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Reg3αconcentrations at day of allogeneic stem cell transplantation
predict outcome and correlate with early antibiotic use
Daniela Weber,
1
Markus Weber,
2
Elisabeth Meedt,
1
Sakhila Ghimire,
1
Daniel Wolff,
1
Matthias Edinger,
1,3
Hendrik Poeck,
1
Andreas Hiergeist,
4
Andr ´
e Gessner,
4
Francis Ayuk,
5
Wolf Roesler,
6
Matthias Wölfl,
7
Sabrina Kraus,
8
Robert Zeiser,
9
Hannah Bertrand,
9
Peter Bader,
10
Evelyn Ullrich,
10-12
Matthias Eder,
13
Sigrun Gleich,
1
Rachel Young,
14
Wolfgang Herr,
1
John E. Levine,
14
James L. M. Ferrara,
14
and Ernst Holler
1
1
Department of Hematology and Oncology, Internal Medicine III, Regensburg University Hospital, Regensburg, Germany;
2
Department of Trauma and Orthopedic Surgery,
Barmherzige Brüder Hospital, Regensburg, Germany;
3
Department of Hematology/Oncology, RCI Regensburg Centre for Interventional Immunology, University and University
Medical Centre of Regensburg, Regensburg, Germany;
4
Institute of Clinical Microbiology and Hygiene, Regensburg University Hospital, Regensburg, Germany;
5
Department
of Stem Cell Transplantation with Research Department Cell and Gene Therapy, Hamburg-Eppendorf University Medical Center, Hamburg, Germany;
6
Department of Internal
Medicine 5, Hematology/Oncology, Erlangen University Hospital, Erlangen, Germany;
7
Pediatric Blood and Marrow Transplantation Program, Children’s Hospital, University of
Würzburg, Würzburg, Germany;
8
Department of Internal Medicine II, Würzburg University Hospital, Würzburg, Germany;
9
Hematology, Oncology and Stem Cell
Transplantation, Department of Medicine I, Faculty of Medicine, Freiburg University Medical Center, University of Freiburg, Freiburg, Germany;
10
Department of Johann
Wolfgang Goethe University, Experimental Immunology, Goethe University, Frankfurt am Main, Germany;
11
Frankfurt Cancer Institute, Goethe University, Frankfurt am Main,
Germany;
12
German Cancer Consortium (DKTK) partner site Frankfurt/Mainz and German Cancer Research Center (DKFZ), Heidelberg, Germany;
13
Department of
Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; and
14
Tisch Cancer Institute, Icahn School of Medicine at
Mount Sinai, New York, NY
Key Points
•Reg3αconcentrations
on day of graft infusion
predict outcome after
ASCT.
•Reg3αconcentrations
are influenced by
intestinal dysbiosis
induced by early AB
use.
Intestinal microbiome diversity plays an important role in the pathophysiology of acute
gastrointestinal (GI) graft-versus-host disease (GVHD) and influences the outcome of
patients after allogeneic stem cell transplantation (ASCT). We analyzed clinical data and
blood samples taken preconditioning and on the day of ASCT from 587 patients from 7
German centers of the Mount Sinai Acute GVHD International Consortium, dividing them
into single-center test (n = 371) and multicenter validation (n = 216) cohorts. Regenerating
islet–derived 3α(Reg3α) serum concentration of day 0 correlated with clinical data as well
as urinary 3-indoxylsulfate (3-IS) and Clostridiales group XIVa, indicators of intestinal
microbiome diversity. High Reg3αconcentration at day 0 of ASCT was associated with
higher 1-year transplant-related mortality (TRM) in both cohorts (P< .001). Cox regression
analysis revealed high Reg3αat day 0 as an independent prognostic factor for 1-year TRM.
Multivariable analysis showed an independent correlation of high Reg3αconcentrations at
day 0 with early systemic antibiotic (AB) treatment. Urinary 3-IS (P= .04) and Clostridiales
group XIVa (P= .004) were lower in patients with high vs those with low day 0 Reg3α
concentrations. In contrast, Reg3αconcentrations before conditioning therapy correlated
neither with TRM nor disease or treatment-related parameters. Reg3α, a known biomarker
of acute GI GVHD correlates with intestinal dysbiosis, induced by early AB treatment in the
period of pretransplant conditioning. Serum concentrations of Reg3αmeasured on the day
of graft infusion are predictive of the risk for TRM of ASCT recipients.
Submitted 5 July 2022; accepted 24 October 2022; prepublished online on Blood
Advances First Edition 9 November 2022; final version published online 3 April 2023.
https://doi.org/10.1182/bloodadvances.2022008480.
Data were analyzed by the authors of this article. All authors confirm access to clinical
trial data upon request.
Data are available on request from the corresponding author, Daniela Weber (daniela.
weber@klinik.uni-regensburg.de).
The full-text version of this article contains a data supplement.
