Peritubular Capillary Inflammatory Cell Accumulation Scoring Dr Ian W. Gibson Associate Professor, Pathology University of Manitoba

Peritubular Capillary Inflammatory Cell Accumulation Scoring - Objectives Review importance of assessment of PTC in renal allograft pathology Illustrate the PTC inflammatory cell accumulation (ptc) score proposed at Banff 2003 conference My experience with applicability & usefulness of proposed ptc score (NB. work in progress)

Peritubular Capillary Lesions in Renal Allograft Biopsies Acute lesions Inflammatory cell accumulation / margination / dilatation (AR) Ig & complement (C4d) deposition (AbMAR) Microthrombi (TMA) Chronic lesions Basement membrane multilayering (CR) Complement (C4d) deposition (AbMCR) Progressive injury & loss (CAN)

Peritubular Capillaritis in Renal Transplants – NEPHROL discussion Nov 1994 Daniel Salomon – ‘A lesion Byron Croker and I found very interesting … the presence of a peritubular capillary lymphocytic infiltrate, usually more prominent at the corticomedullary junction … typically of lymphocytes but also sometimes PMNLs … associated with rejection in over 50 cases biopsies for DGF within 10-14 days post-Tx … also in otherwise classic acute TI rejection at later time points … Should these lesions be studied in the context of the Banff schema?’ Kim Solez – ‘Like acute glomerulitis, peritubular capillaritis is likely to be a very heterogeneous lesion, sometimes due to rejection, sometimes CsA toxicity, sometimes due to ATN … The peritubular accumulation of PMNLs is highly suggestive of AbMAR … accumulation of mononuclear cells can have a number of different etiologies … I myself doubt that peritubular capillaritis will turn out to be useful … except when it involves PMNLs’ Michael Kashgarian – ‘My experience is similar … I have found capillaritis in a number of instances i.e. ATN, CsA toxicity and rejection. It did not help to distinguish rejection from the other entities. We probably should note it without giving it any specific diagnostic significance’

PTC Inflammatory Lesions PMNL accumulation in AbMAR with antidonor HLA class I or II Ab (Halloran et al 1990, 1992, Trpkov et al 1996, Collins et al 1999, Mauiyyedi et al 2002) & ABO incompatibility (Morozumi et al 2001) PMNL accumulation correlates with C4d+ (Magil & Tinckam 2003) Platelet accumulation & PMNLs in AbMAR (Meehan et al 2003)

PTC Inflammatory Lesions Endothelial cell necrosis & mononuclear cell accumulation noted in some cases of AbMAR (Lajoie 1997) Endothelial cell swelling, apoptosis, lysis, fragmentation & marginating degranulated mononuclear cells & PMNLs in AbMAR (Liptak et al, Banff 2003 abstract) Mononuclear cell accumulation & dilatation correlates with C4d+ in CR, but no clear association with interstitial rejection (Regele et al 2002)

C4d+ PTCs with mononuclear inflammatory cell accumulation - “The pathogenic or diagnostic relevance of inflammatory cells accumulating in PTC is currently not clear” (Regele et al J Am Soc Nephrol 13:2371-2380, 2002)

AbMAR, g3 with TMA, severe ATN & PMNLs in PTC Mac387

The Point of Banff Directing the eye / mind of the pathologist to relevant areas of the Bx, which for acute rejection are: glomerulitis – g score interstitial inflammation – i score lymphocytic tubulitis – t score intimal / transmural arteritis – v score By the same principle, in particular for AbMAR, a further highly relevant area is: peritubular capillaritis - ? ptc score

Proposal – Banff ptc score ptc 0 – no significant cortical peritubular inflammatory changes ptc 1 – cortical peritubular capillary with 3 to 4 luminal inflammatory cells* ptc 2 – cortical peritubular capillary with 5 to 10 luminal inflammatory cells* ptc 3 – cortical peritubular capillary with >10 luminal inflammatory cells*

Proposal – Banff ptc score, notes numbers refer to highest number of all types of inflammatory cells (PMNLs, lymphocytes, monocyte / macrophage) * indicates cells are mononuclear only indicate extent of lesion when not diffuse (>50%) indicate if PTCs are dilated exclude cells in PTC from i score (PAS, silver stains optimal) avoid scoring in subcapsular cortex, areas of necrosis / infarction & areas of pyelonephritis

ptc0, PTCs with 1-2 mononuclear cells only

ptc1*, mononuclear cells only

ptc1, PMNLs

7 days post-Tx, AbMAR, ptc2, PMNLs & mononuclear cells

7 days post-Tx, AbMAR, ptc2, PMNLs & mononuclear cells

ptc2, PMNLs & mononuclear cells ptc2, mononuclear cells & PMNLs ptc2, mononuclear cells & PMNLs ptc2*, mononuclear cells only

ptc2*, mononuclear cells only

ptc2*, mononuclear cells only (n=8)

