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Gene Expression Based Categorization of Transplant Pancreas Biopsies. Fu L. Luan M.D. fluan@med.umich.edu University of Michigan. No disclosure to declare. Background. Pancreas transplantation is an effective treatment for patients with type 1 diabetes mellitus;

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gene expression based categorization of transplant pancreas biopsies

Gene Expression Based Categorization of Transplant Pancreas Biopsies

Fu L. Luan M.D.

fluan@med.umich.edu

University of Michigan

background
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;
value of pancreas allograft on patient survival
Value of Pancreas Allograft on Patient Survival

P. Salvalaggio et al. Diabetes Care 32(4): 600-602; 2009

effects of pancreas allograft on cvd risks
Effects of Pancreas Allograft on CVD Risks

Blood Pressure

Total cholesterol

F. L. Luan, et al. Transplantation 84:541-544; 2007

the challenges to maintain a functioning pancreas allograft
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%;
slide9

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;
molecular mechanisms involved in allograft rejection and or failure
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;
hypothesis
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;
materials and methods
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;
technical consideration i
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;
technical consideration ii
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;
slide15

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

slide16

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?

selection of gene markers for the study
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;
cd 20 protein expression in pancreas transplant biopsies
CD 20 Protein Expression in Pancreas Transplant Biopsies

Biopsy with negative CD20 mRNA

Biopsy with positive CD20 mRNA

summary
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;
future direction and potential implication
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;
acknowledgement
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