Benefits and Challenges with Exocrine Drainage Through Native Duodenum in Whole Organ Pancreas Transpolantation

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Oslo, January 2019

BENEFITS AND CHALLENGES WITH EXOCRINE DRAINAGE THROUGH NATIVE DUODENUM IN WHOLE ORGAN

PANCREAS TRANSPLANTATION

Rune Horneland MD

Section for Transplantation Surgery Department of Transplantation Medicine

Oslo University Hospital, Rikshospitalet

Oslo, Norway

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© Rune Horneland , 2019

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-495-5

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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Summary

Background/objectives:

Diabetes mellitus type I (T1DM) is a chronic autoimmune disease with great impact on daily routines, making the patient dependent on lifelong insulin treatment and continuous monitoring of their blood sugar. New technical devices such as sensors and pumps have greatly improved blood sugar control, and achievements in islet cell transplantation over the past two decades now offer long-term glycemic control and 50% insulin independence at 5 years in some series (1). However, islet cell transplantation still requires immunosuppression, and the efficacy is inferior to whole organ pancreas transplantation (PTX), as islet cell transplantation in most cases requires two infusions. Islet cell isolation success rates are in larger series approximately 50%, which in addition to two infusions, translates into four pancreatic grafts per recipient to achieve the outcome reported, and yet whole organ pancreatic transplantation can provide longer insulin independence utilizing only one graft.

Pancreas transplantation is unfortunately hampered with higher complication rate and higher immunological graft loss than kidney (KTX) and liver transplantation (LTX). Despite improved technical success and better immunosuppressive regimens that offer long-term graft survival after PTX, technical failures have been largely unchanged in the past two decades and immunological graft loss is still higher than for KTX and LTX alone. The combined pancreas and kidney transplantation (SPK) have historically demonstrated better

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outcome, less immunological graft loss, and less technical failures than pancreas transplantation alone (PTA) (2), and has therefore been favored compared to the PTA procedure.

Pancreas transplantation alone has become more prevalent, especially after the introduction of induction therapy 15-20 years ago. The lack of biochemical rejection markers and the hesitance to perform biopsies on the pancreas due to fear of complications have made the rejections difficult to monitor, and in particular after PTA where there is no sentinel kidney transplant. Some centers have been using the transplanted duodenal segment as a sentinel organ for rejection surveillance(3-7), like the kidney has been used as an indirect biological rejection marker in SPK, but few have addressed whether there is a reliable concordance of rejection between the duodenal segment and the pancreas parenchyma.

For more than two decades, enteric exocrine drainage of the pancreatic graft has been the gold standard in PTX. In 2012, we modified our previous approach and switched from jejunal exocrine drainage to duodenal exocrine drainage to enable easy endoscopic access to the graft. This facilitated an endoscopic protocol biopsy surveillance program, and armed with a better tool to monitor rejection, we decided to offer more T1DM patients the PTA procedure.

In this thesis, we examined the technical feasibility, benefits and challenges with the new duodeno-duodenostomy (DD) technique and the endoscopic transduodenal biopsy (EUS-Bx) program. We aimed to evaluate whether this translated into improved results and outcome.

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We also addressed the long-standing unresolved question of whether analyses of the duodenum can reliably predict rejection of the pancreatic graft.

Methods:

In paper 1, we performed a retrospective observational cohort study on the initial first 40 consecutive PTX with duodenal exocrine drainage (DD-PTx) from late 2012 to late 2013, and compared them to a cohort of 40 consecutive PTX with jejunal exocrine drainage (DJ-PTX) from early 2012 to late 2013. The main focuses were technical feasibility, benefits, and challenges with regards to the DD-technique and the EUS-procedure.

In paper 2, we evaluated a larger cohort with longer observation time to assess the outcome and investigate whether the changes in our treatment procedures would translate into improved results. We looked at short-term outcome with regards to surgical technical failures, but also long-term outcomes with regards to immunological graft loss. We included 117 consecutive DD-PTX from late 2012 to late 2016 and compared them to a historical cohort of 179 DJ-PTX from early 1998 to late 2012. The retrospective observational cohort study design was also applied for this paper.

For both paper 1 and 2, baseline donor and recipient data were prospectively recorded.

Retrospective, data from the National Renal Registry and the hospital records were merged with the baseline data. Multivariate Cox regression was used to assess associations between transplant modality and patient and graft survival. Patient and graft survival were calculated and plotted according to the Kaplan-Meier method.

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In paper 3, a retrospective analysis of prospectively collected data was applied, as this study was part of a larger clinical trial. We aimed to narrow down paired biopsies of duodenum and the pancreas sampled by EUS in DD-PTX recipients from late 2012 to late 2016. One- hundred and thirteen paired biopsies were analyzed and compared, 97 of these were sampled as protocol biopsies, and 16 due to graft dysfunction. Simple statistical methods were applied to calculate sensitivity, specificity scores, and concordance rates.

Results:

In the first paper, the mean follow-up time in the DD-cohort was 0.84 years compared to 2.2 years in the duodeno-jejunostomy (DJ) cohort. Baseline characteristics were similar, except for twice as many PTA in the DD-cohort (20 vs 10) and a higher body mass index (BMI), 24.7 vs 22.8. We found the DD-anastomosis easy to perform and no graft losses were directly attributed to the choice of exocrine drainage. We experienced a numerically higher incidence of technical failures in the DD-cohort, mainly due to fatal vein thrombosis, and we detected a significant higher rate of vein thrombosis in general (23% vs 5%, p=0.048) in the DD-cohort. In total, we encountered five early graft losses (GL) and three late GL in the DD- cohort, and three early GL and three late GL in the DJ-cohort. A total of 59 EUS procedures were performed in 40 DD-PTX recipients and 59 EUS biopsies from the duodenum, and 43 EUS biopsies of the pancreas were obtained. The graft was easily accessed in all cases and there was a near 100% success rate in obtaining duodenal biopsies. However, despite successful sampling of EUS core biopsies of the pancreas, the biopsy yield in terms of Banff

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diagnosis was disappointing with merely 50% yield. One needle perforation of the duodenum required an open duodenoraphy.

In the second paper, we compared 117 DD-PTX with a mean follow-up time of 2.2 years to 179 DJ-PTX. There was a significantly higher proportion of PTA in the DD-cohort (47% vs 7%), and only 68% of the DJ-PTX received induction therapy vs 100% in the DD-cohort. Defined by coronary angiography, there was a significantly higher proportion of coronary disease in the DJ-population with 29% vs 15% (p=0.003). Cold ischemia time (CIT) went down for the DD- recipients, with a mean CIT of 8.1 hours vs 9.3 hours (p=0.003). We found no difference in graft loss (GL) at 3 months post-transplant between the DD- and the DJ-group (10% vs 7 %).

Reoperation rate was similar, with 32% for both SPK groups and 33% for both PTA groups.

