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The significance of plasma cell infiltrate in acute cellular rejection of liver allografts
Human Pathology (2012) xx, xxx–xxx
www.elsevier.com/locate/humpath
Original contribution
The significance of plasma cell infiltrate in acute cellular rejection of liver allografts Jacob Alexander MDa , Wenjiang Chu MD, PhDb , Paul E. Swanson MDb , Matthew M. Yeh MD, PhDb,⁎ a
Department of Internal Medicine, University of Washington School of Medicine, Seattle, WA 98195-6100, USA Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195-6100, USA
b
Received 7 September 2011; revised 8 December 2011; accepted 9 December 2011
Keywords: Liver transplantation; Graft rejection; Plasma cells
Summary Acute cellular rejections of higher grades of histologic severity are associated with increased risk of graft failure and death after liver transplantation. Plasma cell–rich infiltrates are associated with adverse clinical outcomes in acute renal allograft rejection and in liver allografts without rejection, but there are limited data on plasma cell–rich infiltrates in acute liver allograft rejection. In this study, 59 biopsies of acute liver allograft rejection were confirmed histologically and clinically, independently graded, and the percentage of plasma cells in portal inflammatory infiltrate was objectively assessed using a standardized protocol. Plasma cell infiltrates were observed in 32 (54%) of the specimens, the mean percentage of plasma cells in the infiltrates being 2.97%. Infiltrates with any plasma cells were significantly more common in groups with higher histologic severity of rejection (75% and 100% versus 31% and 48%, P = .006). The mean percentage of plasma cells in the portal infiltrate was also significantly higher in groups with higher histologic severity of rejection (4.95% and 17.82% versus 0.37 and 0.82%, P = .0002). All the biopsies with more than 30% plasma cells in the infiltrate were found to have severe rejection, whereas all with more than 10% plasma cells had either moderate or severe rejection. The association of plasma cell–rich infiltrates with histologic severity of rejection suggests that plasma cell–rich infiltrates could potentially be useful as a marker of severe rejection. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Liver transplantation is the only effective therapy for endstage liver disease. The clinical outcome of liver transplantation has substantially improved during the recent years, with 1 year survival rates currently exceeding 90% and
Abbreviations: ACR, acute cellular rejection; PCAR, Plasma cell–rich acute rejection.
⁎ Corresponding author. Department of Pathology, University of Washington School of Medicine, Box 356100, Seattle, WA98195-6100, USA. E-mail address: [email protected] (M. M. Yeh). 0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2011.12.005
long-term (8 years) survival rates approaching 70% for most indications of liver transplantation [1]. Acute cellular rejection (ACR), which historically has been an important cause of graft failure, occurs in 50% to 75% of allografts posttransplantation [2-4]. However, most of the episodes of ACR in the modern allograft recipients are mild and readily reversed with appropriate immunosuppressive therapy; they are rarely complicated by liver allograft failure or clinically significant structural sequelae [2]. In contrast, episodes of ACR with higher grades of histologic severity remain associated with poorer response to immunosuppressant therapy and increased risk of graft failure from acute or chronic rejection and death [2,4]. Therefore, early identification of
2 allografts with or at risk for higher-grade rejection phenomena is of considerable importance in the clinical management of these patients. Infiltration of the portal tract by a mixture of lymphocytes, neutrophils, and eosinophils with endotheliitis and lymphocytic cholangitis is the histologic hallmark of ACR, and the extent of infiltration (together with the degree of endothelial and bile duct epithelial injury) is the basis of the histologic classification of the severity of ACR [5]. Plasma cell–rich acute rejection (PCAR), characterized by the presence of more than 10% mature plasma cells in the inflammatory cell infiltrate, develops in approximately 5% of the renal allografts with biopsy-proven acute rejection [6,7]. PCAR in this clinical setting is associated with poor outcome, suboptimal response to conventional immunosuppressant therapy, and a need for alternative modalities of treatment such as intravenous immunoglobulins (Ig) [6,8-10]. In addition, the presence of donor-specific IgG anti-human leukocyte antigen antibody has been found to be associated with increased risk of acute rejection, more severe rejection, and lower graft survival in the affected renal allografts [11,12]. Although plasma cell infiltration has been recently documented in ACR in liver allografts, it is not known whether there is any association between plasma cell infiltration and the severity of rejection [13]. Therefore, we performed this study to explore any possible association between the presence or the intensity of plasma cell infiltration and the histologic severity of ACR in the liver allograft.