© 2023 by The American Society of Hematology. Licensed under Creative Commons
Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0),
permitting only noncommercial, nonderivative use with attribution. All other rights
reserved.
REGULAR ARTICLE
1326 11 APRIL 2023 •VOLUME 7, NUMBER 7
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Introduction
Allogeneic stem cell transplantation (ASCT) is a curative treatment
option for a variety of malignant and nonmalignant hematologic
conditions, but a significant risk for life-threatening complications
remains. Acute graft-versus-host disease (aGVHD) is the major
cause of morbidity and mortality. Gastrointestinal (GI) involvement
of aGVHD is a major therapeutic challenge, particularly in cases
refractory to high-dose corticosteroids, the standard therapy of
aGVHD. The mortality of patients with severe steroid-refractory
aGVHD of the GI tract is still high (60%-80%), either due to
GVHD itself or owing to infectious complications as a result of
prolonged immunosuppression.
1-3
Growing evidence shows a
crucial role of intestinal microbiota in the pathophysiology of
aGVHD.
4,5
Although ASCT is often associated with a loss of
intestinal microbiome diversity, this loss is even more pronounced
in patients that develop intestinal aGVHD. Changes in microbiota
composition mainly consist of a reduction in protective commensal
bacteria such as Clostridiales and an overgrowth of potentially
pathogenic bacteria such as enterococci.
5-7
Intestinal dysbiosis
including a reduction in protective metabolites such as short chain
fatty acids (SCFAs), indoles, etc, is associated with inflammatory
conditions in the GI tract.
8-10
Several single-center studies
4,5,9,10
and 1 large multicenter study have confirmed a relationship
between microbiota disruption and poor patient outcome after
ASCT independent of geographic and institutional variations.
11
Early and prolonged antibiotic (AB) exposure, conditioning
regimen toxicity, as well as an impaired oral intake have been
identified as major risk factors of microbiome disturbance.
8
It is
therefore of great interest to identify biomarkers of aGVHD that
reflect damage to the GI tract as early as possible and before onset
of intestinal symptoms. Regenerating islet–derived 3α(Reg3α)is
an antimicrobial peptide primarily produced by Paneth cells (PCs),
which protects the GI epithelium from gram-positive bacteria.
aGVHD damages the intestinal mucosa resulting in an increase in
Reg3αconcentration in the bloodstream.
12
Here, we test the hypothesis that intestinal damage indicated by
Reg3αserum concentrations
13
can be detected as early as day
0 (day of transplantation) before onset of alloreactivity in a multi-
center study including 587 patients undergoing ASCT. Further-
more, we investigated Reg3α, the serum biomarker of acute GI
GVHD and indicator of PC damage, measured on the day of
transplantation as predictor of transplant-related mortality (TRM).
Methods
Study design
This analysis used the Mount Sinai Acute GVHD International
Consortium (MAGIC) database and biorepository for a simulta-
neous prospective assessment of clinical data as well as biomarkers
to develop early, biomarker-based therapeutic strategies. In 2016, 7
German SCT centers constituted MAGIC Germany and followed a
rigorous probe study design funded by the German Jose-Carreras
Leukemia Foundation (DJCLS 01 GVHD/2016) in order to
improve the biomarker-based risk scores
14
through a combination
with microbiome parameters. This database of MAGIC Germany
comprises data of 587 patients undergoing ASCT in Regensburg
(n = 371) and 6 further German transplant centers (Hamburg,
n = 119; Erlangen, n = 45; Würzburg; n = 18; Freiburg, n = 8;
Frankfurt, n = 9; and Hannover, n = 17;) (supplemental Table 1).
Inclusion criteria were hematologic diseases requiring ASCT and
receipt of T-cell–repleted grafts. We first analyzed a test cohort of
371 ASCT recipients enrolled between 2008 and 2020 in
Regensburg to define a benchmark for Reg3αregarding 1-year
TRM. We then validated these findings in a multicenter cohort
consisting of 216 patients who underwent transplant between
2012 and 2021 in 6 MAGIC Germany sites (excluding Regens-
burg). All further analyses were performed using the entire cohort.
With approval by the ethics committee of the MAGIC consortium
(number 21-2521-101) and after receipt of written informed consent,
blood samples of all 587 patients were collected before start of
conditioning therapy (within 3 days before conditioning) and on the
day of SCT, which was defined as the time period between days −1
to +1. Urinary and fecal specimens were collected at day +7 range
(days +2 to +10). All specimens were stored at −80 ◦C until analysis.
During the course of ASCT, patients commonly received prophy-
lactic ABs from the start of conditioning until engraftment, but the
type of prophylactic AB differed between the centers. In Regens-
burg, ciprofloxacin 500 mg twice daily and metronidazole 400 mg 3
times daily were administered orally until March 2012 and oral
rifaximin 200 mg twice daily thereafter to control the emergence of
vancomycin-resistant enterococci. Erlangen also used rifaximin 200
mg twice daily for gut decontamination; patients in Frankfurt,
Freiburg, and Würzburg received no AB prophylaxis at all.
