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Original Clinical Science—General

Association of Alemtuzumab Induction With a Significantly Lower Incidence of GVHD Following Intestinal Transplantation: Results of 445 Consecutive Cases From a Single Center

Vianna, Rodrigo MD, PhD1; Farag, Ahmed MD, PhD1,2; Gaynor, Jeffrey J. PhD1; Selvaggi, Gennaro MD1; Tekin, Akin MD1; Garcia, Jennifer MD3; Pierce, Conlan MD1; Beduschi, Thiago MD1

Author Information
doi: 10.1097/TP.0000000000003111
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Abstract

INTRODUCTION

Intestinal transplantation is the only definitive therapy for patients with irreversible intestinal failure who face life-threatening complications from ongoing use of total parenteral nutrition. Short-term graft and patient survival following intestinal transplantation have markedly improved over the past 20 years.1-3 Important contributing factors include advances in immunosuppression, with particular use of antilymphocyte agents (ie, rabbit antithymocyte globulin [rATG] or alemtuzumab) as induction immunosuppression for prevention (and treatment) of acute cellular rejection (ACR).4 Refinements in surgical technique and increased experience in overall patient management have also been important contributors to the currently more favorable outcomes.

In addition to using tacrolimus (TAC) as the mainstay maintenance drug to prevent the occurrence of ACR,5-8 the polyclonal antibody rATG (thymoglobulin) combined with the chimeric anti-CD20 monoclonal antibody rituximab has been used as dual induction therapy at our center since 2013.9 rATG contains antibodies to a variety of T-cell and plasma cell/B-cell antigens and may have a protective effect against reperfusion injury in solid organ transplantation.10,11 Rituximab was added to rATG with the goal of efficiently removing B cells in an attempt to minimize possible injury caused by preformed and de novo donor-specific antibodies developed against the intestinal graft.

Graft versus host disease (GVHD) also remains a major cause of morbidity and mortality following intestinal transplantation. The incidence of biopsy-proven GVHD in previous reports has ranged from 5.6% to 19.2%,12-16 with these 5 studies consistently showing in univariable analysis that a modified or full multivisceral (MV) (versus isolated intestine [I] or combined liver-intestine [LI]) transplant was associated with a higher risk of developing GVHD. Native splenectomy was also associated in univariable analysis with a higher risk of developing GVHD in 2 of these studies.12,13 However, we are unaware of any multivariable analysis of risk factors for the development of GVHD being reported among intestinal transplant recipients. Furthermore, while it has been suggested that alemtuzumab may be useful in preventing/treating the development of GVHD following hematopoietic stem cell transplantation,17-23 we are unaware of any clinical report in intestinal transplantation that has suggested its use for this reason.

In order to determine the multivariable influence of baseline demographics, transplant-specific characteristics, and different induction regimens on the incidence rate of developing GVHD following intestinal transplantation at our center, we analyzed all intestinal transplants performed since establishment of the intestinal transplant program in 1994. Results of this observational study are presented here.

MATERIALS AND METHODS

Patients and Immunosuppression

Our cohort of 445 consecutive intestinal transplant recipients at the Miami Transplant Institute during 1994–2017 was followed prospectively through March 15, 2019 (date of last follow-up). Over the years, the center institutional review board approved each immunosuppression protocol used for these patients; all patients gave written informed consent before enrollment.

Recipients were divided into 5 induction groups. Group 1 (1994–1997) comprised 44 recipients who received no/old induction therapy (high-dose corticosteroids only in 34, anti-CD3 monoclonal antibody OKT3 in 7, and cyclophosphamide in 3). Group 2 (1998–2011) comprised 159 recipients who received an anti-CD25 monoclonal antibody (daclizumab in 156 and basiliximab in 3). Daclizumab (2 mg/kg) was given on postoperative days 0, 7, and 14 and then every 2 weeks during the first 3 months posttransplant; thereafter, daclizumab dose was reduced to 1 mg/kg every 2 weeks for the following 3 months and then stopped. Basiliximab was given in a standard dose of 10 mg intravenously twice on postoperative days 0 and 4, because the 3 recipients were small children (<35 kg). Group 3 (2001–2011) comprised 113 recipients who received alemtuzumab, with 2 different schedules being used: (a) 0.3 mg/kg ×4 (preoperatively, immediately posttransplant, and on postoperative days 3 and 7) and (b) 30 mg ×2 (on postoperative days 1 and 4). The first schedule of 4 alemtuzumab doses was given to patients transplanted before April 2004; the second schedule of just 2 alemtuzumab doses was given to patients transplanted since April 2004. Group 4 (2006–2012) comprised 34 recipients who were scheduled to receive 3 doses of rATG as induction therapy (total planned rATG dose: 5 mg/kg, with 2.0 mg/kg being given on postoperative d 0, and 1.5 mg/kg being given on postoperative d 2 and 4). However, the actual number of rATG doses that these patients received was uneven, because 12 of 34 received only the first dose, and 3 of 34 patients received only 2 doses.

