Currently, one the challenges of durable glucose management after pancreas transplantation is the ability to accurately and expediently diagnose early rejection to prevent unnecessary damage to the graft. This assessment is further complicated in combined organ transplantation. Simultaneous pancreas and kidney (SPK) transplantation accounts for most of the utilized pancreas grafts. However, both grafted systems (kidney and pancreas) are not subject to equal immunologic pressures even though they are theoretically presented with similar environments. Historically, the diagnosis of pancreatic rejection was assumed to be tethered to the presence of concomitant kidney rejection. The two organs were believed to reject in tandem. As such, many centers use sentinel biopsy of the kidney to determine rejection in the one or both organs. More recently, many studies have called into question this management strategy as there appears to be increasing evidence that both organs can reject independently of one another.

The presence of two organs allows for many combinations of rejection: both organs may undergo acute rejection known as concordant rejection or one organ may undergo acute rejection independently of the other organ known as discordant rejection. Kidney or pancreas rejection may occur in any time frame after SPK and can greatly affect graft survival. In order to clinically determine rejection in SPK recipients we monitor serum amylase, lipase, glucose, creatinine and proteinuria. Abnormalities in these labs in conjunction with clinical changes often are the indication for biopsy to determine the presence of rejection. More importantly, histology dictates treatment regimen and course. Invariably, SPK recipients sometimes present with normal creatinine and renal function, but with abnormal pancreatic enzymes. In most cases, the kidney biopsy would precede any discussion of pancreas biopsy due to the aforementioned notion of concordant rejection between organs. Certainly, biopsy proven rejection in the kidney with pancreatic enzymatic leak would necessitate aggressive anti-rejection therapy given the high likelihood of pancreatic involvement. However, many times these renal biopsies would be normal and lead to a quandary of how vigorously to pursue further evaluation of the pancreas.

Pancreas graft biopsy is not a common practice, but has been reported to be performed percutaneously, transcystoscopically if the pancreas was anastomosed to bladder, endoscopically, and laparoscopically in a few small series. Most centers use Interventional Radiology for CT guided pancreas biopsy. More importantly, there have been reported cases of complications for pancreas biopsies, mainly stemming from intraabdominal bleeding requiring surgical intervention \[1\]. In an effort to reduce potential patient morbidity from pancreatic biopsy, non-invasive tools like AlloSure that assesses donor-derived cell-free DNA (dd-cfDNA) and AlloMap; a gene expression-profiling test may provide an attractive alternative.

Currently dd-cfDNA analysis (AlloSure, CareDx®) in the kidney has shown promise. While the gold standard at present remains histologic assessment of kidney tissue, this may be changing as acceptance of dd-cfDNA grows. Dd-cfDNA analysis is advantageous as it is a noninvasive, less costly, and a potentially safer way to assess allograft rejection. Additionally, the easier accessibility of testing dd-cfDNA can enable more frequent testing, which can elucidate a more accurate progression of rejection rather than a biopsy which is only a snapshot in time. For renal analysis the recommended dd-cfDNA cutoff value is 1.0% to diagnose active rejection (positive and negative predictive values are 61% and 84%). While it seems reasonable that discordant rejection may apply to similar levels of dd-cfDNA seen in kidney alone rejection, concordant rejection levels of dd-cfDNA are unclear.

Notably, the technology behind Allosure has provided several insights into cellular injury from a variety of milieu. In the study by Shen et al., the dd-cfDNA level in deceased donors (44.99%) was significantly higher than that in the living donors (10.24%) at initial time, P \< 0.01. Dd-cfDNA level in delayed graft function (DGF) recipients was lower (23.96%) than that in non-DGF (47.74%) at the initial time, P = 0.89 (19.34% in DGF and 4.46% in non-DGF on the first day, P = 0.17). There was a significant correlation between dd-cfDNA level at initial time and serum creatinine (r2 = 0.219, P = 0.032) and warm ischemia time (r2 = 0.204, P = 0.040). DGF patients experienced a slower decline than non-DGF patients, but both groups had a rapid decline in dd-cfDNA post-transplant.

