Gene Expression Based Categorization of Transplant Pancreas Biopsies

1. Gene Expression Based Categorization of Transplant Pancreas Biopsies Fu L. Luan M.D. fluan@med.umich.edu University of Michigan

2. No disclosure to declare

3. Background • Pancreas transplantation is an effective treatment for patients with type 1 diabetes mellitus; • Successful pancreas transplant establishes long-term normoglycemia with no risk of hypoglycemia; • Potential benefit on improvement of diabetic related end-organ damage and cardiovascular risk factors; • Potential benefit on extending patient survival;

4. Value of Pancreas Allograft on Patient Survival P. Salvalaggio et al. Diabetes Care 32(4): 600-602; 2009

5. Effects of Pancreas Allograft on CVD Risks Blood Pressure Total cholesterol F. L. Luan, et al. Transplantation 84:541-544; 2007

6. Pancreas Transplants, by Transplant Type,1998-2007

7. Unadjusted 1-Year, 3-Year, 5-Year, and 10-Year Pancreas Graft Survival

8. The Challenges to Maintain a Functioning Pancreas Allograft • Complication related to graft and vascular thrombosis accounts for about 20% of early graft failure; • Acute rejection as cause of graft failure within the first year was reported at around 20%; • Chronic rejection as cause of graft failure within the first year was reported at around 19%;

9. Pancreas allograft biopsy is the gold standard; • Clinical indication remains subtle; • Maryland classification, and lately Banff classification provide guidance for clinicians; • Response to the treatment varies;

10. Molecular Mechanisms Involved in Allograft Rejection and/or Failure • Large amount of information available on molecular mechanisms involved in kidney allograft rejection and/or failure; • Microarray technology has allowed better correlation of sets of gene expression with transplant renal outcome; • Little is known about molecular mechanisms involved in pancreas allograft rejection or failure;

11. Hypothesis • Pancreas allograft displays similar molecular mechanisms in acute and chronic rejection, and/or allograft failure; • Pancreas allograft exhibits unique molecular markers inherent to it’s organ specificity; • The pattern of molecular expression in pancreas allograft may correlates with the allograft outcome;

12. Materials and Methods • 26 pancreas transplant biopsy and 4 human pancreas specimens (unaffected area of tumor pancreatectomies); • All specimens were processed with fixation in formaldehyde and paraffin-embedding ; • Maryland classification for histological diagnosis; • Patient management was individualized;

13. Technical Consideration (I) • The formaldehyde-fixed, paraffin-embedded tissue samples were cut in 5 µm sections; • De-paraffinization was performed and followed by rehydration; • The sections (5 slides for each sample) were scraped off the slides and harvested in appropriated lysis buffer; • The total RNA was extracted using the phenol chloroform protocol and reverse-transcribed into cDNA;

14. Technical Consideration (II) • TaqMan® Low Density arrays (TLDA) technique was employed for parallel analysis of different mRNAs in samples; • The cDNA expression value of each sample was compared with other samples following the delta CT technique and the expression of target genes was normalized to a calibrator; • Real time RT-PCR expression values were analyzed with DChip using 2D hierachical clustering for samples as well as for genes;

15. S1 ? S2 Consider the expression profiles for the samples and define a similarity, e.g correlation Can we group these patients and/or genes based on the expression? S3 Unordered values, coded from low (green) to high (red) • Assess similarity between all combinations • 2) Merge the two with the highest similarity • 3) Repeat 1) and 2) until nothing left to merge S1 S2 S3 S1 S2 S3

16. Initiate a perturbation by randomly removing one sample • Re-cluster • Compare the sample composition of the resulting clusters ? If there is a clear structure (long branches) we probably capture some effect Non-random data should be robust to perturbations How meaningful are those groupings?

17. Selection of Gene Markers for the Study • Molecules involved in rejection processes, both acute and chronic, were obtained from the various literatures in kidney transplantation; • Molecules specific to pancreas, down-regulated during disease processes and up-regulated during regeneration processes, were obtained by searching publically available dataset at http://www.ncbi.nlm.nih.gov/geo and http://www.betacell.org; • Molecules considered “house keeping genes” were chosen;

20. Demographics of Study Population

21. Clinical Phenotypes and Clustering

23. CD 20 Protein Expression in Pancreas Transplant Biopsies Biopsy with negative CD20 mRNA Biopsy with positive CD20 mRNA

24. Expression of Some of Selected Molecular Markers

25. Expression of Some of Selected Molecular Markers

26. Summary • The first study looking into molecular expression in transplant pancreas specimen; • Technique feasibility of obtaining RNA of good quality using paraffin preserved pancreas biopsy specimen; • Existence of variable up- and down-regulation of molecular markers; • Corresponding expression of protein in the biopsy specimens; • Apparent correlation of expression pattern with observed clinical outcome;

27. Future Direction and Potential Implication • Need for a validation study involving large sample size; • Need for definition of additional molecular markers; • Need for more detailed correlation between expression patterns and transplant outcome; • Finally, prospective molecular study of pancreas allograft using protocol biopsy; • Potential guidance for target therapeutic intervention based on variable molecular expression in the tissue;

28. Acknowledgement Laboratory Pathology Henry Appelman, M.D. Joel Greenson, M.D. Transplant Silas Norman, M.D. • Matthias Kretzler, M.D. • Fabian Trillsch, M.D.* • Anna Henger,* • Felix Eichinger, * Currently in Germany