Hamburg and Hannover used prophylactic ciprofloxacin 500 mg
twice daily and levofloxacin 400 mg daily in addition to metroni-
dazole 400 mg 3 times daily, respectively (supplemental Table 1).
Treatment of neutropenic fever/infections with additional systemic
broad-spectrum ABs was comparable between the single-center
test cohort and the multicenter validation cohort. All patients
received first- and second-line treatment of neutropenic fever/
infections according to international guidelines
15,16
and the clinical
criteria for initiation of first- and second-line ABs were comparable
between centers. Piperacillin/tazobactam at a thrice daily dose of
4.0/.5 g was used as empiric first-line therapy, whereas mer-
openem 1.0 g thrice daily and vancomycin 1.0 g twice daily served
as second-line therapy. In cases of penicillin intolerance, patients
received alternative first-line treatment with ceftazidime. In the test
and validation cohorts, 93.9% and 95.4% of patients, respectively,
received additional AB treatment beyond prophylactic regimens.
Children received a similar but weight-adapted AB regimen.
To investigate the effect of dysbiosis caused by initiation of sys-
temic broad-spectrum ABs, we classified patients into 2 groups
according to the initiation of additional ABs: the early group began
AB treatment before the day of ASCT and the late group began AB
therapy on or after the day of transplant. In the test cohort, 39.9%
of patients received early AB treatment compared with 66.2% in
the validation cohort (P< .001). However, the incidence of
documented bacteremia or sepsis was even less in the early AB
group. Further patient characteristics are listed in Table 1.In
Regensburg and at all German MAGIC sites patients with unre-
lated donor grafts routinely received antithymocyte globulin (ATG)
as part of GVHD prophylaxis before ASCT in contrast to patients
treated with grafts from related donors. Fever represents a com-
mon side effect of ATG use and thus often favors the use of ABs
before graft infusion.
11 APRIL 2023 •VOLUME 7, NUMBER 7 Reg3αAT DAY 0 PREDICTS TRM AFTER ASCT 1327
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The primary cause of death was assigned according to a published
hierarchy. For cause of death analysis, the previously published
hierarchy of death classification according to the MAGIC con-
sortium was used.
17
Reg3αserum concentrations were analyzed by enzyme-linked
immunosorbent assay, as previously described and are reported
in nanograms per milliliter (ng/mL).
12
Urinary 3-indoxylsulfate (3-IS) and creatinine concentrations were
determined by reverse-phase liquid chromatography electrospray-
ionization tandem mass spectrometry, as previously described.
5
16S ribosomal RNA (rRNA) gene copy numbers of Clostridium
cluster XIVa species were determined in fecal DNA preparations by
real-time quantitative polymerase chain reaction, as previously
described.
10
Bioinformatics and data analysis
Continuous data are presented as mean (± standard deviation).
Group comparisons were performed by Mann-Whitney Utests
owing to nonnormal data distribution. Absolute and relative
frequencies for categorical data are compared between study
groups by χ
2
or Fisher exact tests. All hypotheses were tested with
a two-sided 5% significance level. Receiver operating character-
istic curve plots were generated and the area under the curve was
assessed in both cohorts to evaluate the prediction of 1-year TRM
by Reg3αserum concentrations on the day of graft infusion. The
Youden Index was calculated to define a Reg3αthreshold value.
Factors associated with high Reg3αserum concentrations were
assessed using logistic regression analysis. Kaplan-Meier analysis
was performed to assess TRM, and Cox regression was used for
multivariable assessment of risk factors. Competing-risk analysis
18
for Reg3α-related TRM, infectious TRM, and relapse was per-
formed using software package R version 3.2.2 (The R Foundation
of Statistical Computing, Vienna, Austria). IBM SPSS Statistics 25
(SPSS Inc, Chicago, IL) was used for all other analyses.
Results
Reg3αserum concentrations at day 0 are
associated with TRM
First, we analyzed the ability of Reg3αserum concentrations at day
of transplantation to predict 1-year TRM by creating a receiver
operating characteristic curve in the test cohort. The area under
the curve was 0.64 (95% confidence interval [CI], 0.56-0.72;
P= .001). A threshold of 110 ng/mL was found to have the highest
Youden Index in terms of sensitivity and specificity. Using this
threshold, Kaplan-Meier survival plots showed a significant differ-
ence between patients with high vs those with low Reg3αvalues in
both the test (33.3% vs 11.8%, P< .001) and validation cohorts
(21.3% vs 8.4%, P= .009) (Figure 1).
