach for clinical routine remains to be determined. One recent retrospective analysis demonstrated that either complete colonoscopy including intubation of the terminal ileum as performed by 62% of responding centers or sigmoidoscopy combined with upper endoscopy as performed by 3 additional centers resulted in the highest diagnostic yield [8]. Regardless of histological results, most centers would assume that GVHD had been present if symptoms of GI GVHD responded to empirical steroid treatment. Of note, histological grade of GVHD in GI biopsies correlated with prognosis in a prospective trial evaluating biomarkers of GVHD [9]. Although the benefit of endoscopic evaluation of suspected GI GVHD is broadly accepted among transplantation facilities, biopsies of a skin rash after engraftment are less established, with only 35% of the centers performing a skin biopsy before initiation of treatment and 50% performing it after failure of first-line therapy. This result is in contrast to another survey [7] and may be explained by the fact that the case scenario described was suggestive of a drug rash. A retrospective analysis on the impact of skin biopsies in chronic GVHD indicated a significant (7%) rate for misdiagnosis of GVHD, resulting in inadequate treatment [4]. Although clinical symptoms and laboratory findings of hepatic GVHD are of unspecific nature, only 32% of the centers would perform a liver biopsy at onset and another 41% after failure of first-line treatment of suspected liver involvement. Bleeding risks associated with the procedure as well as the low specificity of histological results of liver biopsies performed within the first month after allo-hsct can have an impact on these clinical decisions [10]. However, the sensitivity of liver biopsies at later time points Figure 4. Applied second-line treatment in steroid-refractory GI agvhd. ranged between 78% and 100%, although as potential bias, the retrospective nature of the analysis has to be kept in mind [2,11]. Percutaneous biopsies reveal a better quality of the biopsy material, whereas transvenous biopsies have the advantage of a lower bleeding risk in thrombocytopenic patients [12]. Results of the survey concerning treatment of agvhd reflect the lack of established therapeutic standards, especially for salvage therapy of steroid-refractory disease but also demonstrate the impact of published evidence on clinical routine because first-line treatment is applied in a more homogeneous fashion in most allo-hsct centers due to available controlled data compared with second-line treatment, which is mainly based on retrospective analyses and phase II trials [5]. The central role of steroids in first-line treatment is confirmed by the results of our survey. Their use is based on two trials published in the early 1980s [13,14], and steroids have remained the mainstay since then [15-17]. Isolated cutaneous GVHD stage 1 is treated with either topical or systemic steroids at a dose below 1 mg/kg/day of prednisolone, whereas in stage 3, skin involvement systemic steroids are administered by all centers. Although steroids are the backbone of GVHD treatment, their optimal dose and circadian distribution remain controversial [15-18]. Furthermore, doses of steroids below 2 mg/kg/day may aid in sparing toxicity despite similar efficacy as has been shown retrospectively in 733 patients with mainly grades I or II agvhd [19]. Most responding allo-hsct centers (83%) would apply a steroid dose ranging between 1 and 2.5 mg/kg/day of prednisolone or methylprednisolone in agvhd grade III. Of interest, steroid doses above 2.5 mg/kg/day are applied only by a few centers in exceptional situations based on a randomized trial comparing initial corticosteroid doses of 2 and 10 mg/kg/day for the first 5 days revealing no improvement in response rates by dose escalation of steroids [20]. The same conclusion was drawn from a retrospective analysis performed on behalf of the EBMT [21]. Of interest, UDCA as adjunct treatment of liver GVHD was applied by 58% of responding centers, although controlled data are only available for its prophylactic use [22,23]. Upper GI GVHD was treated by one-half of centers with a steroid dose below 1 mg/kg/day, and a significant proportion applied additional topical steroids based on retrospective reports [24,25]. Severe GI GVHD is treated with steroid doses ranging between 1 and 2.5 mg/kg/day by the vast majority of centers (90%), and one-third used topical steroids in addition. Of interest, due to the increased mortality of severe GI GVHD, 6 allo-hsct centers (19%) would apply an additional immunosuppressive agent based on previous reports [26,27]. All centers would continue CNI based on a randomized trial