2. Materials and methods We performed a retrospective review of all patients with clinical, laboratory, and histologic features of ACR in the liver allograft who had undergone liver biopsies between January 2000 and December 2004. The study protocol was reviewed and approved by the Human Subject Division at the University of Washington, Seattle, WA. Cases with evidence of autoimmune hepatitis or overlap syndrome with autoimmune hepatitis in the native liver were excluded because autoimmune hepatitis by itself is associated with marked plasma cell infiltration [14]. All liver biopsies were obtained by a 15-gauge Klatskin needle, using the Menghini technique. Serial 5-m sections taken from formalin-fixed and paraffin-embedded tissues were stained with hematoxylin and eosin and were reviewed by a single pathologist (M. M. Y.) who was blinded to the clinical data. Cases that did not meet the histologic criteria for ACR and biopsy specimens containing less than 5 portal tracts were excluded. De novo autoimmune hepatitis was identified and excluded based on the overall histologic and laboratory features. The histologic features specifically evaluated for distinguishing de novo autoimmune hepatitis from ACR included the extent
J. Alexander et al. and severity of interface hepatitis, the extent of bile duct injury, and the presence of venous endotheliitis. The presence of diffuse or severe interface hepatitis or marked lobular inflammation favored a diagnosis of de novo autoimmune hepatitis, whereas the presence of extensive bile duct injury or venous endotheliitis favored a diagnosis of ACR [15,16]. The severity of ACR was graded according to the Banff criteria and thus classified into 4 groups: focal mild, mild, moderate, and severe [5]. To standardize the assessment of plasma cells in the portal areas with variable intensities of inflammation, the degree of plasma cell infiltrate was calculated as the percentage of total inflammatory infiltrate: total number of portal plasma cells and total number of portal inflammatory cells including mononuclear cells, neutrophils, and eosinophils were serially counted in the 5 most cellular portal areas, and the mean percentage of plasma cells was calculated for each biopsy (Fig. 1). The counting and calculation were performed by an independent observer (W. C.) who had no knowledge of the clinical data or severity of ACR diagnosed. Discrete variables were compared among the 4 groups using the χ2 test; the same in the group with focal mild and mild rejections were compared with that in the group with moderate and severe rejection using Fisher exact test. Continuous variables were compared among the 4 groups using the Kruskal-Wallis test; the same in the group with focal mild and mild rejections were compared with that in the group with moderate and severe rejection using the Mann-Whitney U test. The Pearson correlation coefficient was calculated to estimate the correlation between mean percentage of plasma cells in portal inflammatory infiltrate and time since transplantation. Association of mean percentage of plasma cells with risk of death or retransplantation in the whole cohort was evaluated using the Cox proportional hazards model. P b .05 was considered as significant.
3. Results Fifty-nine liver biopsies satisfied the study criteria. Characteristics of the study population, classified according to the histologic severity of rejection, are shown in Table 1. The age distribution of patients in all 4 groups were similar; however, severe rejection occurred exclusively in males and were documented later in the course than the less severe grades of rejection, although it should be noted that there were only 4 biopsies in this study subset. Plasma cells were present in the portal inflammatory infiltrate in 32 (54%) of the specimens. Specimens with any number of plasma cells were progressively more common among groups with higher histologic severity of rejection (Table 1). This difference was statistically significant when compared among all 4 groups (P = .01), as well as when
Significance of plasma cell infiltrate in ACR
3
Fig. 1 Inflammatory cell infiltrates in portal regions. Hematoxylin and eosin–stained liver biopsy specimens showing inflammatory cell infiltration in the portal areas. A, Typical inflammatory cells in ACR with a mixture of lymphocytes, neutrophils, and eosinophils (original magnification ×200). BD indicates bile duct. B, ACR with prominent plasma cell infiltration—low magnification (original magnification ×20). C, ACR with prominent plasma cell infiltration—intermediate magnification (original magnification ×100). D, ACR with prominent plasma cell infiltration—high magnification (original magnification ×200).