It should be noted that alemtuzumab was introduced in 2001 as a tolerance induction protocol; however, because of its initially poor results in young children, starting in August, 2002, its use was limited to patients at least 4 years of age at transplant.24 Since August 2002, most of the patients who received daclizumab induction (group 2) were children, whereas most of the patients who received alemtuzumab induction (group 3) were adults. In total, the percentage of adults in groups 2 and 3 was 15.1% (24/159) versus 74.3% (84/113), respectively. In addition, only 3 of 159 of group 2 patients were transplanted since 2009 (3 young children who received basiliximab); thus, most of the children transplanted during 2009–2011 belonged to group 4.

Group 5 (2013–2017) comprised 95 recipients who received rATG/rituximab induction therapy, with a total rATG dose of 10 mg/kg divided into 5 equal doses given to each patient on postoperative days 0, 2, 4, 6, and 8 and a single dose of rituximab (150 mg/m2) given on postoperative day 1. The rationale in using a total rATG dose of 10 mg/kg was to achieve sufficiently high immunosuppressive protection against rejection risk without concomitantly increasing infection risk.9 In addition, because of concern that patients not receiving a full MV graft may be at higher ACR risk, basiliximab (40 mg) was additionally given to the subset of group 5 patients who received either an I or modified MV (MMV) transplant once every 4 weeks ×3 starting on postoperative day 14.

Maintenance immunosuppression consisted of TAC and corticosteroids (tapered off by 6–9 mo posttransplant) except in patients who received alemtuzumab induction (group 3), with whom TAC alone was planned to be used. Target TAC trough levels during the first 3 months and beyond 3 months posttransplant were 15–20 and 10–15 ng/mL for patients transplanted during 1994–1997, and 12–16 and 8–12 ng/mL for patients transplanted during 1998–2012. For patients transplanted during 2013–2017 (group 5), target TAC trough levels during the first 3 months and beyond 3 months posttransplant were 9–15 and 5–9 ng/mL, respectively.

In the attempt to achieve reduced TAC dosing in group 5 while simultaneously avoiding ACR occurrence, a mammalian target of rapamycin inhibitor was combined with TAC as maintenance in 68 of 95 patients, with 25 of 68 receiving TAC/sirolimus and 43 of 68 receiving TAC/everolimus. The mammalian target of rapamycin inhibitor was not scheduled to start until after 30 days posttransplant to allow for proper wound healing to occur.

Schedules for the treatment of ACR episodes and nonimmunosuppressive prophylactic therapy have been described elsewhere.25

Diagnosis and Treatment of GVHD

GVHD was diagnosed based on clinical symptoms at presentation, followed and confirmed by histopathologic findings of the involved organ(s): skin, native large intestine and/or rectum, native liver, lungs, and bone marrow. In group 5, donor chimerism levels in the peripheral blood were used to prospectively track each intestinal transplant case, with samples being routinely drawn weekly during the first 3 months posttransplant and monthly thereafter. An increase in the percentage of donor-derived lymphocytes in the recipient’s blood, especially when matched with clinical symptoms, aided in the clinical diagnosis of GVHD.

No consistent GVHD treatment strategy was performed over the years. In some cases, a strategy of immunosuppression reduction/withholding was performed; in other cases, treatment with an antilymphocyte agent (either rATG or alemtuzumab) was given.

Statistics

Frequency distributions were determined for baseline categorical variables, and the mean along with SE were calculated for baseline continuous variables. Tests of association among baseline variables were performed using Pearson (uncorrected) chi-square tests and ordinary (2-sided) t tests.

