# CMV Serostatus of Donor-Recipient Pairs Influences the Risk of CMV Infection/Reactivation in HSCT Patients

Authors: Emilia Jaskula; Jolanta Bochenska; Edyta Kocwin; Agnieszka Tarnowska; Andrzej Lange
Journal: Bone Marrow Research
 (2012)

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## Abstract

CMV donor/recipient serostatus was analyzed in 200 patients allografted in our institution from unrelated (122 patients) donors and 78 sibling donors in the years 2002–2011 in relation to posttransplant complications. On a group basis independently of the CMV serostatus of donor-recipient pairs sibling transplantations and those from unrelated donors that matched 10/10 at allele level had a similar rate of CMV reactivation (17/78 versus 19/71,P=ns). The rate of CMV reactivation/infection was higher in patients grafted from donors accepted at the lower level of matching than 10/10 (18/38 versus 36/149, P=0.008). The incidence of aGvHD followed frequencies of CMV reactivation in the tested groups, being 40/156 and 25/44 in patients grafted from sibling or unrelated donors that 10/10 matched and in those grafted from donors taht HLA mismatched, respectively (P=0.001). Regarding the rate of reactivation in both groups seropositive patients receiving a transplant from seronegative donors had more frequently CMV reactivation as compared to those with another donor-recipient matching CMV serostatus constellation (22/43 versus 32/143, P=0<0.001). Multivariate analysis revealed that seropositivity of recipients with concomitant seronegativity of donors plays an independent role in the CMV reactivation/infection (OR=2.669, P=0.037; OR=5.322, P=0.078; OR=23.034, P=0.023 for optimally matched and mismatched patients and the whole group of patients, resp.).


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## Body

## 1. Introduction

Donor-recipient matching for unrelated hematopoietic stem cell transplantation (HSCT) in addition to human leukocyte antigens (HLA) includes CMV serostatus of the donor and recipient to facilitate the decision [1, 2].In the clinical practice the presence of CMV IgM antibodies is suggestive of the active infection/reactivation and the presence of IgG antibodies indicates prior infection and shows CMV immunological competence of individuals [3–5]. Unfortunately, it is very suggestive that IgG CMV antibody positive individuals harbor CMV in a latent form and their blood products are infective for CMV incompetent recipients [6]. In the present era of specific anti-CMV chemotherapy the significant impact of pretransplant donor seropositivity on the patient outcome is controversial—reviewed in the Boeckh and Nichols paper [7]. However, recipient CMV serostatus still remains an important risk factor of the patient outcome [8, 9].HSCT involving pairs in which both donor and recipient lack CMV IgG antibodies is associated with a lower transplant mortality [10]. In the latter situation we are dealing with a donor-recipient pair in which probably neither donor nor recipient has CMV in alatent form. On the other hand, positivity of both donor and recipient should also favor the HSCT outcome—both donors and recipients likely have CMV in a latent form but the immune system of the donor should have a memory of CMV infection, which facilitates the immune response to CMV posttransplant. However, Ljungman et al. [11] in the megafile analysis showed that the latter important observation seems to be valid only for the unrelated donor transplantation setting.To add new information to the still disputable association between the CMV donor/recipient serostatus with the outcome of transplantation the present study was undertaken.


