Clinicopathologic Analysis of Acute Vascular Rejection Cases After Renal Transplantation

Clinicopathologic Analysis of Acute Vascular Rejection Cases After Renal Transplantation T. Shimizu, T. Tanabe, K. Omoto, H. Ishida, and K. Tanabe ABSTRACT Introduction. Histopathologic change of acute vascular rejection (AVR) is characterized by intimal arteritis and transmural arteritis. In this report, we discuss the clinicopathologic analysis of AVR cases after renal transplantation. Patients. AVR was diagnosed in 28 renal transplant recipients followed up in our institute between January 2003 and November 2010. Results. Among 28 cases of AVR, 18 were mild (v1 in Banff 07 classification), 8 were moderate (v2), and 2 were severe (v3). Interstitial inflammation was present in 25 biopsy specimens. Moderate to severe tubulitis (t2–t3) was present in 10 biopsy specimens and transplant glomerulitis in 17; peritubular capillaritis was in 25 of the 28 biopsy specimens. C4d deposition in peritubular capillaries was observed in 11/28 cases. By using assays with plastic beads coated with human leukocyte antigen (HLA) in the 28 cases, we detected circulating anti-HLA alloantibody in 18 patients, among which 11/28 were donor-specific. Acute antibody-mediated rejection was diagnosed in 6 cases. Among AVR cases, 19/28 displayed steroid-resistant rejection (SRR) requiring greater anti-rejection therapy (ART), including muromonab CD3 injection, gusperimus injections, plasmapheresis, intravenous immune globulin, and/or rituximab injections. Twenty of 28 patients recovered renal allograft function after ART, and 26/28 grafts are functioning. Among the 2 cases of graft loss, only 1 patient lost his graft due to AVR. Conclusions. In some cases, AVR might be provoked by anti-donor antibodies. The prognosis of the graft exhibiting AVR was relatively good using available immunosuppression. ISTOPATHOLOGIC changes of acute vascular rejection (AVR) are characterized by intimal and transmural arteritis.1 Intimal arteritis is defined in the Banff working classification 1993 as intimal thickening with inflammation of the arterial subendothelial space ranging from rare intimal inflammatory cells to necrosis of the endothelium with deposition of fibrin, platelets, and inflammatory cells.1 The cellular infiltrate in intimal arteritis is always composed of lymphocytes and monocytes. The severity of intimal arteritis is determined by the number of affected vessels as well as by the intensity of the individual lesions; mild degrees of intimal arteritis can be extremely focal.1 Transmural arteritis as defined in the Banff classification 1993 describes an injury with inflammation of the whole arterial wall including the media, necrosis of medial smooth muscle cells, fibrin insudation, and cellular infiltration with mononuclear as well as polymorphonuclear leukocytes.1 In the Banff 97 classification, quantitative criteria

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for intinmal arteritis (v score) are defined as follows: v0, no arteritis; v1, mild-to-moderate intimal arteritis in at least 1 arterial cross section; v2, severe intimal arteritis with at least 25% luminal area lost in at least 1 arterial cross section; and v3, transmural arteritis and/or arterial fibrinoid change and medial smooth muscle necrosis with lymphocytic infiltration in vessels.2 In the Banff’09 classification, AVR fall into 1 of 4 categories: acute T-cell–mediated rejection (ATMR) Type IIA, ATMR Type IIB, ATMR Type III, and acute antibody-mediated rejection (AAMR) Type III.3 In this report, we have presented a clinicopath-

From the Department of Urology, Tokyo Women’s Medical University, Tokyo, Japan. Address reprint requests to Tomokazu Shimizu, MD, Department of Urology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: [email protected] © 2012 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 44, 230 –235 (2012)

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ologic analysis and an examination of the prognosis of AVR cases after renal transplantation.