As of the last follow-up date (March 15, 2019), median follow-up among 59 transplant cases in induction group 5 who were alive with functioning grafts was 43 months (range, 16–68 mo) posttransplant. Because only 10 of 59 of these patients were followed beyond 60 months posttransplant, statistical evaluation was restricted to the first 60 months posttransplant for all transplanted cases in this study. Graft loss was defined as the date of intestinal graft failure (graft removal) or death, whichever occurred first, with the underlying cause of (triggering event leading to) graft loss being determined in each case.26

Differences in freedom-from-GVHD development were compared by the log-rank test, with time-to-failure curves generated using the Kaplan-Meier method. In this analysis, patients were censored at the time of graft loss (or at the time of being lost to follow-up, if it occurred). Graft survival following GVHD development was estimated using the Kaplan-Meier technique, and to allow for a more complete presentation of the results, select Kaplan-Meier comparisons of graft survival were also performed. P values ≤0.05 were considered to be statistically significant.

Stepwise Cox regression was utilized to identify the significant multivariable predictors of the hazard rate of developing GVHD during the first 60 months posttransplant. Baseline variables that were considered for their prognostic value included demographics, transplant-related information, and type of induction received (Table 1). Testing the validity of the Cox model proportional hazards assumption was performed by considering the inclusion of time by covariate interaction effects. Nelson–Aalen cumulative hazard plots were used to visually show any observed changes in covariate effects over time, with the focus being on the slopes of the curves.

TABLE 1. - Distributions of selected baseline variables (N = 445)
Baseline variable Mean ± SE if continuous; percentage with characteristic if categorical
Recipient age (y) 18.8 ± 0.9 (N = 445)
Median, 8.8; interquartile range, 1.4–34.5
Recipient age (y)
 <5 189/445 (42.5%)
 5–17 65/445 (14.6%)
 ≥18 191/445 (42.9%)
Recipient sex
 Female 219/445 (49.2%)
 Male 226/445 (50.8%)
Recipient race/ethnicity
 White (nonHispanic) 285/445 (64.0%)
 Black (nonHispanic) 80/445 (18.0%)
 Hispanic 74/445 (16.6%)
 Asian 6/445 (1.3%)
Original diagnosis
 Congenital disorder 122/445 (27.4%)
 Inflammatory bowel disease 22/445 (4.9%)
 Malignancy 34/445 (7.6%)
 Motility disorder 31/445 (7.0%)
 Rejection 34/445 (7.6%)
 Trauma 21/445 (4.7%)
 Vascular 136/445 (30.6%)
 Other 45/445 (10.1%)
CMV status
 D−/R− 142/445 (31.9%)
 D−/R+ 85/445 (19.1%)
 D+/R− 116/445 (26.1%)
 D+/R+ 102/445 (22.9%)
Donor age (y) 11.4 ± 0.6 (N = 424)
Median, 6.0; interquartile range, 1.1–18.0
Intestinal transplant status
 Primary 391/445 (87.9%)
 Retransplant 54/445 (12.1%)
Transplant type
 I 125/445 (28.1%)
 LI 38/445 (8.5%)
 MMV 39/445 (8.8%)
 MV 243/445 (54.6%)
Underwent native splenectomy
 No 163/445 (36.6%)
 Yes 282/445 (63.4%)
Native pancreaticoduodenal complex removed
 No 164/445 (36.9%)
 Yes 281/445 (63.1%)
Received a kidney
 No 405/445 (91.0%)
 Yes 40/445 (9.0%)
Received a large bowel
 No 191/445 (42.9%)
 Yes 254/445 (57.1%)
Received a liver
 No 164/445 (36.9%)
 Yes 281/445 (63.1%)
Received a pancreas
 No 142/445 (31.9%)
 Yes 303/445 (68.1%)
Received a spleen
 No 357/445 (80.2%)
 Yes 88/445 (19.8%)
Received a stomach
 No 166/445 (37.3%)
 Yes 279/445 (62.7%)
In hospital (vs at home) before transplant
 No 256/423 (60.5%)
 Yes 167/423 (39.5%)
Induction type
 Received no/old induction 44/445 (9.9%)
 Received anti-CD25 159/445 (35.7%)
 Received alemtuzumab 113/445 (25.4%)
 Received rATG (pre-2013) 34/445 (7.6%)
 Received rATG/rituximab (since 2013) 95/445 (21.4%)
CMV, cytomegalovirus; D, Donor; R, recipient; anti-CD25, antiinterleukin-2 receptor α chain (daclizumab or basiliximab); I, isolated intestine; LI, liver-intestine; MMV, modified multivisceral; MV, multivisceral; rATG, rabbit antithymocyte globulin (thymoglobulin); SE, standard error.