compared between the group with focal mild and mild rejection and the group with moderate and severe rejection (P = .006). The mean percentage of plasma cells in the portal inflammatory infiltrate was progressively higher among groups with higher histologic severity of rejection (Table 1 and Fig. 2). The differences in the percentage of plasma cells among the 4 groups were statistically significant (P = .0014). In addition, the difference in plasma cells between the group with focal mild and mild rejection and the group with moderate and severe rejection was also statistically significant (P = .0002). There was no correlation between percentage of plasma cells and time since transplantation (correlation coefficient, r = 0.15). Plasma cells comprised more than 10% of the inflammatory cells in 5 (9%) of the specimens. Two of the latter specimens, both with 30% plasma cells, had severe rejection, whereas the other 3, with plasma cells between 10% and 30%, had moderate rejection. A total of 7 (12%) mortalities were observed in the cohort, the proximate cause of death in all being infectious
complications or gastrointestinal bleed. A total of 4 (7%) retransplantations were performed—1 for chronic rejection, another for ischemic cholangiopathy, and the other 2 for ACR itself. One of the patients who underwent retransplantation (for chronic rejection) died subsequently (of septicemia). On Cox proportional hazards analysis, there was a statistically significant association between the mean percentage of plasma cells in the portal inflammatory infiltrate and risk of death or retransplantation (hazard ratio, 1.068; 95% confidence interval, 1.0101.128; P = .0201).
4. Discussion In this study, we found that both the presence of plasma cells in the portal inflammatory infiltrate and the intensity of plasma cell infiltration (expressed as a percentage of the total absolute inflammatory cell count) were significantly
4
J. Alexander et al. Table 1
Characteristics of the patients and histologic findings
No. of biopsies Age (y) Sex (male: female) Days since transplantation Etiology Hepatitis B Hepatitis C Alcoholic liver disease NASH Primary biliary cirrhosis Primary sclerosing cholangitis Budd-Chiari syndrome Fulminant hepatic failure Hereditary hemorrhagic telangiectasia Cryptogenic cirrhosis Liver function tests ALT (U/L) AST (U/L) Alkaline phosphatase (U/L) Total bilirubin (mg/dL) INR Biopsies with plasma cells in portal infiltrate Percentage of plasma cells in portal infiltrate Biopsies with no plasma cells
Total
Mild focal (A)
Mild (B)
Moderate (C)
Severe (D)
59 56 (19-69) 37:22 72 (7-3542)
16 52 (23-62) 8:8 42 (9-423)
23 58 (19-69) 12:11 36 (7-3386)
16 56 (35-69) 13:3 106 (8-1507)
4 64 (50-67) 4:0 998 (659-3542)
2 (3%) 20 (34%) 5 (9%) 8 (14%) 9 (15%) 7 (12%) 1 (2%) 2 (3%) 2 (3%)
1 5 0 1 3 3 1 1 1
0 8 3 3 3 3 0 1 1
1 6 2 2 2 1 0 0 0
0 1 0 2 1 0 0 0 0
3 (5%)
0
1
2
0
128 (12-1655) 75 (7-939) 167 (35-1795) 1.6 (0.5-24.4) 1.1 (0.8-1.7) 32 (54%)
82 (12-619) 29 (10-282) 105 (51-471) 1.2 (0.5-12.0) 1.1 (0.9-1.4) 5 (31%)
95 (18-903) 41 (7-654) 239 (35-160) 1.4 (0.5-24.4) 1.0 (0.8-1.6) 11 (48%)
160 (43-1655) 149 (24-662) 234 (84-1795) 2.8 (0.8-22.0) 1.2 (0.8-1.7) 12 (75%)
444 (236-1090) 351 (253-939) 335 (86-1216) 6.1 (1.1-10.0) 1.2 (0.9-1.2) 4 (100%)
0.23 (0.00-30.00)
0.00 (0.00-2.55)
0.00 (0.00-8.48)
0.90 (0.00-25.46)
19.63 (2.02-30.00)
27 (46%)
11 (69%)
12 (52%)
4 (25%)
0
NOTE. Age, days since transplantation, liver function tests, and percentage of plasma cells in the portal infiltrate are expressed as median, with range in parenthesis. All other parameters are expressed as count, with percentage in parenthesis. Abbreviations: NASH, nonalcoholic steatohepatitis; ALT, alanine aminotransferase; AST, aspartate aminotransferase; INR, international normalized ratio.
associated with the histologic severity of ACR in the liver allograft. All specimens with more than 10% plasma cells had either moderate or severe grade of rejection.
