Antibody-mediated rejection: a prototype for antibody-mediated diseases Phil Halloran Alberta Transplant Applied Genomics Centre Edmonton, Canada Disclosures PFH has shares in TSI a university spinoff company with an interest in molecular diagnostics Acknowledgements: The current Edmonton ATAGC team Andre Baretto Jessica Chang Collaborators: Konrad Famulski Arthur Matas Luis Hidalgo Michael Picton Anna Hutton Jon Bromberg Vido Ramassar Daniel Seron Jeff Reeve Jeffery Venner Declan de Freitas Gunilla Einecke Alexandre Loupy Joana Sellares Administration Michelle Ryan Novartis Pharmaceuticals Corporation Stromedix, Inc. Bristol-Myers Squibb Astellas Pharma Inc. Roche Molecular Systems, Roche Canada Alberta Health Services University Hospital Foundation Roche Organ Transplantation Research Foundation Genome Canada Alberta Advanced Education and Technology Canada Foundation for Innovation Alberta Heritage Foundation for Medical Research Muttart Chair in Clinical Immunology Special thanks to our clinical collaborators Special thanks to our patients
Antibody-mediated kidney rejection The leading cause of organ transplant loss Type 1 (early-presensitized) Type 2 (late-de novo, usually after one year) Active or inactive (microcirculation inflammation) At least 50% is currently missed (C4d ve) Molecular correlates: Rejection transcripts : largely IFNG effects Donor-specific antibody (DSA) associated transcripts NK cell transcripts Endothelium transcripts Transcripts associated with progression and scarring Learning points about ABMR based on lessons from kidney transplant biopsies The target is the microcirculation endothelium May be active or inactive: large dynamic range If severe, it compromises blood flow: ischemia Usually operates as a stress on the microcirculation that can be accommodated for variable periods on time Time-dependent lesions: transplant glomerulopathy Often currently missed: C4d is unreliable No truly specific histologic features Probably need molecular pathology readouts There is something special about class II No clear evidence that autoantibody can mediate ABMR Requires biopsy diagnosis: DSA alone is not specific Unmet need Evidence-based medicine Precision diagnosis
The unmet need: pathologists admit that histology is not good enough In 2003 Furness et al wrote that agreement between pathologists using the same lesions and rules was not only poor, but resistant to improvement by training.... international variation in histologic grading is large, under-recognized, recognized, difficult to improve, and almost certainly of major clinical relevance. Urgent steps are required to improve this area of clinical practice. * Thus improvement will NOT come from histology alone. *Furness PN, Taub N, Assmann KJ, Banfi G, Cosyns JP, Dorman AM, et al. International variation in histologic grading is large, and persistent feedback does not improve reproducibilit Am J Surg Pathol 2003;27(6):805-10. Reproducibility of assessment of histological features P N Furness and N Taub for the Convergence of European Renal Transplant Pathology Assessment Procedures (CERTPAP) Project KI 60:1998, 2001 Feature Kappa Tubulitis Banff grade 0.17 Tubulitis per 10 HPF 0.16 Tubular atrophy 0.29 Glomeruli: number present 0.53 Early type of allograft glomerulitis 0.21 Number of glomeruli completely sclerosed 0.47 Mesangial matrix increase 0.12 Glomeruli with segmental sclerosis 0.13 Chronic allograft glomerulopathy 0.11 Mononuclear cell interstitial infiltration 0.34 Interstitial fibrosis 0.30 Number of arterial cross sections 0.19 Arteriolar hyaline thickening 0.11 Endothelial cell activation arterial 0.21 Endothelial cell activation venous 0.10 Neutrophils in peritubular capillaries 0.13 Intimal arteritis 0.35 Arteriolitis 0.24 Fibrous intimal thickening 0.36 Breaks in arterial elastica 0.22 Inflammatory cells in intima in chronic fibrosis 0.34 Kappa values: A measure of how much 2 observers agree compared to chance: Perfect = 1 Chance = 0 Evidence-based medicine requires real evidence US Preventive Services Task Force (USPSTF) Systems to stratify evidence by quality have been developed, such as this one by the USPSTF for ranking evidence about the effectiveness of treatments or screening (or diagnosis?): Level I: at least one properly designed randomized controlled trial. Level II-1: well-designed controlled trials without randomization. Level II-2: well-designed cohort or case-control analytic studies, preferably from more than one center or research group. Level II-3: multiple time series with or without the intervention. Dramatic results in uncontrolled trials Level III: Opinions of respected authorities, based on clinical experience, descriptive studies, or reports of expert committees. Histology is here: Opinion and consensus is not good enough to guide precision medicine for serious illnesses