MATERIALS AND METHODS From January 2003 to November 2010, we diagnosed 28 renal transplant recipients to show AVR upon renal allograft biopsies. These 28 patients were retrospectively reviewed by clinical record and were enrolled in this study. The immunosuppression mainly consisted of methylprednisolone (MP), cyclosporine (CYA) or tacrolimus (TAC), and mycophenolate mofetil (MMF). In some cases basiliximab and rituximab were used as induction therapy. The renal allograft biopsy was performed for the diagnostic analysis of graft dysfunction and proteinuria, or as a protocol procedure. The biopsy specimens were examined using light, electron, and immunofluorescence microscopy. They were scored according to the Banff 2009 classification.3 AVR was diagnosed using light microscopy based on intimal arteritis and transmural arteritis. C4d staining was performed on all biopsies with available frozen tissue. Linear staining of peritubular capillaries (PTC) for C4d with an intensity of very mild or greater was graded as minimal positive (C4d1 in Banff 2007 classification), focal positive (C4d2), or diffuse positive.4 Patient sera obtained in the peri-biopsy period were screened for anti– human leukocyte antigen (HLA) class I and class II antibodies by assays using plastic beads coated with HLA antigens (Luminex Technology, One Lambda, VEITAS, Tokyo, Japan). The study was approval by our ethics committee.

Table 1. Clinical Characteristics of Patients (n ⴝ 28) With AVR Diagnosed by Biopsy Mean

Recipient factors Age at kidney transplantation (y) Gender (M/F) Donor factors Age at donation (y) Gender (M/F) Transplantation factors No. of transplantations (1/2) ABO (COM/INCOM/MM) Living/deceased donor (n) HLA-AB mismatches HLA-DR mismatches Immunosuppression TAC/CYA (n) MMF (n) Basiliximab/rituximab (n) Characteristics at time of biopsy Posttransplantation time (median, d) Episode biopsy/protocol biopsy (n) Serum creatinine (median, mg/ dL) Proteinuria (ⱖ1, dipstic) (yes/ trace/no)

Minimum-Maximum

46.8 17/11

23–69

56.9 15/13

38–67

25/3 14/10/4 27/1 2.00 1.14

0–4 0–2

27/1 28 14/10 52.0

Score in Banff Classification

No. of Cases

v0:v1:v2:v3 i0:i1:i2:i3 t0:t1:t2:t3 g0:g1:g2:g3 ptc0:ptc1:ptc2:ptc3 C4d0:C4d1:C4d2:C4d3

0:18:8:2 3:11:10:4 13:5:8:2 11:10:2:4 3:9:15:1 16:0:5*:7†

Abbreviations: v, arteritis; i, interstitial inflammation; t, tubulitis; g, glomerulitis; ptc, peritubular capillaritis; C4d, C4d deposition in PTC. *Includes 3 cases of ABO incompatible renal transplantation. † Includes 5 cases of ABO incompatible renal transplantation.

RESULTS

The background characteristics of the 28 patients with AVR are shown in Table 1. The mean age at renal transplantation was 46.8 years including 17 male and 11 female subjects. The mean donor age was 56.9 years. Twenty-seven patients received living-related and 1 an deceased donor kidney. Fourteen patients were ABO-compatible cases, 10 were ABO-incompatible, and 4 were ABO minor mismatches. The mean HLA-AB and HLA-DR mismatches were 2.00 and 1.14, respectively. AVR was diagnosed at a median of 52.0 days posttransplantation (range, 9 – 454), with a mean serum creatinine (s-Cr) level of 2.23 mg/dL. Proteinuria was observed in 12 patients at the time of biopsy. The histopathology of the 28 allograft biopsy specimens with AVR is shown in Table 2. Of the 28 AVR biopsy specimens, 18 were mild (v1 in Banff 2007 classification), 8 moderate (v2), and 2 severe (v3). Interstitial inflammation (i1–i3) was present in 25 biopsy specimens, moderate to severe tubulitis (t2–t3) was present in 10, transplant glomerulitis (g1– g3) was present in 17, and peritubular capillaritis (ptc1–ptc3) was present in 25/28 biopsy specimens. C4d deposition in PTC was observed in 11/28 cases as either focal (C4d2; n ⫽ 5) or diffuse staining (C4d3; n ⫽ 7). All 28 patients had sera available for anti-HLA analysis in the peribiopsy period (Table 3). Alloantibodies were detected in 18 patients: both anti-HLA class I and class II Table 3. The Status of Circulating Anti-HLA Antibodies in 28 AVR Cases With Available HLA Analysis in the Peri-biopsy Period N

9–454

23/5 2.23

Table 2. The Histopathology of 28 Allograft Biopsy Specimens With AVR

1.08–5.95

12/6/10

Abbreviations: M, male; F, female; COM, ABO compatible; INCOM, ABO incompatible; MM, minor mismatch.