Fig. 2 Scattergram showing percentage of plasma cells in portal inflammatory infiltrate in each grade of severity of acute rejection.
ACR, reported to occur in 50% to 75% of the allografts, is one of the most common complications after liver transplantation [2-4]. Although ACR, irrespective of organ type, is predominantly a T-cell–mediated immunologic process, recent studies have identified evidence that humoral immunity is important in certain cases of acute rejection in renal allografts. Interstitial/peritubular infiltrates containing more than 10% mature plasma cells are present in approximately 5% of acute renal allograft rejections and are associated with a poor response to therapy with pulse steroids and immunosuppressants and an increased risk of graft failure and death [6,7,9]. Graft loss associated with plasma cell–rich acute rejection is significantly higher (52% versus 20%) and earlier (median time to graft loss, 6.3 months versus 21.5 months) than in classic acute rejection [7,9]. As in the renal transplant setting, infiltration of B lymphocytes, macrophages, and plasma cells, as well as portal capillary deposition of C4d, has been demonstrated in acute liver allograft rejection [13,17]. In addition, the presence of a relatively high proportion of plasma cells has recently been described in inflammatory cell infiltrates in liver allografts without evidence of acute or chronic rejection
Significance of plasma cell infiltrate in ACR and found to be associated worse outcomes in this setting [18,19]. However, there have been no previous reports of a similar phenomenon in liver allografts with histologic evidence of rejection, nor has there been any study on the association between the presence or intensity of plasma cell infiltration and the histologic severity of rejection. Our finding of plasma cell infiltration in liver allografts with ACR is complementary to the concept of PCAR in renal allografts, as well as to the previously reported association of plasma cell infiltration with worse outcomes after liver transplantation. As can be seen in Table 1, ACR with moderate to severe plasma cell infiltrates were noted to occur later in the posttransplant course in patients in our series. This finding is considered analogous to the plasma cell–rich rejection in kidney allografts, which occurs late after transplantation [6,10]. Taken together, these observations raise the interesting and novel possibility that plasma cells could have a significant pathogenic role in the allograft liver, both in presence of and in the absence of evidence of rejection. Distinguishing PCAR from de novo autoimmune hepatitis is often a vexing diagnostic problem because of the similarity in the histologic features between the 2 entities [20]. The term plasma cell hepatitis encompasses both PCAR and de novo autoimmune hepatitis and is commonly used when the distinction between the 2 is difficult. There is no single diagnostic test or histologic feature that has been found to consistently discriminate between these 2 conditions. However, the relative prominence of bile duct damage and venous endotheliitis and lack of marked interface hepatitis or lobular inflammation in ACR is useful in distinguishing the 2 conditions. Both bile duct damage and venous endotheliitis are characteristic of ACR and can be quite prominent, whereas these findings are minimal or absent in autoimmune hepatitis. On the other hand, interface hepatitis and lobular inflammation are prominent features of autoimmune hepatitis, but quite mild in ACR [15]. The current practice is to make a diagnosis of ACR when typical laboratory and histologic features of autoimmune hepatitis are absent, and plasma cell infiltrates are present in association with other features of ACR such as prominent bile duct damage and endotheliitis [19]. We have adhered to this convention and restricted this study to patients who had unequivocal evidence of ACR, without any features suggestive of de novo autoimmune hepatitis. We acknowledge that the current study has some limitations. This is a retrospective study with a mediumsized number of patients and did not have sufficient power to detect the association of plasma cell infiltration with clinical outcomes. Lack of a control group without ACR may be considered as a limitation, although the groups with different degrees of plasma cell infiltration served as internal controls. Not using specialized stains for plasma cells could also be considered a limitation, but the appearance of the plasma cells on the routine hematoxy-
5 lin-eosin staining is distinctive enough that this is unlikely to have affected the diagnostic accuracy. In conclusion, both the presence of plasma cells in the portal inflammatory infiltrate and the intensity of plasma cell infiltration are significantly associated with the histologic severity of ACR in the liver allograft. These findings suggest a potential role for plasma cells in the pathophysiology of ACR in the liver allograft and define a robust additional histologic marker of liver allografts with or at risk for highgrade rejection. Further prospective studies to confirm this observation and to evaluate whether plasma cell infiltration could be predictive of clinical outcomes such as response to therapy, graft failure, and patient survival are warranted.
