Late Acute Rejection After Liver Transplantation: The Western Canada Experience

Alnoor Rdmji, * Eric M. Yosbidu, * Vincent G. Buin,' Normun M. Knetemun," Cburles H. Scudumore, ' Mung M. MU,' Urs P. Steinbrecber, * Kluus S. Gu@eund,l

Siefiied R. Erb, * Nilufdr Purtovi,' Stephen 'W: Cbung, fJdmes Sbupiro," und Winnie KS. Won?

Acute rejection usually occurs within 3 months posttrans- plantation. Most centers reduce immunosuppression over 6 to 12 months to minimize opportunistic infection, malignancy, and drug toxicity. Pretransplant disease and low immunosuppression have been reported in associa- tion with late acute rejection (LAR). The objective of this study was to determine the incidence, predictive factors, and outcomes of LAR via retrospective review of adult liver transplant recipients in Western Canada from 1989 to 2000. LAR was defined as biopsy-proven acute rejec- tion occurring more than 180 days posttransplantation. Patient characteristics, immunosuppression, and out- come were determined. Both a univariate and multiple logistic regression analysis were performed. LAR occurred in 97 (23%) of 415 patients more than 180 days posttrans- plantation. Median follow-up was 402 days (range, 180 to 3 137 days); 79% of LAR episodes were graded mild. At the time of LAR, 33% were on a steroid taper. A total of 73% of LAR episodes were treated with pulse intravenous ste- roids, and 5% were steroid-resistant. In the univariate analysis, patients undergoing transplantation for viral eti- ologies and older age were associated with less LAR. Immunosuppression was significant in a multiple logistic regression model, but not with a proportional hazards model. On multivariate analysis, only patients undergo- ing transplantation for viral etiologies remained resistant to LAR (hazard ratio, 0.52; range, 0.34 to 0.93, P = .02). There was a trend toward increased chronic rejection in patients who developed LAR (P = .04). LAR is common and occurs after more than 1 year posttransplantation. Patients undergoing transplantation for viral etiologies seem to have a lower risk of LAR. There may be an increased risk of chronic rejection in those developing LAR. (Liver Trunspl2002;8:945-951.)

P osttransplant survival has continued to improve such that both patients and clinicians can antici-

pate a l-year survival of 85% and 3-year survival of 78Yo.l With improved patient and graft survival, increasing attention is being focused on long-term maintenance immunosuppression with a view to minimizing the adverse effects of immunosuppressive drugs. Although acute rejection continues to be a fre- quent posttransplantation most acute rejec- tion occurs in the first 3 months posttransplantation3 and more patients die of infection than rejection, sug- gesting a possible historic trend toward overimmuno- suppression.5 In the past decade, studies of the newer

immunosuppressants, such as tacrolimus and myco- phenolate mofetil, have reported lower incidence, milder severity, and less steroid-resistant acute rejec- tion.6-8 Accordingly, most centers now wean patients from immunosuppression in the first year posttrans- plantation and attempt to discontinue steroids in the first 6 months.9 In most cases, a significant degree of graft immunotolerance occurs, possibly through the mechanism of microchimerism, l0 and maintenance immunosuppression can be successfully tapered to min- imal levels.

Acute rejection occurring after the immediate and early posttransplantation period is termed late acute rejection (LAR); however, there is no uniform defini- tion. Despite the fact that there are reports of patients who are able to completely withdraw from immuno- suppression without adverse effects,11,12 there is a con- tinued risk of rejection in patients who have had tapered from immunosuppression. Importantly, LAR can be associated with a risk of graft loss and morbidity. l3>I4 In previous investigations of LAR, multiple factors have been assessed, searching for possible associations. Underlying liver disease and cyclosporine levels have been reportedly associated with LAR2,5,l5; however, published studies have failed to consistently show an association between rejection and HLA compatibility,

From the *Departments ofMedicine and fSurgey, the University of British Columbia, Vancouver, BC; the #Department of Pharmacy, the Vancouver General Hospital and the British Columbia Transplant Soci- eg, Vancouver, BC; and the Departments of §Medicine and JJSurgey, the University ofAlberta, Edmonton, AB, Canada.

Results of this study were presented in posterform at the 50th Annual Meeting of the American Association for the Study of Liver Disease, October 2000, D a l h , X .