Cox regression analysis in the combined test and validation cohorts
identified Reg3αat the time of ASCT as an independent prog-
nostic factor for 1-year TRM (hazard ratio, 2.9; 95% CI, 1.8-4.8;
P< .001) as well as patient age (hazard ratio, 4.0; 95% CI, 1.7-9.4;
P= .002). Other key parameters such as sex, type/stage of dis-
ease, donor type, and Karnofsky Index were not predictive
(Table 2). Competing-risk analysis confirmed the association of
high Reg3αconcentrations at day 0 and TRM when relapse was
considered as competing risk (P< .001) (Figure 2). TRM was
higher in the high-Reg3αgroup owing to GI GVHD (11.0% vs
5.3%, P= .005), infectious complications (8.8% vs 2.4%,
P< .001), as well as other reasons (8.1% vs 2.7%, P= .001)
(Table 3; supplemental Figure 1). In contrast, preconditioning
Reg3αconcentrations showed no correlation to 1-year TRM (log
rank = 0.22) (supplemental Figure 2). This was also confirmed in
Cox regression analysis (supplemental Table 2).
Early AB treatment independently correlates with
day-0 Reg3αconcentrations
Intravenous application of broad-spectrum ABs before the day of
ASCT correlated with higher Reg3αconcentrations at day
0 (112.1 ± 132.4 ng/mL) compared with late or non-AB treatment
(63.7 ± 53.3 ng/mL, P< .001), whereas no difference was found
for pretransplant Reg3αconcentrations (42.9 ± 45.1 ng/mL vs
41.6 ± 52.0 ng/mL; nonsignificant) (Figure 3). Similarly, patients
that had received ciprofloxacin or combined ciprofloxacin/metro-
nidazole had higher Reg3αserum concentrations (106.4 ± 140.3
ng/mL) than patients receiving rifaximin or no prophylactic gut
Table 1. Anthropometric characteristics of the test (n = 371) and
validation (n = 216) cohorts
Test cohort,
n = 371, %
Validation
cohort,
n = 216, % P
Total,
n = 587, %
Patient age, y
>50 68.2 62.5 .16 66.1
Patient sex
Female 35.8 36.6 .86 36.1
Type of underlying disease
Acute leukemia 51.8 48.1 .40 50.4
Disease stage
Late 37.5 34.3 .46 36.4
Conditioning .09
RIC 85.3 79.6 83.2
Standard 14.4 19.0 16.1
Other 0.3 1.4 0.7
GVHD prophylaxis <.001
CNI/MTX 64.0 20.4 47.8
CNI/MMF 18.7 73.6 39.1
Other 17.3 6.0 13.1
Karnofsky Index
≥90% 61.8 71.90 .03 64.9
Donor
Family (no ATG) 30.0 25.0 .42 28.2
Gut decontamination <.001
Rifaximin/no ABs 87.1 37.7 68.7
Cipro/cipro + metro 12.9 62.3 32.3
Early systemic ABs
Before day of ASCT 39.9% 66.2% <.001 49.7%
CNI, calcineurin inhibitors; cipro, ciprofloxacin; metro, metronidazole; MMF, mycophenolate
mofetil; MTX, methotrexate; RIC, reduced intensity conditioning.
1328 WEBER et al 11 APRIL 2023 •VOLUME 7, NUMBER 7
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decontamination (79.1 ± 80.6 ng/mL, P= .01). However, the effect
of early systemic broad-spectrum ABs was independent of type of
gut decontamination. Reg3αconcentrations at day of trans-
plantation were also higher in patients aged >50 years (95.3 ±
115.6 ng/mL) than in younger patients (72.8 ± 73.4 ng/mL,
P= .002). Higher Reg3αserum concentrations were measured at
day of transplantation in patients with unrelated donors (99.3 ±
113.6 ng/mL) compared with related donors (59.3 ± 69.3 ng/mL,
P< .001). This seemingly contradictory result points toward the
differential use of ATG treatment, leading to an earlier use of sys-
temic ABs owing to febrile episodes. Of the patients with unrelated
donors, 54% were treated with ABs and had signs of cytokine
release syndrome after ATG, whereas only 39% of patients with
related donors were treated systemically (P= .001). In addition,
Reg3αconcentrations (94.4 ± 92.7 ng/mL) were higher in patients
with a Karnofsky Index of <90% than in patients with a Karnofsky
Index of ≥90% (73.9 ± 76.6 ng/mL, P= .008). Multivariable
analysis confirmed the independent correlation of high Reg3α
serum concentrations at day 0 and early systemic ABs (odds ratio,
3.1; 95% CI, 2.0-4.8; P< .001), whereas type of gut decontami-
nation, patient age, donor type, and Karnofsky Index revealed no
independent correlation (Table 4).
