Schemas for Histopathological Diagnosis of Rejection: Kidney Schemas for Histopathological Diagnosis of Rejection: Kidney Kim Solez, M.D.

Slide 2 Having fun creating order out of chaos. Bird formations at edge of Iguassu Falls! The Banff consensus process is like that!

Slide 3 Consensus Generation Online, Not Just Face to Face, Role of Protest. A good example of successful use is the World Wide Web Consortium. “We reject kings, presidents, and voting. We believe in rough consensus and running code.” David Clark (MIT) “Consensus stops the majority ruling the minority and is more consistent with anarchist principles.” Anarchist FAQ. ConsensUs: Computer-moderated Structured Discourse. obus/consensus.html obus/consensus.html obus/consensus.html FacilitatePro – Online collab. tool. A good example of successful use is the World Wide Web Consortium. “We reject kings, presidents, and voting. We believe in rough consensus and running code.” David Clark (MIT) “Consensus stops the majority ruling the minority and is more consistent with anarchist principles.” Anarchist FAQ. ConsensUs: Computer-moderated Structured Discourse. obus/consensus.html obus/consensus.html obus/consensus.html FacilitatePro – Online collab. tool.

Slide 4 Consensus Generation Online, Not Just Face to Face, Role of Protest, Alternate Views. (Possible if Banff participants knew I researched this stuff as a science, my facilitator role would be less effective. So shhh! Mum’s the word!)

Slide 5 History of the Banff Classification, Antecedents before 1991! Hard to know where to begin! Always building unusual things, assisted, inspired by female friends - muses. Three-story shack in back yard, age 9. Hard to know where to begin! Always building unusual things, assisted, inspired by female friends - muses. Three-story shack in back yard, age 9. Lorraine Racusen and I have worked together since she joined me as a fellow in Lorraine Racusen and I have worked together since she joined me as a fellow in I left for Chairmanship in Pathology at University of Alberta in Edmonton in I left for Chairmanship in Pathology at University of Alberta in Edmonton in Consensus generation experience: Future of Pathology/Laboratory Medicine in Canada Consortium, gave Canadian laboratory physicians political clout. Consensus generation experience: Future of Pathology/Laboratory Medicine in Canada Consortium, gave Canadian laboratory physicians political clout. ISHLT Heart Classification published in Lorraine and I started working on Banff classification in early ISHLT Heart Classification published in Lorraine and I started working on Banff classification in early 1991.

Slide 6 Lorraine Racusen in 1998 and today.

Slide 7 Banff Classification: Milestones 1991 First Conference 1991 First Conference 1993 First Kidney International publication 1993 First Kidney International publication 1995 Integration with CADI 1995 Integration with CADI 1997 Integration with CCTT classification 1997 Integration with CCTT classification 1999 Second KI paper. Clinical practice guidelines. Implantation biopsies, microwave Second KI paper. Clinical practice guidelines. Implantation biopsies, microwave Classification of antibody-mediated rejection 2001 Classification of antibody-mediated rejection Regulatory agencies participating Regulatory agencies participating 2003 Genomics focus, ptc cell accumulation scoring 2003 Genomics focus, ptc cell accumulation scoring 2005 Gene chip analysis. Elimination of CAN, identification of chronic antibody-mediated rejection Gene chip analysis. Elimination of CAN, identification of chronic antibody-mediated rejection First meeting far from a town called “Banff” – La Coruna, Spain First meeting far from a town called “Banff” – La Coruna, Spain.

Slide 8artistcitizen entrepreneur BBC Creativity “connecting with audiences”

We need to connect with audiences too! If we do it right we will be changing the face of medicine!

Someday the percutaneous biopsy will be replaced by some superior noninvasive approach lacking the sampling error, invasiveness, relative non-specificity of current diagnostic assessment.

“Wow, then we will be out of a job!” vs. “Hey that will be really exciting to practice pathology like that!”

The replacement of the invasive percutaneous biopsy approach by a noninvasive molecular biology/genomics approach has analogy in major political change. The replacement of the invasive percutaneous biopsy approach by a noninvasive molecular biology/genomics approach has analogy in major political change.

In the 80s one knew that sometime apartheid in South Africa would end and the Berlin Wall would come down but would that happen in a day, a year, a decade, a century?

Also have to be prepared for changes that could not be predicted, like the fall of the Soviet Union. The unexpected change that alters everything!

We now await similar positive tumultuous changes in the field of transplantation. And life will be better after than before.

We need the right approach.

"Possess the right thinking, in this you must never lapse." Splinter - the ninja master/talking rat - Teenage Mutant Ninja Turtle movie (1990)

Slide 18 Banff Conferences on Allograft Pathology 1991-?

Global consensus generation while maintaining intellectual freedom.

Like the mosh pit at a great rock concert. No partner, the ultimate in individuality, dangerous, but when the music is good everyone dances in sync and life is good.

