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Parmjeet Randhawa Professor, Division of Transplantation Department of Pathology University of Pittsburgh ROLE OF ALLOGRAFT BIOPSY IN THE MANAGEMENT OF.

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Presentation on theme: "Parmjeet Randhawa Professor, Division of Transplantation Department of Pathology University of Pittsburgh ROLE OF ALLOGRAFT BIOPSY IN THE MANAGEMENT OF."— Presentation transcript:

1 Parmjeet Randhawa Professor, Division of Transplantation Department of Pathology University of Pittsburgh ROLE OF ALLOGRAFT BIOPSY IN THE MANAGEMENT OF TRANSPLANTED PATIENT

2 OPTIMAL RENAL ALLOGRAFT BIOPSY TECHNIQUES US guidance: serious AEs including death 2 cores obtained with 18 gauge needle ideal Evaluate adequacy of sample in biopsy suite Saving frozen tissue desirable for AMR, necessary for diagnosis ICGN EM is needed to characterize glomerular disease & demonstrate PTC-BMD

3 ADEQUACY CRITERIA FOR RENAL ALLOGRAFT BIOPSY 10 gloms, 2 arteries, examined in 7 slides Suboptimal biopsies can be diagnostic Cortex-severe tubulitis with no glomeruli -single artery with intimal arteritis Medulla-BKVN -diffuse C4d

4 GENERAL APPROACH Determine if it is adequate Low power grade i, ci, ct, cv Medium to high power evaluation needed to score g, t, v, cg, ah Synthesize findings & correlate clinical data

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6 THE BANFF SCHEMA FOR RENAL ALLOGRAFT PATHOLOGY REJECTION RELATED CATEGORIES -Acute or chronic antibody mediated rejection -Acute or chronic T-cell mediated rejection NON-REJECTION RELATED -Acute tubular necrosis -Drug toxicity, Donor derived pathology, -Infections, Recurrent disease -Technical & vascular complications

7 ACUTE REJECTION Acute T-cell mediated rejection Acute cellular rejection

8 ACUTE T-CELL MEDIATED REJECTION Th IL-2 CTL Granzyme B Perforin GMP-17 (TIA-1) Fas Ligand CD95 Th MM TNF  IFN  B anti  HLA Ab IL-4IL-10 APC Th IL-2Allorecognition Direct Direct Indirect IndirectIL-15 Dr. David Rush

9 ACUTE T-CELL MEDIATED REJECTION An alloimmune reaction mediated by cell mediated immunity Subclinical with normal creatinine Laboratory evidence of graft dysfunction Severe cases may show fever, graft tenderness, leukocytosis and eosinophilia

10 HISTOLOGIC CRITERIA FOR DIAGNOSIS OF ACUTE T-CELL MEDIATED REJECTION Predominantly mononuclear infiltrate Presence of parenchymal damage Occurrence of subendothelial inflammation in venules, arteries

11 GRADING OF ACUTE TCMR-1 Severity of inflammation Intensity of tubulitis Disruption of tubular architecture Presence of arteritis

12 GRADING OF INTERSTITIAL INFLAMMATION Limit assessment to cortex unless medulla alone sampled Ignore areas in continuity with capsule <10% area is assigned grade 0 Grades 1, 2, 3 are 10-25, 26-50, >50% Area is evaluated not the intensity

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14 INFLAMMATION IN AREAS OF SCARRING

15 IMPLICATIONS OF LESION SCORING IN AREAS WITH FIBROSIS DeKAF study 265 bx from 7 centers (7.5+/-6.1 y) 72% biopsies had tatr scores>0(conventional t = 0) 50% had iatr scores >0 (conventional i = 0) Inclusion of modified scores in data analyses yielded prognostic information Matas et al. Am J Transplant 2010: 10: 315

16 GRADING OF TUBULITIS Define area of maximal involvement Avoid tubules cut tangentially Grades 0-3 (0, 1-4, 5-10, >10) Look for disrupted tubules (2 foci, i2, i3) Atrophic tubules historically ignored Do not overlook non-atrophic areas or lymphocytes with blast transformation

17 GRADING OF INTIMAL ARTERITIS Look closely if interstitial hemorrhage, glomerulitis, or PTC inflammation Caveat added to report if < 4 arteries Grade v1 (<25%) (mild to moderate) Grade v2 (>25%) (severe) Grade v3 fibrinoid change, necrosis, transmural i (transmural intimal arteritis)