Stepwise logistic regression to determine the significant multivariable predictors of the likelihood of receiving alemtuzumab induction (ie, being in induction group 3) (yes/no) was also performed along with resulting propensity scores.27 Propensity scores are typically used as a way to control for the effects of any unbalanced distributions of other potentially important baseline prognosticators existing between 2 study groups (ie, selection bias). By controlling for these imbalances in the statistical analysis, the “adjustment for propensity scores” approach attempts to ensure that an unbiased comparability exists between the 2 study groups (in this case, receiving versus not receiving alemtuzumab induction). Here, the final Cox model for GVHD incidence was rerun after controlling for the propensity of receiving alemtuzumab induction.

Last, because no meaningful differences in any of the clinical outcomes were observed among the 3 less intensive induction therapy groups (ie, groups 1, 2, and 4), for the sake of clarity, these 3 groups were combined in the presentation of all results.

RESULTS

Baseline Characteristics

Baseline characteristics are shown in Table 1. Mean age at transplant was 18.8 years (median age, 8.8 y; interquartile range, 1.4–34.5 y), with African-Americans and Hispanics comprising 18.0% (80/445) and 16.6% (74/445), respectively; retransplant cases comprised 12.1% (54/445). The percentage of recipients who received I, LI, MMV, and full MV allografts were 28.1% (125/445), 8.5% (38/445), 8.8% (39/445), and 54.6% (243/445), respectively. The percentage who underwent a native splenectomy was 63.4% (282/445), and the percentage who received a donor spleen was 19.8% (88/445), respectively. Of note, native splenectomy was performed in only 2.5% (4/163) of I/LI recipients versus in 98.6% (278/282) of MMV/MV recipients (P < 0.000001). In 2 I cases having a native splenectomy, these 2 cases were retransplants of previously failed MV grafts. Similarly, the donor spleen was transplanted into no I/LI cases versus 31.2% (88/282) of MMV/MV cases.

Propensity to Receive Alemtuzumab Induction

Stepwise logistic regression found that 3 baseline factors were associated in a multivariable fashion with a significantly higher propensity to receive alemtuzumab induction (ie, induction group 3) (listed in order of selection) (Table 2): adult recipient (P < 0.000001), did not receive a liver (ie, transplant type I or MMV) (P < 0.000001), and received a spleen (P < 0.000001). Once these 3 variables were controlled, no other variables offered additional prognostic value (P > 0.05). The multivariable model’s fit was quite strong, with the distribution of resulting propensity scores being highly significantly different between not receiving and receiving alemtuzumab (P < 0.000001; Table 3).

TABLE 2. - Logistic regression model (via stepwise regression) for the propensity to receive alemtuzumab induction (overall, 113/445)
Variable a,b Selected logistic model c
P Coefficient ± SE
Adult recipient (≥18 y of age) <0.000001 1.963 ± 0.291
Transplant type I or MMV <0.000001 1.509 ± 0.274
Received a spleen <0.000001 1.813 ± 0.352
aVariables selected into the linear regression model were defined as follows: adult recipient = {1 if recipient ≥18 y of age at transplant, 0 otherwise}; transplant type I or MMV = {1 if transplant type = I or MMV, 0 otherwise}; received a spleen = {1 if recipient received a spleen, 0 otherwise}.
bThe intercept term ± SE for this 3 variable model was −1.722 ± 0.284 (P < 0.000001).
cThe 3 selected variables are listed by order of selection into the logistic regression model.
I, isolated intestine; MMV, modified multivisceral; SE, standard error.

TABLE 3. - Distribution of alemtuzumab induction propensity score by induction type (P < 0.000001)
Alemtuzumab induction Induction type
Propensity score Nonalemtuzumab Alemtuzumab
<0.10 127/332 (38.3%) 3/113 (2.7%)
≥0.10, <0.20 97/332 (29.2%) 20/113 (17.7%)
≥0.20, <0.50 56/332 (16.9%) 21/113 (18.6%)
≥0.50 52/332 (15.7%) 69/113 (61.1%)

Analysis of GVHD Incidence

GVHD was observed in 8.8% (39/445) of the transplanted cases during the first 60 months posttransplant. Median time-to-GVHD development (range) was 1.5 months (0.5–17.3 mo) posttransplant; 100% of the GVHD episodes (39/39) occurred during the first 18 months posttransplant. Patients had the following sites of GVHD: skin (N = 21), skin/GI (N = 6), GI/rectum (N = 4), skin/liver (N = 4), skin/lung (N = 2), skin/rectum (N = 1), and skin/bone marrow (N = 1).