Anti-HLA antibodies Negative Positive Anti-HLA class I antibodies alone Anti-HLA class II antibodies alone Both Anti-HLA class I and class II antibodies Donor-specific anti-HLA antibodies Negative Positive Anti-HLA class I antibodies alone Anti-HLA class II antibodies alone Both Anti-HLA class I and class II antibodies

10 (36%) 18 (64%) 9 3 6 17 (61%) 11 (39%) 5 5 1

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Table 4. The Histopathology of 18 Allograft Biopsy Specimens With v1 and the Association of AVR With Anti-HLA Antibody and C4d Deposition in PTC Banff Classification Score

Anti-HLA Antibody

Case No.

v

i

t

g

ptc

C4d

Class I

Class II

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0 0 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3

0 0 0 0 0 1 1 1 0 0 2 2 2 2 3 0 2 2

0 3 1 1 3 0 2 1 3 2 1 1 0 3 1 0 1 3

1 2 1 1 1 1 2 1 2 2 1 2 1 2 2 2 2 2

3* 2 3* 3* 0 0 3 3* 2 3* 0 0 0 0 0 0 0 2*

N P P N N N P N P P P N P N P N P P

N N P N P N P N N N P N N N N P N P

DSA

N N N N Class N Class N Class Class Class N N N N N Class Class

II I ⫹ II I I I

I II

Abbreviations: P, positive; N, negative. *ABO incompatible renal transplantation cases.

demonstrated PTCtis in their biopsy specimens, glomerulitis in 8, and PTC C4d deposition in 6. AAMR was diagnosed in 6 cases by histopathologic features of PTCtis and/or glomerulitis with circulating antibodies to donor HLA class I and class II (DSA) and with focal to diffuse C4d deposition in PTC in renal allograft biopsy specimens using immunofluorescence study. Tables 6 and 7 show the associations of AVR with anti-rejection therapy (ART), s-Cr level at biopsy, after ART, and at present, as well as renal allograft prognosis. Among the AVR cases, 19/28 showed steroid-resistant rejection (SRR) requiring muromonab CD3 (OKT3), intravenous immune globulin (IVIg), gusperimus (DSG), or rituximab injections and/or plasmapheresis, (Tables 6 and 7). In v1 cases, SRR was observed in 11/18; in v2 and v3

antibodies in 6, anti-HLA class I alone in 9, and class II alone in 3 patients (Table 3). Of the 18 subjects with anti-HLA antibodies, 11 showed donor-specific anti-HLA antibodies (DSA) against class I alone (n ⫽ 3), class II alone (n ⫽ 2), and both class I and II (n ⫽ 1; Table 3). The histopathology of the 28 allograft biopsy specimens showing AVR and its association with anti-HLA antibodies and C4d deposition in PTC are demonstrated in Tables 4 and 5. There was no correlation between the severity of arteritis, of interstitial inflammation, and/or tubulitis. Moderate-to-severe tubulitis combined with moderate-tosevere interstitial inflammation were seen in only 9/28 cases. Peritubular capillaritis (PTCtis) was seen in 25 cases with 10 positive for C4d deposition in PTC (Tables 4 and 5). Of the 11 patients who displayed DSA at the biopsy times 10/11

Table 5. The Histopathology of 10 Allograft Biopsy Specimens With v2 and v3 and the Association of AVR With Anti-HLA Antibody and C4d Deposition in PTC Banff Classification Score

Anti-HLA Antibody

Case No.

v

i

t

g

ptc

C4d

Class I

Class II

19 20 21 22 23 24 25 26 27 28

2 2 2 2 2 2 2 2 3 3

0 1 1 1 2 2 2 3 1 1

0 0 0 0 0 1 2 3 1 2

0 0 1 0 1 1 0 0 0 0

2 0 2 2 2 2 0 3 1 0

0 0 3 0 0 0 2* 0 0 2*

N P N N N N P P P P

N N P P N N N P P N

*ABO incompatible renal transplantation cases.

DSA

N N Class Class N N N N Class Class

II II

II I

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Table 6. The Association of AVR v1 Cases With the ART, s-Cr Level at Biopsy, After ART, and at Present, and Renal Allograft Prognosis Case No.