18 GRADING OF TCMR IN BANFF SCHEMA Borderline: criteria higher grade not met Type IA: t2 (i2 or i3) Type 1B: t3 (i2 or i3) Type IIA: v1 (any i or t score) Type II B: v2 Type III: transmural inflam/fibrinoid PTC C4d indicates concurrent AMR

19 BORDERLINE CHANGES SUSPICIOUS FOR ACUTE REJECTION Looks like rejection but criteria I, II not met i1 with any t grade OR t1 with any i Most often t1 tubulitis and i1 inflammation For biopsies with i2,3 look for t2,t3,v1 Theoretically i1,t2,t3 & i2,i3,t1 allowed Some cases evolving or treated higher AR

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21 SHOULD BIOPSIES WITH BORDERLINE CHANGES BE TREATED AS REJECTION? Scheweitzer et al. 58% CR, 30% PR Saad et al. 63% CR, 13% PR Dooper et al. 24% definite rejection Gaber et al. 8/8 (100%) CR

22 REASONS FOR HETEROGENEITY IN RESPONSE TO TREATMENT Borderline changes SUSPICIOUS for AR Non responsive cases may be non-immune (dehydration, ATN, CNI, infection) AMR, underlying CR Responsive cases may be early stage TCMR Examples of sampling error where biopsy did not sample more significant i or t lesions

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24 Type 1B

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28 INTIMAL ARTERITIS NOT ALWAYS TCMR 56% had donor specific antibodies 33% had PTC C4d diffuse or focal Can be pure AMR, pure TCMR, or mixed AMR-TCMR Sis et al Am J Transplantation 10: 421

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31 GRADING OF ACUTE REJECTION IS IMPORTANT FOR PROGNOSIS Banff 1 = 93% CR, 5 yr GS 67% Banff 2 = 79% CR, 5 yr GS 56% Banff 3 = 47% CR, 5 yr GS 32%

32 HISTOLOGIC GRADING OF INDIVIDUAL LESIONS Graft Loss t1 tubulitis0/360% t2 tubulitis0/360% t3 tubulitis10/3628% v1 arteritis11/3631% v2 arteritis11/1861% v3 arteritis11/11100%

33 IMMUNOHISTOCHEMICAL STUDIES IN ACUTE REJECTION Not necessary for Dx (except C4d) May occasionally help d/d PTLD vs AR Intraglomerular CD68 worse prognosis CD20 not correlation AMR or response CD4/CD8/CD68: stage of evolution, patient selection, immunosuppression.

34 MOLECULAR DIAGNOSIS TCMR MDx potentially valuable non-invasive tool Dx TCMR:sensitivity & specificities of 70-90% Disagreements in an average of 20% Reflect sampling issues, arbitrary grading thresholds & low frequency diagnoses Provides information that is complementary

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36 . ACUTE TUBULAR NECROSIS ACUTE TUBULAR INJURY ACUTE KIDNEY INJURY

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42 CALCINEURIN INHIBITOR TOXICITY 1.Cyclosporine 2.Tacrolimus

43 CALCINEURIN INHIBITOR CAN CAUSE FUNCTIONAL TOXICITY Elevation in serum creatinine Blood levels Tac/Csa may be elevated Biopsy shows no specific pathology Reduction of dosage restores serum creat Graft dysfunction attributed to vasospasm

44 CALCINEURIN INHIBITORS CAN CONTRIBUTE TO ATI CNI induced AKI confirmed in animals Clinically, prolonged cases of DGF can recover once calcineurin inhibitors switched Likely mechanism is reversal of drug induced vasoconstriction

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46 TUBULAR VACUOLIZATION IS NOT SPECIFIC FOR CNI Use of plasma expanders (dextran, mannitol), radiocontrast media, IVIG preps containing sucrose as a stabilizer, hyperosmolar sucrose infusions Frequently seen in context of ACR Occurs in donor biopsies Patients maintained on Azathioprine Attributed to CNI by exclusion

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49 OTHER CAUSES OF MYOCYTE VACUOLIZATION Amphotericin B therapy Use of vasopressors Injury secondary to cholesterol emboli Acute cellular rejection with intimal arteritis