Kaplan-Meier freedom-from-GVHD curves by transplant type (Figure 1) show that the hazard rate of developing GVHD was significantly higher among MMV and MV versus I and LI transplant types (P = 0.0002). Actuarial estimates of freedom-from-GVHD at 18 months posttransplant were 97.0%, 100.0%, 77.8%, and 85.2% for I, LI, MMV, and MV transplant types, respectively (Figure 1); in fact, only 3 of the combined 163 I and LI recipients were observed to develop GVHD.

FIGURE 1.
FIGURE 1.:
Kaplan-Meier freedom-from-graft vs host disease (GVHD) curves by 4 transplant types (isolated intestine [I], liver-intestine [LI], modified multivisceral [MMV], and multivisceral [MV]) during the first 18 mo posttransplant.

Freedom-from-GVHD curves by induction group (Figure 2) shows a significantly higher hazard rate of developing GVHD among the 4 nonalemtuzumab induction groups combined in comparison with receiving alemtuzumab induction (P = 0.01). The actuarial estimate of freedom-from-GVHD at 18 months posttransplant was 95.6% for the alemtuzumab induction group versus 87.2% for induction groups 1, 2, and 4 combined versus 85.0% for induction group 5, respectively. While 3 of 113 patients who received alemtuzumab induction developed GVHD, only 1 of 113 developed GVHD during the first 6 months posttransplant. In fact, the cumulative hazard plot in Figure 3 shows that the hazard rate of developing GVHD (slope of the curve) was significantly lower among alemtuzumab induction recipients but only during the first 6 months posttransplant (P = 0.003). Beyond 6 months posttransplant, there appeared to be no further beneficial effect of having received alemtuzumab induction (P = 0.45).

FIGURE 2.
FIGURE 2.:
Kaplan-Meier freedom-from-graft vs host disease (GVHD) curves by 3 induction therapy groups (other vs alemtuzumab vs rabbit antithymocyte globulin [rATG]/rituximab) during the first 18 mo posttransplant.
FIGURE 3.
FIGURE 3.:
Nelson–Aalen cumulative hazard plot of the hazard rate of developing graft vs host disease (GVHD) comparing patients who received vs did not receive alemtuzumab induction.

Excluding the 113 recipients who received alemtuzumab induction, freedom-from-GVHD curves by transplant type (I/LI versus MMV/MV) and liver inclusion (no/yes) (Figure 4) show a significant protective effect of liver inclusion (P = 0.003, stratified log-rank test comparing LI versus I in 1 stratum, and MV versus MMV in the other stratum). The actuarial estimate of freedom-from-GVHD at 18 months posttransplant was 100.0% versus 95.4% for LI versus I and 84.1% versus 55.6% for MV versus MMV transplant types, respectively.

FIGURE 4.
FIGURE 4.:
Excluding patients who received alemtuzumab induction, Kaplan-Meier freedom-from-graft vs host disease (GVHD) curves by modified multivisceral (MMV)/multivisceral (MV) (no vs yes), and liver inclusion (no vs yes) (ie, liver-intestine [LI] vs isolated intestine [I] as 1 stratum, and MV vs MMV as the other stratum) during the first 18 mo posttransplant.

Three multivariable predictors were selected into the Cox model indicating a significantly higher hazard rate of developing GVHD (listed by order of selection) (Table 4): transplant type MMV or MV (P = 0.00003), not receiving alemtuzumab induction (P = 0.004), and not receiving a liver (P = 0.009). Once the 3 selected variables were controlled, none of the other variables listed in Table 4 offered additional prognostic value (P > 0.15). For instance, a native splenectomy was associated with a significantly higher hazard rate of GVHD development in univariable analysis (P = 0.002); however, as stated above, a native splenectomy was much more likely to be performed among MMV/MV versus I/LI recipients. Thus, once the 3 selected baseline variables in Table 4 were controlled, the multivariable P to include native splenectomy was not significant (P = 0.30). Also of note, recipient age, donor age, and receiving a donor spleen were not associated with the hazard rate of developing GVHD in either univariable or multivariable analysis (Table 4). In addition, Table 5 shows that these Cox model results remained unchanged after controlling for the propensity to receive alemtuzumab induction.

