To the Editor:
Blood or marrow transplantation can cure patients with transfusion-dependent thalassemia
(TDT) [1] and normalize their long-term health-related quality of life [2]. In regions
where TDT is most prevalent, 38–60% of transplant candidates may find a fully matched-related
donor [3], thus a substantial proportion of patients require alternative donors. The
use of posttransplant cyclophosphamide (PTCy) as well as ex vivo T-cell depletion
methods have allowed to safely perform transplants across HLA barriers [4–6]. Because
of its simplicity and inexpensiveness, PTCy seems a particularly attractive option
for centers in lower-middle-income countries (LMIC) in South-East Asia where TDT is
the most frequent life-threatening noncommunicable disorder of childhood and a major
financial burden to families and health care systems [7]. Outcomes using partially
matched-related donors (PMRD) with the PTCy approach have been shown to be comparable
with those using unrelated or related fully matched donors [5, 8], but unrelated donors
in LMIC are often unavailable and/or unaffordable. In the PMRD transplantation context
the relevance of donor-specific antibodies (DSA) is well established [9], less so
that of different degrees and types of HLA mismatches which, in fact, are not generally
considered relevant [10].
We retrospectively assessed the impact of DSA as well as that of Host vs. Graft (HVG)
set ups, i.e., when the recipient is homozygous for one or more HLA specificities
while the donor is not, so that for those HLA specificities the recipient has the
potential to react towards the unshared allele of the donor but not vice versa. A
total of 28 consecutive partially matched-related BMTs where analyzed, these were
performed between March 2017 and December 2018 in three centers in India: The South-East
Asia Institute for Thalassemia in Jaipur, Rajasthan (20 cases), the People Tree Hospitals
in Bangalore, Karnataka (seven cases), and the Care Institute for Medical Sciences
in Ahmedabad (one case). Selection criteria included a diagnosis of TDT, lack of a
fully matched-related donor, age at BMT <15 years (range 1.5–13.5 years, median 5.3),
no significant hepatosplenomegaly (<2 cm from costal margin) or serum ferritin >5000 ng/mL
pre-BMT. None of the patients underwent a liver biopsy and thus would be considered
Pesaro class I–II given the absence of hepatomegaly [1]. A common online electronic
medical record system and collaboration platform where data were entered prospectively
on a daily basis (BMTPlus®, Jagrity Innovations, Bangalore, India, www.bmtplus.net)
[11], and a single transplant approach approved by centers’ IRB was used. The preparative
regime, modified from Anurathapan at al. [5], is outlined in Supplementary Fig. 1.
All caretakers provided informed consent to share personal data as well as for the
BMT procedure. All patients received G-CSF-primed (5 µg/kg twice daily from day −5
to −1) bone marrow with a total nucleated cell dose ranging from 12.1 to 52.9 × 108/recipient
kg (median 16.3). Post-BMT all blood products were irradiated with ≥25 Gy. Autologous
marrow was cryopreserved in all cases. Chimerism was monitored at least at 1, 2, 4,
and 8 months by molecular (STR) analysis or Y chromosome cytogenetics or fluorescent
in-situ hybridization when informative. All patients and immediate family members
were HLA-typed by sequence-based high-resolution typing confirmed in two independent
samples in different laboratories. All but one patient were evaluated for DSA status.
Data were collected and analyzed on 31 May 2019. Fisher’s exact tests were used to
compare proportions and Mann–Whitney nonparametric test were used to compare continuous
distribution values. Kaplan–Meier survival curves were compared using the log-rank
(Mantel–Cox) test. All P values are two-tailed. Statistical analysis was performed
using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, California,
USA, www.graphpad.com.