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51 GIANT MITOCHONDRIA Association with CNI confirmed in rats Lesion of limited diagnostic utility, since it is uncommon, not readily recognized, & may needs EM to exclude lysosomes Not specific: described in azathioprine Rx, glomerulonephritis, acute tubular necrosis

52 . THROMBOTIC MICROANGIOPATHY

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57 LABORATORY ABNORMALITIES Thrombocytopenia Schistocytes on PBF Elevated serum bilirubin Low serum haptoglobin Presence of free Hb in plasma

58 COMMON DRUGS ASSOCIATED WITH THROMBOTIC MICROANGIOPATHY Cyclosporine Tacrolimus Sirolimus?

59 LESS COMMONLY USED DRUGS Vasopressors: dopamine Antimicrobials: penicillin, metronidazole Anti-inflammatory: penicillamine Anti-epileptic: phenytoin Chemotherapeutic agents: mitomycin

60 THROMBOTIC MICROANGIOPATHY IS MAY BE OF IMMUNE ORIGIN Antibody mediated rejection T-cell mediated ac rej with intimal arteritis. Ac rejection treated with OKT3 Autoimmune disorders with circulating autoab (ACA, APA, ANCA)

61 INFECTIONS ARE IN IMPORTANT CAUSE OF TMP Gastroenteritis (E.coli, Shigella) CMV infection (damage to endothelium) HCV (anti-cardiolipins) Parvovirus infection (targets endothelium) HIV infection (TTP syndrome)

62 RECURRENT GENETIC HUS Recurrent rate depends on mutation 50% for CFH, CFI (inhibitors complement) -liver-kid transplant may be curative Not expected MCP/CD46 (present in donor kidney, will catalyse breakdown of C3b, C4b normally) -extrarenal activation of complement -chimerism in glomerular capillaries -

63 OTHER BIOCHEMICAL DEFECTS ASSOCIATED TMP Deficiency of anti-thrombin III Protein C Protein S Factor V mutation (resistance to Protein C) Homocysteinemia (B6, B12 deficiency)

64 CNI ASSOCIATED ACUTE ARTERIOLOPATHY Intimal edema and fibrin Necrosis of myocytes leaves behind empty basement membranes bags Filled up by knot like protein deposits Knots fuse to form ring of pearls

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66 ARTERIOLONECROSIS Fibrinoid change in arteriolar wall Collapse of distal glomeruli Healing by onion skin change

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69 CHRONIC GRAFT DYSFUNCTION Evolves slowly Usually begins > 3m post-tx Typically progressive Rate of decline, may be punctuated by periods of stability

70 MAGNITUDE OF PROBLEM

71 CAUSES OF CHRONIC GRAFT DYSFUNCTION REJECTION RELATED CATEGORIES -Chronic active T-cell mediated rejection -Cronic active antibody mediated rejection NON-REJECTION RELATED CATEGORIES -Calcineurin inhibitor toxicity -Glomerulonephritis -Recurrence of original disease -Donor disease (age, hypertension) -RA stenosis, reflux nephropathy, recurrent UTI/BKN -Technical & vascular complications h

72 CHRONIC REJECTION (BANFF 1991) No distinction made between chronic active TCMR or chronic AMR Chronic Transplant Glomerulopathy New onset arteriosclerosis (not pre- existing donor disease)

73 CHRONIC TRANSPLANT GLOMERULOPATHY Reduplication of GBM Subendothelial translucent material Mesangial interposition (tram track MST) Suggested increase in capillary wall thickness of >2 fold, at least 3 capillary loops Ivanyi 2001; Mod Pathol 14:1200

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78 DIAGNOSTIC CRITERIA FOR CHRONIC REJECTION (BANFF 1991) Chronic Transplant Glomerulopathy New onset arteriosclerosis (exclude pre- existing donor disease) - intimal fibrosis without elastosis - fibroelastosis may develop after prolonged graft dysfunction

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80 Dr Robert Colvin, ATC 2006 R. Colvin

81 DIFFERENCES BETWEEN GRAFT AND ENDEMIC ATHEROSCLEROSIS 1.Extent of involvement (diffuse vs proximal) 2.Type of luminal compromise distinctive 3.Calcification is late & less common in GAS. 4.Differences in biochemical composition (Apo-A,E, biglycans vs Apo-B, decorin) Differences are relative & not absolute