Supported in part by an unrestricted research grant fiom Fujisawa-

Address reprint requests to Eric M. Yoshida, MD, FRCPC, Division of Gastroenterology, Vancouver General Hospital, 100-2647 Willow St, Vancouver, BC, V52 3P1, Canada. Telephone: 604-875-5371; FAX: 604-875-5447; E-mail: eyoshida@interchange. ubc.ca

Liver Diseases

Canada, Inc.

Copyright 0 2002 by the American Association for the Study of

1527-6465/02/0810-0014$35.00/0 doi:l 0.1053/jlts.2002.34969

Liver Transplantation, Vol8, No l0 (October), 2002:pp 945-951 945

946 Ramji et a1

cytomegalovirus (CMV) mismatch, gender, and preser- vation injury.I6-l9 Overall, there have been relatively few studies of LAR after liver transplantation. Immu- nosuppression, in particular, has not been well studied in this context. The schedule of long-term immunosup- pressive maintenance therapy remains largely empiric, and center-specific. Moreover, within most centers, patients are treated with the same immunosuppression schedules and the underlying assumption is that all grafts have similar risks of rejection.

The last study of LAR was performed at one of our two centers in 1993,12 and LAR continues to occur despite the greater variety of available immunosuppres- sive agents since that time. We decided to review LAR in Western Canada to determine its incidence, predic- tive factors, and outcome. In particular, we were inter- ested in the immunosuppression regimen.

Patients and Methods

A retrospective chart review was performed on all liver trans- plant recipients from the two transplant centers in Western Canada, Edmonton and Vancouver, between 1989 and March 2000. The study period incorporated the conversion from gelatin capsule cyclosporine (Sandimmune; Nomartis- Canada, Domal, QC) to microemulsion cyclosporine (Neoral; Nomartis-Canada) in 1993 and the introduction of tacrolimus (Prograf; Fujisawa-Canada, Markham, ON), mycophenolate mofetil (Cellcept; Roche-Canada, Missis- sauga, O N ) in 1996, and sirolimus (Rapamune; Wyeth-Ay- erst Canada, Toronto, ON) in 1999. Muromonab-CD3 (Othoclone OKT3; Janssen-Ortho, Toronto, ON) was used for initial postoperative induction immunosuppression in Vancouver from 1989 and discontinued in early 1996. Simi- larly, Minnesota ALG as an initial induction agent was dis- continued in Edmonton in 1993.

LAR was defined as clinically suspected (on the basis of elevated liver enzymes) and biopsy-proven acute rejection, occurring at or more than 180 days after transplantation. Exclusion criteria were age less than 18 years, and a follow-up period of less than 6 months. In cases in which a patient had more than one LAR, only the first biopsy-proven rejection was counted as an event.

Demographic characteristics collected included recipient age, gender, underlying liver disease at transplantation, C M V status, and induction and maintenance immunosuppression. Underlying liver disease was categorized into autoimmune, viral, alcoholic, or primary nonviral hepatoma. Transplanta- tion for toxin-induced metabolic diseases, fulminant hepati- tis, and cryptogenic states were combined. The autoimmune category included primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and autoimmune hepatitis. Viral etiology included hepatitis B, hepatitis C, concurrent alcoholic liver disease with hepatitis B or C, and hepatocellu-

lar carcinoma secondary to hepatitis B or C. Donor charac- teristics of age, gender, and CMV status were also collected.

Severity of LAR was categorized as histologically mild, moderate, or severe acute rejectionz0; biochemical measures were included. Immunosuppression and steroid taper, includ- ing drug dosages and drug serum levels at rejection, and changes within the prior 8 weeks were noted. Patients were followed up for response to rescue therapy. Early acute rejec- tion (EAR) was defined as histologically confirmed acute rejection occurring less than 180 days posttransplantation. Chronic rejection (CR) was diagnosed by standard histologic criteria.21

Patients were followed up through the last visit or com- munication with the transplantation clinics. Immunosup- pression regimen and serum levels within 2 months of final follow-up were considered. Further duration of steroid dis- continuation or last steroid dose was recorded. Patients were followed up for progression to chronic rejection, recurrent or de novo liver disease, malignancy, graft failure with retrans- plantation, and death.