TABLE 4. - Cox model for the hazard rate of developing GVHD during the first 60 mo posttransplant (39 events): selected Cox model via stepwise regression
Univariable Multivariable model
Baseline variablea P P Coefficient ± SE Hazard ratio (95% CI)
Recipient age 0.37
Recipient age ≥5 y 0.45
Recipient age ≥18 y 0.21
Male recipient 0.25
Black (nonHispanic) recipient 0.67
Hispanic recipient 0.56
Intestinal retransplant 0.32
CMV antibody status: D+/R− 0.56
Donor age 0.86
Transplant type I 0.003
Transplant type LI 0.09
Transplant type MMV 0.04
Transplant type MV 0.01
Transplant type MMV or MV 0.0002 (√) 0.00003 2.633 ± 0.672 13.91 (3.73-51.96)
Received donor liver (LI or MV) 0.11 (√) 0.009 −1.107 ± 0.429 0.33 (0.14-0.77)
Received donor spleen 0.46
Received donor large bowel 0.40
Received native splenectomy 0.002
In hospital pretransplant 0.71
Received other/no induction 0.36
Received alemtuzumab induction 0.01 (√) 0.004 −1.712 ± 0.635 0.18 (0.05-0.63)
Received rATG/rituximab induction 0.15
Propensity to receive alemtuzumab 0.21
(√) represents selection into the Cox model.
aVariables included in the Cox model were defined as follows: transplant type MMV or MV = {1 if transplant type = MMV or MV, 0 otherwise}; received alemtuzumab induction = {1 if recipient received alemtuzumab induction, 0 otherwise}; received donor liver (LI or MV) = {1 if transplant type = LI or MV, 0 otherwise}. The order of selection for the 3 baseline variables selected into the Cox model via stepwise regression was as follows: transplant type MMV or MV, received alemtuzumab induction, and received donor liver (LI or MV). Once the 3 selected variables were controlled, none of the other variables listed in Table 4 offered additional prognostic (P > 0.15).
CI, confidence interval; GVHD, graft vs host disease; I, isolated intestine; LI, liver-intestine; MMV, modified multivisceral; MV, multivisceral; rATG, rabbit antithymocyte globulin; SE, standard error.

TABLE 5. - Cox model for the hazard rate of developing GVHD during the first 60 mo posttransplant (39 events): final Cox model, controlling for propensity to receive alemtuzumab induction
Baseline variablea Multivariable model Hazard ratio (95% CI)
P Coefficient ± SE
Transplant type MMV or MV 0.00004 2.693 ± 0.690 14.78 (3.82-57.17)
Received alemtuzumab induction 0.008 −1.644 ± 0.658 0.19 (0.05-0.70)
Received donor liver (LI or MV) 0.03 −1.274 ± 0.597 0.28 (0.09-0.90)
Propensity to receive alemtuzumab 0.69 −0.474 ± 1.199 0.62 (0.06-6.52)
aVariables included in the Cox model were defined as follows: transplant type MMV or MV = {1 if transplant type = MMV or MV, 0 otherwise}; received alemtuzumab induction = {1 if recipient received alemtuzumab induction, 0 otherwise}; received donor liver (LI or MV) = {1 if transplant type = LI or MV, 0 otherwise}; propensity to receive alemtuzumab (continuous variable).
CI, confidence interval; GVHD, graft vs host disease; LI, liver-intestine; MMV, modified multivisceral; MV, multivisceral; SE, standard error.

Table 6 shows (as reflected in Figure 3) that the protective effect of induction group 3 (alemtuzumab) disappeared over time. Specifically, after controlling for the effects of transplant type (I/LI versus MMV/MV) and liver inclusion (no/yes) in the Cox model, there was a significant protective effect of receiving alemtuzumab induction during the first 6 months posttransplant (P = 0.009) that apparently disappeared beyond 6 months posttransplant (P = 0.73).