Microbiota disruption by early AB treatment
correlates with Reg3αrelease
Prolonged suppression of commensals by systemic AB treatment
before day −3 in relation to ASCT resulted in even higher Reg 3α
100
Test cohort
80
60
40
20
0
0246
Time post-transplant (months)
Probability of transplant-related mortality (%)
81012
Reg3α serum concentrations
at day of allogeneic
SCT
> 110 ng/ml - censored
≤ 110 ng/ml - censored
> 110 ng/ml
≤ 110 ng/ml
A
Validation cohort
100
80
60
40
20
0
0246
Time post-transplant (months)
Probability of transplant-related mortality (%)
81012
Reg3α serum concentrations
at day of allogeneic
SCT
> 110 ng/ml - censored
≤ 110 ng/ml - censored
> 110 ng/ml
≤ 110 ng/ml
B
Figure 1. Kaplan-Meier survival plots for 1-year TRM in
relation to Reg3αserum concentrations at the time of
ASCT. Kaplan-Meier survival plots show a significant
difference between patients with high and those with low
Reg3αserum values according to a threshold of 110 ng/mL in
both the test (n = 371, P< .001) (A) and validation cohorts
(n = 216, P= .009) (B).
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concentrations than in AB treatment after day −3 (114.4 ± 115.8
ng/mL vs 75.4 ± 95.1 ng/mL, P< .001) (Figure 4). The adminis-
tration of carbapenems, known to be the strongest suppressors of
commensals, was associated with high Reg3αconcentrations
(139.2 ± 143.4 vs 70.7 ± 62.1 ng/mL, P< .001) compared with all
other types of AB treatment.
Urinary 3-IS concentrations on day +7, as a marker for microbiome
diversity, were reduced (5.7 ± 18.9 ng/mL) in patients with high
Reg3αserum concentrations on day 0, whereas patients with low
Reg3αconcentrations on day 0 had higher urinary 3-IS concen-
trations (21.7 ± 20.4 ng/mL, P= .04; correlation coeffi-
cient, −0.21, P= .001). Similarly, stool copy numbers of the
commensal Clostridiales group XIVa revealed lower values in
patients with high Reg3αconcentrations (4.9 × 10
8
± 1.5 × 10
9
copy numbers) than in patients with low Reg3αconcentrations
(1.4 × 10
9
± 2.7 × 10
9
copy numbers, P= .004; correlation
coefficient, −0.15, P= .04).
Older patient age is the only independent variable
correlating with high Reg3αconcentrations before condi-
tioning. Analyzing parameters associated with high Reg3αserum
concentrations in the preconditioning situation we identified high
patient age (P= .002) and low Karnofsky Index (P= .005) in
univariate analysis. However, only patient age remained as inde-
pendent parameter correlating with Reg3αserum concentrations
in multivariable analysis (Table 5).
Discussion
Here, we report, to the best of our knowledge, for the first time, that
early Reg3αserum concentrations measured on the day of trans-
plant predict long-term outcomes of recipients of ASCT: increased
concentrations of Reg3αare associated with increased TRM as
well as poor overall survival at 1 year. Notably, not only GVHD-
induced deaths but also deaths due to infections and organ tox-
icities were increased in the high-Reg3αgroup. This could be
explained by indirect effects of intensified immunosuppression in
these patients or by direct effects of microbiota damage because
anti-infectious defense and bacterial translocation as well as
epithelial integrity are also directly influenced by the microbiome.
19
Multivariable analysis that included established clinical risk factors
showed early AB treatment to be the strongest predictor of early
Reg3αrelease. We observed that high Reg3αconcentrations
correlated with low concentrations of urinary 3-IS, a metabolic
marker indicating an absence of commensal bacteria, as well as
decreased stool copy numbers of Clostridiales;
5
therefore, these
data indicate an association between intestinal dysbiosis and
elevated Reg3αconcentrations at day 0. This correlation is unlikely
related to a preexisting microbiome disturbance because we found
no difference in microbiome diversity before conditioning.
Early intestinal dysbiosis and pretransplant conditioning toxicity
might damage intestinal mucosa and reduce intestinal epithelial
integrity. In addition, pretransplant conditioning may facilitate
epithelial injury by dysbiosis. Furthermore, the impaired repair
capacity of epithelial cells and intestinal stem cells as a conse-
quence of PC damage may sensitize the epithelial cells for GVHD-
induced damage. Increased bacterial translocation by intestinal
dysbiosis and epithelial injury may further enhance immunostimu-
lation and GVHD development. Recently, Reddy et al
20
reported
that a balanced interaction between immune and tissue tolerance
can modulate the immunopathology of aGVHD. The capacity of the
parenchymal tissue to maintain intestinal homeostasis and to
contribute to tissue resilience and regeneration is an important
factor to mitigate GVHD severity and to influence disease
outcome.