## 2. Materials and Methods

Two hundred patients (F/M: 91/109; 26 and 174 patients were below and above 16 years of age, resp.) allografted from unrelated donors (122 patients), and 78 from sibling (SIB) donors in our institution in the years 2002–2011 were studied. One hundred and seventy-five suffered from hematological malignancies acute myeloid leukemia (AML;n=67), chronic myeloid leukemia (CML; n=24), acute lymphocytic leukemia (ALL; n=39), other lymphoproliferative disorders (n=23), myeloproliferative diseases (n=10), and myelodysplastic syndromes (n=12). The others were transplanted because of anemias (10 patients) and immunodeficiencies (14 patients) and osteopetrosis (n=1).One hundred and five and 95 patients received myeloablative (based on busulfan and cyclophosphamide) and reduced intensity conditioning (reduced busulfan dose or melphalan plus fludarabine and antithymocyte globulin (ATG)), respectively. Unrelated donor transplanted patients and those on reduced intensity conditioning received ATG (10 to 12.5 mg/kg b.w. cumulative dose, 125 patients) or alemtuzumab (90 mg as a dose, 38 patients) as a part of the conditioning regimen. All patients were on cyclosporin A with a dose adjusted to the blood CsA trough a level to 200 ng/L. CMV serostatus, age, gender, underlying disease, donor source, and HLA match as well as conditioning regimen (reduced or myeloablative) are given in Table1.Table 1
Patient characteristics.
Number of patients
200
Age
(median, range), yrs
34, 1–60
Adults> 16 yrs
174
Children≤ 16 yrs
26
Recipient gender
Female
91
Male
109
Donor gender
Female
83
Male
116
Donor
Sibling
78
Unrelated HLA matched (10/10 at the allele level),
78
Mismatched, at the allele or low resolution levels up to two mismatches
44
Transplant material
Bone marrow (BM)
28
Peripheral blood progenitor cells (PBPC)
172
Diagnosis
Hematological malignancies (HM)
175
Chronic myeloid leukemia (CML)
24
Chronic lymphocytic leukemia (CLL)
5
Acute myeloid leukemia (AML)
67
Acute lymphocytic leukemia (ALL)
39
Other HM
40
Anemias and immunodeficiencies
24
Osteopetrosis
1
Conditioning regimen
Myeloablative
105
Reduced intensity conditioning (RIC)
95
Acute GvHD, grades
0
114
I
21
II
26
III
16
IV
23
Chronic GvHD
Extensive
38
Limited
33
EBV≥100 DNA copies/105 cells
45/187
CMV≥100 DNA copies/105 cells
54/187
HHV6≥100 DNA copies/105 cells
34/187
Polyoma (JC/BK)
19/33
CMV IgG serostatus
Recipients
CMV IgG negative
32
CMV IgG positive
168
Donors
CMV IgG negative
66
CMV IgG positive
132
Recipient/donor CMV serostatus
Recipient CMV IgG (+)/donor CMV IgG (+)
118
Recipient CMV IgG (−)/donor CMV IgG (−)
18
Recipient CMV IgG (+)/donor CMV IgG (−)
48
Recipient CMV IgG (−)/donor CMV IgG (+)
14The patients were routinely followed for clinical outcome in one-week intervals until 30 days posttransplant, then monthly until one year post-transplant and as well as when clinical symptoms were suggestive of CMV, EBV, or HHV6 reactivation or any other serious post-transplant complications including relapse or GvHD. Out of 200 patients studied viral CMV, EBV and HHV6 DNA copies in blood were determined in 187 recipients transplanted after the year 2003.The Zeus Scientific, Inc. (NJ, USA), IgG and IgM ELISA test system was used for qualitative detection of CMV-specific antibodies in donors’ and recipients’ plasma. The ELISA kit was used according to the manufacturer’s instructions. Briefly, microtiter plates, precoated with inactivated CMV antigen, were incubated with the recipient or donor plasma. Bound IgG or IgM was detected with peroxidase labeled anti-IgG and anti-IgM antibodies by the addition of the color substrate and reading by spectrometry. Results were interpreted as seropositive or seronegative as per the manufacturer’s instructions.DNA was extracted from peripheral blood (QiAmp Blood Kit; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The numbers of CMV, EBV, and HHV6 DNA copies in peripheral blood cells were determined using real-time PCR and Light Cycler II (Roche, Mannheim, Germany). The sequences of the PCR primers and the probe were selected from theBALF5 region of EBV, the US17 region of CMV, and the U67 region of HHV6. PCRs were performed as described by Jaskula et al. [12].


### 2.1. Statistical Analysis

Statistical analysis was performed using the CSS Statistica for Windows (version 10.0) software (Stat-Soft Inc., Tulsa, OK). Univariate analyses were performed by the Fisher exact test. Logistic regression was used for the multivariate analysis, and alog-rank test to analyze the survival probability. Differences between samples were considered to be significant whenP<0.05 and those between 0.05 and 0.1 were indicative of a trend.


## 2.1. Statistical Analysis

Statistical analysis was performed using the CSS Statistica for Windows (version 10.0) software (Stat-Soft Inc., Tulsa, OK). Univariate analyses were performed by the Fisher exact test. Logistic regression was used for the multivariate analysis, and alog-rank test to analyze the survival probability. Differences between samples were considered to be significant whenP<0.05 and those between 0.05 and 0.1 were indicative of a trend.


