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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
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 2011. 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 ¼2, 0.5 to 1.0 mg/kg/day n ¼10, above 1.0 to 2.5
mg/kg/day n ¼19) without or with topical agents (steroids n ¼10; calcineurin inhibitors n ¼3). 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 ¼14) and
extracorporeal photopheresis (n ¼10). Our survey also demonstrates that clinicians chose salvage therapies
for steroid-refractory aGVHD based on their centers’own clinical experience.
Ó2013 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. Histor-
ically, aGVHD has been diagnosed clinically, and the extent of
clinical symptoms defines its organ and overall severity [1].
However, none of these is specific for aGVHD, and relevant
differential diagnoses such as allergies, infections, or toxic-
ities 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 clin-
ical 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
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,
93053 Regensburg, Germany.
E-mail address: daniel.wolff@ukr.de (D. Wolff).
1083-8791/$ esee front matter Ó2013 American Society for Blood and Marrow Transplantation.
http://dx.doi.org/10.1016/j.bbmt.2013.01.018
Biol Blood Marrow Transplant 19 (2013) 767e776
American Society for Blood
ASBMT
and Marrow Transplantation
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 retrospec-
tive analyses. Consequently, no accepted treatment algo-
rithm 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-HSCTcenters 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, digi-
talized, 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 histopatho-
logical 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 2011. 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. Twenty-
one of 34 centers (62%) would perform a colonoscopy
before initiation of treatment, including 8 centers perform-
ing 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 endos-
copy 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 treat-
ment. 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 diar-
rhea 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 Fluorodeoxyglucose-
positron 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 abnormali-
ties 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 there-
after. 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 iso-
lated 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 fol-
lowed by steroids alone. Four centers would just perform
a liver biopsy and wait for the result before starting corti-
costeroid 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%. Twenty-
six 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
D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e776768
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 ¼1), 0.5 to 1 mg/kg/day (n ¼3), or 1 to 2.5 mg/kg/day
(n ¼4), respectively. Topical calcineurin inhibitors (CNIs)
with (n ¼2) or without topical steroids (n ¼1) 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 centersstated they would performa skin and 2 a liver
biopsy. Responses to therapeutic strategies are shown in
Figure 2A. Eighteencenters (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
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 addi-
tional center would perform a skin biopsy, sigmoidoscopy,
and gastroscopy. Eight centers would only perform colono-
scopy. Two centers would combine colonoscopy with
gastroscopy. Seven centers would only perform sigmoidos-
copy. 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 pro-
gressing 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 colono-
scopy, 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
Figure 1. (A) Treatment of isolated cutaneous aGVHD grade I. (B) Treatment of
isolated cutaneous aGVHD stage 3.
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.
D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e776 769
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 ¼1),
pentostatin (n ¼1), etanercept (n ¼2), or extracorporeal
photopheresis (ECP) (n ¼1). Four centers would only
administer high-dose steroids above 2.5 mg/kg/day pred-
nisolone. Others would use combinations of high-dose
steroids with alemtuzumab at 10 mg (n ¼2), mesenchymal
stromal cells (n ¼1), etanercept (n ¼6), ECP (n ¼1), or
basiliximab (n ¼1). 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 ¼4) or sigmoidoscopy (n ¼5), 4 centers
would perform only a sigmoidoscopy, 6 only a colonoscopy,
whereas 9 centers would not perform histological confir-
mation 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 methylpredniso-
lone, 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 colono-
scopy. 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 immu-
nosuppressive agents for first-line treatment of aGVHD.
Results are listed in Table 1. One center each mentioned
occasional use of antithymocyte globulin (ATG), mesen-
chymal stromal cells, and tacrolimus.
Second-Line Treatment
First, we focused on the time point of initiation of second-
line 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 manifesta-
tions responded to methylprednisolone at 2 mg/kg/day,
enteral budesonide, MMF, and CsA but who continued to
suffer from diarrhea. GI GVHD had been confirmed histo-
logically 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 treat-
ment 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 treat-
ment remain to be established. This is reflected by the
Figure 3. (A) Treatment of multiorgan aGVHD grade IV. (B) Treatment of late-
onset aGVHD grade III involving the skin and gut. (C) Treatment of overlap
syndrome including GI involvement.
D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e776770
diversity of diagnostic and therapeutic approaches docu-
mented 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 prospec-
tive trial evaluating biomarkers of GVHD [9].
Although the benefit of endoscopic evaluation of sus-
pected 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 misdiag-
nosis 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
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, espe-
cially for salvage therapy of steroid-refractory disease but
also demonstrate the impact of published evidence on clin-
ical 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 treat-
ment, 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 predniso-
lone, 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 circa-
dian 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 retro-
spectively 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 immu-
nosuppressive agent based on previous reports [26,27]. All
centers would continue CNI based on a randomized trial
Table 1
Applied Treatment Options for First-Line Therapy of aGVHD
Treatment Option Never Used Considered in General
but Not Applied yet
Rarely Used Frequently Used Always Used Not Mentioned
Budesonid/Beclomethason 0 0 13 75 6 6
MMF 26 0 46 22 0 6
Steroid-pulse 20 6 42 22 0 10
ECP 35 10 29 16 0 10
Etanercept 42 3 29 13 0 13
Sirolimus 61 6 20 3 0 10
Everolimus 62 6 16 3 0 13
Alemtuzumab 58 6 26 0 0 10
Rituximab 68 3 16 0 0 13
Values are percents.