6 772 D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e776 Table 2 Frequency of Applied Treatment Options in Second-Line Therapy of Steroid-Refractory agvhd Treatment Option Never Used Considered in General Rarely Used Frequently Used Always Used Not Mentioned but not Applied yet MMF ECP Basiliximab Pentostatin UVB/UVA1/PUVA Etanercept Steroid-pulse Alemtuzumab ATG-Fresenius Everolimus Infliximab Sirolimus Methotrexate ATG Thymoglobulin Rituximab UVB indicates ultraviolet B; UVA, ultraviolet A; PUVA, psoralen and ultraviolet A irradiation. Values are percents. demonstrating better response rates in the CsA arm compared with corticosteroids [28]. Severe rapidly evolving multiorgan agvhd after allo- HSCT with an HLA-mismatched donor remains clinically challenging as indicated by the heterogeneity of the applied first-line therapeutic strategies reported in our survey. Only one-third of centers would apply steroids alone, with doses ranging between 1 and 2.5 mg/kg/day, and 4 additional centers would use higher doses of steroids. Centers applying combined first-line treatment in this situation most frequently used the tumor necrosis factor blocking agent etanercept. However, when asked about applied treatment options for first-line therapy of agvhd in general, MMF and ECP reportedly were more frequently used in clinical routine than etanercept. Frequent administration of MMF can be explained by the fact that the Clinical Trials Network trial NCT demonstrated an improved response rate in patients receiving MMF, although the trial was never designed to prove the superior efficacy of MMF [29]. Most likely due to its favorable risk profile, ECP was applied by one-half of centers as part of first-line treatment, with 16% reporting frequent use, despite lacking evidence in this setting because most patients reported so far were given ECP as second-line therapy [30]. However, by far most frequently mentioned additional agent applied in clinical routine for first-line therapy represents topical steroids for GI GVHD, which are applied by the vast majority of responding allo-hsct centers due to the existing evidence on response and improved survival [25,31-33]. The frequent use of etanercept in combination with high-dose steroids demonstrates the impact of limited published evidence on clinical practice because etanercept has been shown to speed up response in first-line treatment, although the trial did apply a case-matched control only [26]. A less frequently used immunosuppressive agent in firstline therapy is alemtuzumab based on 2 retrospective surveys [34,35] and 1 prospective study [36] including 18 steroid-refractory patients reporting a median complete response (CR) rate of 33%. Of note, in the only prospective trial, no long-term survival was achieved [36]. Mammalian Target of Rapamycin inhibitors are rarely applied in first-line treatment, supported by a small retrospective analysis evaluating sirolimus and tacrolimus without steroids [37]. Treatment of late-onset agvhd after withdrawal of immunosuppressive prophylaxis consists of steroids alone as stated by one-fourth of centers and steroids in combination with a CNI as reported by one-half of the centers. Neither retrospective nor prospective data supporting a specific approach in late-onset agvhd are available, because before the National Institutes of Health consensus this patient group Table 3 Frequency of Applied Treatment Options after Second-Line Therapy of Steroid-Refractory agvhd Treatment Option Never Used Considered in General Rarely Used Frequently Used Always Used Not Mentioned but not Applied yet ECP MMF Pentostatin UVB/UVA1/PUVA Steroid-pulse Etanercept ATG-Fresenius Infliximab Basiliximab Alemtuzumab Rituximab Sirolimus Everolimus ATG Thymoglobulin Mesenchymal stromal cells Methotrexate UVB indicates ultraviolet B; UVA, ultraviolet A; PUVA, psoralen and ultraviolet A irradiation. Values are percents.