Slide 21 Two future phases in the relationship between renal biopsies and management of the renal allograft recipient In the short term, the rigorous quantitation and internationally-agreed-upon evaluation of renal biopsies via the Banff Classification, which has proven itself quite useful in the early post-transplant period, will be extended to apply fully to late graft biopsies In the short term, the rigorous quantitation and internationally-agreed-upon evaluation of renal biopsies via the Banff Classification, which has proven itself quite useful in the early post-transplant period, will be extended to apply fully to late graft biopsies In the long term,perhaps years or decades away, the processes of acute and chronic rejection will be so well understood mechanistically that a test for specific markers in blood or urine will completely replace the percutaneous biopsy as a means of diagnosing these conditions In the long term,perhaps years or decades away, the processes of acute and chronic rejection will be so well understood mechanistically that a test for specific markers in blood or urine will completely replace the percutaneous biopsy as a means of diagnosing these conditions

Slide 22 Other Causes of Kidney Scarring – Why “CAN” does us a disservice! Hypertensive vascular disease. Hypertensive vascular disease. Chronic calcineurin inhibitor toxocity. Chronic calcineurin inhibitor toxocity. Obstruction. Obstruction. Chronic polyoma virus infection. Chronic polyoma virus infection. Donor origin vascular disease. Donor origin vascular disease. Chronic bacterial infection. Chronic bacterial infection. Recurrent or de novo glomerular disease Recurrent or de novo glomerular disease Recurrent or de novo vascular disease. Recurrent or de novo vascular disease.

Slide 23 Chronic scarring in Banff Classification – CAN gone. 5. Interstitial fibrosis and tubular atrophy (nephron loss), cause unknown. (Every attempt should be made to assign cases to known etiologies from other categories 2, 4, and 6.".... With interstitial fibrosis and tubular atrophy" Assignment to this cause-unknown category is a last resort.) 5. Interstitial fibrosis and tubular atrophy (nephron loss), cause unknown. (Every attempt should be made to assign cases to known etiologies from other categories 2, 4, and 6.".... With interstitial fibrosis and tubular atrophy" Assignment to this cause-unknown category is a last resort.) Grade I Mild interstitial fibrosis and tubular atrophy (  25% of cortical area Grade I Mild interstitial fibrosis and tubular atrophy (  25% of cortical area Grade II Moderate interstitial fibrosis and tubular atrophy (26-50% of cortical area) Grade II Moderate interstitial fibrosis and tubular atrophy (26-50% of cortical area) Grade III Severe interstitial fibrosis and tubular atrophy/ loss (  50% of cortical area) Grade III Severe interstitial fibrosis and tubular atrophy/ loss (  50% of cortical area)

Slide 24 Antibody Mediated Rejection in Banff Classification 2. Antibody-mediated rejection Rejection due, at least in part, to documented anti-donor antibody (‘suspicious for’ if antibody not demonstrated) 2. Antibody-mediated rejection Rejection due, at least in part, to documented anti-donor antibody (‘suspicious for’ if antibody not demonstrated) Acute Acute ATN-like – C4d +, minimal inflammation ATN-like – C4d +, minimal inflammation Capillary- margination and/or thromboses, C4d + Capillary- margination and/or thromboses, C4d + Arterial – v3, C4d + Arterial – v3, C4d + Chronic active Chronic active PTC basement membrane multilayering. chronic transplant glomerulopathy (cg 1-3, mm 1-3), C4d + PTC basement membrane multilayering. chronic transplant glomerulopathy (cg 1-3, mm 1-3), C4d +

Slide 25 T Cell Mediated Rejection 4. Acute/active cellular rejection may coincide with categories 2 and 5 Type (Grade) Histopathological findings IA Cases with significant interstitial infiltration (>25% of parenchyma affected) and foci of moderate tubulitis (>4 mononuclear cells/tubular cross section or group of 10 tubular cells) IB Cases with significant interstitial infiltration (>25% of parenchyma affected) and foci of severe tubulitis (>10 mononuclear cells/tubular cross-section or group of 10 tubular cells) IIA Cases with mild to moderate intimal arteritis (v1) IIB Cases with severe intimal arteritis comprising >25% of the luminal area (v2) III Cases with ‘transmural’ arteritis and/or arterial fibrinoid change and necrosis of medial smooth muscle cells (v3) 4. Acute/active cellular rejection may coincide with categories 2 and 5 Type (Grade) Histopathological findings IA Cases with significant interstitial infiltration (>25% of parenchyma affected) and foci of moderate tubulitis (>4 mononuclear cells/tubular cross section or group of 10 tubular cells) IB Cases with significant interstitial infiltration (>25% of parenchyma affected) and foci of severe tubulitis (>10 mononuclear cells/tubular cross-section or group of 10 tubular cells) IIA Cases with mild to moderate intimal arteritis (v1) IIB Cases with severe intimal arteritis comprising >25% of the luminal area (v2) III Cases with ‘transmural’ arteritis and/or arterial fibrinoid change and necrosis of medial smooth muscle cells (v3)

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Slide 29 More than half of transplant biopsies in 2005 do not show rejection! Calcineurin inhibitor toxicity most common entity. Scoring/classification system must deal with all entities, not just rejection! New onset hyaline arteriolar thickening (ah) a sign of calcineurin inhibitor toxicity.