TABLE 6. - Cox model for the hazard rate of developing GVHD during the first 60 months posttransplant (39 events): final Cox model including the alemtuzumab × time interaction effects
Multivariable model
Variablea P Coefficient ± SE Hazard ratio (95% CI)
Transplant type MMV or MV 0.0001 2.613 ± 0.671 13.64 (3.66-50.81)
Received alemtuzumab (during first 6 mo) 0.009 −2.704 ± 1.034 0.07 (0.009-0.51)
Received alemtuzumab (beyond 6 mo) 0.73 0.325 ± 0.943 1.38 (0.22-8.79)
Received donor liver (LI or MV) 0.01 −1.081 ± 0.429 0.34 (0.15-0.79)
aVariables included in the Cox model were defined as follows: transplant type MMV or MV = {1 if transplant type = MMV or MV, 0 otherwise}; received donor liver (LI or MV) = {1 if transplant type = LI or MV, 0 otherwise}; received alemtuzumab (during first 6 mo) = {1 if recipient received alemtuzumab induction and time ≤ the first 6 mo posttransplant, 0 otherwise}; received alemtuzumab (beyond 6 mo) = {1 if recipient received alemtuzumab induction and time >6 mo posttransplant, 0 otherwise}.
CI, confidence interval; GVHD, graft vs host disease; LI, liver-intestine; MMV, modified multivisceral; MV, multivisceral; SE, standard error.

It should be noted that 0 of 54 versus 3 of 59 of the alemtuzumab-induced patients who received 4 versus 2 induction doses of alemtuzumab had developed GVHD; however, this observed difference was not statistically significant (P = 0.13).

Although not shown in Table 4, none of the original diagnosis categories as depicted in Table 1 were associated with a significantly higher hazard rate of GVHD development. However, it should be noted that the 22 patients with intestinal atresia (a subcategory of congenital disorder) had a significantly higher GVHD rate both in univariable (P = 0.002) and multivariable (P = 0.0001) analysis. Among I/LI recipients, the observed incidence of GVHD was 0.7% (1/153) versus 20.0% (2/10) among those without versus with intestinal atresia; among MMV/MV recipients, the observed incidence of GVHD was 11.9% (32/270) versus 33.3% (4/12) among those without versus with intestinal atresia. However, because of the relatively small sample of patients having intestinal atresia as their original diagnosis, this variable was not included into the Cox model results in Tables 4–6.

Death Due to GVHD

The observed percentage of patients who subsequently died of GVHD following its occurrence was 33.3% (13/39). Figure 5 shows that the actuarial percentage of patients who died of GVHD was 42.6% ± 9.2% at 18 months following its occurrence. An additional 13 patients had graft loss following GVHD of other causes, including 5 patients who died of infection (3 due to sepsis, 2 due to pneumonia). Cox stepwise regression of the hazard rate of death due to GVHD following its occurrence yielded no significant predictors (P > 0.05).

FIGURE 5.
FIGURE 5.:
Kaplan-Meier freedom-from-death due to graft vs host disease (GVHD) following GVHD development (all patients combined).

Graft Survival by Induction Group

In a recent report,25 we showed that while the hazard rate of graft loss due to rejection was significantly lower during the first 6 months posttransplant among the more intensive induction therapy groups (alemtuzumab and rATG/rituximab), the hazard rate of graft loss due to rejection beyond 6 months as well as the hazard rates of graft loss due to infection and graft loss due to other causes were each higher for induction group 3 (alemtuzumab) in comparison with induction group 5 (rATG/rituximab). Thus, Figure 6 shows that graft survival was significantly more favorable for patients in induction group 5 versus all other induction groups combined (P = 0.0002), with no noticeable difference in graft survival observed between induction group 3 (alemtuzumab) versus the other/no induction groups (1, 2, and 4) combined (P = 0.55).

FIGURE 6.
FIGURE 6.:
Kaplan-Meier graft survival curves by 3 induction therapy groups (other vs alemtuzumab vs rabbit antithymocyte globulin [rATG]/rituximab) during the first 60 mo posttransplant.

DISCUSSION

We believe that this is the first report in intestinal transplantation to show the results of a multivariable analysis of predictors of GVHD development. Other studies either did not contain a sufficient number of events12,13 or were too small in size14,15 to be able to perform such an analysis. Cox stepwise regression found that 3 baseline variables were associated with a significantly higher GVHD hazard rate: MMV/MV transplant type, not receiving alemtuzumab induction, and not receiving a donor liver. The favorable effect of alemtuzumab induction remained even after controlling for the propensity to receive alemtuzumab induction, indicating that these results were most likely not due to selection bias.