References [1] Roberts MS, Angus DC, Bryce CL, Valenta Z, Weissfeld L. Survival after liver transplantation in the United States: a disease-specific analysis of the UNOS database. Liver Transpl 2004;10:886-97. [2] Demetris AJ, Ruppert K, Dvorchik I, et al. Real-time monitoring of acute liver-allograft rejection using the Banff schema. Transplantation 2002;74:1290-6. [3] Martinez OM, Rosen HR. Basic concepts in transplant immunology. Liver Transpl 2005;11:370-81. [4] Wiesner RH, Demetris AJ, Belle SH, et al. Acute hepatic allograft rejection: incidence, risk factors, and impact on outcome. Hepatology 1998;28:638-45. [5] Demetris AJ, Batts KP, Dhillon AP, et al. Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 1997;25:658-63. [6] Desvaux D, Le Gouvello S, Pastural M, et al. Acute renal allograft rejections with major interstitial oedema and plasma cell–rich infiltrates: high gamma-interferon expression and poor clinical outcome. Nephrol Dial Transplant 2004;19:933-9. [7] Meehan SM, Domer P, Josephson M, et al. The clinical and pathologic implications of plasmacytic infiltrates in percutaneous renal allograft biopsies. HUM PATHOL 2001;32:205-15. [8] Adrogue HE, Soltero L, Land GA, Ramanathan V, Truong LD, Suki WN. Immunoglobulin therapy for plasma cell–rich rejection in the renal allograft. Transplantation 2006;82:567-9. [9] Charney DA, Nadasdy T, Lo AW, Racusen LC. Plasma cell–rich acute renal allograft rejection. Transplantation 1999;68:791-7. [10] Gartner V, Eigentler TK, Viebahn R. Plasma cell–rich rejection processes in renal transplantation: morphology and prognostic relevance. Transplantation 2006;81:986-91. [11] Halloran PF, Schlaut J, Solez K, Srinivasa NS. The significance of the anti-class I response. II. Clinical and pathologic features of renal transplants with anti-class I–like antibody. Transplantation 1992;53: 550-5. [12] Lobo PI, Spencer CE, Stevenson WC, Pruett TL. Evidence demonstrating poor kidney graft survival when acute rejections are associated with IgG donor-specific lymphocytotoxin. Transplantation 1995;59:357-60. [13] Krukemeyer MG, Moeller J, Morawietz L, et al. Description of B lymphocytes and plasma cells, complement, and chemokines/receptors in acute liver allograft rejection. Transplantation 2004;78:65-70. [14] Abe M, Onji M, Kawai-Ninomiya K, et al. Clinicopathologic features of the severe form of acute type 1 autoimmune hepatitis. Clin Gastroenterol Hepatol 2007;5:255-8. [15] Tripathi D, Neuberger J. Autoimmune hepatitis and liver transplantation: indications, results, and management of recurrent disease. Semin Liver Dis 2009;29:286-96.
