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Zinc Transporter 8 Autoantibodies Increase the Predictive Value of Islet Autoantibodies for Function Loss of Technically Successful Solitary Pancreas Transplant

Occhipinti, Margherita1; Lampasona, Vito2; Vistoli, Fabio3; Bazzigaluppi, Elena4; Scavini, Marina5; Boggi, Ugo3; Marchetti, Piero1; Bosi, Emanuele5,6,7

doi: 10.1097/TP.0b013e31822ae65f
Clinical and Translational Research

Background. Despite the success rate of solitary pancreas transplantation in type 1 diabetes with preserved kidney function has greatly improved in recent years, a residual proportion of failures persists.

Methods. With the aim of investigating autoimmunity as an unrecognized cause of graft failure, we measured autoantibodies to glutamic acid decarboxylase (GADA), insulinoma-associated protein 2 (IA-2A) and the recently discovered zinc transporter 8 antigen (ZnT8A) in 25 recipients of technically successful solitary pancreas transplantation.

Results. The overall pancreas graft survival was 92%, 88%, and 80% at 2, 4, and 6 years, respectively. Fourteen patients (56%) had one or more autoantibodies before transplantation, with no effect on subsequent pancreas graft outcome. After transplantation, major autoantibody changes (serum conversion from negative to positive, spreading from one to multiple autoantibodies, or titer increase) were observed in 5 of 25 recipients: in four patients, the autoantibody change was followed by the loss of graft function (95% sensitivity, 80% positive predictive value), with a significantly lower graft survival compared with patients without autoantibodies (P<0.0001). The addition of ZnT8A to GADA and IA-2A increased the number of identified autoantibody changes from three to five of 25 recipients and the number of predicted graft function loss from two to four out of five graft losses.

Conclusions. Detection of major autoantibody changes after technically successful solitary pancreas transplantation is predictive of subsequent loss of graft function. ZnT8A in addition to GADA and IA-2A are a useful marker to be included in the screening panel of posttransplant immune monitoring.

1 Department of Endocrinology & Metabolism, Cisanello University Hospital, Pisa, Italy.

2 Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy.

3 Division of General & Transplant Surgery, Cisanello University Hospital, Pisa, Italy.

4 Laboraf Diagnostica e Ricerca San Raffaele, Milan, Italy.

5 Diabetes Research Institute, San Raffaele Scientific Institute, Milan, Italy.

6 Vita Salute San Raffaele University, Milan, Italy.

The authors declare no funding or conflicts of interest.

7 Address correspondence to: Emanuele Bosi, M.D., San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy.


M.O. participated in research design, the performance of the research, data analysis, and the writing of the manuscript. V.L. participated in research design and contributed new analytic tools. F.V. participated in research design and the performance of the research. E.B. contributed new analytic tools. M.S. performed data analysis and participated in the writing of the manuscript. U.B. participated in research design and the performance of the research. P.M. participated in research design, the performance of the research, and the writing of the manuscript. E.B. participated in research design and the writing of the manuscript.

Received 27 April 2011. Revision requested 29 May 2011.

Accepted 23 June 2011.

Recent advances in surgical procedure, immunosuppressive treatment, and posttransplant clinical management have significantly improved the clinical outcome of solitary pancreas transplantation in patients with type 1 diabetes without end-stage renal disease (1). In facts, although the initial experience with solitary pancreas transplant was burdened by a high rate of death (2) and early failure due to graft thrombosis and other surgical complications (3), in the most recent series the incidence of early failure was reduced close to that of simultaneous pancreas and kidney transplantation (1, 3). Nonetheless, the long-term function of solitary pancreas grafts is still lower than that of simultaneous pancreas and kidney transplantation in patients with type 1 diabetes and end-stage renal disease. Although not demonstrated, it is assumed that the excess loss of function of solitary pancreas grafts is due to immunological causes (1).

Recurrence or recrudescence of islet autoimmunity represent a barrier to overcome to achieve successful beta-cell transplantation in human type 1 diabetes, performed as a whole pancreas or isolated islet transplantation (4–7). Circulating islet autoantibodies are the only easily measurable markers of type 1 diabetes-specific autoimmunity. Although islet autoantibodies do not play an active role in the process of β-cell destruction, they are accurate markers of the underlying autoimmune process and, to no surprise, they are detected in the large majority of patients with type 1 diabetes at the time of diagnosis (8). Furthermore, islet autoantibodies are predictive of the development of type 1 diabetes in susceptible individuals (i.e., family members of patients with type 1 diabetes) where their predictive value is associated with the titer and number of positive autoantibodies (9, 10).