Numerically, more DD-PTX recipients suffered from early GL (9% vs 6%) and thrombosis was the main cause (8% vs 4%). Similar patient survival was achieved in all four groups at 3 months (98-100%), 1 year (93-100%) and 3 years (93-100%). Graft survival (GS) were superior in the SPK-groups compared to the PTA-groups, and GS in SPKDD group vs PTADD

group at 3 months 1 year and 3 year respectively were 90% vs 89%, 90% vs 74% and 83% vs 64%. Pancreas rejection was the most common cause for GL in all groups and was the only significant factor that differed in GL between PTADD and SPKDD (p=0.004).

In the third paper, we obtained 113 paired biopsies from the duodenal segment and the pancreatic graft by EUS in 67 PTX recipients (27 SPK, 40 PTA). We compared the histological findings in the duodenal biopsies to the pancreatic biopsies and classified them according to (i) no rejection (ii) intermediate and (iii) rejection. In 113 all cause biopsies, we found a 9%

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sensitivity of the duodenum to predict a rejection in pancreas and a 96% specificity to exclude rejection in pancreas, given that intermediate duodenal biopsy findings were classified as negative. In the protocol biopsy subgroup of 97 paired biopsies, the corresponding sensitivity and specificity were 18% and 98%.

Conclusions, implications and future perspectives:

Duodenal exocrine drainage in PTx is safe and enables easy endoscopic access to the pancreatic graft for both interventions, duodenal and transduodenal EUS biopsies for rejection monitoring and other pathological diagnostic purposes. The EUS procedure is safe and offers an important alternative tool to sample pancreas biopsies.

The retrocolic position of the pancreatic graft in combination with the DD-anastomosis did not reduce the rate of technical graft loss and complications and did not improve outcome after PTX in the short- and medium-term perspective. This could be attributed to the technique itself, learning curve, less experienced surgeons, and other confounding factors may contribute to the inferior results.

We found a low sensitivity and a high specificity for the predictive value of the duodenum for rejections in the pancreas. Due to high discordance rate and the fact that the majority of pancreas rejections took place along with normal findings in the duodenum, we recommend a pancreas biopsy to verify rejection of the pancreas.

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This study has provided data on safety and outcome of duodenal drainage in PTX and the potential benefits of a protocol biopsy program. We have also addressed the issue with discordant rejections between the duodenum and the pancreas, and our finding strongly suggest that pancreas biopsies should be performed in order to rule out or confirm a rejection of the pancreas. In our study, we have also shown that duodenal exocrine drainage in PTX is safe and that it should no longer be a controversial surgical approach, despite the jejunal drainage being by far the most common technique. Future studies and preferably randomized controlled trials will be needed to determine whether this approach is indeed equal to established techniques, and if the DD-technique and the EUS-guided biopsies proves valuable and translates into improved long-term outcome.

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Acknowledgements

This work was conducted at the Section for Transplant Surgery in the Department of Transplantation Medicine, Oslo University Hospital, Rikshospitalet from January 2013 to January 2018. The research was performed concurrently with my full time clinical position as a transplant surgeon at the same department. This project was completed without any external funding.

First and foremost, I would like to thank my principle supervisor, Dr. Einar M. Aandahl for his incredible support and help throughout this entire process, which at many times felt like a rollercoaster. Initially co-supervisor from 2013-2016, he succeeded to principle supervisor in 2016 when my former principle supervisor Dr. Ole Øyen resigned. Einar Martin is the only supervisor in this project who has been involved the entire time, making him the only supervisor who knows all of the details from start to end. He is the person who I could always rely on during difficult times and for advice on what move to make at various crossroads I came to. I am so grateful for his patience, guidance, and how he would always make time to help me revise any manuscript and advocate on my behalf. We have a long history not only as good friends since medical school, but also as colleagues for more than 10 years in the transplant department. I could not ask for a better supervisor; this work would not have been possible without him.

I would also like to acknowledge my former supervisors Dr. Ole Øyen and Dr. Aksel Foss. Ole was there to kick-start my project and clinical trial in 2012, and was my principle supervisor until he resigned from his position in 2016. It was his enthusiasm and vast clinical and academic knowledge that got me excited and involved in both pancreas transplants and

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research. As a young fellow, he mentored me in the clinical and surgical techniques of pancreas transplantation, which later led me to take over his responsibility of the pancreas transplant program. Aksel was the chair of the transplant department and my co-supervisor from 2013-2016. He pushed me to initiate and perform this PhD, as he strongly believes that research and clinical work go hand in hand. Initially it felt like a stressful undertaking, but today I am very grateful for both Aksel and Ole's encouragement and dedication to this advancement in my career. I would also like to thank my co-supervisors Professor Pål-Dag Line, Dr. Håkon Haugaa, and Professor Tor-Inge Tønnessen who stepped in when Ole and Aksel both resigned. I am thankful for their relentless support and for their guidance.

This project would not have been possible without my colleagues who, alongside with my supervisors and myself, transplanted all of the study patients and were loyal to all study procedures: Bjarte Fosby, Trygve Thorsen, Bjørn Helge Lien, Morten Skauby, Morten Hagness, Harald Hugenschmidt, Jon Solheim, Kristine Fasting, Ammar Khan, Steinar Guvåg, William Indrevåg, Casper Beiske, and Olav Liavåg. I am thankful for their steadfast support of my clinical study over the years, and their help with enrolling patients, performing surgeries, and providing data for research. Furthermore, for their understanding over the past few months when I have been less present in my clinical job due to finalizing my Thesis and Defense.

Clinical research like this is always dependent on collaborative partners and departments for referrals of patients, follow-ups, and daily support in postoperative care. I want to note my appreciation for our collaborative partners who made this project possible: the staff of the Transplant Department, the staff of the Nephrology Department, the staff of The Endoscopy Department, the staff of the Pathology Department, and the staff of the Radiology

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Department. In particular, I am very thankful for the tireless work and positive attitudes of both Cecilie Rogne Jorvang from the Nephrology Observation Unit and Helga Sørhøy from the Nephrology Physiology Laboratory. With their associates, they managed all of the logistics with protocol biopsies and follow-ups, and have been absolutely vital for making this project possible.

The nurses at the Transplant department have been invaluable in this work as well, in particular Tone Vidnes who implemented study routines and logistics. I would like to thank all of the nurses for their support, for treating all of our patients in an exceptional way, and for looking after the surgeons as well.

Additionally, I want to express my gratitude to the secretaries at my department who have expertly dealt with logistics and made sure the data for this research was available to me.

Big thanks to our close partners at the Nephrology Department for always looking after all of the Pancreas Transplant patients and for their contributions to this project, especially Dr.

Jørn Petter Lindahl and Professor Trond Jenssen. Special thanks to Dr. Vemund Paulsen and Professor Lars Aabakken from the Endoscopy Department who enthusiastically listened to my new ideas (that may have sounded both crazy and unconventional at that time), supported my project, and who designed and developed the EUS-technique for endoscopic pancreas biopsies.

In the Pathology Department I want to extend my thanks to Dr. Krzysztof Grzyb for providing solid histology reports and his contributions to all three papers. The high quality treatment of Pancreas Transplant patients would not be possible without the outstanding help from our radiologists, especially Dr. Knut Brabrand, Dr. Trygve Syversveen, Dr. Audun Berstad and all of the interventional radiologists. And lastly, no surgery can be performed without the

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service and support from our amazing colleagues at the anesthesiology department, in particular Dr. Håkon Haugaa, Gisle Kjøsen, Kristina Rydenfelt, and Professor Tor Inge Tønnessen, who were all involved in this project by virtue of the Micro dialysis study in these patients.