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86 CLASSIFICATION OF CHRONIC REJECTION (BANFF 2005) Chronic active T-cell mediated rejection - chronic graft arteriopathy (intimal thickening +/- inflamm+/- neointima) - absence of C4d and DSA Chronic active antibody mediated rejection -chronic tissue injury (cg, ci, ptcdd) - with C4d and DSA

87 CHRONIC TXGM IS FRQUENTLY DUE TO CHRONIC AMR 30-70% biopsies DSA (mostly class II) 20-40% have C4d in peritubular capillaries ENDAT seen on microarray analyses Significance of C4d –ve cg+ biopsies? AMR, TCMR, TMP, MPGN

88 REDUPLICATION OF PERITUBULAR CAPILLARY BASEMENT MEMBRANES Needs EM: single PTC with 7 layers 3 PTC’s with 5-6 layers Duplication >60-75% of circumference Thus defined, lesion will only rarely occur in other diseases: TMP, reflux, Phenacetin kidney (repeated endothelial injury)

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90 CHRONIC CALCINEURIN INHIBITOR TOXICITY Cyclosporine Tacrolimus

91 PATTERNS OF TISSUE INJURY IN CHRONIC CNI TOXICITY Arteriolar lesions Striped Interstitial fibrosis/tubular atrophy Chronic phase thrombotic microangiopathy Ischemic glomerulopathy Focal segmental glomerulosclerosis Juxtaglomerular hyperplasia Tubular vacuolization and calcification

92 ARTERIOLAR LESIONS 1-3 layers smooth muscle No complete internal elastic lamina Diameter < 1/3 rd of a glomerulus

93 ARTERIOLAR HYALINOSIS Deposition of glassy material Initially in endothelial layer Followed by circumferential extension Medial involvement

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95 ARTERIOLAR HYALINOSIS Donor disease Diabetes mellitus Hypertension PSS Chronic thrombotic microangiopathy Radiation

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98 . INTERSTITIAL FIBROSIS IN CALCINEURIN INHIBITOR TOXICITY

99 PATTERNS OF FIBROSIS Striped fibrosis: alternating areas of atrophic AND normal or even hypertrophic renal parenchyma Diffuse interstitial fibrosis: more extensive and uniformly distributed collagen deposition

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101 GLOMERULONEPHRITIS IN THE ALLOGRAFT KIDNEY Donor transmitted glomerulonephritis De-novo glomerulonephritis Recurrence of original disease Morphologic spectrum similar to native kidneys

102 DONOR TRANSMITTED GLOMERULONEPHRITIS IgA nephropathy SLE MPGN

103 DE-NOVO GLOMERULONEPHRITIS IN THE ALLOGRAFT KIDNEY Work up infection, neoplasms, autoimmune Review medication (captopril, hydralazine) Elicit past h/o horse ATG for TCMR Recently C4d + cases MGN recognized Most frequently, however, no cause found

104 DE-NOVO ANTI-GLOMERULAR BASEMENT MEMBRANE DISEASE Seen in patients with Alport’s (<15%) Exposure to Goodpasture’s antigen Often subclinical but may cause graft loss Histology: proliferative or crescentic GN ELISA, IF confirmation antiGBMab

105 Briganti et al 2002; NEJM 347: 103 RECURRENT GLOMERULONEPHRITIS 3rd most frequent cause of graft loss at 10y (1=chr rej, 2=death with functional graft) Accounts for 8.4% of all lost grafts overall Range %, highest if male sex, primary disease FSGS or MGN

106 Hariharan et al 1999; Transplantation 68:635 RECURRENT GLOMERULAR DISEASE Focal segmental glomerulosclerosis 3.4y Membranoproliferative GN 3.6y Membranous nephropathy 3.3y IgA nephropathy 4.4y Life of avg CRTX 13y, LRD 21y

107 Hariharan et al 1999; Transplantation 68:635 RATES OF GRAFT FAILURE Focal segmental glomerulosclerosis 65% Membranoproliferative GN 66% Membranous nephropathy 44% IgA nephropathy 41% Diabetes mellitus 53% HUS/TTP 63%

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111 MANAGEMENT OF PATIENTS TRANSPLANTED FOR FSGS Early dx & intervention best prognosis Ur.prot/creat ratio OD to disch, wx4, mx12, Ratio > 0.5 consider 24 hr measurement Puria >2g/day consider biopsy confirmation Plasmapheresis removes permeability factor