Univariate and multivariate analysis were conducted for predictive factors and outcome measures of LAR. Categorical complications of LAR (chronic rejection, recurrent disease, malignancy, retransplantation, and death) were compared between patients with and without LAR by Chi-squared or Fisher’s exact test. Student’s t-test or Wilcoxon rank sum test was used to analyze continuous variables. Because of the vari- ation in follow-up duration of the cohort, predictive (categor- ical variables) factors are determined by examining LAR-free survival using univariate and multivariate Cox regression. Multivariate analysis was also performed using multiple logis- tic regression, both forward and backward stepwise. The level of significance for a two-tailed test was set at .05 (P < . O S ) . Analyses were performed using the computer statistical soft- ware package SAS, version 8.0 (SAS Inc, Cary, NC).

Results

During the study period, 524 transplantations were performed in 506 recipients. One hundred nine trans- plantations were excluded because of a follow-up of less than 6 months from transplantation or because the recipient was not an adult (age at transplant less than 18 years). Four hundred fifteen transplantations in 397 recipients were reviewed for episodes of LAR, with median follow-up of 1180 days (range, 18 1 to 3756 days). The study population consisted of 234 (56%) male and 181 (44%) female patients. The most com- mon indications for transplantation were end-stage liver disease secondary to chronic viral hepatitis (33%). Hepatitis C accounted for 105 (25.5%) transplanta- tions, hepatitis B for 27 (6.5%), and hepatitis B and C coinfection for 2 (0.5%). One hundred twenty-two (29.5%) patients underwent transplantation for auto- immune diseases, with the majority (16. l YO) being for

Late Acute Rejection in Western Canada 947

TaMo l. Reasons for Liver T r ~ ~ p ~ ~ ~ ~

No. of Patients (%) Etiology of Liver Disease (N = 415)

Viral 136 (32.8) Chronic HBV 18 (4.4) HBV + hepatocellular carcinoma 8 (1.9) HBV + HCV 2 (0.5) HBV + alcohol 1 (0.2) HBV + HDV 1 (0.2) HCV 81 (19.5) HCV + alcohol 18 (4.3) HCV + hepatocellular carcinoma 6 (1.5) Acute HAV 1 (0.2)

Immune-mediated 122 (29.4) Autoimmune hepatitis 23 (5.5) Primary biliary cirrhosis 67 (16.1) Primary sclerosing cholangitis 32 (7.7)

Alcoholic liver disease 46 (11.1) Alcohol 44 (1 0.6) Alcohol + hepatocellular carcinoma 2 (0.5)

Others 111 (26.7) Retransplantation 26 (6.3) Cryptogenic 29 (7.0) Fulminant liver failure 7 (1.7) Miscellaneous 49 (11.8)

PBC. Alcoholic liver disease accounted for 46 (1 1.1 Yo) transplantations (Table 1).

In this group of 4 15 transplantations, there were 97 episodes of LAR (23%). LAR occurred at a median posttransplantation time of 402 days (range, 180 to 3 137 days). The posttransplantation period between 6 months and 1 year accounted for 43% of LAR episodes. At LAR, 32 (33%) of patients had undergone a steroid taper within the prior 8 weeks, 15 (16%) were not on any steroids, and 48 (50%) had a steroid dose of pred- nisone 5 mg or less daily. In those who developed LAR, 17 (l 8%) had had subtherapeutic cyclosporine or tacrolimus levels at least once during the preceding 8 weeks, 4 in the tacrolimus group (level 5 5 ng/mL) and 13 in the cyclosporine group (level 5 100 ng/mL). When evaluated by biopsy, 79% of LAR episodes were graded as mild, 18% as moderate, and 3% as severe rejection. The majority of LAR episodes (70%) were treated with intravenous steroids (i.e., pulse methyl- prednisolone), and the rest of the rejection episodes were treated with an increase in oral prednisone or a change in calcineurin inhibitor agent (i.e., cyclosporine to tacrolimus). Six percent of LAR episodes were ste- roid-resistant and required OKT3.