I want to thank all of my friends for their support and help over the years, especially my international friends; Beth van Fossan and Jae McGuire for always supporting me and Jacquelyn Marie Wells and Anna Aaker for both supporting me and for proof reading all my papers and reports. Deep thanks to all my local friends who have always been there for me, even in difficult times, in particular Jøran Grønstad, Anine Kleven and Martin Ott.

Finally, I am grateful to my parents Aslaug and Jens Håkon Horneland for their lifelong support and love. They adopted me from Korea at age five to their loving home in Etne, Norway. Without them, I would likely not have become a surgeon or accomplished my PhD today.

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List of Abbreviations

ACR Acute Cellular Rejection AMR Antibody Mediated Rejection APC Antigen Presenting Cells ASA Acetylsalicylic Acid

ATG Anti-Thymocyte Globulin AZA Azathioprin

BMI Body Mass Index

BPAR Biopsy Proven Acute Rejection CIT Cold Ischemia Time

CMV CytoMegaloVirus CNI Calcineurin Inhibitor CS Corticosteroids

CT Computed Tomography CyA Cyclosporine A

DBE Double Balloon Enteroscopy DD Duodeno-Duodenostomy

DD-PTX Pancreas Transplantation with Duodeno-Duodenostomy DJ Duodeno-Jejunostomy

DJ-PTX Pancreas Transplantation with Duodeno-Jejunostomy DSA Donor Specific Antibodies

ERCP Endoscopic Retrograde Choledocho-Pancreaticografi EUS Endoscopic Ultrasound

FDA U.S. Food&Drug Administration GL Graft Loss

GS Graft Survival

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HLA Human Leukocyte Antigen IAA Islet Autoantibodies

KTX Kidney Transplantation

LMWH Low Molecular Weight Heparin LTX Liver Transplantation

MHC Major Histocompatibility Complex MMF Mycophenolate Mofetil

PAI Pancreas After Islets PAK Pancreas After Kidney PRA Panel Reactive Antibodies PS Patient Survival

PTA Pancreas Transplantation Alone PTX Pancreas Transplantation

PVD Portal Venous Drainage RCT Randomized Controlled Trial rePTX Repeat Pancreas Transplantation SPK Simultaneous Pancreas and Kidney S-PTX Solitary Pancreas Transplantation SVD Systemic venous Drainage

T1DM Diabetes Mellitus type 1 Tac Tacrolimus

TCMR T-Cell Mediated Rejection TF Technical Failure

UNOS United Network for Organ Sharing

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List of Papers

The present work is based on the following papers:

1. Horneland R, Paulsen V, Lindahl JP, Grzyb K, Eide TJ, Lundin K, Aabakken L, Jenssen T, Aandahl EM, Foss A, Øyen O. Pancreas transplantation with enteroanastomosis to native duodenum poses technical challenges - but offers improved endoscopic access for scheduled biopsies and therapeutic interventions. Am J Transplant. 2015 Jan;15(1):242-50

2. Lindahl JP, Horneland R, Nordheim E, Hartmann A, Aandahl EM, Grzyb K, Haugaa H, Kjøsen G, Åsberg A, Jenssen T. Outcomes in Pancreas Transplantation With Exocrine Drainage Through a Duodenoduodenostomy Versus Duodenojejunostomy. Am J Transplant. 2018 Jan;18(1):154-162

3. Nordheim E, Horneland R, Aandahl EM, Grzyb K, Aabakken L, Paulsen V, Midtvedt K, Hartmann A, Jenssen T. Pancreas transplant rejection episodes are not revealed by biopsies of the donor duodenum in a prospective study with paired biopsies. Am J Transplant. 2018 Jan 9

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1.Introduction to the Thesis

1.1 Background and indications for pancreas transplantation

In 1922, when Banting and Macleod treated a 14-year-old diabetic boy with type 1 diabetes (8), their discovery changed the course of diabetes mellitus type I (T1DM) from a fatal disease to a chronic disease. For this discovery, they were awarded the Nobel Prize in Physiology/Medicine in 1923 "for the discovery of insulin." Banting shared the award with his colleague, Dr. Charles Best, and by receiving the prize at the age of 32 made Banting the youngest Nobel laureate in the area of Physiology/Medicine.

As diabetes became a chronic disease, secondary complications to diabetes became evident.

Longstanding diabetes is well known to cause various complications due to hyperglycemia including nephropathy, retinopathy, neuropathy and cardiovascular disease. Pancreas transplantation (PTX) aims to substitute the insulin-producing β-cells that are lost due to organ specific autoimmunity by completely restoring insulin dependence with glucose control and normoglycemia, and thereby counteract the development of secondary complications and even in some cases ameliorate them. Type I diabetes mellitus (T1DM) has been the main indications for pancreas transplantation (PTX), but type II diabetes mellitus and benign pancreatic disease with β-cell insufficiency including pancreatectomy, have lately gained more interest and acceptance. Overall numbers of PTX in the US have declined recent years (9), but type II diabetes mellitus as indication for PTX has increased and constituted 12% of SPK, 7% of the PAK and 1% of PTA performed in the US 2011-16 (10). According to

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the UNOS (United Network for Organ Sharing), T1DM patients need to be dependent on exogenous insulin and have a C-peptide of 2ng/mL (equivalent to 0.67 pmol/L) or less in order to be eligible for pancreas transplantation.

Recurrent severe hypoglycemia and unawareness, often combined with brittle diabetes, represents a subgroup of T1DM. This condition can occur prior to the development of secondary complications to diabetes. The annual prevalence of severe hypoglycemia within the T1DM population is estimated to be 30%, with several factors such as long disease duration increasing its incidence (11). Given that conservative treatments have been tried and not been successful, these patients are typically candidates for pancreas transplantation alone (PTA) or pancreatic islet transplantation (12), with several criteria discriminating between the two treatment modalities.

In Norway, T1DM has with few exceptions been the one and only indication for PTX (13-15).

Until 2011, the combined indication of diabetes and diabetes nephropathy represented approximately 90% of all the PTX performed. Since 2012, there has been an intentional increase in numbers of pancreas transplantation alone (PTA) in Norway. Since 2013, PTA reached an average of 50% of the total numbers of PTX and the PTA procedure is now the most frequent. The total numbers of PTX in Norway averaged 10 PTX annually from 1983 to 2011, but since 2012 to 2017 the average annual number reached 28, which equals 5.4 per million population (per January 1st 2018, source http://ww.ssb.no/befolkning). That puts Norway at the very top globally in terms of number of PTX per million population.