Six out of twenty-five patients (24%) had a rejection, with no significant differences
in terms of sex, maternal vs. paternal donor, or cell dose between patient who rejected
and those who did not (see Table 1). A total of 4 patients out of 27 evaluated for
DSA where positive with mean fluorescent intensity >2000, 2/6 in the rejection group
(33%) and 2/21 in the nonrejection one (9%). In the rejection group 3/6 (50%) had
a HVG set up (patient characteristics are summarized in Table 2). Actuarial rejection
proportion went from 5% in patients with neither DSA-positivity nor HVG set up, to
56% in those with either one, see Supplementary Fig. 2. There was no overlap between
DSA-positive patients and those with HVG set up. Among patients who did not reject
4/22 (18%) had a HVG set up, all with unilateral homozygosities at the A locus, in
2 the donor was DRB1 and DQB1 compatible, in 1 was also B and C compatible, and in
1 was DRB1 and DQB1 unilaterally homozygous, thus there was a concomitant HVG and
GHV set up. Both DSA-positive patients who did not reject had a GVH set up. None of
the patients who rejected had a GVH set up. Of the 21 thalassemia-free patients, 18
(86%) have >95% donor chimerism, 2/22 (9%) of engrafted patients developed grade III
or IV GHVD, no case of extensive chronic GHVD has been observed so far. One patient
died of grade IV GVHD and had a GVH set up, otherwise there was no apparent correlation
between GVH set up and actual occurrence of GVHD. At a median follow up of 13 months
(range 5.7–26.4) transplant-related mortality was 24% vs. 17% in DSA+ HVG+ and DSA−
HVG− patients respectively with a P value of 0.53.
Table 1
Data summary of patients who rejected vs. those who did not
Patients who rejected
Patients who did not reject
P
Total patients
6
22
Median age (range)
6.5 (3.4–10)
4.4 (1.5–13.5)
0.40
Sex
4 males, 2 females
16 males, 6 females
1
Donor
5 mother, 1 father
13 mother, 6 father
1
Marrow cell dose ×108/kg
17.3 (16–21.2)
16.2 (12.1–52.9)
0.52
Marrow white cell count/µL
55,445 (31,730–75,600
68,215 (34,790–252,100)
0.15
Table 2
Patients characteristics
UPN
Age at BMT
Sex
Donor
FU
Pre-BMT Blood Tx
Cell dose (×108/kg)
Day to ANC 500
Day to plt 20,000
DSA
HLA notes
DSA + or HVG +
Latest donor chimerism
Rej
Acute GVHD
CMV activation
Status
Notes
INA16034
4.3
F
Mother
26.4
79
23
17
16
Neg
A compatible
No
100
No
2
Yes
A&TF
INA15054
2.2
M
Father
25.0
28
15.38
17
15
Neg
A compatible
No
61
No
0
Yes
A&TF
INA15007
3.9
F
Mother
17.2
69
18.5
14
12
Not done
B & DPB1 compatible
No
100
No
1
No
A&TF
INA15020
3.4
M
Mother
18.4
57
21.7
24
15
Neg
No
99
No
2
No
A&TF
INA12018
8.9
M
Mother
13.7
120
15.4
19
20
Neg
DRB1, DQB1 & DPB1 compatible
No
100
No
0
No
A&TF
Possible IFD
INA13107
8
M
Mother
16.2
90
20.5
26
14
Neg
DQB1 GVH vector
No
100
No
0
No
A&TF
MAS
INA12057
9.4
M
Mother
NA
201
16.05
26
35
Neg
DRB1, DQB1 & DPB1 compatible
No
100
No
0
No
TRM
BK virus HC, ICH
INA17022
5.1
F
Mother
13.0
50
13.7
24
18
Neg
No
100
No
1
No
A&TF
INE16041
5.9
F
Mother
12.2
117
52.9
18
18
Neg
B, C, DRB1 & DQB1 GVH vector
No
97
No
0
No
A&TF
INE17035
13.5
M
Mother
NA
147
12.14
20
25
Neg
DRB1, DQB1 & DPB1 compatible
No
97
No
0
No
TRM
Probabble IFD
INA10055
11.5
M
Father
11.2
198
13.28
24
27
Neg
DRB1, DQB1 & DPB1 compatble, A HVG vector
No
100
No
2
No
A&TF
INA17041
2
M
Mother
10.3
20
14.12
13
16
Neg
A, C, DRB1, DQB1 & DPB1 compatible
No
100
No
2
No
A&TF
INE15024
3
M
Mother
9.5
36
44.4
14
18
Neg
DRB1 compatible, DQB1 GVH vector
No
100
No
0
No
A&TF
AFC17001
3.4
M
Mother
8.5
57
18.4
NA
NA
Neg
DPB1 compatible
No
0
d + 30
0
No
A&T
PGF-PP, Probable IFD
INA11012
9.5
F
Mother
7.5
204
16
17
39
Neg
C compatible
No
100
No
0
No
A&TF
2nd BMT after MRD rejection, MAS, SOS
INA13015
10.7
M