7 D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e would have been classified as chronic GVHD [6]. Retrospective analyses using the National Institutes of Health definitions revealed significantly increased transplantationrelated mortality, whereas Kuzmina and colleagues in a prospective study observed no significant increase in nonrelapse mortality in patients with late-onset agvhd [38-40]. With regard to overlap syndrome, reported treatment is comparable with treatment of late-onset agvhd, implicating the acute component of overlap syndrome drives the choice of immunosuppressive agent. Overall, the current standard first-line treatment of agvhd consists of steroids with a dose between 1 and 2.5 mg/kg, and any additional treatment besides topical steroids for intestinal manifestations remains experimental due to the lack of prospective trials [5]. It has to be acknowledged that the results of standard first-line treatment in grades III and IV agvhd are disappointing, and prospective trials evaluating new treatment options in this situation are warranted. In contrast to first-line treatment, second-line therapy of steroid-refractory agvhd is rather heterogeneous in terms of choice and sequence of agents administered. This reflects the lack of controlled data, various definitions applied for steroid refractoriness, and the deficiencies regarding study design and conduct as well as patient selection and response evaluation [5]. Moreover, response rates of different treatment options appear relatively homogeneous with one-third of patients showing CR, indicating the biology of the disease may influence the outcome more than the provided treatment. With respect to the time interval for initiation of second-line treatment, 75% of responding centers would intensify immunosuppression if agvhd manifestations failed to improve with first-line treatment applied for 1 week, which is in accordance with the American Society of Blood and Marrow Transplantation guidelines [5]. The most frequently reported agent in our survey used for second-line treatment was MMF, which can be explained by the availability of 2 small phase II trials and retrospective analyses and by its relatively good tolerability [41-48]. Of note, CR rates were relatively low (15% to 31%) in 2 prospective trials, and overall survival ranged between 16% and 33% [46,47]. Furthermore, an increased risk for infectious complications and GI toxicity has been reported [41-44,46,48]. The second most frequently applied second-line treatment option reported in our survey is ECP, which was used by 70% of centers in clinical practice. Of note, recently published data from the American Society of Blood and Marrow Transplantation on high-quality prospective and retrospective studies revealed ECP as the second most frequently reported treatment besides horse ATG and emphasized the favorable safety profile of ECP with low risks for viral reactivation, other toxicities, and interactions with other drugs [5]. Currently, approximately 300 patients with steroidrefractory agvhd have reportedly been treated with ECP, with numbers increasing during the last years [30,49-61]. Overall responses (complete and partial resolution) of cutaneous, hepatic, and GI manifestations were observed in 75%, 47%, and 58% of patients, respectively. Median overall survival of patients was 60% (range, 37.5% to 85%). Another frequently used second-line agent applied according to our survey is etanercept, which has been evaluated as a single agent only in a retrospective analysis showing a CR in 4 of 13 patients [62]. Susceptibility to infection after etanercept may not be as high as seen with infliximab [62-64], which is a chimeric human antietumor necrosis factor-a-igg1k antibody [65,66]. In several retrospective reports on the use of infliximab in steroid-refractory agvhd [67-73], higher response rates were observed in intestinal GVHD (70% to 85%) and pediatric patients (82%) [73]. Awareness should be directed toward the potential consequences of partial immune paralysis from tumor necrosis factor neutralization, as higher incidence of bacterial (77% to 81%), viral (32% to 67%), and fungal (19% to 48%) infections have been observed in patients treated with infliximab [67,70,73-75]. In our survey, second-line use of pentostatin was reported by 46% of centers based on a prospective phase I trial [76] and two retrospective analyses [77,78]. Bolanos-Meade and colleagues reported a CR in 14 of 22 eligible patients (63%), although overall survival at 1 year was only 26% [76]. In retrospective analyses on patients with overall grades III/IV agvhd of the GI tract, CR rates were 38% (9 of 24) and 70% (17 of 23) and overall survival rates at 2 years 17% and 43%, respectively [77,78]. One-third of the responding centers applied basiliximab in treatment of steroid-refractory agvhd based on 2 prospective phase II trials with 40 patients achieving CR rates ranging from 17% to 53% and 2 retrospective case series