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1.2 Epidemiology of diabetes type I

According to International Diabetes Federation (IDF) Diabetes Atlas (16), it was estimated that 451 million adults (ages 18-99) were diabetic and that almost half of them were undiagnosed. The projection for 2045 is an increase to 693 million. In addition, an estimated 374 million people have impaired glucose tolerance . In 2017 alone, approximately 5 million adults died worldwide due to diabetes and the global healthcare cost was estimated to be

$850 billion USD. In Norway, the incidence of T1DM among children aged 0-14 during 2004- 2012 was 32.7 per 100.000 person-years (17) and the prevalence of diabetes in Norway was in 2004 estimated to be somewhere between 90-120.000 patients (18). Only a highly selected fraction of these patients will be considered for a PTX and even fewer will be listed.

1.3 Categories of pancreas transplantation

There are two main groups of PTX, the solitary pancreas transplantations (S-PTX) and the simultaneous pancreas- and kidney transplantations (SPK). The SPK procedure is the most common, and warrants eligibility for a kidney transplant (Figure 1).

The S-PTX procedure is subdivided into Pancreas Transplantation alone (PTA), Pancreas after Kidney Transplantation (PAK), Pancreas after Islet Transplantation (PAI) and finally the small, but increasing group of repeat PTX (rePTX), also commonly denoted pancreas after PTA/PAK/SPK/PAI (PAPTA, PAPAK, PASPK, PAPAI).

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In the United States (US), the combined procedure accounts for approximately 70% (9), followed by PAK and PTA. PTA is the less prevalent representing only 10% of the total number of PTX performed when excluding the more rare procedures like PAI and rePTX.

Figure 1. There are two main groups of pancreas transplantation, the solitary pancreas (left) and the simultaneous pancreas and kidney (right)

1.4 Donor selection

The outcome after PTX depends on many factors. Multiple studies have shown that donor age and donor body mass index (BMI) is the two most important parameters correlated with negative outcome (19, 20). Donor BMI >30 and donor age <55 are generally upper acceptance criteria; however, comorbidity like history of pancreatitis, vascular cause of death, kidney failure and HbA1C levels are important parameters in order to make a valid

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donor assessment. Score systems like the Pancreas Donor Risk Index (PDRI) may be helpful and combines these parameters with cold ischemia time (CIT) to generate a risk index for graft failure. A pancreas donor risk index score of 1.24 or higher is associated with graft failure in both univariate and multivariate analysis with a concordance index of 0.69 (21).

The selection criteria for an adequate pancreas donor are much stricter than for liver and kidney transplantation, and more in line with those used for thoracic organ donors. Donor medical records, age, BMI, laboratory values as creatinine, amylase, lipase, HbA1C, and C- peptide are important for donor assessment, but still the most important acceptance criteria is based on direct visual assessment of the pancreas by an experienced transplant surgeon regarding degree of fatty infiltration, fibrosis, parenchyma definition, trauma, and anomalies (22).

As a result, pancreas donors are a scarce resource worldwide. Donation after brain dead (DBD) is standard practice in all solid organ transplants. Donation after cardiac death (DCD) has lately become more popular, but donor after cardiac death in PTX is still controversial despite that recent studies report acceptable results and outcome (23) and it is likely that donor after cardiac death will increase the PTX donor pool in the future.

1.5 Recipient selection

The only indication for PTX in Norway is T1DM, with or without concomitant kidney failure, but internationally diabetes type 2 has increasingly been accepted for PTX. In light of limited access to pancreatic grafts suitable for transplantation, only a small percentage of the

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diabetic population can ever be offered a PTX. In line with most centers, Norway limits the procedure for adults (in Norway 18 years and up), as PTX involves a non-negligible surgical procedure and is not a lifesaving treatment in the immediate perspective. In addition, it requires strong motivation and adherence to ensure a successful long -erm outcome. Upper age limit varies, but most centers would be hesitant to accept recipients above 60 years.

Along with age limit, we have practiced an upper BMI limit of 30. Diabetes type 1 should be confirmed and C-peptide should be less than 0.67 pmol/L. Traditional diabetic therapy with insulin should have been thoroughly tested and reported on whenever a diabetic patient is referred for PTX. General recipient evaluation for organ transplant applies to PTX as well such as cardiovascular examination, functional lung tests, immunological testing, psychological evaluation, radiological mapping of vascular status, chest x-ray, prior medical history like cancer, tobacco use, and other surgical procedures.

Due to its nature, some particular conditions apply to the PTX-recipient evaluation. Diabetes and uremia is well known for its long-term vascular and coronary complications; therefore, coronary angiography is usually mandatory upon recipient approval. A coronary ejection fraction of less than 30% is an absolute contraindication in our center. A CT-scan and CT angiography of abdomen is mandatory in any diabetic patient with uremia and in Norway also in any recipient above 40 years old. High grade of vascular pathology is usually considered a contraindication and vascular bypasses are contraindicated prior to a PTX.

Previous abdominal surgery is a relative contraindication, and must be considered when evaluating the recipient. Other diabetic associated complications like peripheral neuropathy, retinopathy, autonomous neuropathy like gastroparesis, limb amputation and chronic

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diabetic ulcers are all conditions that need to be thoroughly examined and treated if possible and should be assessed when the final approval for PTX is to be made by the clinicians in a multidisciplinary meeting.

The higher rejection rate and the higher immunological graft loss and the lack of reliable biochemical rejection markers in PTX, makes most centers reluctant to accept high grade of Panel Reactive antibodies (PRA) and preformed donor specific antibodies (DSA). In our center, we have allowed for PRA up to 60%, but we have not accepted preformed DSA as DSA in PTX has shown strong correlation with rejection and graft loss (24).

Motivation and potential benefit from PTX has to be taken in consideration when evaluating the recipient. Failure to achieve glycemic control with traditional insulin therapy and hypoglycemia unawareness in T1DM is associated with high risk of hypoglycemia and disease related morbidity and mortality (25). Real-time glucose monitoring has shown to improve awareness, but with no change in HbA1c (25) as most of these patients are more worried about severe hypoglycemia than moderate hyperglycemia. Clarke and Gold score are frequently used to quantify hypoglycemic unawareness (26) and a high score strongly suggests a benefit from either islet transplantation or PTX. Generally, a daily insulin requirement of more than 40 units exclude the patient from islet transplantation, and if the primary goal is insulin independence and/or hyperglycemia is the typical symptom, then they are likely to benefit from PTX.

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1.6 Evolution of surgical approaches in pancreas transplantation

1.6.1 Early history of pancreas transplantation – the first era

Since Kelly and Lillehei at University of Minnesota Hospital introduced pancreas transplantation (PTX) to ameliorate type I diabetes December 17^th 1966 (27), the surgical technique has been subject to constant changes. The surgical approach has evolved through different eras, and earlier concepts have been revisited in many cases. All of the eras have been characterized by different approaches for management of the venous and exocrine drainage of the graft and different combinations thereof (Figure 2).

In the early years, the procedure was hampered with a high rate of complications and graft loss due to technical failures and rejections. The introduction of Cyclosporine A (CyA) for clinical use in the early 1980s and U.S. Food&Drug Administration (FDA) approval in 1983, revolutionized organ transplantation and PTX became a procedure that could potentially offer long-term graft survival (28). Despite major improvements and refinement for more than three decades, PTX, both solitary and in combination with a kidney transplant, are still associated with more surgical complications and early graft loss (GL) than kidney transplantation alone (KTX). In the 1980s, technical failures caused more than 25% early GL (29). Current GL due to technical failure is now approximately 7-10% (30, 31), yet the relaparotomy rate can reach up to 30% (20, 32-34).

