Significance of the total i-score Michael Mengel Alberta Transplant Applied Genomics Centre University of Alberta, Edmonton, Canada

Lorraine Racusen & Kim Solez 10% 25% 0% The Banff-Consensus Lorraine Racusen & Kim Solez

Cellular rejection Granzyme B

Banff i- and t-score Table 4 - Quantitative Criteria for Mononuclear Cell Interstitial Inflammation ("i") Scores i0 - No or trivial interstitial inflammation (<10% of unscarred parenchyma) i1 - 10 to 25% of parenchyma inflamed i2 - 26 to 50% of parenchyma inflamed i3 - >50% of parenchyma inflamed Do not consider for i-score: subcapsular infiltrates perivascular infiltrates fibrotic areas areas of tubular atrophy ?nodular infiltrates Table 2 - Quantitative Criteria for Tubulitis ("t") Score (applies to tubules no more than mildly atrophic) t0 - No mononuclear cells in tubules t1 - Foci with 1 to 4 cells/tubular cross section or 10 tubular cells t2 - Foci with 5 to 10 cells/tubular cross section t3 - Foci with >10 cells/tubular cross section, or the presence of at least two areas of tubular basement membrane destruction accompanied by i2/i3 inflammation and t2 tubulitis elsewhere in the biopsy. Do not consider for t-score: moderately to severe atrophic tubules ?mild atrophic tubules in areas of tubular atrophy and fibrosis ?tubules in areas with minor inflammation Racusen L. et al., Kidney Int. 1999 Feb;55(2):713-23.

subcapsular perivascular

Infiltrates in areas of fibrosis and tubular atrophy

nodular Infiltrates

How do people score? (Poll at the 2007 Banff meeting) (0.1 - 6 mm)

Infiltrate type p  0.05 Mengel et al. Am J Transplant. 2007 Feb;7(2):356-65.

Infiltrates and allograft function p  0.05 Mengel et al. Am J Transplant. 2007 Feb;7(2):356-65.

Infiltrates and outcome Mengel et al. Am J Transplant. 2007 Feb;7(2):356-65.

A relationship between inflammation and progression of IF/TA?

Inflammation as risk factor for progression of IFTA

Progression of ci-score and Inflammation

How much graft inflammation is significant? normal fibrosis fibrosis+ i=1 fibrosis+ i >1 Reading protocol biopsies versus reading clinical biopsies. The criteria are different. In setting clinical criteria we want to treat those pathologic findings that are of clinical significance. At the same time, we do not want to over-treat with the fear that treatment maybe worse than disease. Protocol biopsies put “teeth” under the arbitrarily decided criteria that define disease. For example, the criteria for cellular AR is based on arbitrary counts of cells in tubules. These data suggest that any degree of inflammation is relevant. What about the inflammation that occurs in areas of fibrosis. Clearly, this is difficult to interpret. However, as Nankivell has suggested not all fibrotic areas have inflammation so if they have, what does it mean? p<0.001 Cosio FG et al AJT, 5:2464, 2005

Scoring inflammation in renal allograft biopsies 100% Cortex 5% 3% absolute scoring 40% i-IFTA 10% i-Banff nodular perivascular subcapsular 60% IFTA compartment 40% non-scarred compartment relative scoring according to current Banff rules 25% = Banff i-score 1 “67% i-IFTA”

Infiltrates and time in BFC p<0.0001 p<0.0001

Relationship of total i-score to other Banff lesions Sis B. 2009 AJT, in press

Relationship of total i-score to other Banff lesions

Banff i- and total i-score and diagnosis: interstitial infiltrates are not disease specific * i-score total i-score *p<0.05 * * * % cortex with infiltrate * *

correlations between gene expression and Banff scores Gene sets (Spearman correlation, p<0.001) Banff-i-score t-score total-i-score T-cell associated 0.534 0.484 0.741 γ-Interferon induced 0.532 0.441 0.703 Kidney parenchyma associated -0.296 -0.303 -0.536 Injury and repair associated 0.379 0.355 0.645 B-cell associated 0.281 0.279 0.660

Correlation with PBTs is independent of time post transplant Biopsies taken ≤6 months post Tx i-score t-score total i-score T cell associated transcripts 0.633 0.608 0.726 gamma-interferon inducible transcripts 0.587 0.493 0.68 Kidney parenchymal transcripts -0.217 -0.185 -0.322 Injury inducible transcripts 0.023 -0.018 0.191 Immunoglobulin transcripts 0.259 0.36 0.276 B-cell associated transcripts 0.336 0.428 0.516 Biopsies taken ≤1 year post Tx i-score t-score total i-score T cell associated transcripts 0.699 0.635 0.771 gamma-interferon inducible transcripts 0.652 0.529 0.719 Kidney parenchymal transcripts -0.323 -0.240 -0.383 Injury inducible transcripts 0.066 -0.048 0.207 Immunoglobulin transcripts 0.437 0.495 0.457 B-cell associated transcripts 0.475 0.501 0.611

Defining a molecular threshold for pathological inflammation

The total i-score is superior in reflecting the molecular inflammatory burden AUC total i-score 0.85 i-score 0.73 p=0.012 AUC total i-score 0.82 i-score 0.58 p=0.001 C D AUC total i-score 0.86 i-score 0.86 p=0.9 AUC total i-score 0.97 i-score 0.91 p=0.7

t0-cases with high total inflammatory burden have also significantly higher other Banff scores *p<0.05

Prognostic value of Banff i- and total i-score versus diagnosis ABMR TG TCMR,GN Borderline CNIT ATN Other IFTA NOS total i-score AUC = 0.81 i-score AUC = 0.65 ← increasing ti/i-scores total vs. i-score p=0.012

Banff i- and total i-score and allograft survival allografts with ≥IFTA grade I (n=88) C D i-score <25% i-score >25% p=0.599 p=0.002 total i-score <25% total i-score >25% A i-score <25% i-score i-score >25% p=0.058 B total i-score <25% total i-score total i-score >25% p<0.0001 all allografts (n=104)

Conclusions about new total-i-score Comprises primarily two major inflammatory compartments: i-Banff (non-scarred) i-IFTA (scarred) reflects better the molecular burden of inflammation and tissue injury more robust predictor of allograft survival

Proposal for total i-score Test reproducibility for i-Banff, i-IFTA, and total i-score: if feasible, reporting of the different inflammatory compartments might allow to design new clinical trials Incorporate into the Banff-classification as a prognostic lesion either as ti-score alone or together with i-Banff and i-IFTA

Acknowledgements Special thanks to our clinical collaborators Kara Allanach Dina Badr Sakarn Bunnag Patricia Campbell Jessica Chang Gunilla Einecke Konrad Famulski Luis Hidalgo Anna Hutton Zija Jacaj Deborah James Bruce Kaplan Bert Kasiske Nathalie Kayser Daniel Kayser Daniel Kim Rob Leduc Arthur Matas Vido Ramassar Jeff Reeve Gui Renesto Joana Sellares Banu Sis Lin-Fu Zhu Stromedix, Astellas Roche Molecular Systems, Roche Canada Alberta Health Services University Hospital Foundation Roche Organ Transplant Research Foundation Genome Canada/Genome Alberta University of Alberta Alberta Ministry of Advanced Education and Technology Canada Foundation for Innovation Canadian Institutes of Health Research Kidney Foundation of Canada Alberta Heritage Foundation for Medical Research Muttart Chair in Clinical Immunology, Canada Research Chair in Life Sciences Special thanks to our clinical collaborators Special thanks to our patients