20
A balanced microbiome with high percentage of commensal bac-
teria like Clostridiales and their protective metabolites such as
Table 2. Cox regression analysis for 1-year TRM in the study cohort
PHazard ratio
95% CI for
hazard ratio
Reg3αserum concentrations, ng/mL
≤110 vs >110 <.001 3.2 1.9-5.2
Patient age, y
≤50 vs >50 .003 3.8 1.6-9.2
Patient sex
Female vs male .8 1.1 0.6-1.8
Type of underlying disease
Acute leukemia vs no acute leukemia .7 0.9 0.5-1.5
Stage of disease
Early vs late .2 1.4 0.9-2.4
Donor type
Related (no ATG) vs nonrelated (ATG) .6 1.1 0.8-1.6
Karnofsky Index, %
<90 vs ≥90 .4 0.8 0.5-1.4
Conditioning regimen
RIC vs standard .7 0.9 0.4-1.8
Parameters included in the model: Reg3αserum concentration, patient’s sex, patient’s age,
type of underlying disease, stage of disease, donor type, Karnofsky Index, and conditioning
regimen.
0246
Time post-transplant (months)
Probability of transplant-related and
relapse-related mortality (%)
81012
100
80
60
40
20
0
low Reg3a relapse
high Reg3a relapse
low Reg3a TRM
high Reg3a TRM
Figure 2. Competing-risk analysis for 1-year TRM and relapse in relation to
high Reg3αconcentrations. Competing-risk analysis confirmed the association of
high Reg3αconcentrations at day 0 and TRM when relapse was considered as
competing risk.
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SCFA is of great importance for intestinal homeostasis and
epithelial integrity.
21,22
In several single-center studies, the detri-
mental impact of microbiome disruptions on outcome of ASCT
recipients was demonstrated.
4,5,10,23
Recently, even a large-scaled
multicenter study showed patterns of microbiota disruption and,
particularly, a dominance of enterococci to be associated with poor
clinical outcomes independent of institutional practices and
geographic locations.
11
Several factors, however, can affect this
sensitive equilibrium; for example, the use of systemic broad-
spectrum ABs has a profound and long-lasting effect on the
composition and functionality of human microbiota resulting in
higher susceptibility to inflammatory conditions.
24
In ASCT, early
beginning of AB treatment before day 0 significantly affects clinical
outcomes and GVHD-associated TRM.
9,10,24
As fever is a common
side effect of ATG use, when applied before unrelated donor
transplantations, it frequently triggers immediate AB treatment,
which facilitates early microbiome disruptions and thus increases
the risk for transplant-related morbidity and mortality. In this context
and on the basis of careful clinical monitoring of septicemia, a more
restrictive use of broad-spectrum ABs during ATG therapy could
be considered; for example, by delaying start of ABs in the first 12
hours after ATG-related fever, which is frequently triggered by
cytokine release.
To date, Reg3αrelease was considered to be a consequence of
GVHD-induced PC damage, in which GVHD was enhanced by
early dysbiosis and loss of protective metabolites. Our data, how-
ever, suggest that Reg3αrelease can occur as a direct conse-
quence of early dysbiosis in the period of pretransplant
conditioning independently from T-cell activation. PCs are
described to play an important role in the effector phase of GI
GVHD; destruction of PCs due to cytotoxic T-cell damage was
correlated with high disease severity and poor treatment
response.
25-27
PCs are crucial in maintaining intestinal homeosta-
sis and promote the regeneration of the intestinal epithelium as well
as the modulation of microbial flora by production of antimicrobial
peptides like α-defensins and Reg3α.
28-33
In line with PC damage,
Reg3αwas described as a biomarker at onset of GVHD and sys-
temic Reg3αrelease explained by microscopic breaches in the
mucosal epithelial barrier permitting Reg3αto traverse into the
systemic circulation.
12,26
According to our observation, early
Reg3αmeasurements already on the day of transplant may be
used as a valuable clinical parameter predicting the outcome of
ASCT recipients. Whereas detailed microbiome analyses like 16S
rRNA sequencing are expensive, time consuming, and thus usually
not practicable in clinical routine, the analysis of Reg3αis well
established and provides real-time results. Although previous
findings indicate an indirect effect of dysbiosis on PCs via loss of
immunoregulation and a subsequently increased crypt and PC
destruction by alloreactive T cells, our data suggest that intestinal
mucosal damage induced by early dysbiosis after AB treatment
during conditioning toxicity might directly cause PC damage and
contribute to epithelial barrier disruption that favors transmission of
Reg3αinto the blood. Because Reg3αconcentrations before
conditioning therapy neither correlated with TRM nor underlying
Table 3. Overview of survival and causes of death according to
MAGIC hierarchy in relation to Reg3αserum concentrations at
day of ASCT
Low Reg3α, % High Reg3α, % Total, %
Survival 79.4 64.0 75.8
Relapse 10.2 8.1 9.7
GVHD*5.3 11.0 6.6
Infection 2.4 8.8 3.9
Others†2.7 8.1 3.9
*Organ-specific rates of GVHD: GI GVHD, 87.2%; liver GVHD, 19.0%; and skin GVHD,
71.4%.