The first PTX by Kelly was performed as a duct ligated segment pancreas to the left iliac vessels along with a kidney on the right side (27). Nevertheless, when Lillehei proceeded

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after Kelly with the second PTX in 1967, the duodenal segment was preserved and used to create an external stoma of the distal part of the duodenum, and hence internal enteric drainage was avoided (35). He used this approach for the first four of his PTX patients, but converted to internal enteric drainage by means of a roux-y loop on the following patients.

Interestingly, his case number three on March 3^rd 1968 was a pancreas transplant alone (PTA) and became the first PTA (35). Because of a high rate of complications related to the duodenal segment and the enteric anastomosis, enteric drainage was not reestablished as standard procedure until three decades later. This was prior to the introduction of CyA and high doses of steroids were believed to contribute to the complications related to the duodenal segment and the enteric anastomosis. The following decade was characterized with experimental surgery and many reports of small series with different technical approaches aimed to solve the issues with exocrine drainage, enteric complications, graft pancreatitis, and exocrine fistulas. In 1978, Dubernard et al. reported on a segmental pancreatic graft with neoprene obliterated pancreatic duct, thereby avoiding the need for exocrine drainage (36). This procedure turned out to be the most widely used approach for the next decade, but was associated with a high rate of exocrine pancreatic fistulas and a low rate of long-term insulin independence due to chronic inflammation and fibrosis in the graft, which eventually led to the next era in PTX.

Figure 2. The evolution of surgical techniques in pancreas transplantation. From left to right: The duct occluded segmental graft – the first era. The bladder drained pancreatic graft – the second era. The enteric drained pancreatic graft – the third era. The duodenal drained pancreatic graft – the fourth era?

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1.6.2 The Bladder Drained Pancreatic Graft – the second era

In 1987, the duct occluded technique of PTX was gradually abandoned when Nghiem and Corry (37) reported on nine consecutive bladder drained SPKs with duodeno-cystostomy.

Nghiem and Corry also implanted the pancreatic graft to the right iliac vessels unlike the duct occluded segmental grafts that were mostly implanted on the left side. However, the bladder drained technique with duct to bladder was first described in 1983 by Sollinger et al.

At the time, the bladder drained pancreatic grafts were associated with fewer surgical complications than the enteric drained grafts, and it quickly became more popular and was the preferred technique until the late 1990s. Some reported that monitoring rejection of the graft was reliable through assessment of the exocrine function by means of urine sampling, and some centers still use this approach (38). In addition, Nakleh et al. reported on endoscopic pancreas biopsies under direct visualization through transurethral cystoscopic access (3). Due to heavy loss of bicarbonate, metabolic disorders, and chemical cystitis, and other urinary complications, enteric conversion was often needed. Conversion rate reports vary between 6-17% (39-41), and some even report up to a 50% (2) conversion rate. Despite urinary complications, bladder drainage has demonstrated long-term graft survival

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comparable to enteric drainage in the succeeding era, and in some reports bladder drainage even has a favorable outcome (30).

1.6.3 The Enteric Drained Pancreatic Graft – the third era

Originally, Lillehei used the enteric drainage with roux-loop-deviation in his first report (35).

Overall improvements in organ preservation, surgical technique and immunosuppressive therapies diminished the risk of enteric anastomotic failure, and allowed the enteric approach to be revisited in the early 90s in order to avoid the urinary complications with bladder drainage. The FDA approval of Tacrolimus (Tac) for clinical use in 1994, and Mycophenolate Mofetil (MMF) were both important milestones in the transition to the third era. Initially, the enteric diversion with roux-y-loop was used, but the Stockholm group reported in 1996 on a simplified technique for enteric drainage with direct side-to-side anastomosis to jejunum without the roux-loop (42), demonstrating a more physiological exocrine drainage without any increase in surgical complications. Nevertheless, both techniques with enteric drainage either by a roux-loop or directly to the small intestine have been used up to date, and the superiority in terms of outcome has yet to be decided. Since the late 90s, enteric drainage has become the preferred method and is considered the gold standard of surgical technique in PTX, and about 90% percent adhere to this approach since 2010 (43). However, enteric drainage has been combined with multiple variations of surgical access, positioning in the abdomen and vascular anastomoses, and connection sites on the jejunum or ileum, all with similar outcomes.

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1.6.4 The Duodenal or Gastric Drained Pancreatic Graft – the fourth era?

Along with the enteric drainage to jejunum or ileum, both rejection monitoring by measuring amylase activity in the exocrine excretion and endoscopic access to the pancreatic graft for biopsies was diminished or lost. At best, graft duodenum could be reached with double balloon enteroscopy (DBE) for duodenal mucosa biopsies.(4). However, the possibility to perform transduodenal pancreatic core biopsies was lost. In relation to this, Margreiter et al.

(4) pointed out the unresolved issue of whether duodenal mucosa biopsies would be representative for rejection monitoring of the pancreatic graft. This question is particularly relevant for solitary PTX (S-PTX) compared to SPK due to the lack of a simultaneous reporter kidney. In addition, S-PTX are suffering from a higher graft loss rate than SPK, which is believed to be partly caused by rejection (22, 24).

This issue led some centers to introduce duodenal and gastric exocrine drainage (44-51).

Both offer easy endoscopic access and the possibility for transduodenal pancreatic parenchyma biopsies, in addition to mucosa biopsies and intervention like stenting of the pancreatic duct. The reports vary from case reports to the large report of Walter et al. who reported on 125 duodenal drained PTX in 2014. The duodenal drainage offers the most physiological handling of the exocrine juice. Gastric drainage is technically feasible; however there are no reports on long-term follow up on the consequence of a continuous pancreatic fluid outflow into the stomach. Both Perosa et al. and Walter et al. have suggested the possibility to perform transduodenal ultrasound guided endoscopic pancreasbiopsies (EUS- Bx). However, Walter et al. reported only four EUS, all of them successful in terms of

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endoscopic access, biopsy yield and complications, whereas Perosa et al. did not report any performed EUS.

1.6.5 Living Donor PTX and Portal Venous versus Systemic Venous Drainage

In 1979 the first segmental living donor PTX was performed by Sutherland (52). Due to the nature of this surgery and the potential risk for post donation diabetes mellitus, living donor PTX has not become a standard procedure, despite that some small series and case reports have shown acceptable results (53-56). Living donor PTX is beyond the scope of this thesis and will not be further elaborated in this report.

Currently, approximately 90% of PTX are enteric drained, and more than 80% of these are employed with systemic venous drainage (SVD) (49). Systemic venous drainage to cava or iliac vein had been the default approach since the introduction of PTX, but in 1992, Shokouh- Amiri et al. reported on a small series of PTX with portal venous drainage (PVD) (57). Later studies have advocated in favor of portal venous drainage PVD compared SVD in order to achieve more physiological insulin delivery (58). The proponents of PVD claim that SVD may cause hyperinsulinemia and disturbances in lipid metabolism; however, Petruzzo et al.

showed no significant differences concerning fasting insulin level, C-peptide, cholesterol, and triglyceride levels between the two techniques (59). Portal venous drainage has been combined with both bladder drainage and enteric drainage including duodenal drainage (49). Since the PVD is considered technically more challenging than SVD, the SVD has gained more popularity than the PVD. Nevertheless, studies show equal results regarding rejection, complication rates and outcome (58).