112 DE NOVO FOCAL SEGMENTAL GLOMERULOSCLEROSIS FSGS in patient tx for another cause Common finding in ISTA Most cases months to years after Transplant -Ischemic glomerulopathy secondary to ah -Hyperfiltration injury, a compensatory response to loss renal mass

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116 RECURRENT METABOLIC DISEASES Diabetes mellitus Amyloidosis LCDD Lipoprotein glomerulopathy Fabry’s disease

117 RECURRENT DIABETIC NEPHROPATHY Electron microscopy thickening 6 months Arteriolar hyalinosis 2 years Nodular glomerulosclerosis 18% by 13 years Rate of progression slow 10 year graft survival 32% vs 52% in one study Graft loss attributable entirely to DM<10%

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120 RELATIVE IMPORTANCE OF VARIOUS CAUSES OF CHRONIC GRAFT DYSFUNCTION Non-Immune Causes 80% Chronic Rejection 20% 1/3-1/2 C4d+

121 NON-IMMUNE FACTORS Unclassified 37% Drug Toxicity 14% Recurrent disease 16% De novo GN 13% 2% if EM DNA-MA

122 ASSIGNING PRIMARY CAUSE OF CGD HAS LIMITATONS ah often simplistically taken as CNIT - virtually all patients hypertensive - 1/3 rd are diabetic - variable proportion are ECD of age >50 C4d +ve CGD cases are classified as CAMR -many have had prior episodes of TCMR -most will have hypertension &/or diabetes

123 CHRONIC ALLOGRAFT NEPHROPATHY Coined at 1991 Banff Meeting - to recognize multifactorial nature CGD - inability to determine etiology in all cases Widely & variably used to denote a process assumed to have no prev/therapeutic measures Banff 2005 proposed elimination & suggested ISTA-NOS (no evidence of specific pathology)

124 GRADING OF CAN/ISTA NOS Interstitial fibrosis (ci 0-3) ( 50) Tubular atrophy (ct 0-3) (0, 50) Arteriosclerosis (cv 0-3) (% luminal occl) Arteriolar hyalinosis (ah 0-3) (#, severity) Tx glomerulopathy (cg 0-3)( 50)

125 PROGNOSTIC IMPORTANCE OF HISTOLOGIC GRADING

126 GROUPING OF LESIONS INTO CLUSTERS Tubulo-interstitial inflammation (i, t) Microcirculation lesions (g, v, ptc, cg) Scarring hyalinosis lesions (ci, ct, cv, ah) Based on 234 unselected biopsies graded for acute and chronic Banff lesions Sis et al. Am J Transplant 2010: 10: 421

127 UTILITY OF HISTOLOGIC CLUSTERS IN DeKAF STUDY Two different analyses performed Only g, i, mm, ct, cv, ah scores used in 1 analysis All scores including i-atr,t-atr used in 2 nd analysis Six clusters difft prognosis 18 m GS 96% best cluster  75% worst cluster Matas et al. Am J Transplant 2010: 10: 315

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129 PATHOGENESIS OF CAN/ISTA NOS Multifactorial parenchymal INJURY Exhausted REPARATIVE ABILITY due to impaired tub regen or vascular remodeling Persistence of injury inflammation, progressive fibrosis, and GS Self-perpetuating hyperfiltration injury culminating as graft failure

130 CAUSES OF RENAL ALLOGRAFT DYSFUNCTION REJECTION RELATED CATEGORIES -Acute & chronic Ab mediated rejection -Acute & chronic T-cell mediated rejection NON-REJECTION RELATED CATEGORIES -ATN, CNIT, Infections, GN, Recurrent native disease, donor derived pathology

131 RENAL BIOPSY REPORTS SHOULD USE STANDARDIZED TERMINOLOGY Example of non-standardized report: Allograft kidney, needle biopsy: Interstitial inflammation and tubulitis c/w ac acute rejection (?TCMR, AMR, ?grade) Patchy interstitial fibrosis & tubular atrophy

132 A STANDARDIZED BIOPSY REPORT Diagnosis: acute T-cell mediated rejection Grade severity: Banff grade 1 (g0,i2,t3,v0) Worse tubulitis compared to prior biopsy Chronicity: severe interstitial fibrosis (cg2, ci3, ct3, cv2) We use standardized Banff scoring templates -saves time & facilitates large databases

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