Univariate analysis of patient characteristics of those who developed LAR were compared with those who

remained LAR-free during the study period (Table 2). Etiology of liver disease at transplantation was a signif- icant factor. Those patients undergoing transplantation with a pretransplant diagnosis of viral liver disease were less likely to develop LAR (hazard ratio, 0.55; range, 0.33 to 0.91). The age of a patient was also a predictive factor for LAR. The patients who developed LAR were younger, with a mean age of 45.7 years, compared with 49. l years in those who did not develop LAR (P = .O 1). Patients undergoing transplantation at one of the ten-

ters (Alberta) were less likely to develop LAR compared with those at the other center (hazard ratio, 0.66; range, 0.44 to 0.98). Recipient gender and CMV status and EARS were not predictive of the development of LAR. In those who had an episode of EAR (n = 203), treat- ment with intravenous steroids was associated with lower risk of LAR (hazard ratio, 0.41; range, 0.23 to 0.71).

In the analysis of maintenance immunosuppression, cyclosporine was compared with tacrolimus. Immuno- suppression either at the time of transplantation or at the time of LAR (or at follow-up for those who did not develop LAR) were predictive of LAR only if LAR was categorized as a binary variable (i.e., present or absent) and analyzed by logistic regression. In this analysis,

Table 2. Demographic Characterkics md Predictive Factors €or LAR: Univariatt: Analysis, LAR-Free Sunrival

(Cox Regression)

Hazard Ratio P Patient Characteristics (95% CI) Value

Viral liver disease 0.55 (0.33-0.91) .02 Transplantation site 0.66 (0.44-0.98) .04 Male donor 1.04 (0.62-1.72) .89 Male recipient 0.80 (0.54-1.19) .27 Young recipient (age 5 45 y) 1.18 (0.79-1.76) .41 Recipient CMV positivity 0.92 (0.60-1.40) .69 Use of intravenous steroids in

FAR 0.41 (0.23-0.71) .002

Cyclosporine regimen at time of transplantation* 1.30 (0.57-2.97) .54 Cyclosporine regimen at time of LAR* 0.36 (0.19-0.69) .002 Cyclosporine maintenance regimen at last Follow-up* 1.60 (0.87-2.94) . l 3

Abbreviations: LAR, late acute rejection; CMV, cytomegalo- virus; EAR, early acute rejection. *Risk of LAR with cyclosporine versus tacrolimus regimens at three observation points: initial time of transplantation time of LAR, and time of last follow-up (i.e., long-term mainte- nance regimen).

Immunosuppression:

948 Ramji et al

LAR was more likely to develop in patients who were treated with maintenance cyclosporine compared with tacrolimus, 28% versus 14% respectively (P = .006). When the data set was analyzed with a Cox propor- tional hazards model, in which the likelihood of LAR- free survival over time was examined, immunosuppres- sion was not predictive of LAR (hazard ratio, 1.6; range, 0.87 to 2.94). In the study cohort, we note that patients treated with tacrolimus had a shorter median follow-up when compared with patients treated with cyclospor- ine, 844 days versus 1429 days (P < .OOl) . In the sub- group of patients who developed LAR, the LAR-free survival was longer in those who were on cyclosporine at the time of LAR (hazard ratio, 0.36; range, 0.19 to 0.69). Kaplan-Meier plots of LAR-free survival accord- ing to various predictive factors are shown in Figure 1 .

The independence of predictive factors was analyzed using multivariate analysis: etiology at transplantation, age, gender, presence of prior EAR, and maintenance immunosuppression. O n multivariate analysis, only patients undergoing transplantation with a pretrans- plantation diagnosis of viral liver disease (hazard ratio, 0.52; range, 0.34 to 0.93, P = .O2) were found to be less likely to develop an episode of LAR.

Patients were followed up for CR, recurrent liver disease, biliary complications, graft loss, and death. In patients with an LAR episode, CR developed in 9 (9%), whereas CR developed in only 12 (4%) patients who did not have an LAR episode. Overall the number of cases of CR that developed is low. There was no increased risk of recurrent liver disease, biliary compli- cations, graft loss, or death in patients who had experi- enced an LAR episode (Table 3).