10 weeks post-Tx, AbMAR, ptc3, mononuclear cells & PMNLs

2 days post-Tx, ptc3, PMNLs only, n=14

ptc3, mononuclear cells & PMNLs ptc3*, mononuclear cells only (n=11) ptc3*, mononuclear cells only (n=12)

Banff IB AR (g1,i2,t3,v0,ptc3*)

Banff IIA AR, i3,t3,v1,ptc3, eosinophil-rich & int. hemorrhage

Banff IIA AR, i3,t3,v1,ptc3, eosinophil-rich, int. hem. & C4d +

ptc2 & C4d + ptc2 & C4d + ptc2 & C4d - ptc2 & C4d -

LCA CD3 CD68 CD68

Longitudinally sectioned PTC, c/w ptc2*

Peritubular (ptc) versus interstitial (i) infiltrates

Peritubular (ptc) versus interstitial (i) infiltrates

Peritubular capillaritis (ptc2 & ptc3) with dilatation

Venule with inflammatory cell accumulation

Vasa recta infiltrates in medulla

Applicability of ptc score The ptc score, as proposed at Banff 03, is applicable to adequate renal allograft Bxs It does require more time / dedication to the LM examination Assessment of extent of Bx involved is challenging, suggest limiting to diffuse (>50%) versus focal (<50%) Avoid scoring, or make allowance for, longitudinally sectioned PTCs Assessment of dilatation very subjective PTCs must be distinguished from venules & vasa recta

Diagnostic utility of ptc score The ptc score does not equate with specific diagnoses, but is helpful to include in overall assessment of an allograft Bx, as part of clinicopathological correlation The ptc score often appears to correlate with overall inflammatory ‘activity’, but in occasional allograft Bxs peritubular inflammation can be the predominant lesion Distinguishing ptc score with & without (*) PMNLs is worthwhile, as literature does associate PMNLs with AbMAR, but seeing some PMNLs does not make a diagnosis of AbMAR The ptc score is helpful for comparison of sequential Bxs, e.g. assessing responses to therapy The ptc score, like all Banff scores, should be useful in assessing allograft Bxs for clinical trials Reproducibility of ptc score needs to be tested / proven

ptc scoring useful for comparison of sequential biopsies A case of AbMAR g1i2t1v0 ptc2 g0i2t1v0 ptc1 g0i0t1v0 ptc3 g0i2t1v0 ptc2 g0i1t1v0 ptc2 Creatinine (mmol/L) Days Post-Transplant Clinical data: P Nickerson

Peritubular capillaritis in early renal allograft is associated with the development of chronic rejection and chronic allograft nephropathy Aita et al Clin Transplant 19 (Suppl.14):20-26 2005 Evaluated PTCitis (using Banff 03 ptc score) in early allografts with ACR (n=15), AbMAR (n=8), ptc 1/2 only (n=16) & no AR/ptc0 (n=14) & in subsequent later Bxs ptc score highest in AbMAR group (“in early allografts, ptc scores of 2 or 3 are highly suggestive of acute/active rejection by humoral immunity”) In some cases, PTCitis persisted, & ptc score gradually increased during follow-up >30% of cases with ptc1/2* only in early Bx progressed to CR, suggesting need to monitor for ptc1/2 in early allografts & follow-up closely to prevent later development of CR Data proved the applicability of the Banff 03 ptc score; regarded as “reliable” & “adequate for evaluating the severity of PTCitis”

ptc 1* (ACR) ptc 2 (AbMAR) ptc 3 (AbMAR) Peritubular capillaritis in early renal allograft is associated with the development of chronic rejection and chronic allograft nephropathy Aita et al Clin Transplant 19 (Suppl.14):20-26 2005 ptc 1* (ACR) ptc 2 (AbMAR) ptc 3 (AbMAR)

Peritubular capillaritis in early renal allograft is associated with the development of chronic rejection and chronic allograft nephropathy Aita et al Clin Transplant 19 (Suppl.14):20-26 2005