## 3. Results and Discussion

The presence of >100 CMV, EBV, and HHV6 DNA copies per 105 blood cells (clinically significant [12, 13]) was detected in 29%, 24%, and 18% of patients, respectively. Sixty out of 100 patients having during the observation period one or more reactivation events of one or more examined herpes viruses died. The mortality rate was lower in the group of patients lacking reactivations/infections (32 out of 87 patients), which resulted in a better five-year survival (59% versus 37%,   P=0.018, Figure 1).Figure 1
Overall survival in the groups of patients having and lackingherpes virus (CMV and/or EBV and/or HHV6) reactivations/infections.Patients receiving transplantation from the CMV IgG seronegative donors tended to suffer more frequently from CMV infection/reactivation after HSCT as compared to those grafted from CMV seropositive donors (23/59 versus 31/127,P=0.055, Figure 2(b)). This association was valid for seropositive and seronegative recipients. However, the highest risk of CMV reactivation was when seropositive recipients were transplanted from the seronegative donors (22/43 versus 32/143, P<0.001 Figure 2(a)). In contrast, CMV negative serostatus of both the donor and the recipient was associated with the lowest rate of the CMV reactivation (1 out of 16 patients) as compared to other recipient (R)/donor (D) CMV IgG serostatus relations, being 22/43 versus 28/115 versus 3/12, (P<0.001) for R+/D−, R+/D+, and R−/D+, resp. (Figure 2(a)). We also found that aGvHD (grade > I) was more frequently seen in patients receiving grafts from IgG negative donors (28/66 versus 36/132, P=0.036, Figure 2(c)). However, donor serostatus did not affect the survival of HSCT recipients (Figure 3).CMV reactivation/infection with respect to donor/recipient CMV serology (a) and donor CMV IgG status independently of the serostatus of recipients (b). Acute GvHD in patients transplanted from CMV IgG negative and CMV IgG positive donors (c) (R: recipient, D: donor, “+”: CMV IgG positive, and “−”: CMV IgG negative).
(a)
(b)
(c)Figure 3
Survival of HSCT patients in the groups stratified according to CMV donor-recipient serostatus constellation (R: recipient, D: donor, “+”: CMV IgG positive, and “−”: CMV IgG negative).In addition to the factors associated with the serostatus of donors and recipients, a lack of optimal donor/recipient HLA matching was associated with a higher risk of grade > I aGvHD (25/44 versus 40/156,P<0.001) and with a higher rate of CMV reactivation/infection (18/38 versus 36/149, P=0.008). CMV reactivation was also more frequently seen in patients who were over 16 years old at the time of transplantation (52/165 versus 2/22, P=0.042, Table 2) and in those having CMV IgG antibodies before transplantation (50/159 versus 4/28, P=0.073, Table 2).Table 2
Univariate analysis of risk factors for aGvHD and CMV reactivation/infection event(s) in patients post-alloHSCT.
Variable
aGvHD
P value
CMV absence
CMV presence
P value
≤grade I
>grade I
Infection/reactivation until 1 year post-HSCT
Donor/recipient HLA match
Matched
116
40
P
<
0.001
113
36
P
=
0.008
Mismatched at the allele or  low resolution levels up to two mismatches
19
25
20
18
Source of HSCT
PBPC
111
61
P
=
0.029
112
48
P
=
0.496
BM
24
4
21
6
Type of donor
SIB
65
13
P
<
0.001
61
17
P
=
0.074
MUD
70
52
72
37
Conditioning regimen
RIC
67
28
P
=
0.450
60
30
P
=
0.202
Myeloablative
68
37
73
24
Donor CMV IgG
CMV IgG−
38
28
P
=
0.036
36
23
P
=
0.055
CMV IgG+
96
36
96
31
Recipient CMV IgG
CMV IgG−
19
13
P
=
0.307
24
4
P
=
0.073
CMV IgG+
116
52
109
50
Donor-recipient IgG CMV serology
R−/D−
10
8
15
1
R+/D−
28
20
P
=
0.159
21
22
P
<
0.001
R−/D+
9
5
9
3
R+/D+
87
31
87
28
R−/D−, R+/D−, R−/D+, R+/D+
106
44
P
=
0.115
111
32
P
<
0.001
R+/D−
28
20
21
22
Donor/recipient gender
Male to male, female to female, and male  to female