Discussion

The majority of acute rejection (40% to 60%) occurs in the first few months after liver transplantation,3,6J and most centers aim to taper off steroids in the first 6 months posttransplantation and minimize mainte- nance immunosuppression within the first year. Although this reduction in the immunosuppression regimen is driven by the need to reduce the risks of immunosuppression, including opportunistic infec- tion, malignancy, and drug toxicity, it is also clear that LAR can occur. In our study, we found a 23% incidence of LAR, with more than half of these episodes occurring more than 1 year posttransplantation. Other studies have reported lower rates of LAR (7% to 10%).2,22~3 Our finding of a higher rate of LAR may be related to the longer median follow-up time in our study, because more than 50% of LAR cases occurred after the first

1 .oo

0.95

0.90

0.85

0.80

0.75

0.70

0.65

0.60

0 1000 2000 3000

Days Post Transplant

0.95 ‘moom 0.80

0.75

0.70

0.65 ’, cyclosporine

I v

I I I -~~ts n

0 1000 2000 3000

Days Post Transplant

Figure 1. Kaplan-Meier plots of LAR-free survival based on univariate precictors. (A) Etiology of liver disease (haz- ard ratio, 0.55; 0.33-0.91; P = .02). (B) Immunosuppres- sion (at time of LAR for those with LAR and at last fol- low-up evaluation for those with no LAR) (hazard ratio, 1.60; 0.87-2.04; P = .14).

year posttransplantation and the mean follow-up time was longer in those cases in which LAR developed, compared with those in which it did not. The differing incidences may also be reflective of different definitions ofwhat constitutes LAR (i.e., more than 6 v 12 months posttransplantation) or greater center-specific histo- logic surveillance of these patients.

In our study we found that the etiology of liver disease at the time of transplantation was predictive of LAR. Patients undergoing transplantation for viral liver disease had fewer LAR episodes as determined by mul- tivariate analysis (hazard ratio, 0.52). Previous stud- ies5,l5 have also described lower rates of acute rejection in those undergoing transplantation for viral liver dis-

Late Acute Rejection in Western Canada 949

Complication LAR (%) NO LAR (Yo) (n = 97) (n = 318) Odds Ratio (95% CI) P Value

Chronic rejection Recurrent disease Malignancy Retransplantation Death

2.58 (1.05-6.33) 4.13 (0.52-33.0) 0.93 (0.30-2.91) 1.68 (0.61-4.61) 0.95 (0.48-1.90)

.04

. l8

.9 1

.3 1

.89

ease. The mechanism by which patients with primary viral disease seem to be less likely to develop LAR is unclear. Some have suggested a persistent defect in cell- mediated immunity.5,*5,23 It is also accepted that patients undergoing transplantation for hepatitis C who are treated for rejection have a higher rate of disease r e c ~ r r e n c e . ~ ~ In patients with hepatitis C and elevated liver biochemistry, sometimes only clearly moderate to severe episodes of acute rejection are treated and histo- logically mild episodes on a background of graft hepa- titis may be managed expectantly. Furthermore, the biochemical and histologic findings in recurrent hepa- titis C and mild rejection can be similar, thereby creat- ing an element of uncertainty in the actual diagnosis. Theoretically, possible episodes of rejection may be cat- egorized as grafi hepatitis, especially if they do not lead to treatment with increased immunosuppression, thereby creating a classification bias. In our cohort, we think that this is unlikely because disease recurrence was similar in patients with and without LAR. LAR epi- sodes in our patients also resolved with therapy, which would be atypical for viral grafi hepatitis. Autoimmune liver diseases have previously been reported to be a risk factor for increased acute rejection, particularly in the first 6 months po~ttransplantation.59~5 It has been sug- gested that this is secondary to a hyperactive immune system. In our study, we could not detect a statistically significant increased risk of LAR in this group of patients. When our cohort was categorized into immune versus nonimmune etiology, there was a trend toward increased LAR in the autoimmune group, but this did not reach significance (hazard ratio, 1.5; P = 3).

In our cohort, patients with EAR did not have an increased risk of LAR. This finding is consistent with findings that have been reported previously.2 Interest- ingly, we found that patients treated with intravenous steroids for EAR had a lower risk of LAR. This may suggest that in some patients who do not develop EAR, either immunologic anergy may not be completely

achieved with standard maintenance immunosuppres- sion despite normal liver biochemistry, and the graft is at risk of immunologic rejection when immunosup- pressive medications are tapered, or an episode of EAR somehow increases the likelihood of later immunologic tolerance. LAR as a reflection of incomplete immuno- logic anergy/tolerance is supported by reports that LAR has been associated with subtherapeutic levels of cyclo- sporine in up to 50% of patients with In our patients, we found that at the time of LAR, 33% of patients had undergone a recent prednisone taper, and 18% had had subtherapeutic levels of immunosuppres- sion during the prior 8 weeks. We were not, however, able to show that the overall maintenance steroid dose was different in patients developing LAR. In terms of steroid-resistant rejection, our cohort showed a rela- tively low rate, with only 6% requiring OKT3. There is little published information on steroid-resistant LAR