Figure 4. Applied second-line treatment in steroid-refractory GI aGVHD.
D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e776 771
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
NCT00224874 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 eta-
nercept in combination with high-dose steroids demon-
strates 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 first-
line 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 eval-
uating 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
but not Applied yet
Rarely Used Frequently Used Always Used Not Mentioned
ECP 3 10 36 39 6 6
MMF 10 0 48 26 6 10
Pentostatin 29 10 42 13 3 3
UVB/UVA1/PUVA 32 10 36 6 3 13
Steroid-pulse 32.5 3 32.5 26 0 6
Etanercept 25.5 3 38.5 23 0 10
ATG-Fresenius 36 6 32 13 0 13
Infliximab 48.5 6 19.5 13 0 13
Basiliximab 48 10 16 13 0 13
Alemtuzumab 29.5 6 48.5 10 0 6
Rituximab 42 13 29 10 0 6
Sirolimus 44 10 26 10 0 10
Everolimus 42 13 26 6 0 13
ATG Thymoglobulin 58.5 3 19.5 6 0 13
Mesenchymal stromal cells 48 16.5 16.5 6 0 13
Methotrexate 61 10 23 0 0 6
UVB indicates ultraviolet B; UVA, ultraviolet A; PUVA, psoralen and ultraviolet A irradiation.
Values are percents.
Table 2
Frequency of Applied Treatment Options in Second-Line Therapy of Steroid-Refractory aGVHD
Treatment Option Never Used Considered in General
but not Applied yet
Rarely Used Frequently Used Always Used Not Mentioned
MMF 6 3 33 46 6 6
ECP 3 17 32 32 6 10
Basiliximab 46 13 13 19 3 6
Pentostatin 35 13 33 10 3 6
UVB/UVA1/PUVA 32 3 39 23 0 3
Etanercept 26 6 39 19 0 10
Steroid-pulse 35 0 42 13 0 10
Alemtuzumab 42 10 32 13 0 3
ATG-Fresenius 39 6 23 13 0 19
Everolimus 52 10 19 13 0 6
Infliximab 35 13 32 10 0 10
Sirolimus 42 13 29 10 0 6
Methotrexate 58 13 13 6 0 10
ATG Thymoglobulin 42.5 6 29.5 3 0 19
Rituximab 45 13 26 3 0 13
UVB indicates ultraviolet B; UVA, ultraviolet A; PUVA, psoralen and ultraviolet A irradiation.
Values are percents.
D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e776772
would have been classified as chronic GVHD [6]. Retro-
spective analyses using the National Institutes of Health
definitions revealed significantly increased transplantation-
related 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 treat-
ment 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 eval-
uation [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 treat-
ment. 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 treat-
ment option reported in our survey is ECP, which was used
by 70% of centers in clinical practice. Of note, recently pub-
lished data from the American Society of Blood and Marrow
Transplantation on high-quality prospective and retrospec-
tive studies revealed ECP as the second most frequently re-
ported treatment besides horse ATG and emphasized the
favorable safety profile of ECP with low risks for viral reac-
tivation, other toxicities, and interactions with other drugs
[5]. Currently, approximately 300 patients with steroid-
refractory aGVHD have reportedly been treated with ECP,
with numbers increasing during the last years [30,49-61].
Overall responses (complete and partial resolution) of cuta-
neous, 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 eval-
uated 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
-IgG1
k
antibody [65,66]. In several retro-
spective 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 bacte-
rial (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 pentostatinwas 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 daclizu-
mab, 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 ¼21) 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 micro-
angiopathy in combination with a CNI [87-89]. In a retro-
spective 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 second-
line 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 associ-
ated 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,
D. Wolff et al. / Biol Blood Marrow Transplant 19 (2013) 767e776 773
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
[96,98,10 0].
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 [104-106], long wavelength ultravi-
olet A (340-400 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, well-
designed clinical trials systematically evaluating available
treatment options are urgently needed as well as identifi-
cation 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, Old-
enburg, 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 037703 STEMDIAGNOSTICS.
Conflict of interest statement: D.W. received a research
grant from Novartis and Therakos, a lecture fee from Frese-
nius, and serves as consultant for Falk-Pharma. H.B. received
a lecture fee and travel support from Fresenius. S.K. received
a research grant and honoraria from Hospira. H. G. received
lecture fees from Therakos Inc. A.L., J.H., G.H., R.-G.M., M.S.,
S.M., M.E., and F.A. declare no conflict of interest.
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