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1.7 The recipient operation

Regardless of type of exocrine drainage, venous drainage and arterial supply, most PTX in the modern era are performed in a similar manner. Successful PTX are dependent on a good quality pancreas graft, that is carefully procured and well preserved (60). Typically, the graft needs to be meticulously prepared on a backtable in cold saline solution. The donor duodenum is shortened with suture machine (stapler) to the correct length and oversewn with resorbable suture. The staple line of the cut surface of the mesenteric vessels is oversewn with non-resorbable vascular suture. If the spleen is not removed in situ, it is now carefully removed and all vessels from the tail to the spleen are ligated. The portal vein is mobilized and elongated if needed. In most cases, the arterial supply is reconstructed with a Y-iliac-arterial graft from the same donor, but in some cases the aortic patch can be used if it is provided with the graft. Finally, all redundant tissue is removed, preferably with a hemostatic sealer and the graft is flushed and tested for any leakages (60).

Most PTX are performed through a midline incision. The cava inferior (or a mesenteric vein) and the common right iliac artery are exposed. Care is taken not to harm the right ureter.

Then the portal vein of the graft is implanted end-to-side to cava inferior/right iliac vein/mesenteric vein. The next step is to implant the aortic patch of the graft or the common leg of the Y-graft reconstruction end-to-side to the recipient’s right iliac artery.

After reperfusion, the most prominent bleeders are sealed before the duodenal segment is connected to the smaller intestine or duodenum by a side-to-side anastomosis or by means of a roux-y-loop. At last, meticulous hemostasis is performed. If SPK, then the kidney is usually implanted on the left side according to standard local procedure. Before closure, one

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or two active suction drains are placed next to the grafts. As with all organ transplants, cold ischemia time (CIT) is correlated to the results. Both Rudolph et al. and Finger et al. found CIT >20 hours to be an independent risk factor for early graft loss, in addition Rudolph et al.

found that CIT >12 hours to negatively impact long-term-graft survival. This was most pronounced for donors older than 25 years and with BMI above 25 (30, 61).

1.8 Transplant Immunology

1.8.1 The Immune System

The immune system is designed to protect the host from harmful pathogens or their biologic products (toxins). In vertebrates, the immune system is comprised of two arms, the innate immune system (nonspecific) and the adaptive immune system (specific), representing the first and the second line of the immune defense. If a pathogen breaches the physical barriers of a host, the innate immune system responds with an immediate, but non-specific immune response, acting by directly attacking the pathogen or by inducing an inflammatory immune response that recruits other immune cells.

Evolution has provided almost all living organisms, including plants and invertebrates with an innate immune system that consist of primary defense mechanisms encoded in the germline. They include defense systems such as the complement system, Toll-like receptors, phagocytic cells, humoral inflammatory mediators, and both surface and cellular barriers.

The innate system has two basic ways of immune recognition; recognition of microbial antigens and recognition of missing self-antigens.

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Recognition of microbial antigens is based on identifying pathogens by pattern recognition receptors that recognize components which are common among a wide group of microorganisms and not specific for a particular microorganism or antigen, and this recognition activates innate immune effector cells (62). Recognition of missing self-antigens and activation of the innate immune system is based on recognition of self-molecules on normal healthy cells. Whenever these self-molecules are not expressed or recognized in the setting of infected cells or damaged cells, it triggers an immune response. The innate immune system responds to pathogens in a generic way. It does not adapt, it is not antigen specific, and it does not provide long-lasting immunological memory; however, the innate immune system is in most organisms the main defense system.

If the pathogens also evade the first line of defense, the immune system employs the second line of defense, the adaptive immune system which is activated by the innate response to the pathogen. This second line of the immune system is antigen specific and improves its recognition and targets the response to the specific pathogen. When the threat has been eliminated, this learned immune attack is retained as an immunological memory by preservation and clonal expansion of the antigen specific cells. This allows the adaptive immune system to respond faster and more efficient next time the pathogen is encountered (63, 64).

The innate immune cells act as a cellular barrier and include phagocytes, innate lymphoid cells, mast cells, macrophages, dendritic cells, neutrophils, eosinophils, basophils, and natural killer cells. These cells attack and eliminate pathogens through direct contact or by

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engulfing them, a feature called phagocytosis. This allows the innate immune system to communicate with and activate the adaptive immune system through processing and presentation of microbial peptide antigens to effector cells of the adaptive immune system.

Adaptive immune cells are mainly B and T-cells with several functional subgroups.

Endogenous and intracellular peptides are bound to human major histocompatibility complex (MHC) class I molecules, also known as human leukocyte antigen (HLA) class 1 on the surface of all nucleated cells and presented to T cells, whereas exogenous and extracellular peptides are presented by MHC class II (HLA class 2) on the cell surface of antigen presenting cells (macrophages and dendritic cells).

The antibody receptors on the B cell surface on the other hand, have the ability to recognize the pathogens itself and trigger a humoral response directly. The complete B-cell population represents the antibody repertoire of the host (65). Both B and T cell receptors recognize specific peptide antigens. Autoreactive T cells are eliminated during the maturation process that takes place in the thymus, whereas autoreactive B cells are eliminated or inactivated by complex processes in both the bone marrow and secondary lymphatic tissues. These processes are crucial to avoid autoimmune diseases.

Alloreactivity, the immune response against an organ transplant is mostly T cell dependent, but involves and triggers all components of the immune system, hence the main target for immunosuppressive drugs are T cells and T cell signaling molecules. Along with ABO blood group antigens, the HLA molecules represent the most important alloantigens.

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T cells recognize alloantigens through the direct pathway and the indirect pathway (66).

Through the direct pathway, T cells directly recognize the intact non-self HLA molecules on the surface of donor cells. The indirect pathway describes the T-cells ability to recognize non-self (donor) HLA molecules that have been processed and now presented as peptide antigens by self-HLA molecules on the surface of antigen presenting cells (APC), like dendritic cells and macrophages (Figure 3).

Figure 3. a) In the direct pathway, which is important in the early phase of allorecognition of host antigens and graft rejection, polyclonal recipient T cells recognize intact donor major histocompatibility complex (MHC) molecules directly via their T cell receptors (TCRs). b) By contrast, the indirect pathway is oligoclonal and dependent on a restricted set of T cells that display a specific repertoire of TCRs. These T cells recognize only a limited number of dominant peptides that are displayed on the MHC of recipient antigen-presenting cells (APCs), and they play an important part in late and chronic rejection. The indirect pathway is also responsible for the alloantibody responses seen in patients who have received organ transplants. Adapted with permission from Yang, Transplant Genetics and Genomics, Nature Reviews Genetics volume 18, (2017).