In the three large clinical tacrolimus versus cyclo- sporine studies,GJ,25 a lower risk of EAR was reported in the tacrolimus arm. When we looked at tacrolimus versus cyclosporine use as a variable for increased risk of LAR, we found tacrolimus was less likely to be associ- ated with LAR on logistic regression analysis, but this same finding was not present when the data were ana- lyzed using a Cox proportional hazards model. An important difference between these two statistical mod- els is the temporal factor. A multiple logistic regression model does not take time into account and assumes that all events occur at a similar time point. The propor- tional hazards model assesses the risk of the events occurring over time. It could be argued that because tacrolimus is a slightly newer immunosuppression agent than cyclosporine, these patients would have .a shorter follow-up period. Patients who were on tacroli- mus therapy could therefore be favorably biased toward an apparent lower risk of rejection. Because the median time to LAR in our study was 400 days and the median time of follow-up for the tacrolimus group was 844. days (1429 days for the cyclosporine group), this seems less

Ramji et al

likely. Because the proportional hazards model assessed a relatively small number of events over a long period of time, it is also possible that a failure to find a significant difference reflected a type I1 error. Another aspect of our study was that the time period included a period when Canadian centers where changing the cyclospor- ine formulation from the gelatin capsule (Sandim- mune) to the microemulsion preparation (Neoral). These two differing formulations of cyclosporine were grouped together in our analysis, but it is possible that because there are pharmacokinetic differences between the products, our analysis may have had some bias against cyclosporine. With regard to the two other newer immunosuppressive agents, mycophenolate mofetil and sirolimus, there were not enough patients to allow any meaningful analysis.

Another interesting aspect of this study was that on univariate analysis, the transplant center seemed to affect the risk of LAR, with one center experiencing more LARS than the other. We note that there was no difference in the type of immunosuppression, primary indication for transplantation, or length of follow-up between the two transplant centers. There may be sev- eral reasons for this apparent difference in specific-spe- cific risk of LAR. Half of the patients at one of the centers (Alberta) were followed up by a regional clinic in another city where liver biopsy was less readily avail- able. This introduces the possibility of surveillance bias in which a lower risk of LAR may be a result of under- diagnosis when the definition of rejection is made on histologic grounds. Another possibility is that the spe- cific center that experienced a greater likelihood of LAR may have been more willing to biopsy patients on the basis of abnormal liver biochemistry, thereby increasing the chances of a positive diagnosis. Because the overall outcome between the two centers in terms of patient and graft survival is similar, this may suggest that very mild cases of LAR, which are more likely to be missed if patients/physicians wish to avoid liver biopsies, may not be a significant prognosticator of poor long-term out- comes. We note that in the early 1990s, Dousset et a126

reported that some cases of biopsy-confirmed mild acute rejection may resolve spontaneously without any change in immunosuppressive therapy. Undoubtedly, some cases of biopsy-unconfirmed acute rejection may behave similarly.

Lastly, we found that LAR was associated with increased chronic rejection (odds ratio, 2.58; P = .04), although the sample size was small, which may explain why we could not show an increase in recurrent disease, retransplantation, or death in LAR patients. Backman et a127 have shown that the most common causes of late

graft loss (more than 1 year posttransplantation) are chronic rejection and recurrent hepatitis; therefore, an association between LAR and CR could be a concern, making predictive factors for significant LAR impor- tant to assess.

In summary, LAR is common and occurs beyond 1 year posttransplantation. Patients undergoing trans- plantation for viral hepatitis seem to have a lesser risk of LAR, although the reasons for this are not clear. There may be an increased risk of chronic rejection in some patients in whom LAR develops; therefore, continuous monitoring of immunosuppression and liver biochem- istry appears to be a perpetual necessity.

Acknowledgments

The authors gratefully thank the nursing staff of the liver transplant clinics of the Vancouver General Hospital (Uni- versity of British Columbia), the University of Alberta, and the University of Calgary for their dedicated support of these patients.

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