Why histology diagnoses are not a gold standard Lack of objective prospective studies! Uses non-specific lesions with low kappa values Requirement for subjective interpretation of rules e.g. too atrophic Incorrect rules e.g. ABMR Requirement for C4d staining: 2/3 are negative Isolated v-lesion=tcmr: 80% wrong often ABMR Inherent ambiguity Borderline Changing times since 1991: altered probabilities Less rejection, much more injury Lessons from 2 prospective consented studies of indication biopsies Genome Canada: 2005 present: 403 biopsies, 315 patients INTERCOM: 2011-present: 300 biopsies in 264 patients plus Paris collaboration (Alexandre Loupy): presensitized Hannover: 105 protocol biopsies Edmonton heart transplants: 106 biopsies Contents History of ABMR: clinical phenotype Molecular phenotype and diagnosis Prognosis and natural history Relationship to non-adherence
Peter B Medawar and Peter Gorer Peter Medawar Peter Gorer: died 1961 Historical accident Did the death of Peter Gorer cause transplant to underestimate the role of alloantibody for 40 years? But Paul Terasaki kept the flame alive Transplantation 49:85-91,1990 Description of antibody mediated rejection: a microcirculation disease with DSA distinct from T cell-mediated rejection
Alloantibody Mediated Rejection: alloantibody-mediated attack by cells on the microcirculation PTC Inflammatory Cells The disease is microcirculation inflammation (g, ptc) but lesions are non-specific C4d has been useful but many false ve, some false +ve Clin Exp Immunol 86: 464-470,1991 Association of capillary C4d staining with severe rejection, suggesting a role for antibody in post transplant rejection syndromes C4 splits into C4d b A marker, not a mechanism? a1 a3 a2(=c4d) a4 Thioester bond covalently binds tissue H. E. Feucht. Complement C4d in graft capillaries -- the missing link in the recognition of humoral alloreactivity. Am.J.Transplant. 3 (6):646-652, 2003 g
J Am Soc Nephrol 12: 574 582, 2001 2001 Association of capillary C4d staining with TG and chronic rejection TG is a time dependent feature: Rare before 1 year But lesions are not specific Double contours in transplant glomerulopathy TG JASN 13: 2371-2380, 2002: The full C4d+ve phenotype: Peritubular capillaritis, transplant glomerulopathy
AJT 9:2520-2531,2009: 2531 2009 Many kidneys with late C4d negative ABMR fail but are being misdiagnosed: the disease is really microcirculation inflammation with DSA C4d+ve ABMR C4d-ve ABMR (nonadherence!) Recurrent GN AKI, TCMR, CNI, etc Outcomes in 315 biopsied patients: Time of biopsy affects risk: time-dependent diseases 403datalock = 315 Patients p= 1.11e-06 Einecke G, et al. Am J Transplant. 2009;9(11):2520-3251. The molecular microscope A central diagnostic system that uses microarray measurement of transcripts in a renal biopsy core to interpret the state of a kidney transplant Molecular Uses predefined measurements to estimate: Microscope Probability of TCMR Probability of ABMR Extent of acute kidney injury (AKI) Extent of atrophy-scarring Prognosis (Interesting point: no signal from CNI toxicity )
AJT 9:1802-1810, 2009: The transcripts associated with all rejection (ABMR, TCMR, mixed) are mostly IFNG-induced transcripts (CXCL9/10/11, GBPs, MHC, INDO, etc) AJT 9:1802-1810, 2009 Separation of rejection from non-rejection but revealed interesting apparent histology false positives e.g. pretreatment Reeve et al AJT 9:1802-1810, 1810, 2009 Many of the genes selected by association with rejection were IFNG-induced, suggesting that IFNG is produced by both in TCMR (effector T cells) and ABMR (by NK cells triggered through Fc receptors?)
AJT 9: 2312-2323, 2009: Endothelial l transcripts t are increased in late C4d +ve but are also increased in many late cases that are C4d negative: C4d -ve ABMR Sis al AJT 9:2312-2323, 2323, 2009 Endothelial transcripts identified from the literature were abnormal in many diseases but provided some separation of ABMR from TCMR: proof of concept Normal kidney TCMR ABMR AJT 10:1812-1822,2010: DSA-associated transcripts: Association of ABMR with NK markers and IFNG effects as well as microcirculation transcripts
Hidalgo et el AJT 10:1812-1822,20101822,2010 In late indication biopsies the DSASTs included endothelium NK, and IFNG-induced transcripts Problem: some sharing with effector T cells AJT 13:645-655,2013 TCMR score correlates with tubulitis and interstitial infiltrate, and with unanimity among pathologists A minority of borderline, and some cases of PVN actually have TCMR TCMR score indicates many errors in inflamed biopsies: false positives due to early or late tissue injury and isolated v lesions No effect on graft survival When one pathologist diagnoses TCMR, another agrees only 45% of the time Reeve et al AJT 13:645-655,2013