In previous studies, the posttransplant reappearance or increase in the titer of autoantibodies against glutamic acid decarboxylase (GADA) and insulinoma-associated protein 2 (IA-2A) was associated with graft failure after simultaneous pancreas and kidney (11) or islet transplantation (12). Recently, antibodies against the zinc transporter 8 (ZnT8A) have been identified as an additional marker of type 1 diabetes-specific autoimmunity (13), and their potential in identifying recurrence of autoimmunity after pancreas transplantation has been suggested by at least one of three recently reported cases (14).

In view of the relevance of recurrent autoimmunity as a possible, although often unrecognized, cause of graft failure in solitary pancreas transplantation, we investigated the autoimmune response against islet autoantigens by measuring GADA, IA-2A, and ZnT8A titers in patients with type 1 diabetes who had received a solitary pancreas transplantation, and we assessed their ability to predict graft function outcome.

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Total posttransplant follow-up was 142.9 patient-year, with a median of 6.0 patient-year (interquartile range: 0.8–8.0). The probability of solitary pancreas graft function was 92% (95% CI: 72–98) at 2 years, 88% (95% CI: 67–96) at 4 years, and 80% (95% CI: 58–91) at 6 years.

Autoantibodies above the threshold for positivity were found in 14 (56%) of 25 patients before pancreas transplantation: of these, 8 cases had one autoantibody (six had GADA, one had IA-2A, and one had ZnT8A), five cases had two autoantibodies (four had GADA and IA-2A, one had IA-2A and ZnT8A) and one case had all three autoantibodies. The survival of solitary pancreas graft function was similar among patients with or without islet-specific autoantibodies before transplantation (Fig. 1, upper panel).



During posttransplant follow-up, autoantibody levels remained unchanged in 20 of the 25 patients: they persisted at undetectable concentrations during follow-up in 10 of 11 antibody-negative patients and remained relatively stable in 10 of 14 patients in whom GADA, IA-2A, or ZnT8A were detected before transplantation. In five patients, major changes in the autoantibody profile were observed after transplantation (Fig. 2): in one case, there was a serum conversion from negative to positive IA-2A and ZnT8A at 6 months after transplantation; in two cases, there was spreading from GADA positivity only to ZnT8A positivity at 6 and 24 months; and in the two remaining cases, there was a significant increase of the GADA titer at 6 months in one case and of GADA, IA-2A and ZnT8A titers at 24 months in the other case. In four of five patients with major changes of the autoantibody profile, the change was followed by the loss of solitary pancreas graft function 3 to 45 months after detection of the autoantibody changes. In the remaining patient, solitary pancreas graft function was still maintained 67 months posttransplantation and 61 months after the autoantibody change was detected. In this transplant series, there was one case of solitary pancreas graft loss of function that occurred in a patients who was persistently autoantibody negative (Table 1). Therefore, all the autoantibody changes combined as one marker of future loss of pancreas graft function, showed a sensitivity of 95% (95% CI: 75.1–99.9), a specificity of 80% (95% CI: 28.4–99.5), and a positive predictive value of 80% (95% CI: 28.4–99.5). Survival of solitary pancreas graft function was significantly lower (P<0.0001) after the detection of an autoantibody change (Fig. 1, lower panel).





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Our findings confirm previous observations on the value of islet autoantibody monitoring for predicting graft function loss after solitary pancreas transplantation, and extends those observations with the measurements of ZnT8A, a recently identified marker of islet autoimmunity, providing additional insights into the matter of posttransplant recurrence or recrudescence of autoimmunity.

The first confirmatory finding is the lack of influence of the pretransplant autoantibody status on functional outcome of pancreas transplantation. In facts, the survival of the solitary pancreas transplant was not affected by the presence of islet autoantibodies, their titers or possible combinations of different autoantibodies before transplantation. This observation is in agreement with our previous reports (11, 15) and further clarifies a matter still controversial in earlier studies (16–20).

The second confirmatory finding is the predictive value of major autoantibody changes for future pancreatic transplant failure. This evidence is further strengthened by the availability of ZnT8A as an additional marker of islet autoimmunity. Indeed, the addition of ZnT8A to GADA and IA-2A in the screening panel increased the number of identified autoantibody changes (defined as serum conversion, spreading, or increasing titers) from three to five of 25 cases and the number of predicted graft function loss from two to four of five cases.

In this series, only one case of solitary pancreas loss of function was not preceded by an autoantibody change, whereas in only one case an autoantibody change was not followed by the loss of function of the solitary pancreas graft over a follow-up of 67 months. Therefore, thanks to the addition of ZnT8A, the use of autoantibody changes as a marker of future beta-cell failure predicted the loss of solitary pancreas graft function with a sensitivity of 95%, a specificity of 80%, and a predictive value of 80%.