Banff v Score

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Anti-Rejection Therapy

SP SP SP SP SP SP SP SP SP SP SP SP SP SP SP SP SP SP

s-Cr (mg/dL) at Biopsy

s-Cr (mg/dL) After ART

s-CR (mg/dL) at Present

Allograft Prognosis

1.18 4.06 1.08 1.31 2.90 1.56 5.95 1.23 1.35 1.56 1.80 1.11 4.94 1.98 1.79 1.92 1.45 1.19

0.77 1.36 1.18 1.01 1.16 1.55 1.44 0.99 1.16 1.55 1.90 0.96 2.79 1.57 1.42 1.42 2.07 1.12

0.77 1.53 1.25 1.21 1.38 1.14 0.83 0.87 2.89 1.10 1.94 1.98 3.72 1.40 1.66 1.21 1.42 1.70

Function Function Function Function Function Function Function Function Function Function Function Function Function Function Function Function Function Function

⫹ IVIg

⫹ PEX ⫹ IVIg ⫹ PEX ⫹ IVIg ⫹ PEX ⫹ DSG ⫹ ⫹ ⫹ ⫹ ⫹

DSG ⫹ RIT OKT3 DSG ⫹ RIT OKT3 ⫹ PEX PEX

⫹ DSG ⫹ PEX

Abbreviations: ART, anti-rejection therapy; SP, steroid pulse therapy; PEX, plasmapheresis, OKT3, muromonab CD3 injection; DSG, gusperimus injection; IVIg, intravenous immune globulin; RIT, rituximab injection.

cases, SRR was observed in 8/10 cases. Twenty of 28 patients recovered renal allograft functions after ART, and 26/28 have functioning grafts. Graft loss was observed in 2 cases, 1 of whom lost his graft due to BK nephropathy, and the other due to AVR despite ART with SP, DSG injections, and plasmapheresis (Table 7). The patient and graft survivals with versus without AVR between January 2003 and November 2010 are compared in Figures 1 and 2. There were no significant differences between the 2 groups with regard to patient survival (Fig 1). The graft survival was slightly worse with versus without AVR, but the difference was not significant (Fig 2).

DISCUSSION

Histopathologic changes of AVR are characterized by intimal arteritis and transmural arteritis.1 In the Banff 2009

classification, AVR has 4 categories: ATMR Type IIA, ATMR Type IIB, ATMR Type III, and AAMR Type III.3 In this study, there was no correlation between the severity of arteritis and interstitial inflammation and/or tubulitis. Interstitial inflammation (i1–i3) was present in 25 biopsy specimens (89%), but moderate-to-severe tubulitis combined with moderate-to-severe interstitial inflammation was seen in only 9/28 cases. Indeed, in 13 cases tubulitis was not demonstrated in the allograft biopsy specimen; Thus AVR is not always accompanied by tubulitis. In this study, AAMR was diagnosed in 6 subjects. The other 9 cases, including 5 DSA-positive ones without C4d deposition in PTC and 4 DSA-negative ones with C4d deposition in PTCs, were considered to be suspicious for AAMR. Because PTCtis was seen in 25/28 cases and glomerulitis was seen in 17 cases, many of the AVR cases may be provoked by anti-donor antibodies. In these AVR

Table 7. The Association of AVR v2 and v3 Cases With the ART, s-Cr Level at Biopsy, After ART, and at Present, and Renal Allograft Prognosis Case No.

Banff v Score

19 20 21 22 23 24 25 26 27 28

2 2 2 2 2 2 2 2 3 3

Anti-Rejection Therapy

SP SP SP SP SP SP SP SP SP SP

*Graft loss due to BK nephropathy.

