Supplementary Online Material
Patients and Methods
Patients and Transplantation
Patients were identified from a database of 718 consecutive patients receiving allogeneic HCT in
Calgary from January 1999 to March 2010. The rationale for starting in 1999 is that
antithymocyte globulin (Thymoglobulin- Genzyme, Cambridge MA) was routinely used for
GVHD prophylaxis at our institution since 1999 (Russell, J.A. et al.: Biol Blood Marrow
Transplant 2007, 13:299-306). Also in approximately 1999, fludarabine and busulfan became
the preferred conditioning regimen and filgrastim-mobilized blood stem cells became the
preferred graft source. Moreover, our management of infectious complications of GVHD
improved at approximately that time or thereafter. Specifically, preemptive therapy of
cytomegaloviral disease started at our institution in 1999 and mold-active antifungals became
available soon thereafter (caspofungin was Health Canada-approved in 2001 and voriconazole in
2004). Moreover new immunosuppressive therapies became available: mycophenolate mofetil
was approved in 1998 and daclizumab in 2000. The reason for ending follow up in March 2010
was that we wished to determine the long-term survival, with minimum follow up of 2 years.
Conditioning was typically with fludarabine (250 mg/m2), busulfan (approximately 12.8 mg/kg
IV, pharmacokinetically adjusted) and ATG (4.5 mg/kg). Additional GVHD prophylaxis was
with methotrexate (day 1, 3, 6, and 11) and cyclosporine from day -1 until 3 to 6 months posttransplant (longer in case of chronic GVHD) (Russel, J.A. et al.: Biol Blood Marrow Transplant
2007, 13:299-306). Conditioning of some patients included total body irradiation (4 cGy)
(Russel, J.A. et al.: Biol Blood Marrow Transplant 2007, 13:814-821).
Acute GVHD Treatment and Response to Treatment
Acute GVHD (aGVHD) was graded by the Keystone Consensus criteria (Przepiorka, D. et al.:
Bone Marrow Transplant 1995, 15:825-828). Patients were classified as steroid-refractory if
they had no improvement or worsening of their overall aGVHD grade despite 2 mg/kg of
prednisone or methylprednisolone (for a length of time determined by the attending physician),
or if they had a recurrent flare of aGVHD while tapering or off corticosteroids that did not
respond to incrementing or re-starting the corticosteroids. Patients were classified as steroidresponsive if they had complete and sustained resolution of aGVHD with only corticosteroid
therapy. Steroid-refractory patients were typically treated with ATG (Thymoglobulin) at a dose
of 2.5 mg/kg every other day for four doses, while the cyclosporine was held for 14 days and
then reinstituted, and corticosteroids were continued. Response to ATG was evaluated at 4
weeks and 12 weeks and classified as complete (CR, achieving aGVHD grade 0), partial (PR,
improvement in overall grade without deterioration in a single organ) or no response (NR, no
change, worsening of overall grade, or improvement in overall grade but deterioration in a single
organ).
Literature Review of Survival after Grade 3-4 aGVHD
Medline search was performed using subject heading “graft vs. host disease” and subheadings
“mortality” and “therapy”. Studies cited in 2 recent reviews were also reviewed (Pidala J et al:
Biol Blood Marrow Transplant 2010, 16:1504; Martin PJ et al: Biol Blood Marrow Transplant
2012, 18:982). Prospective as well as retrospective studies, regardless of therapy, were included
if the outcome of patients with grade 3-4 (Consensus grading) or CIBMTR grade C-D aGVHD
could be ascertained. These could be studies that either enrolled patients with any grade of
aGVHD in which the outcomes of the patients with grade 3-4 aGVHD was presented or studies
that included only patients with grade 3-4 aGVHD. Studies were included if they presented 10
or more patients and if surviving patients were either followed for at least 1 year (after transplant
or after the initiation of therapy for aGVHD, whichever was given) or if Kaplan-Meyer survival
estimate at >2 years was presented. Studies were excluded if they did not provide the length of
follow-up time for surviving patients, unless Kaplan-Meyer survival estimate at >2 years was
presented. If studies presented data for individual patients with variable follow-up times, those
who were alive at the conclusion of the study but followed for less than one year were excluded
while the remaining patients were included in the review.
Supplementary Table 1. Patient Characteristics.