Reeve et al AJT 13:645-655,2013 TCMR score V-lesions alone are not reliable for diagnosing TCMR: Only 5/24 were actually molecular TCMR AJT 13:645-655,2013 TCMR diagnosed by the TCMR score (microarray) does not affect future graft survival Death censored survi ival probability 0.6 0.8 1.0 0.0 0.2 0.4 BFC403 dataset: 315 patients TCMR Other Dx Edmonton Genome Canada study and INTERCOM: unpublished 2012 TCMRprob<0.1 N=279 F=71 TCMRprob>0.1 N=36 F=10 TCMRprob >0.1 TCMRprob <0.1 0 500 1000 1500 2000 2500 Event time (days)
April 2013 AJT Algorithm for the ABMR classifier Sellares et al April 2013 AJT Sellares et al April 2013 AJT
The ABMR score correlates with microcirculation lesions in the 403 indication biopsies ABMR score > ABMR score Gamma 0.2 [n=90] <0.2 [n=313] statistic B Median time of biopsy post transplant (months) 63 10 0.47*** Mean histologic lesion scores Peritubular capillaritis: ptc-score 1.41 0.23 0.81*** ABMR related lesions TCMR related lesions TCMR/ ABMR related lesion Glomerulitis: g-score 0.84 0.16 0.77*** Transplant glomerulopathy: cgscore 1.15 0.18 0.80*** Interstitial inflammation i-score 0.6 0.43 0.21* Tubulitis t-score 0.66 0.63 0.15 Intimal arteritis v-score 0.16 0.10 0.29 Interstitial fibrosis: ci-score 1.70 1.08 0.50*** Atrophy-scarring related lesions Tubular atrophy: ct-score 1.72 1.15 0.52*** Arterial fibrous intimal 1.38 1.11 0.23** thickening: cv-score Arteriolar hyalinosis: ah-score 1.41 0.88 0.36*** April 2013 AJT The ABMR score correlates with DSA positivity at the time of biopsy ABMR score DSA positive (Class II or I/II) HLA antibody not demonstrably donor-specific (NDSA) HLA antibody negative Not done B TOTAL > 0.5 40 (35) 5 0 0 45 0.2-0.5 23 (20) 4 14 4 45 < 0.2 61 (40) 61 151 40 313 TOTAL 124 (95) 70 165 44 403 April 2013 AJT The positive ABMR score more powerfully predicts subsequent graft loss than any histologic definition of ABMR Univariable results LL Hazard Ratio UL p-value Molecular ABMR score B 1.50 1.76 2.06 5x10-12 Histology-DSA ABMR (C4d+/-/mixed) 2.33 3.71 5.90 3x10-8 Histology-DSA C4d+ ABMR(both alone and mixed) 1.2 2.4 4.7 0.01 Histology-DSA C4d- ABMR (both alone and mixed) 2.0 3.3 5.5 2x10-6 Multivariable results C Molecular ABMR score B 1.21 1.53 1.91 2.6x10-4 Histology-DSA ABMR (C4d+/-/mixed) 0.93 1.79 3.43 0.08 April 2013 AJT
ABMR diagnosed by the ABMR Score (microarray) severely reduces future graft survival Death censored surv vival probability 0.6 0.8 1.0 0.0 0.2 0.4 ABMRprob<0.2 N=247 F=44 ABMRprob>0.2 N=68 F=37 Other diagnoses ABMR ABMRprob<0.2 ABMRprob>0.2 BFC403 dataset: 315 patients 0 500 1000 1500 2000 2500 Event time (days) Attribution of failures in the Genome Canada study
TCMR score Histologic TCMR Borderline Failures ABMR score Histologic ABMR Non adherence Mixed unpublished Figure 1b. Distribution of attributed causes of failure in failed grafts [n=60] p1 PVN 7% Medical conditions 10% Missing Data 5% 65% ABMR, probable ABMR, or mixed GN 13% ABMR 47% Nonadherent 48% Adherent 52% Mixed 5% Probable ABMR 13% Some late ABMR/mixed is due to non-adherence BUT what about the rest? Sellares et al AJT 12: 388, 2012 Progress is slow 1990: Recognition of ABMR as a microcirculation process distinct from TCMR, an interstitial-epithelial epithelial process 1991: Description of C4d staining 1998: ABMR meeting in Montreal 2001: Uptake by Banff required C4d: error 2009: Recognition of C4d negative ABMR 2011: continuing failure of histology consensus 2013: description of molecular ABMR score
Slide 47 p1 no titles usually on figures, just legends phil, 3/24/2011
Patent 1897 Technology adoption is very slow: the story of Rudolph Diesel 1858 1913 Invented the high-compression Diesel internal combustion engine: patented 1897 Thermal efficiency of 50% vs 6% for steam engines Widespread adoption took 60 years Institutional inertia Vested interest resistance Technical challenges Costs of new technology Rudolf Diesel met a mysterious end in September of 1913: boarded SS Dresden on a trip to England his body was found days later floating in the sea Suicide? Financial problems Murder? Strategic and corporate issues Dieselization of railroads: >1950 The problem is compacency Histology is good enough I like my horse Who needs a car? What we have discussed History of ABMR: clinical phenotype Molecular phenotype and diagnosis Prognosis and natural history Relationship to non-adherence
Thank you