†Other: hemorrhage, 22.2%; cardiovascular disease, 14.8%; secondary malignancy,
14.8%; leukoencephalopathy, 22.2%; posttransplant lymphoproliferative disease, 7.4%;
and liver toxicity, 11.1%.
600
400
*
*
*
*
*
**
*
*
*
*
*
*
*
*
*
*
200
Reg3α serum concentration (ng/ml)
0
pre-conditioning day 0
Timepoint of Reg3α measurement
Beginning of systematic AB in
relation to allogeneic
SCT
No AB or AB on/after day 0
AB before day 0
Figure 3. Correlation of Reg3αincrease in relation to
early AB treatment. The increase in Reg3αconcentrations
from the pretransplant situation until day 0 was higher in the
early ABs group than in patients who received late or no AB
treatment (P= .04).
11 APRIL 2023 •VOLUME 7, NUMBER 7 Reg3αAT DAY 0 PREDICTS TRM AFTER ASCT 1331
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disease or treatment-related parameters, we assume that day0
Reg3αrelease does not simply indicate higher vulnerability of
heavily pretreated patients but is induced by AB treatment in the
presence of conditioning toxicity. Several mechanisms could
contribute to this early PC damage: first, there is increasing evi-
dence that conditioning by cytotoxic treatment and irradiation
results in PC apoptosis;
34-36
and second, dysbiosis itself induced
by early use of ABs has been reported to substantially reduce
PCs.
37
PC function and survival are regulated, in part, by interleukin
22 (IL-22) signaling, which requires intact commensal bacteria and
their protective metabolites such as SCFA. Along these lines, Yang
et al demonstrated that SCFA produced by commensal bacteria
like Clostridiales can promote IL-22 production by innate lymphoid
cells and T cells through G protein–coupled receptor 41.
38
In
addition, Ghimire et al showed significant suppression of intestinal
IL-22 by systemic broad-spectrum ABs in patients suffering from
acute GI GVHD.
39
Low numbers of IL-22–secreting innate
lymphoid cells as a consequence of AB treatment as well as
intestinal dysbiosis with loss of microbiota-derived SCFA can lead
to loss of epithelial integrity and again favor Reg3αleakage.
38-40
Because it is not possible to obtain GI biopsies in this early
period of SCT, murine GVHD experiments may help to dissect the
mechanisms of early Reg3αrelease in the future. Interestingly,
recipient age emerged as the only additional risk factor for Reg3α
increase on day 0 and before conditioning. It is known from liter-
ature that age-related changes of intestinal microbiota can promote
intestinal inflammation through the production of proinflammatory
cytokines,
41
and age-related changes in metabolic signaling can
drive the loss of epithelial integrity and homeostasis.
42
In addition,
in murine models, adverse effects of aging reduce the expression
of key PC-related genes as well as their function and thus nega-
tively affect the regenerative ability of the gut epithelium.
43
This study has several limitations, the validation cohort shows a
variable recruitment of patient numbers between centers and
patient heterogeneity. Furthermore, this study is limited to available
parameters obtained in the MAGIC Germany consortium. Detailed
microbiome analyses using 16S rRNA sequencing are missing. In
addition, the current study is only able to show correlations
between the different variables and thus does not allow conclu-
sions regarding causality. A strength of this study is the multicenter
approach including different types of AB prophylaxis applied in the
different MAGIC sites. Although further prospective trials are
required to confirm our results, the current data open a new
perspective on the role of Reg3αin ASCT. High Reg3αserum
concentrations seem to be not only a biomarker for acute GI GVHD
but, even more so, an early indicator of intestinal mucosal damage
exacerbated by pretransplant conditioning toxicity and intestinal
microbiome disruption. Because Reg3αserum concentration on
day 0 was highly predictive for 1-year TRM, Reg3αmight depict a
valuable and clinically useful parameter for early identification of
patients at risk of poor outcome after ASCT. An early disruption of
the intestinal epithelial barrier function might induce a tissue
micromilieu that suppresses tolerance mechanisms, even before
the infusion of the donor graft, and thus might increase the sus-
ceptibility for GVHD development. However, our observations need
to be further elucidated by experimental models focusing on
pathophysiologic pathways.
As we have shown previously, intestinal dysbiosis early in the course
of an ASCT between day 0 and 10 is crucial to the outcome of ASCT
recipients because this time frame represents a vulnerable phase of
Table 4. Logistic regression analysis for parameters correlating with
Reg3αserum concentrations at day of ASCT (significant
parameters of univariate testing)
Risk factor POdds ratio 95% CI
Timing of AB treatment
Early (before day of ASCT) vs late (on or after day of
ASCT)
<.001 3.1 2.0-4.8
Gut decontamination
Rifaximin/no ABs vs cipro/cipro + metro .6 1.1 0.7-2.0
Patient age, y
≤50 vs >50 .3 1.0 1.0-1.0
Donor type
Related (no ATG) vs unrelated (ATG) .4 1.2 0.8-1.6
Karnofsky Index, %
<90 vs ≥90 .3 1.1 0.9-1.5
Parameters included in the model: timing of AB treatment, gut decontamination, patient
age, donor type, and Karnofsky Index.