The increased risk of GVHD among MMV/MV recipients has been consistently suggested by 5 other intestinal transplant studies,12-16 including the previous report from our center by Wu et al.13 In Mazariegos et al,12 the incidence of biopsy-proven GVHD was 4.4% (9/204) for I/LI versus 10.9% (5/46) for MMV/MV recipients (P = 0.09 by Pearson chi-square test). Biopsy-proven GVHD incidence was reported to be 2.4% (1/41) for I/LI versus 29.4% (5/17) for MMV/MV recipients (P = 0.002) in Feito-Rodríguez et al,14 0.0% (0/6) for I/LI versus 25.0% (5/20) for MMV/MV recipients (P = 0.17) in Cromvik et al,15 and 6.5% (4/62) for I/LI versus 19.0% (33/174) for MMV/MV recipients (P = 0.02) in Clouse et el.16 While the logic behind greater removal of native lymphatic tissue and/or addition of greater donor lymphatic tissue with MMV/MV (versus I/LI) transplantation would explain the higher GVHD risk in these recipients, it is unclear at this time as to why liver inclusion might offer a protective role.

While this is the first intestinal transplant study to show a significantly favorable effect of alemtuzumab induction in lowering the hazard rate of developing GVHD posttransplant, a previous in vitro study28 from our center demonstrated a significant reduction in the alloreactive response of deceased donor intestinal lymphocytes by exposing the recipient intraoperatively to alemtuzumab. The authors’ main conclusion was that alemtuzumab use may reduce both graft versus host and host versus graft responses.

Alemtuzumab has also previously been shown to be useful in both preventing and treating GVHD following hematopoietic stem cell transplant.17-23 In one of these studies,17 it was proposed that alemtuzumab “abrogates” GVHD not only by T-cell depletion but also by removing native monocyte-derived dendritic cells (which express CD52 abundantly) and their precursors, thereby mitigating monocyte-derived dendritic cell phagocytosis and presentation of recipient-derived antigens to donor T cells in the inflammatory peritransplant period, limiting GVHD. In 4 relatively small studies using alemtuzumab to treat steroid refractory acute GVHD following hematopoietic stem cell transplant,18,20,21,23 a complete response was achieved in 27.8%–45.8% of patients, with survival being dismal among adults who were not able to achieve a complete response. These results were reasonably consistent with our findings in that a large percentage (at least one-third) of our intestinal transplant cohort who developed GVHD ultimately died of GVHD.

A recent observational study25 using the same cohort with the same length of follow-up found that the more recent induction combination of rATG/rituximab (using a total of 10 mg/kg of rATG during the first 8 d posttransplant) was associated with a significantly more favorable graft survival in comparison with the 4 older induction groups (also shown here in Figure 6); however, no specific unfavorable effect of alemtuzumab induction on overall graft survival was observed in comparison with the 3 other older (and less intensive) induction groups. Because this particular GHVD study was observational in nature (in spite of performing the clinical follow-up of patients prospectively over time), it is not clear what the optimal timing and dose of alemtuzumab might be (and whether/how it might be combined with other induction agents) in the attempt to best protect patients against development of GVHD as well as other adverse events (ACR, infection, etc). Clearly, based on the overall graft survival results, we are certainly not recommending to replace rATG/rituximab with alemtuzumab.

Numerous study limitations did exist. First and foremost is the fact that we are reporting the results of an observational study—the gold standard for reporting comparisons of immunosuppressive regimens would be a randomized clinical trial. Second, while this is one of the largest series of intestinal transplant cases to be reported to date, these patients were transplanted over a long period of time and were heterogeneous in many aspects. The relatively rare occurrence of GVHD limited our ability to determine a complete set of multivariable predictors with good statistical power and may have caused the statistical (large sample) tests to become less reliable (increasing the likelihood of observing spurious associations, unless the type I error was set lower). Third, routine use of donor chimerism (and changes in donor chimerism) levels to prospectively track GVHD occurrence began at our center in 2013; thus, demonstration of its usefulness in predicting (i) GVHD development in advance of biopsy proof and (ii) when to proactively treat in the attempt to limit GVHD severity will await further data.

In summary, we believe that the results presented here may help advance the current state of knowledge about risk factors for GVHD development following intestinal transplantation, with an additional suggestion made for possibly including a small dose of alemtuzumab (or some other immunosuppressive agent that provides successful prophylaxis against GVHD) into current immunosuppression protocols but without overimmunosuppressing the patients, in an attempt to better prevent and/or treat GHVD occurrence following intestinal transplantation.

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