105
54
P
=
0.572
105
44
P
=
0.841
Female to male
29
11
28
10
Donor gender
Male
76
40
P
=
0.543
75
32
P
=
0.747
Female
58
25
58
22
Recipient gender
Male
76
33
P
=
0.544
71
27
P
=
0.747
Female
59
32
62
27
Recipient age
≤16
17
9
P
=
0.824
20
2
P
=
0.042
>16
118
56
113
52
CMV infection/reactivation event within 1 year post-HSCT
CMV−
97
36
P
=
0.025
CMV+
30
24
aGvHD
aGvHD ≤ grade I
97
30
P
=
0.025
aGvHD>grade I
36
24
EBV infection/reactivation event within 1 year post HSCT
EBV−
100
42
P
=
0.204
104
38
P
=
0.263
EBV+
27
18
29
16
HHV6 infection/reactivation event within 1 year post HSCT
HHV6−
103
50
P
=
0.835
110
43
P
=
0.677
HHV6+
24
10
23
11
PBPC: peripheral blood progenitor cells; BM: bone marrow; R: recipient; D: donor; “−”: negative; “+”: positive; ATG: antithymocyte globulin; SIB: HLA-identical siblings; MUD: unrelated donors; RIC: reduced intensity conditioning.Multivariate analysis devoted to the evaluation of the risk factors of aGvHD showed that unrelated donor (OR=2.591, P=0.036) transplantation and HLA mismatch (OR=2.361, P=0.042) appeared as independent and significant factors associated with aGvHD grade > I (Table 3). In spite of the univariate results multivariate analysis did not confirm the role of CMV reactivation and donor serology as independent factors associated with aGvHD (Table 3).Table 3
Multivariate analysis of risk factors for aGvHD (grade > I).
Variable
Coefficient
P value
Odds ratio
95% CI
CMV infection/reactivation event within 1 year post HSCT
0.5473
0.1362
1.7286
0.8415 to 3.5509
CMV IgG in donor serum
0.0290
0.9411
1.0295
0.4762 to 2.2254
Donor-recipient HLA mismatch
0.8591
0.0421
2.3611
1.0310 to 5.4072
Unrelated donor
0.9520
0.0355
2.5909
1.0669 to 6.2921
BM as a source of cells
−0.6177
0.3090
0.5392
0.1640 to 1.7723The next statistical approach was to validate factors associated with CMV reactivation. For that also a multivariate analysis was calculated employing factors as follows: recipient IgG serology, donor-recipient HLA mismatch, transplantation recipient in CMV IgG positive/donor CMV IgG negative serology, type of donors, recipient age, and aGvHD. Among the above factors donor-recipient HLA mismatch (OR=3.499, P=0.016), recipient CMV IgG positive/donor CMV IgG negative serology status constellation (OR=23.030, P=0.023), and recipient age over 16 years (OR=9.865, P=0.007) were found to be significant risk factors of CMV reactivation (Table 4).Table 4
Multivariate analysis of risk factors for CMV reactivation/infection.
Variable
Coefficient
P value
Odds ratio
95% CI
Recipient CMV IgG seronegativity
−0.0761
0.9224
0.9267
0.2000 to 4.2929
Donor/recipient  HLA mismatch
1.2525
0.0155
3.4992
1.2695 to 9.6446
R CMV IgG+/D CMV IgG−
3.1370
0.0227
23.0340
1.5491 to 342.4999
Unrelated donor
0.0021
0.9965
1.0021
0.3904 to 2.5722
aGvHD > 1
0.5363
0.1755
1.7096
0.7870 to 3.7141
Recipient age
>
16
years
2.2890
0.0072
9.8650
1.8606 to 52.3036To further analyze the significance of CMV serology as a risk factor of CMV reactivation similar to that above, analysis was performed for groups consisting of SIB and MUD 10/10 matched and MUD not optimally matched (Tables5 and 6). On agroup basis independently of the CMV serostatus of donor-recipient pairs, sibling transplantations and those from unrelated donors matched 10/10 at allele level had asimilar rate of CMV reactivation (17/78 versus 19/71, P=ns). Notably, the rate of CMV reactivation was higher in patients grafted from donors accepted at the lower level of matching than 10/10 (18/38 versus 36/149, P=0.008). Also when we considered separately the optimal match group (SIB + MUD) the highest risk of CMV reactivation was observed when donors were negative but recipients were positive (12/28 versus 24/120, P=0.015). In MUD HLA mismatched recipients a tendency to the association seropositivity of recipients with concomitant seronegativity of donors with the CMV reactivation/infection was observed (10/15 versus 8/23, P=0.096). Notably in the MUD HLA mismatch group recipient age >16 years was a risk factor for CMV reactivation (17/28 versus 1/10, P=0.009, Table 5). There were no significant associations between aGvHD and variables considered in this paper in the optimally matched group (SIB + 10/10 HLA matched) and in the MUD HLA mismatched group of patients.Table 5