However, most importantly it appears that a recurrence of dd-cfDNA level may be indicative of active rejection \[5\]. In the case report by Hurkmans et al., it was shown that dd-cfDNA was a sensitive biomarker to detect rejection in solid organ transplantation in a renal transplant patient diagnosed with melanoma and taking nivolumab as treatment. Additionally, the utility of Dd-cfDNA has been studied in the pediatric renal transplant population. In the study by Puliyanda et al., biopsies were completed when dd-cfDNA \> 1.0% or when there was high clinical suspicion. 19 of 67 patients had dd-cfDNA testing as part of routine monitoring with a median dd-cfDNA score of 0.37 (IQR: 0.19-1.10). 48 of 67 patients who had clinical suspicion of rejection had median dd-cfDNA score of 0.47 (0.24-2.15). DSA-positive recipients had higher dd-cfDNA scores than those who were negative or had AT1R positivity alone (P = .003). There was no association between dd-cfDNA score and strength of DSA positivity. 7 of 48 recipients had a biopsy with a dd-cfDNA score \<1%; two showed evidence of rejection. Neither DSA nor AT1R positivity was statistically associated with biopsy-proven rejection. However, dd-cfDNA \>1% was diagnostic of rejection with sensitivity of 86% and specificity of 100% (AUC: 0.996, 0.98-1.00; P = .002). In the study by Sigdal et al., urinary dd-cfDNA after kidney transplantation correlates to the apoptotic injury load of the donor organ. Serial monitoring of urinary dd-cfDNA has been shown to be a sensitive proxy and biomarker of acute injury in the donor organ, but fails to have specificity to differentiate between acute rejection and BK virus nephropathy. Therefore, dd-cfDNA is an appropriate and accurate method in lieu of a biopsy to assess allograft rejection and injury. However, one of the limitations of dd-cfDNA is its ability to be detected is inhibited by methylprednisolone.

In addition to dd-cDNA, the quantification of select genes in circulating leukocytes (AlloMap, CareDx®) has potential to determine rejection. AlloMap has been shown to determine the risk for rejection in Cardiac Allograft Rejection Gene Expression Observational Trial (CARGO) and in the subsequent trial known as CARGO II. The current research of AlloMap in multi-organ recipients reflects a national cohort of 18 heart-kidney, 8 heart-liver, 1 heart-lung matched to 54 heart only recipients. AlloMap Heart® is a panel assay of 20 genes, 11 informative and 9 used for normalization and/or quality control, which produces gene expression data used in the calculation of an AlloMap Heart test score - an integer ranging from 0 to 40. Compared with patients in the same post-transplant period, the lower the score, the lower the probability of acute cellular rejection at the time of testing. Recently a multivariable gene-expression signature targeting T-cell-mediated rejection in peripheral blood of kidney transplant recipients was developed. This frugal TCMR-signature was made of (IFNG, IP-10, ITGA4, MARCH8, RORc, SEMA7A, WDR40A).

The gene expression profiles in renal transplant recipients with BKV viremia have been investigated by microarrays. This research analyzed entire blood gene expression profiles of 19 BKV viremia patients matched to 14 patients without BKV viremia and showed a significant higher expression of GRIT, CAT, and NK cell associated transcript. The study results indicated increased activity of cytotoxic T cells and NK cells in both BKV viremia and nephropathy that resembled acute rejection and showed potential involvement of both the innate and adaptive immune system. This study documents to the importance of studying BKV viremia to exclude the diagnosis due to similar gene expression pattern in blood compared to rejection. In other study, gene expression profiles of DSA+ renal transplant recipients were analyzed with and without tissue findings of ABMR and found that DSA+ patients with ABMR has increased expression of activation, regulation, and differentiation of immune cells (T cells, NK cells, leukocytes, and interleukins).

Allosure and Allomap tests were not studied in SPK recipients in detail. Cutoff values for Allosure test could be different in stable SPK recipients compared to kidney transplant recipients due to additional donor tissue (pancreas and duodenum). It is also unknown, what would be the Allosure values in pancreas and/or kidney rejection in SPK recipients. It is also unknow the circulating gene transcripts in SPK recipients with rejection. Using the similar concept of peripheral blood gene expression by Allomap testing, AlloMap Kidney/Pancreas could be created.