6 [16] Guido M, Burra P. De novo autoimmune hepatitis after liver transplantation. Semin Liver Dis 2011;31:71-81. [17] Dankof A, Schmeding M, Morawietz L, et al. Portal capillary C4d deposits and increased infiltration by macrophages indicate humorally mediated mechanisms in acute cellular liver allograft rejection. Virchows Arch 2005;447:87-93. [18] Ward SC, Schiano TD, Thung SN, Fiel MI. Plasma cell hepatitis in hepatitis C virus patients post-liver transplantation: case-control study
J. Alexander et al. showing poor outcome and predictive features in the liver explant. Liver Transpl 2009;15:1826-33. [19] Fiel MI, Agarwal K, Stanca C, et al. Posttransplant plasma cell hepatitis (de novo autoimmune hepatitis) is a variant of rejection and may lead to a negative outcome in patients with hepatitis C virus. Liver Transpl 2008;14:861-71. [20] Demetris AJ, Sebagh M. Plasma cell hepatitis in liver allografts: variant of rejection or autoimmune hepatitis? Liver Transpl 2008;14:750-5.
www.elsevier.com/locate/humpath
Original contribution
The significance of plasma cell infiltrate in acute cellular rejection of liver allografts Jacob Alexander MDa , Wenjiang Chu MD, PhDb , Paul E. Swanson MDb , Matthew M. Yeh MD, PhDb,⁎ a
Department of Internal Medicine, University of Washington School of Medicine, Seattle, WA 98195-6100, USA Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195-6100, USA
b
Received 7 September 2011; revised 8 December 2011; accepted 9 December 2011
Keywords: Liver transplantation; Graft rejection; Plasma cells
Summary Acute cellular rejections of higher grades of histologic severity are associated with increased risk of graft failure and death after liver transplantation. Plasma cell–rich infiltrates are associated with adverse clinical outcomes in acute renal allograft rejection and in liver allografts without rejection, but there are limited data on plasma cell–rich infiltrates in acute liver allograft rejection. In this study, 59 biopsies of acute liver allograft rejection were confirmed histologically and clinically, independently graded, and the percentage of plasma cells in portal inflammatory infiltrate was objectively assessed using a standardized protocol. Plasma cell infiltrates were observed in 32 (54%) of the specimens, the mean percentage of plasma cells in the infiltrates being 2.97%. Infiltrates with any plasma cells were significantly more common in groups with higher histologic severity of rejection (75% and 100% versus 31% and 48%, P = .006). The mean percentage of plasma cells in the portal infiltrate was also significantly higher in groups with higher histologic severity of rejection (4.95% and 17.82% versus 0.37 and 0.82%, P = .0002). All the biopsies with more than 30% plasma cells in the infiltrate were found to have severe rejection, whereas all with more than 10% plasma cells had either moderate or severe rejection. The association of plasma cell–rich infiltrates with histologic severity of rejection suggests that plasma cell–rich infiltrates could potentially be useful as a marker of severe rejection. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Liver transplantation is the only effective therapy for endstage liver disease. The clinical outcome of liver transplantation has substantially improved during the recent years, with 1 year survival rates currently exceeding 90% and
Abbreviations: ACR, acute cellular rejection; PCAR, Plasma cell–rich acute rejection.
⁎ Corresponding author. Department of Pathology, University of Washington School of Medicine, Box 356100, Seattle, WA98195-6100, USA. E-mail address: [email protected] (M. M. Yeh). 0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2011.12.005
long-term (8 years) survival rates approaching 70% for most indications of liver transplantation [1]. Acute cellular rejection (ACR), which historically has been an important cause of graft failure, occurs in 50% to 75% of allografts posttransplantation [2-4]. However, most of the episodes of ACR in the modern allograft recipients are mild and readily reversed with appropriate immunosuppressive therapy; they are rarely complicated by liver allograft failure or clinically significant structural sequelae [2]. In contrast, episodes of ACR with higher grades of histologic severity remain associated with poorer response to immunosuppressant therapy and increased risk of graft failure from acute or chronic rejection and death [2,4]. Therefore, early identification of
2 allografts with or at risk for higher-grade rejection phenomena is of considerable importance in the clinical management of these patients. Infiltration of the portal tract by a mixture of lymphocytes, neutrophils, and eosinophils with endotheliitis and lymphocytic cholangitis is the histologic hallmark of ACR, and the extent of infiltration (together with the degree of endothelial and bile duct epithelial injury) is the basis of the histologic classification of the severity of ACR [5]. Plasma cell–rich acute rejection (PCAR), characterized by the presence of more than 10% mature plasma cells in the inflammatory cell infiltrate, develops in approximately 5% of the renal allografts with biopsy-proven acute rejection [6,7]. PCAR in this clinical setting is associated with poor outcome, suboptimal response to conventional immunosuppressant therapy, and a need for alternative modalities of treatment such as intravenous immunoglobulins (Ig) [6,8-10]. In addition, the presence of donor-specific IgG anti-human leukocyte antigen antibody has been found to be associated with increased risk of acute rejection, more severe rejection, and lower graft survival in the affected renal allografts [11,12]. Although plasma cell infiltration has been recently documented in ACR in liver allografts, it is not known whether there is any association between plasma cell infiltration and the severity of rejection [13]. Therefore, we performed this study to explore any possible association between the presence or the intensity of plasma cell infiltration and the histologic severity of ACR in the liver allograft.