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To ensure adequate activation of the adaptive immune system, the T cells require minimum two independent signals. The antigen specific signal is delivered through interaction between the T cell receptor and a peptide antigen bound to HLA class II on the surface of an APC, and the antigen non-specific signal is delivered via interaction between a costimulatory molecule on the APC and ligands on the T cell (67) (Figure 3). The costimulatory signalling is crucial for the activation of the T cell and represents an important therapeutic target for immunosuppressive drugs in order to inhibit alloreactivity and protect the graft.

1.8.2 Rejection in Pancreas Transplantation

Allograft rejection is a major challenge in solid organ transplantation, and in particular in PTX, even with modern immunosuppressive drugs and better knowledge of donor and recipient matching. After the initial perioperative hit and complications that follows within the first month, rejection is the major cause for graft loss (GL). Studies report a 1-year acute rejection rate of 15-21% (68, 69) and can be divided into acute cellular rejection (ACR), antibody-mediated rejection (AMR), and mixed rejection. Rejection can be further defined as hyperacute, acute or chronic; the latter tend to be mainly chronic AMR which is postulated to be the most important cause for late graft loss in solid organ transplantation (70, 71).

Allograft biopsy is the gold standard for diagnosing a rejection, and a valid diagnosis, can in most cases, be made when combined with a CT scan and antibody measurements (72).

Given the limited pool of eligible pancreas donor and recipients, HLA matching in PTX has been given less priority than HLA matching in kidney transplants. However, studies have shown a correlation between higher HLA mismatch and poorer outcome and development

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of de novo donor specific antibodies (DSA). The risk for rejection is particularly high with class II mismatch and the presence of DSA (73-75).

1.8.3 T-cell mediated rejection (TCMR) in Pancreas and Duodenum

T-cell mediated rejection (TCMR), previously known as acute cellular rejection (ACR) is well studied in transplantation immunology. It can develop within days and at any point after transplantation. The strength of an immune response in general is determined by the binding affinity between the T cell receptor and the MHC class II/alloantigen complex, the balance between costimulatory signals and inhibitory receptor-ligand interactions and the cytokine milieu in the microenvironment (67, 76). However, in a rejection reaction, the recognition of allograft HLA is the initial event that leads to the host’s immunological immune response. The direct pathway is dominating over the indirect pathway early post- transplant, as host T-cells have the ability to recognize an allo-antigen directly and prompt an efficient effector function. This is further propagated when derived APCs expressing donor alloantigen rapidly migrate from the graft to the host´s lymphoid tissues. The number of T-cells that can respond via the direct pathway is much greater than the pool of T-cells that can respond through the indirect pathway (76), which commence when antigens derived from the graft tissue is presented by the recipient´s APCs and sustains the immune response.

The majority of TCMR occurs within, but not limited to the first three months. Accumulative rejection rate within the first year post-transplant has been reported to be ranging from 15- 21% (68, 69). Out of these, the majority are TCMR. The hyperacute type rejection mediated

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with preformed antibodies and characterized by immediate endothelial injury and graft loss are rare these days with mandatory tissue crossmatching and modern immunosuppression.

For both acute and chronic TCMR the targets of the mature effector T-cells are donor derived vascular endothelium, epithelial cells of both the pancreas, the duodenal segment, and in the setting of a SPK the kidney graft.

Risk factors for rejection in pancreas in multivariate analysis include race mismatch, solitary pancreas transplant, younger age, retransplantation and previous episodes of rejection.

Patient survival after rejection is excellent, regardless of ACR or AMR, but 20% ultimately develop graft failure within 1 year after treatment for rejection, mainly due to progression into chronic rejection (68).

According to Banff 2011 classification, acute mild TCMR (grade I) in the pancreas is characterized by active septal inflammation and/or acinar inflammation. In moderate (grade II) TCMR of the pancreas, mild arteritis is also seen. In severe (grade III) TCMR the histological picture shows diffuse acinar inflammation and necrosis and/or moderate to severe arteritis (77) (Table 1). TCMR of the pancreas is entirely a histological diagnosis.

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Table 1. From Drachenberg et al. 2011: Guidelines for the Diagnosis of Antibody-Mediated Rejection in Pancreas Allografts – Updated Banff Grading Schema (77) . Banff pancreas allograft rejection grading schema. (Table 4 in the original article)

There are no standard Banff criteria currently available for grading the severity of intestinal rejection, hence Wu et al. and Remotti et al. made two proposals for a histological grading

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schema (78, 79) in 2003 and 2004 respectively. These have later undergone minor revisions.

Their grading schema includes the major histopathological changes associated with intestinal ACR established from previous reports (80) and include varying degrees of: (i) leukocyte infiltration; (ii) crypt injury; (iii) increase in the number of crypt apoptotic body count (ABC);

and (iv) distortion of villous and crypt architecture (78). The diagnosis of a rejection in small bowel cannot be made without close correlation with clinical and endoscopic findings (79).

Clinical endoscopic information includes: (i) location; (ii) native organ versus graft comparison; and (iii) description of macro pathology. In addition, clinical information regarding active infection is important because the small bowel is very rich in lymphoid tissue and the precise diagnosis of rejection is difficult. This has been a major challenge in relation to multivisceral transplantation, which was introduced and approved by Medicare in 2001 as definitive therapy for short gut syndrome (79). However, transplantation of a small duodenal segment along with the pancreas graft has not shown to follow the same clinical course, and in most cases been secondary to the pancreatic graft in rejection. In line with observations done after simultaneous pancreas and kidney transplantation (SPK), it was believed that both the duodenum and the kidney would serve as a surrogate marker for rejection in the pancreas graft (4, 81), yet a report from Nakleh et al. back in 1995 stated that the duodenum and the pancreas indeed could be rejected independently and hence both organs should be biopsied for reliable diagnosis (3). Both grading schemes are heavily relying upon the number of crypt epithelial apoptosis, and <6 apoptotic body counts/10 crypts are considered normal. Along with higher severity of rejection, crypt deformation and arteritis are seen. In other organ transplants, the entity of AMR is well established, but in the small intestine there are limited data on humoral rejection, and both the frequency and

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clinical relevance of AMR and C4d staining are unknown (79). The grading of rejection is based upon the degree of white blood cell count, inflammation and mucosal injury and are categorized as no ACR, indeterminate ACR, moderate ACR and severe ACR (Table 2 and 3).

Isolated rejection reaction in the duodenum, the pancreas or the kidney graft is a phenomenon that has been known since the bladder drained era (3, 6, 81). Nevertheless, in the enteric drained era, pancreatic core biopsies have been less accessible, hence isolated rejections was an entity that was less studied for two decades. In the absence of an accurate biomarker, biopsies of both the kidney graft and the duodenal mucosa were believed to be valid surrogate markers for rejection in the pancreas. This preconception has in recent years gained more attention and it is now a well-known fact that discordant rejections are a true phenomenon (3, 6, 7, 72, 81-84).