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Slide 31 Non- Circumferential vs. Circumferential hyalinosis

Slide 32 Quantitative Criteria for Arteriolar Hyaline Thickening – Proposed new scoring - Mihatsch 0 = No PAS-positive hyaline thickening 1 = PAS-positive hyaline thickening present in only one arteriole, no circular involvement 2 = PAS-positive hyaline thickening present in more than one arteriole, but no circular involvement 3 = PAS-positive hyaline thickening with circular involvement, independent of the number of arterioles involved

Slide 33 Quantitative Criteria for Arteriolar Hyaline Thickening – Study of Sis et al. (Banff ’05) The severity of ah scored by both criteria, was significantly correlated with serum creatinine at biopsy (p<0.05). Using Banff criteria, the mean rate of pairwise agreement was 57.8% with an overall kappa value of With the newly proposed criteria, the mean rate of pairwise agreement was 70% and the overall kappa value was The mean interslide variation rates using Banff criteria and the new criterion were 30.7% and 36.7%, respectively. The severity of ah scored by both criteria, was significantly correlated with serum creatinine at biopsy (p<0.05). Using Banff criteria, the mean rate of pairwise agreement was 57.8% with an overall kappa value of With the newly proposed criteria, the mean rate of pairwise agreement was 70% and the overall kappa value was The mean interslide variation rates using Banff criteria and the new criterion were 30.7% and 36.7%, respectively. Conclusion: While Banff and the recently proposed criteria for ah scoring resulted in fair to moderate interobserver agreement, the new criterion seems to be more objective and results in better interobserver reproducibility. There is a substantial variation in the distribution and severity of arteriolar lesions in an individual biopsy, therefore, evaluation of more than one section is crucial to determine the severity of arteriolar damage more accurately. Conclusion: While Banff and the recently proposed criteria for ah scoring resulted in fair to moderate interobserver agreement, the new criterion seems to be more objective and results in better interobserver reproducibility. There is a substantial variation in the distribution and severity of arteriolar lesions in an individual biopsy, therefore, evaluation of more than one section is crucial to determine the severity of arteriolar damage more accurately.

Slide 34 Specimen Adequacy – (Banff ’97, ‘05) Minimum Sampling,procedures Unsatisfactory – No glomeruli or arteries Unsatisfactory – No glomeruli or arteries Marginal – 7 glomeruli with an artery Marginal – 7 glomeruli with an artery Adequate – 10 or more glomeruli with at least two arteries Adequate – 10 or more glomeruli with at least two arteries Minimum Sampling: 7 slides – 3 H&E, 3 PAS or silver stains, and 1 trichrome Minimum Sampling: 7 slides – 3 H&E, 3 PAS or silver stains, and 1 trichrome Must do C4d! Must do C4d!

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Slide 36 DNA Microarrays Transcription of many thousands of genes can be measured on one tiny chip Transcription of many thousands of genes can be measured on one tiny chip Define mechanisms of rejection and other complications that arise in transplant kidneys Define mechanisms of rejection and other complications that arise in transplant kidneys

Slide 37 Affymetrix GeneChip® probe array. Image courtesy of Affymetrix.

Slide 38 DNA synthesis (replication) RNA synthesis (transcription) protein synthesis (translation) DNA RNA Protein amino acids From DNA to Protein microarrays real-time RT-PCR

Slide 39 CTL genes in human kidney biopsies normalized vs well functioning transplants Tx1 Tx8 Tx4 Tx5 NR1 NR2 NR3 Tx7 AR2 AR3 AR4 AR6 AR1 AR7 NR5 AR5 Tx2 Tx6 Tx10 Tx9 Tx3

Slide 40 Current Research Microarray analysis of both human & mouse kidney transplants with rejection and other complications Microarray analysis of both human & mouse kidney transplants with rejection and other complications Correlate with Clinical data & Banff lesions Correlate with Clinical data & Banff lesions

Slide 41 Human and Mouse similar genes and similar development The Cell 2002.

Slide 42Outlook Discover patterns in mouse and human kidney tx Discover patterns in mouse and human kidney tx define molecular basis of Banff lesions define molecular basis of Banff lesions develop an array-based Banff classification develop an array-based Banff classification identify blood patterns correlating with biopsy patterns identify blood patterns correlating with biopsy patterns Validate patterns in large scale study Validate patterns in large scale study USA, Canada centers c.f. Matas-Halloran consortium USA, Canada centers c.f. Matas-Halloran consortium Develop new gold standard Develop new gold standard consensus in Banff process consensus in Banff process recognition by FDA: endpoints recognition by FDA: endpoints Develop commercial opportunities: IP, products, services Develop commercial opportunities: IP, products, services Extend Extend Other organ and tissue transplants Other organ and tissue transplants Autoimmune and infectious diseases e.g. hepatitis c Autoimmune and infectious diseases e.g. hepatitis c

Slide 43 Become part of the ongoing discussions Contact: or

Slide 44 Future Banff Meetings: La Coruna, Spain La Coruna, Spain Whistler, British Columbia, Canada Whistler, British Columbia, Canada Paris, France Paris, France Banff, Alberta, Canada Banff, Alberta, Canada Stockholm, Sweden Stockholm, Sweden