Univariate analysis of risk factors for CMV reactivation/infection event(s) in group of SIB and MUD HLA match patients and in group of MUD HLA mismatch patients.
Optimally matched group (SIB+ 10/10 HLA matched) of patients
MUD HLA mismatched group of patients
Variable
CMV absence
CMV presence
P value
CMV absence
CMV presence
P value
Infection/reactivation until 1 year post HSCT
Infection/reactivation until 1 year post HSCT
Source of HSCT
PBPC
95
30
1.000
17
18
0.232
BM
18
6
3
0
Conditioning regimen
Absence of ATG and Campath
33
5
0.167
0.170
ATG
60
22
19
14
Campath
20
9
1
4
RIC
53
23
0.087
7
7
1.000
Myeloablative
60
13
13
11
Donor CMV IgG
CMV IgG−
26
12
0.273
10
11
0.532
CMV IgG+
86
24
10
7
Recipient CMV IgG
CMV IgG−
18
2
0.160
6
2
0.238
CMV IgG+
95
34
14
16
Donor-recipient IgG CMV serology
R−/D−
10
0
5
1
R−/D+
8
2
0.032
1
1
0.18
R+/D−
16
12
5
10
R+/D+
78
22
9
6
R−/D−, R−/D+, R+/D+
96
24
0.015
15
8
0.096
R+/D−
16
12
5
10
Donor/recipient gender
Male to male, female to female, and  male to female
67
23
0.698
13
14
0.485
Female to male
46
13
7
4
Donor gender
Male
67
23
0.569
8
9
0.746
Female
46
13
12
9
Recipient gender
Male
59
21
0.698
12
6
0.112
Female
54
15
8
12
Recipient age
≤16
11
1
0.290
9
1
0.009
>16
102
35
11
17
aGvHD
aGvHD ≤ grade I
88
23
0.123
9
7
0.752
aGvHD > grade I
25
13
11
11Table 6
Multivariate analysis of risk factors for CMV infection/reactivation in group of SIB and MUD HLA match patients and in group of MUD HLA mismatch patients.
Variable
Optimally matched group (SIB+ 10/10 HLA matched) of patients
MUD HLA mismatched group of patients
Coefficient
P value
Odds ratio
95% CI
Coefficient
P value
Oddsratio
95%CI
aGvHD > 1
0.7265
0.1015
2.0679
0.8667 to 4.9336
0.0185
0.9816
1.0187
0.2113 to 4.9106
Recipient CMV IgG seronegativity
0.9058
0.2601
2.474
0.5113 to 11.9711
0.5751
0.6126
1.7773
0.1918 to 16.4703
R CMV IgG+/D CMV IgG−
0.9819
0.0374
2.6695
1.0587 to 6.7314
1.6719
0.0776
5.3222
0.8313 to 34.0733
RIC conditioning regimen
−0.5655
0.1745
0.5681
0.2511 to 1.2849
0.30101
0.7333
1.3513
0.2391 to 7.6375
Recipient age
>
16
years
1.2945
0.2318
3.6492
0.4371 to 30.4624
3.1026
0.0148
22.256
1.8360 to 269.7835Multivariate analysis results of patients optimally matched and separately those not optimally matched were similar and revealed that among factors analyzed for the risk of CMV reactivation seropositivity of recipients with concomitant seronegativity of donors plays an independent role (OR=2.670, P=0.037) for optimally matched and as tendency in HLA mismatched patients (OR=5.322, P=0.078, Table 6).


## 4. Conclusions

The information provided in the present paper shows that IgG negativity in donors favors the outcome of HSCT only when recipients are also CMV IgG negative. The worst is when donor IgG CMV negativity is confronting IgG CMV positivity in recipients. This confirms the importance of CMV IgG positivity likely associated with the immune competence of donors [3–5], which is of a special value in seropositive patients, very likely having CMV in alatent form [6]. Therefore, when recipients are IgG CMV positive the immune competence of donors is required to reduce the risk of CMV reactivation. This observation can be used as one of the factors that should be considered during donor selections for an optimal post-HSCT outcome.



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*Source: 375075-2012-11-22.xml*