including 87 patients with CR rates of 50% to 70% mainly in cutaneous GVHD associated with increased infectious mortality [79-82]. The latter is of concern because daclizumab, which also targets CD25, has been associated with increased infectious mortality in first-line treatment of agvhd and has been withdrawn from the market [83]. Alemtuzumab was applied in second-line treatment by 45% of responding centers based on small cohorts applying a relatively high cumulative dose leading to significant infectious mortality, with a CR rate of 33% and overall survival of 56% within the only prospective phase II trial involving 18 patients [36,84-86]. Schnitzler and colleagues observed a CR rate in 20 patients of 35% using lower doses (weekly dose of 10 mg intravenously) in severe GI GVHD along with an improved overall survival of 50% [34]. This was confirmed by another retrospective analysis [35]. Mammalian Target of Rapamycin inhibitors were used in one-third of responding centers, with sirolimus more frequently applied than everolimus, in which the use in the setting of agvhd has not been published so far. In a phase II trial (n ¼ 21) and 2 retrospective analyses with 22 and 34 patients, CR rates of 24% to 72% and overall survival at 1 year of w40% have been reported but were associated with a considerable rate of transplantation-associated microangiopathy in combination with a CNI [87-89]. In a retrospective analysis from a small cohort of pediatric patients treated with sirolimus and tacrolimus, the incidence of transplantation-associated microangiopathy (exclusively associated with high blood levels of sirolimus) was nearly 50% [90]. Although, historically, in addition to CNI, ATG represented the first-treatment option in steroid-refractory GVHD, only one-third of centers reported use of rabbit ATG in secondline treatment in our survey based on approximately 600 reported cases in mostly retrospective analyses, with only 86 patients included in prospective trials and CR rates ranging from 8% to 36% [17,91-100]. ATG use reportedly was associated with high mortality rates in a number of trials published in the 1990s at a time of limited availability of anti-infectious agents and diagnostic procedures that have considerably improved throughout the last 15 years [91,101,102]. Of note,

8 774 D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e776 in a randomized trial comparing ATG with ABX-CBL (hybridoma-generated purified murine IgM monoclonal antibody, which recognizes the CD147 antigen), a better overall survival for ATG (45% versus 35%) was observed [103]. Because ATG predominantly depletes T cells, Epstein-Barr viruseassociated posttransplantation lymphoproliferative disorders with an incidence of 1% to 25% are of concern [96,98,100]. A treatment offered infrequently by more than one-half of responding centers for therapy of cutaneous agvhd was topical phototherapy. This therapy consisted of psoralen and ultraviolet A irradiation [ ], long wavelength ultraviolet A ( nm ultraviolet A-1) alone [107,108], or narrow band ultraviolet A, achieving CR rates reportedly ranging between 37% and 70% [109,110]. Overall, the results of our survey show that published evidencedalthough often scarcedsignificantly influences clinical management of agvhd, and the most important reason for heterogeneity of second-line treatment results from the lack of controlled data [5]. In addition, the survey underlines that in the absence of prospective trials comparing treatment options in second-line treatment, allo- HSCT centers chose strategies based on local experience and in accordance with the American Society of Blood and Marrow Transplantation guideline [5]. Therefore, welldesigned clinical trials systematically evaluating available treatment options are urgently needed as well as identification of biomarkers associated with prognosis and response because agvhd appears to be rather heterogeneous, and a more risk-specific approach may be more likely to be successful than uniform treatment strategies [111]. ACKNOWLEDGMENTS We thank all participating centers of conferences and surveys that included participants from the allo-hsct centers in Basel, Berlin, Dresden, Duesseldorf, Erlangen, Essen, Frankfurt, Freiburg, Goettingen, Greifswald, Halle, Hamburg, Hannover, Jena, Linz, Mainz, Mannheim, Muenster, Munich (Großhadern and Schwabing), Nuernberg, Oldenburg, Regensburg, Rostock, Tuebingen, Ulm, Vienna, Wiesbaden, Wuerzburg, and Zuerich. We are grateful to the German Registry of Stem Cell Transplantation (DRST), the Austrian Registry of Stem Cell Transplantation (ASCT), and the Swiss National Stem Cell Transplant Registry for providing the information on annual allo-hsct numbers in these countries. Financial disclosure: The conference was supported by the Jose Carreras Foundation project Competence center GvHD Regensburg. H. G. was supported by European Commission Grant STEMDIAGNOSTICS. 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