Future Clinical Trials - PTC Data

PTC mononuclear inflammatory cell accumulation could reflect site of leucocyte trafficking Renkonen et al, ‘Site of influx of inflammatory white cells into a rejecting rat renal allograft’ Transplantation 47:577-579 1989 Renkonen et al, ‘Characterization of high endothelial-like properties of peritubular capillary endothelium during acute renal allograft rejection’ Am J Pathol 137:643-651 1990 Ivanyi et al, Postcapillary venule-like transformation of peritubular capillaries in acute renal allograft rejection. An ultrastructural study’ Arch Pathol Lab Med 116:1062-1067 1992

PTC mononuclear inflammatory cell accumulation could reflect upregulation of adhesion molecules VCAM-1 expressed on PTC endothelium in acute & chronic rejection (Solez et al ‘Adhesion molecules and rejection of renal allografts’ Kidney Int 51:1476-1480 1997)

Chronic PTC Lesions Loss of endothelium & basement membrane thickening / reduplication on PAS in CR (Mauiyyedi et al 2001) Moderate-severe basement membrane multilayering on EM associated with CR & chronic Tx glomerulopathy (Monga et al 1990, Morozumi et al 1997, Drachenberg et al 1997, Ivanyi et al 2000, Gough et al 2001, Vongwiwatana et al 2004) C4d deposition in subset of CR – chronic humoral rejection (Mauiyyedi et al 2001, Regele et al 2002, Ishii et al 2005) BM multilayering correlates with humoral mechanisms (Lajoie 1997, Takeuchi et al 2000) & PTC C4d+ (Mauiyyedi et al 2000, Regele et al 2002, Vongwiwatana et al 2004) Progressive injury, destruction & loss in CR/CAN in association with progressive interstitial fibrosis (Bishop et al 1989, Shimizu et al 2002, Ishii et al 2005)

3 years post-Tx, gradual rise in sCr, cv2 & cg2

3 years post-Tx, gradual rise in sCr, cv2, cg2 & PTC BM multilayering

7/14 had histologic features of CR (cg & cv) Histological analysis of late renal allografts of antidonor antibody positive patients with C4d depositis in peritubular capillaries Aita et al Clin Tranplant 18 (Suppl.11):7-12 2004 Evaluated PTCitis (using Banff 03 ptc score) in late allografts (>1 year) with C4d+ & antidonor Ab + (n=14) 7/14 had histologic features of CR (cg & cv) PTCitis of varying severity observed in all 14 cases: ptc1 (n=3), ptc2 (n=5), ptc3 (n=6) ptc score did not correlate with other Banff score PTC BM multilayering on EM found in 12/14 Severity of multilayering did not correlate with ptc score

Histological analysis of late renal allografts of antidonor antibody positive patients with C4d depositis in peritubular capillaries Aita et al Clin Tranplant 18 (Suppl.11):7-12 2004 ptc3 ptc2 ptc1

Could peritubular capillaritis & capillary basement membrane thickening be the diagnostic indicator of chronic rejection Aita et al Banff 2005 abstract Analysed late renal allograft Bxs (>3 years post-Tx) from 53 patients for ptc score (as per Banff 03) & for PTC BM thickening (using PAS) Compared ptc & ptcbm scores in cases of CAN with & without CR & non-CR/CAN group, analyzed correlation between the two scores & C4d staining The ptc & ptcbm scores correlated significantly (p<0.001) & both scores were highest in group of CAN with CR (in 23/27 cases, both scores were 2 or 3) No correlation between ptc / ptcbm scores & C4d staining Concluded that high ptc / ptcbm scores are highly suggestive of CR, and should be used as diagnostic criteria

Peritubular capillaritis in early renal allograft is associated with the development of chronic rejection and chronic allograft nephropathy Aita et al Clin Transplant 19 (Suppl.14):20-26 2005

3 years post-Tx, ptc2* IF C4d, weak diffuse ICC C4d, focal PTC+

3 years post-Tx, ptc2*

3 years post-Tx, ptc2* & PTC BM multilayering

3 years post-Tx, PTC BM multilayering (up to 6 layers) Serology shows anti-class II donor-specific Ab (DQ6, titre >1:64)

Features of Chronic Humoral Rejection A constellation of findings, including: - Peritubular capillaritis - PTC C4d deposition - PTC BM multilayering - Chronic Tx glomerulopathy Therefore ptc scoring would be helpful in this chronic context, as well as in acute stages of humoral rejection

Peritubular Capillary Inflammatory Cell Accumulation Scoring - Summary Assessment of PTCs in renal allograft pathology is important, and can provide useful diagnostic information The proposed PTC inflammatory cell accumulation (ptc) score is feasible, and there are compelling reasons for inclusion with established Banff scores Much more work is needed on the diagnostic significance of this lesion, and widespread application of the ptc score would facilitate that process