600
*
*
*
*
*
*
*
*
*
*
*
*
*
400
200
Reg3α serum concentration at day of allogeneic
SCT (ng/ml)
0
AB before day -3 AB between day -3
and day -1
Beginning of systemic broad-spectrum antibiotics in relation to allogeneic SCT
AB after day -1 No AB
Figure 4. Correlation of Reg3αserum concentration on day
0 in relation to beginning of AB treatment. Prolonged
suppression of commensals by systemic AB treatment before
day −3 in relation to ASCT resulted in higher Reg 3α
concentrations than with AB treatment after day −3(P< .001).
1332 WEBER et al 11 APRIL 2023 •VOLUME 7, NUMBER 7
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immune reconstitution in which changes in the microbiota could
trigger long-term complications.
5,10
Our data assume that the stage
for a safe ASCT without TRM/GVHD may be set early or even before
the infusion of allogeneic cells and might be positively influenced by
a balanced microbiome composition; for example, by the very
deliberate restrictive use of systemic AB treatment, risk adapted
prophylaxis, or even by interventions restoring microbial integrity
such as fecal microbiota transplantation.
Acknowledgments
The authors thank Heike Bremm, Yvonne Schuhmann, and Tatjana
Schifferstein for collecting and cryopreserving patient specimens
and Heike Bremm and George Morales for performing Reg3α
analyses. Furthermore, the authors thank all colleagues at German
Mount Sinai Acute GVHD International Consortium sites for col-
lecting patient specimens and clinical data.
The German MAGIC consortium was supported by the German
Jose-Carreras Leukemia Foundation (DJCLS 01 GVHD/2016) and
the German Research Foundation (DFG) via SFB/TRR 221 (proj-
ect B13). The Reg3αanalyses performed at Tisch Cancer Institute,
Icahn School of Medicine at Mount Sinai were also supported by a
grant from the National Institutes of Health (P01 CA 039542).
Authorship
Contribution: D. Weber, E.H., J.E.L., J.L.M.F., W.H., and M. Edinger
were involved in conception and design of the study; D. Wolff, M.
Edinger, and H.P. were responsible for collection of specimens;
E.H. and J.L.M.F. were responsible for Reg3αmeasurements; A.H.
and A.G. performed bacterial analysis; S. Gleich and R.Y. were
responsible for data entry in the MAGIC database; M. Weber
contributed to statistical data analysis; F.A., W.R., M. Wölfl,S.K.,
R.Z., H.B., P.B., E.U., and M. Eder were responsible for collection
of patient specimens and clinical data at German MAGIC sites; D.
Weber, E.H., E.M., and S. Ghimire collected and analyzed clinical
data; D. Weber, M. Weber, J.L.M.F., and E.H. wrote the manu-
script; and all authors read and corrected the final draft.
Conflict-of-interest disclosure: E.H. served on the advisory
board for Maat Pharma and Pharmabiom. R.Z. declares honoraria
from Novartis, Incyte, Sanofi, and Mallinckrodt. D. Wolff declares
honoraria from Novartis, Gilead, Takeda, Incyte, Sanofi, and
Mallinckrodt. J.E.L. received royalties and is a coinventor on a graft-
versus-host disease biomarkers patent; received research support
from Biogen, Equillium, Incyte, MaaT Pharma, and Mesoblast; and
received consulting fees from Bluebird Bio, Equillium, Jazz,
Mallinckrodt/Therakos, Mesoblast, and X4 Pharmaceuticals.
J.L.M.F. received royalties and is a coinventor on a graft-versus-host
disease biomarkers patent. P.B. declares research grants from
Neovii, Riemser, and Medac; serves on the advisory board for
Novartis, Cellgene, Amgen, Medac, and Servier; declares honoraria
from Miltenyi, Jazz, Riemser, Novartis, as well as Amgen; and
received patent and royalties from Medac. None of these stated
conflicts of interests are related to this study. The remaining
authors declare no competing financial interests.
ORCID profiles: A.H., 0000-0002-3154-0638; H.B., 0000-
0002-5190-0899; P.B., 0000-0003-4554-0265; E.U., 0000-
0001-8530-1192; J.E.L., 0000-0002-5611-7828.
Correspondence: Daniela Weber, Department of Hematology
and Oncology, Internal Medicine III, University Medical Center,
Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany;
email: daniela.weber@klinik.uni-regensburg.de.
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