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;

• 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

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

Effects of Pancreas Allograft on CVD Risks

Blood Pressure Total cholesterol

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

Pancreas Transplants, by Transplant Type,1998-2007

1998 1999 2000 2001 2002 2003 2004 2005 2006 20070

400

800

1200

1600All Pancreas SPK PAK PTA

Nu

mb

er

of

Tra

nsp

lan

ts

1-Year 3-Year 5-Year 10-Year0%

20%

40%

60%

80%

100% SPK PTA PAK

Un

ad

just

ed

Gra

ft S

urv

iva

l (%

)

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

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%;

• 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

• 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

• 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

• 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)

• 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;

• 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;

Technical Consideration (II)

Unordered values, coded from low (green) to high (red)

?Can we group these patients and/or genesbased on the expression?

Consider the expression profiles for the samples and define a similarity, e.g correlation

S2

S1

S3

S3

1) Assess similarity between all combinations

2) Merge the two with the highest similarity

3) Repeat 1) and 2) until nothing left to merge

S3

S2

S1

S1

S2

?How meaningful are those groupings?

1) Initiate a perturbation by randomly removing one sample

2) Re-cluster3) 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

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;

Gene Official Symbol Full name

C3 Complement Component 3

CCL19, CCL2 and CCL5, CCR5 Chemokine (C-C motif) ligands 19, 2 and 5, receptor 5

CX3CL1 and CXCL10, CXCR3,DARC Chemokine (C-X3-C motif) ligands 1 and 10, Chemokine (C-X-C motif) receptor 3, hemokine receptor (CD234)

FAS and FASLG, GNLY, GZMB and PRF1

TNF receptor and TNF superfamily, member 6, Granulysin, Granzyme B and Perforin

MS4A1, PTGS1 and 2membrane-spanning 4-domains, subfamily A, member 1, CD20, Prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) and 2

ADAM8 and 19, MMP-2, 7 and 9, TIMP1 ADAM metallopeptidase domain 8 and 19, Matrix metallopeptidase 2, 7 and 9, TIMP metallopeptidase inhibitor 1

IL6, and IL10 Interleukin 6 (interferon, beta 2), and 10

EDN1, Serpine1, TNF Endothelin 1, Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1, Tumor necrosis factor

HOXB7, KRT15, OPCML, TUSC4 Homeobox B7, Keratin 15, Opioid binding protein/cell adhesion molecule-like, Tumor suppressor candidate 4

INS, GCG, SST, AMY1A,CCKAR, ELA2A

Insulin, Glucagon, Somatostatin, Amylase, alpha 1A, Cholecystokinin A receptor, Elastase 2A

PDX1, MAF, MAFB, NEUROD1Pancreatic and duodenal homeobox 1(Ipf1), V-maf musculoaponeurotic fibrosarcoma oncogene homolog and B, Neurogenic differentiation 1

POLR2A, PPIA, UBC, 18s rRNA and GAPDH

Polymerase (RNA) II (DNA directed) polypeptide A, Peptidylprolyl isomerase A, Ubiquitin C, 18s ribosomal RNA, Glyceraldehyde-3-phosphate dehydrogenase

Demographics of Study PopulationCluster A

N=7Cluster B (I)

N=12Cluster B (II)

N=7Age (years, S.D.) 37.7 + 6.2 37.3 + 6.3 34.9 + 4.5Sex (male, %) 6 (85.7) 6 (50.0) 5 (71.4)Race (AA, %) 2 (28.6) 1 (8.3) 1 (14.3)Type of transplant SPK (%) PAK (%)

6 (85.7)1 (14.3)

6 (50.0)6 (50.0)

2 (28.6)5 (71.4)

Induction Thymo/OKT3 (%) Basiliximab (%)

4 (57.1)3 (42.9)

3 (25.0)9 (75.0)

1 (14.3)5 (71.4)

CNI CsA (%) Tac (%)

2 (28.6)5 (71.4)

2 (16.7)10 (75.0)

1 (14.3)6 (85.7)

Clinical Phenotypes and Clustering

Cluster AN=7

Cluster B N=19

In=12

II n=7

Pancreas allograft failure (%) 1 (14.8) 8 (66.7) 5 (71.4)

Acute rejection (Maryland) III or lower (%) IV and V (%)

7 (100)0 (0)

7 (58.3)4 (33.3)

5 (71.4)1 (14.3)

Chronic rejection (%) 0 (0) 1 (8.3) 2 (28.6)

Positive CD20 expression (%) 0 (0) 0 (0) 4 (57.1)

CD 20 Protein Expression in Pancreas Transplant Biopsies

Biopsy with negative CD20 mRNA Biopsy with positive CD20 mRNA

Expression of Some of Selected Molecular Markers

Expression of Some of Selected Molecular Markers

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

• 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

Laboratory

• Matthias Kretzler, M.D.

• Fabian Trillsch, M.D.*

• Anna Henger,*

• Felix Eichinger,

Pathology• Henry Appelman, M.D.• Joel Greenson, M.D.

Transplant• Silas Norman, M.D.

* Currently in Germany