Father
6.5
232
16
18
19
Neg
No
100
No
2
No
A&TF
INA13128
7
M
Father
6.1
144
14.3
20
16
Neg
DRB1 & DQB1 GVH vector
No
98
No
0
No
A&TF
INM17025
3.9
M
Mother
5.7
69
23.8
18
19
Neg
B, C, DRB1 & DQB1 compatible, A HVG vector
No
84
No
0
No
A&TF
Responded to DLI for EMC
INA17009
2.1
M
Father
NA
12
16.26
19
18
Neg
A, C, DPB1 GVH vector
No
NA
No
4
No
TRM
Severe GVHD-Sepsis
INA14043
3.5
M
Father
10.9
60
19.13
23
16
Pos
DRB1 & DQB! GVH vector, A HVG vector
Yes
100
No
2
Yes
Dead
Death unrelated to BMT
INA16078
5.8
M
Mother
18.9
115
16.3
NA
NA
Neg
C compatible, DRB1 HVG vector
Yes
0
d + 29
0
No
A&T
PGF-EAR
INE16076
4.2
F
Mother
18.3
76
14.6
21
29
Neg
DRB1 & QB1 compatible, A HVG vector
Yes
100
No
0
Yes
A&TF
INA16041
5.5
M
Mother
NA
108
20.16
21
19
Pos
C compatible
Yes
3
d + 43
0
No
TRM
SGF
INC15040
8.3
F
Mother
14.2
175
16
NA
NA
Pos
Yes
0
d + 25
0
Yes
A&T
PGF-PP, Provem IFD
INA17120
1.5
M
Mother
14.1
12
18.9
20
36
Pos
A GVH vector
Yes
100
No
1
No
A&TF
INE17044
10
F
Father
13.0
170
21.21
35
54
Neg
DRB1 & DPB1 HVG vector
Yes
0
d + 20
0
No
A&T
PGF-PP, SOS
INA17021
7.3
M
Mother
11.8
90
16.21
NA
NA
Neg
A HVG vector
Yes
0
d+25
0
No
A&T
PGF-PP
INA15003
4.6
M
Mother
NA
86
17.4
NA
NA
Neg
A HVG vector
Yes
100
No
3
No
TRM
MAS
FU follow-up, Rej rejection, A&TF alive and thalassemia-free, A&T alive with thalassemia,
TRM transplant-related mortality, MAS macrophage activation syndrome, HC hemorrhagic
cystitis, ICH intracranial hemorrhage, IFD invasive fungal disease, PGF primary graft
failure, PP persistent pancytopenia, SOS synusoidal obstructive syndrome, EMC early
mixed chimerism (<day +60), EAR early autologous reconstitution, SGF secondary graft
failure
Thalassemia seems an ideal model to study the role of immunogenetic factors in the
HVG direction because of its homogeneity, functional immune system and exposure to
multiple transfusions resulting in higher potential for rejection compared with the
hematological malignancy context. The observation that HLA HVG disparities can affect
rejection in thalassemia patients has been previously reported in the unrelated setting
[12] but not in the haploidentical-related one. A factor which might have contributed
to not having identified this HLA vector effect previously in haploidentical BMT is
that high-resolution typing is not routinely employed for related donor identification
[13], while high-resolution mismatches may have the same clinical significance as
low-resolution ones [14]. In fact, the identification of a HLA vector is based on
the assumption of true homozygosity of one or more HLA alleles.
We believe that even if PTCy is quite effective in inducing tolerance, some degree
of escape still remains since GHVD is not infrequent albeit generally mild and manageable.
The same maybe true in the HVG (rejection) direction.
In conclusion, these findings may have important practical implications for the selection
of partially matched donors for nonmalignant conditions in which rejection is a potential
issue. With all the limitations of a small case series, in our experience the presence
of a HVG set up in the context of thalassemia seems as impactful on rejection as that
of DSA. The potential implication is that in this context it might be advisable to
get high-resolution HLA typing and possibly consider the use of unrelated donors in
the presence of unilateral recipient’s HLA homozygosities. This occurrence maybe more
frequent in populations with high consanguinity or close ethnicity, like in the Indian
subcontinent. The impact of HLA vectors in the HVG or GVH direction in haploidentical
transplantation for thalassemia may deserve to be assessed in larger studies.
Supplementary information
Supplemental Fig 1
Supplemental Fig 2