Although our sample size is small, the implication of our observation may be of relevance. The most obvious interpretation is that major posttransplant autoantibody changes reflect the recurrence or recrudescence of type 1 diabetes-associated autoimmunity. Confirming this hypothesis would require concomitant graft biopsy studies that are not available in this series. However, a recent report on three well-documented cases of recurrent diabetes after simultaneous pancreas-kidney transplantation showed that the loss of function of the transplanted beta-cell mass was mediated by autoreactive CD4 T-cells, and preceded by the detection of autoantibody changes similar to those observed in our study (14). An intriguing interpretation would be that posttransplant autoantibody changes reflect not only the reactivation of humoral autoimmunity but also of autoreactive memory T cells (6).

The analysis of large series of solitary pancreas transplantation documents a residual loss of function among technically successful cases, that is assumed to be immune-mediated (1, 3). Our findings suggest that the contribution of autoimmunity to the loss of function of a solitary transplanted pancreas may be more important than previously estimated, possibly accounting for a large fraction of the events. In this context, the increased sensitivity and predictive value provided by ZnT8A, suggest that ZnT8 is a relevant autoantigen in the process of posttransplant exacerbation of type 1 diabetes-specific autoimmunity. Therefore, including ZnT8A in the panel of autoantibodies will increase the accuracy and predictive value of posttransplant autoantibody monitoring of graft function after solitary pancreas transplant.

In conclusion, our study demonstrates that recurrent autoimmunity detected as major autoantibody changes after a technically successful solitary pancreas transplantation in patients with type 1 diabetes is a clinically relevant phenomenon, possibly responsible for a large proportion of graft failures until now considered of unknown origin. The occurrence of a major autoantibody change among patients receiving immunosuppression further emphasizes the relative inability of current immunotherapies to control the autoimmune response against beta-cells, as also suggested by the yet elusive results obtained by immune treatments in type 1 diabetes of recent onset (21). Therefore, innovative approaches to prevent the exacerbation of the memory autoimmune response are needed to achieve long-term success of solitary pancreas transplantation.

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We studied 25 nonuremic patients (9 men, 16 women; age [mean±SD], 36.4±7.7 years; body mass index [mean±SD], 23.7±2.7 kg/m2) with type 1 diabetes (duration [mean±SD], 25.0±8.2 years) receiving solitary pancreas transplantation at the Division of General and Transplant Surgery of the Cisanello University Hospital in Pisa (Italy). This series was a convenience sample of solitary pancreas transplants performed at the same Institution between the years 2001 and 2004. Patients included in this study had no procedure-related surgical complications and had serum samples before transplantation and during posttransplant follow-up available for autoantibody measurements. Pancreas grafts were donated by heart-beating donors selected for ABO blood compatibility and negative cross-match. Human leukocyte antigen type was not considered as a criterion for donor-recipient matching. The whole pancreas with the duodenum was transplanted, with enteric drainage of exocrine secretion and vascular portal drainage, as previously described (22). Immunosuppression included basiliximab as induction, and then mycophenolate, tacrolimus, and low dose steroid as chronic treatment. Serum samples for autoantibody measurements were obtained before transplantation and 6, 12, 24, 36 months thereafter.

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Autoantibody Assays

GADA, IA-2A, and ZnT8A were measured by radiobinding and immunoprecipitation assays as previously described (8, 23, 24). The thresholds for positivity in each assay corresponded to the 99th percentile of control subjects, equivalent to three arbitrary units for GADA, one arbitrary unit for IA-2A and five arbitrary units for ZnT8A. In the last Diabetes Autoantibodies Standardization Proficiency workshop held in 2009, these assays had the following sensitivities and specificities: GADA 66% and 97%, IA-2A 58% and 98%, and ZnT8A 68% and 99%, respectively.

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Statistical Analysis

Failure of pancreas transplant was defined as the need to restore exogenous insulin administration to treat hyperglycemia. A rise in islet autoantibody titers during the posttransplant follow-up was defined as (a) serum conversion when in a patient with no serological markers of islet autoimmunity at least one islet autoantibody became detectable; (b) increasing titers when the titer of an already positive islet autoantibody at least doubled; (c) spreading, when the serum conversion of additional autoantibodies occurred. Confidence intervals for proportions were computed using the binomial method. The probability of having a functioning pancreas transplant was estimated according to Kaplan-Meier for patients with or without autoantibodies to islet autoantigens before pancreas transplantation (Fig. 1, upper panel). The comparison of survival probability between patients with or without autoantibodies was performed using the log-rank test. The probability of having a functioning pancreas transplant was estimated according to Kaplan-Meier for all patients, with the rise of islet cell autoantibodies as a time-varying covariate (Fig. 1, lower panel). The comparison of survival probability before and after a posttransplant rise in islet autoantibodies was performed using the Mantel-Byar test to account for dependencies. Statistical analysis was performed using Stata version 10.1 (Stata Corp., College Station, TX).

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Solitary pancreas transplant; Islet autoantibodies; Type 1 diabetes; ZnT8 autoantibodies

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