Table 2. From Wu et al. 2003: A Schema for Histologic Grading of Small Intestine Allograft Acute Rejection (78). Histologic criteria for grading of small bowel acute rejection. (Table 2 in the original article)

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Table 3. From Remotti et al. 2012: Small Bowel Allograft Biopsies in the Management of Small-Intestinal and Multivisceral Transplant Recipients (79). Histologic Grading of Acute Cellular Rejection in Small-Intestine Allografts.

1.8.4 Antibody mediated rejection (AMR) and donor specific antibodies (DSA)

Humoral rejection, caused by preformed donor specific antibodies (DSA) and/or de novo DSA has gained increasingly more attention the past decade due to its frequency and negative impact on long-term graft survival in pancreas transplantation and lack of effective drug treatments. Antibodies against HLA epitopes and ABO antigens are the most studied and considered the most important, yet there are wide arrays of antibodies against other antigens of uncertain clinical relevance (75). It is believed that AMR is the most important contributor to late graft loss in pancreas transplantation (77). Approximately 10% of PTX recipients have been reported to experience an AMR within the first year and 7 % had in the same report experienced a mixed rejection type with TCMR. Some recipients also experienced rejection episodes of both types (68).

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In response to increasing knowledge of AMR as a separate entity, the 2011 Banff meeting outlined extensive guidelines for diagnosis of acute and chronic AMR of the pancreas (77).

The diagnosis of AMR in pancreas allograft is, as opposed to TCMR, dependent not only on histology findings, but a combination of immunohistopathological and serological findings consisting of: (i) positive donor specific antibodies (DSA); (ii) morphological evidence of tissue injury in histology specimen; and finally, (iii) positive staining for C4d where >5% is considered positive (68) (Table 1).

The presence of 2 or 3 out of 3 features are consistent with an acute AMR, whereas 1 out of three diagnostic features raise suspicion and require exclusion of an AMR. In addition, there is a histological grading of AMR into mild, moderate and severe AMR (Table 4). Despite that there will in most cases be a mixed histological picture, since there is no evidence that direct antibody mediated mechanisms produce rejection outside of the vasculature (85).

Whenever a rejection is suspected, a DSA screening is warranted in addition to a pancreatic core biopsy. Some centers also advocate for protocol screening for de novo DSA. However, currently there is no consensus on what to do in response to de novo DSA in the setting of a normal pancreas allograft function, regardless, in most cases it would raise awareness and lead to increased immunosuppression (72). The presence of fibrosis is normally a sign of chronic tissue injury, and (i) fibrosis along with criteria of (ii) acute AMR, without any signs of (iii) acute TCMR, is defined as chronic active AMR (77) (Table 1).

The level of DSA are correlated to the degree of C4d staining, rejection rate and clinical outcome, but the mean fluorescence intensity (MFI) cut-off level of clinical relevance is not

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known, and it probably varies whether there are DSA towards class I HLA or class II HLA, in addition different HLA antibodies within class I and class II may have different immunogenicity potential (68, 75). Antibodies against HLA class II antigens are believed to have a greater clinical relevance and a greater severe negative impact on graft survival than HLA class I (75). Hence, knowing the high immunological response to a pancreas graft, most centers would not allow preformed DSA, which normally translates into a default mean fluorescence intensity cut-off level <1000. There are few data quantifying the risk of preformed DSA versus de novo DSA, but DSA per se is known to be an independent risk factor for graft failure (24, 86).

Table 4. From Drachenberg et al. 2011: Guidelines for the Diagnosis of Antibody-Mediated Rejection in Pancreas Allografts – Updated Banff Grading Schema (77). Histological grading of acute antibody-mediated rejection. (Table 2 in the original article)

1.8.5 Immunosuppression in Pancreas Transplantation

Immunosuppression regimens of PTX have changed along with drug development and optimization of the combination of drugs. The introduction of CyA as the first calcineurin Inhibitor (CNI) in the early 1980s dramatically increased the number of solid organ transplants performed worldwide (28) and CyA quickly became the cornerstone of the

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immunosuppressive cocktails for PTX. In addition to steroids and CNI, azathioprine (AZA) remained a mainstay in the maintenance immunosuppressive treatment until the mid-90s.

However, acute rejection was higher in SPKs compared to KTX alone.

The next trend came in early 2000s, when induction therapy was widely introduced.

Currently, 80% of SPK recipients receive induction therapy (87) with a T-cell depleting antibody (e.g. anti-thymocyte globulin or alemtuzumab) and only 10% receive a non- depleting antibody agent such as an interleukin-2 receptor inhibitor (e.g. basiliximab) (87, 88). About 10-15% receive no induction therapy. Polyclonal rabbit anti-thymocyte globulin (ATG) is used in about half of all antibody induction regimens whereas monoclonal antibody induction with alemtuzumab (anti-CD52 antibody) is used in 19% of SPK recipients (89). Two randomized trials (RCT) were performed in 2003 testing anti-T-cell antibody induction versus no induction combined with Tacrolimus (Tac), MMF and steroids (CS) as maintenance agents. Both studies showed a lower acute rejection rate during the first year with induction therapy, but no differences between the groups after a 3-year follow up (90, 91). Stratta et al. performed an RCT study in 2014 comparing the two most commonly used antibody induction agents in SPK, ATG versus alemtuzumab combined with standard maintenance regimen of Tac, MMF and CS. A single dose alemtuzumab (30 mg) and multiple dose ATG (total of 5-6 mg/kg) provided similar patient and graft outcomes with no major differences in morbidity (87), but the hospital cost of a single dose of alemtuzumab was significantly lower than 6mg/kg ATG (92). Studies reporting on non-T-cell-depleting IL-2 receptor antibodies show comparable graft survival to depleting-T-cell antibody induction, but the rate of acute rejection is higher (92).

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Tacrolimus (Tac) was first FDA approved for liver transplantation in 1994, and in the late 90s Tac gradually replaced CyA also in PTX despite that an FDA approval for Tac in PTX has never been issued. Currently more than 80% of PTX recipients receive a Tac based regimen along with the purine synthesis inhibitor mycophenolate mofetil (88, 93). In the EURO-SPK 001 RCT that was comparing Tac with CyA, the Tac group showed lower rates of acute rejection and the graft survival (GS) was better in the Tac group (94).

Mycophenolate mofetil (MMF) was FDA approved in 1995 and rapidly replaced AZA as the adjuvant agent to CNI. MMF has been shown to decrease the risk of interstitial fibrosis and tubular atrophy (IFTA), and based on randomized trials MMF is more effective than AZA in preventing acute rejection during the first year (92). Enteric coated mycophenolate sodium is an alternative to MMF. Studies in KTX recipients have suggested that gastrointestinal adverse events may be improved after switching to enteric coated mycophenolate sodium, which delays the gastrointestinal absorption. Clinical studies have shown that enteric coated mycophenolate sodium is equally effective as MMF in preventing acute rejection in KTX recipients (92).

There have been a few small sample retrospective reports on the use of sirolimus in SPK recipients where sirolimus has been used in combination with CNI, or as a substitute for CNI.

These reports showed excellent short-term outcomes with low rates of acute rejection, but long-term follow-up data in RCT is warranted (92).

Benefits and Challenges with Exocrine Drainage Through Native Duodenum in Whole Organ Pancreas Transpolantation

Oslo, January 2019