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

DSG ⫹ RIT DSG ⫹ RIT DSG DSG ⫹ PEX DSG ⫹ OKT3 ⫹ IVIg OKT3 ⫹ IVIg ⫹ RIT OKT3 ⫹ PEX ⫹ IVIg ⫹ RIT DSG ⫹ PEX

s-Cr (mg/dL) at Biopsy

s-Cr (mg/dL) After ART

s-CR (mg/dL) at Present

Allograft Prognosis

2.96 1.54 1.36 2.12 2.72 1.80 1.67 5.37 1.77 2.76

1.45 1.37 1.50 1.73 1.82 1.66 1.40 3.29 1.99 3.40

1.59 1.40 1.31 2.01 1.31 0.66 1.69 7.11 1.81 8.47

Function Function Function Function Function Function Function Graft loss* Function Graft loss

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SHIMIZU, TANABE, OMOTO ET AL

Fig 1. Patient survival with vs without AVR. Renal transplantations were performed between January 2003 and November 2010.

cases, some DSA-negative cases and/or negative deposition of C4d in PTC cases may be provoked by non-HLA antibodies. Some recent reports describe non-HLA antiendothelial cell antibodies (AECA). Major histocompatibility complex class I chain-related antigen A (MICA) and B (MICB) have been identified to be targets for AECA associated with hyperacute rejection and early graft loss.5– 8 Autoantibodies against angiotensin II receptor type I have been reported recently in renal allograft recipients with severe vascular rejection but not anti-HLA antibodies.9 The graft prognosis after AVR is influenced by many factors, including the degree of vasculitis, its causes the therapeutic strategies, as well as the timing.10 Koike et al

classified AVR into 3 groups: favorable, relatively poor, and poor prognosis groups,11 demonstrating that the v score according to the Banff scheme was higher among the poor versus the favorable and relatively poor prognosis groups, albeit not statistically significantly.11 In our study, the prognosis of grafts exhibiting AVR was relatively good. Twenty of 28 patients recovered renal allograft function and 26/28 transplants are functioning. As for our 2 cases of transmural arteritis, which were v3 according to the Banff scheme, were SRR; in 2 cases there was 1 graft loss and the other had deteriorated graft function. We compared the patient and graft survivals with versus without AVR, showing no significant differences in the

Fig 2. Graft survival with versus without AVR. Renal transplantations were performed between January 2003 and November 2010.

AVR AFTER RENAL TRANSPLANTATION

former and a slight albeit not significant difference in the latter. We speculated that the graft prognosis with AVR was relatively good using MP, CYA or TAC, and MMF, as well as available ART. In conclusion, AVR was usually accompanied by interstitial inflammation, but not always by tubulitis. In some cases, AVR may be provoked by anti-donor antibodies. Isolated v-lesions were seen in 2 cases. Many cases of intimal arteritis could not be resolved by SP, requiring more intensive ART. The prognosis grafts exhibiting AVR was relatively good using available immunosuppressive protocols. REFERENCES 1. Solez K, Axelsen RA, Benediktsson H, et al: International standardization of criteria for the histologic diagnosis of renal allograft rejection: the Banff working classification of kidney transplant pathology. Kidney Int 44:411, 1993 2. Racusen LC, Solez K, Colvin RB, et al: The Banff 97 working classification of renal allograft pathology. Kidney Int 55:713, 1999 3. Sis B, Mengel M, Colvin RB, et al: Banff ’09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups. Am J Transplant 10:464, 2010

235 4. Solez K, Colvin RB, Racusen LC, et al: Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant 8:753, 2008 5. Sumitran-Karuppan S, Tyden G, Reinholt F, et al: Hyperacute rejections of two consecutive renal allografts and early loss of the third transplant caused by non-HLA antibodies specific for endothelial cells. Transpl Immunol 5:321, 1997 6. Cerilli J, Brasile L: Endothelial cell alloantigens. Transplant Proc 12(supp 1):37, 1980 7. Stastny P: Endothelial-monocyte antigens. Transplant Proc 12(suppl 1):32, 1980 8. Sumitran-Holgersson S, Wilczek HE, Holgersson J, et al: Identification of the nonclassical HLA molecules, MICA, as targets for humoral immunity associated with irreversible rejection of kidney allografts, Transplantation 74:268, 2002 9. Dragun D, Muller DN, Brasen JH, et al: Angiotensin II type 1-receptor activating antibodies in renal-allograft rejection. N Engl J Med 352:558, 2005 10. Nickeleit V, Vamvakas EC, Pascual M, et al: The prognostic significance of specific arterial lesions in acute renal allograft rejection. J Am Soc Nephrol 9:1301, 1998 11. Koike J, Yamaguchi Y, Horita S, et al: Clinical and pathological assessment of acute vascular rejection in the transplant kidney. Clin Transplant 15(suppl 5):41, 2001