Median Age,
Years (Range)
Steroid Responsive
(N=12)
49
(27-61)
Steroid Refractory
(N=38)
46
(19-65)
P-Value*
1.0
Sex
(%)
Female
Male
4 (33)
8 (67)
14 (37)
24 (63)
Diagnosis
(%)
Acute Leukemia
MDS
CML
CLL
Other
7 (58)
3 (25)
0
0
2 (17)
19 (50)
3 (8)
6 (16)
4 (10)
6 (16)
HLA-Matched Sibling
MUD
HLA-Mismatched
Unknown
3 (25)
6 (50)
3 (25)
0 (0)
15 (39)
9 (24)
13 (34)
1 (3)
Stem Cell Source
(%)
Peripheral Blood
Bone Marrow
11 (92)
1 (8)
33 (87)
5 (13)
Conditioning
Regimen
(%)
FLU+BU+ATG
FLU+BU+ATG+
TBI
Other**
4 (33)
8 (67)
21 (55)
15 (39)
0 (0)
2 (5)
22
(11-90)
33
(12-121)
3
11 (92)
21 (55)
4
1 (8)
17 (45)
Donor (%)
Median Posttransplant day of
aGVHD onset
(range)
aGVHD Overall
Grade
(%)
0.45
0.27
0.41
1.0
0.23
0.11
0.03
0 (0)
3 (8)
1.0
Skin Only
0 (0)
1 (2)
1.0
Liver Only
3 (25)
17 (45)
0.32
GI Only
8 (67)
11 (29)
Skin + GI
0.03
0 (0)
1 (2)
1.0
Skin + Liver
1 (8)
4 (11)
1.0
Liver + GI
0 (0)
1 (2)
1.0
Skin + GI + Liver
*All P-values calculated via two-tailed Fisher’s exact test except for median post-transplant day of GVHD onset and
median age - both calculated via Mann-Whitney U-test.
** Etoposide + total body irradiation + ATG in one patient, and Cyclophosphamide + total body irradiation + ATG
in one patient.
Abbreviations: MDS=myelodysplastic syndrome, CML=chronic myeloid leukemia, CLL=chronic lymphocytic
leukemia, MUD=HLA-A,B,C,DRB1,DQB1-matched unrelated donor.
aGVHD Organs
Involved (%)
Supplementary Table 2. Therapies given prior to or in conjunction with ATG in steroidrefractory aGVHD patients.
Therapies Prior to ATG
No.
Corticosteroids Only
22
+ MMF
9
+ Tacrolimus
1
+ Daclizumab
1
+ MMF and Daclizumab
1
Therapies in Conjunction with ATG
Corticosteroids Only
28
+ Mesenchymal stromal cells (Prochymal,
Osiris, Columbia, MD) or Placebo*
6
* Double-blind, randomized trial. Randomization status on individual patients is not available,
however, it is irrelevant as the trial was negative (Martin, P.J. et al.: Biol Blood Marrow
Transplant 2010, 16(2):S169-S170; Baron F & Storb R: Biol Blood Marrow Transplant 2012,
18(6):822-840).
Supplementary Table 3. Overall response and response by organ to ATG at four and twelve
weeks in patients with steroid-refractory graft-versus-host disease.
Organ
4 Weeks
12 Weeks
CR
PR
NR
Death
CR
PR
NR
Death
11 (73)
1 (7)
1 (7)
2 (13)
7 (47)
0 (0)
0 (0)
8 (53)
Skin
(%)
6 (21)
9 (31)
7 (24)
7 (24)
8 (28)
1 (3)
4 (14)
16 (55)
GI (%)
1 (17)
1 (17)
3 (50)
1 (17)
1 (17)
1 (17)
0 (0)
4 (66)
Liver
(%)
5 (15)
9 (26)
13 (38)
7 (21)
5 (15)
2 (6)
8 (23)
19 (56)
Overall
(%)
Supplementary Table 4. Primary cause of death in steroid-responsive and in ATG-treated
steroid-refractory aGVHD patients.
Steroid-Responsive
ATG-Treated SteroidRefractory
aGVHD
0
3
aGVHD + Infection
0
6
Infection
3
11*
Chronic GVHD
4
3
Relapse
3
3
Other
0
3**
Unknown
0
1
* Including 2 cases of post-transplant lymphoproliferative disorder.
** Cerebral bleeding in one case, myocardial infarction in one case, and thrombotic
microangiopathy in one case
Supplementary Figure 1. Survival plot for patients with steroid-refractory and steroidresponsive grade 3-4 acute graft-versus-host disease.
100
80
60
Steroid-Responsive
Steroid-Refractory
40
20
0
0
1000 2000 3000 4000 5000
Days from aGVHD onset