2. Materials and methods We performed a retrospective review of all patients with clinical, laboratory, and histologic features of ACR in the liver allograft who had undergone liver biopsies between January 2000 and December 2004. The study protocol was reviewed and approved by the Human Subject Division at the University of Washington, Seattle, WA. Cases with evidence of autoimmune hepatitis or overlap syndrome with autoimmune hepatitis in the native liver were excluded because autoimmune hepatitis by itself is associated with marked plasma cell infiltration [14]. All liver biopsies were obtained by a 15-gauge Klatskin needle, using the Menghini technique. Serial 5-m sections taken from formalin-fixed and paraffin-embedded tissues were stained with hematoxylin and eosin and were reviewed by a single pathologist (M. M. Y.) who was blinded to the clinical data. Cases that did not meet the histologic criteria for ACR and biopsy specimens containing less than 5 portal tracts were excluded. De novo autoimmune hepatitis was identified and excluded based on the overall histologic and laboratory features. The histologic features specifically evaluated for distinguishing de novo autoimmune hepatitis from ACR included the extent
J. Alexander et al. and severity of interface hepatitis, the extent of bile duct injury, and the presence of venous endotheliitis. The presence of diffuse or severe interface hepatitis or marked lobular inflammation favored a diagnosis of de novo autoimmune hepatitis, whereas the presence of extensive bile duct injury or venous endotheliitis favored a diagnosis of ACR [15,16]. The severity of ACR was graded according to the Banff criteria and thus classified into 4 groups: focal mild, mild, moderate, and severe [5]. To standardize the assessment of plasma cells in the portal areas with variable intensities of inflammation, the degree of plasma cell infiltrate was calculated as the percentage of total inflammatory infiltrate: total number of portal plasma cells and total number of portal inflammatory cells including mononuclear cells, neutrophils, and eosinophils were serially counted in the 5 most cellular portal areas, and the mean percentage of plasma cells was calculated for each biopsy (Fig. 1). The counting and calculation were performed by an independent observer (W. C.) who had no knowledge of the clinical data or severity of ACR diagnosed. Discrete variables were compared among the 4 groups using the χ2 test; the same in the group with focal mild and mild rejections were compared with that in the group with moderate and severe rejection using Fisher exact test. Continuous variables were compared among the 4 groups using the Kruskal-Wallis test; the same in the group with focal mild and mild rejections were compared with that in the group with moderate and severe rejection using the Mann-Whitney U test. The Pearson correlation coefficient was calculated to estimate the correlation between mean percentage of plasma cells in portal inflammatory infiltrate and time since transplantation. Association of mean percentage of plasma cells with risk of death or retransplantation in the whole cohort was evaluated using the Cox proportional hazards model. P b .05 was considered as significant.
3. Results Fifty-nine liver biopsies satisfied the study criteria. Characteristics of the study population, classified according to the histologic severity of rejection, are shown in Table 1. The age distribution of patients in all 4 groups were similar; however, severe rejection occurred exclusively in males and were documented later in the course than the less severe grades of rejection, although it should be noted that there were only 4 biopsies in this study subset. Plasma cells were present in the portal inflammatory infiltrate in 32 (54%) of the specimens. Specimens with any number of plasma cells were progressively more common among groups with higher histologic severity of rejection (Table 1). This difference was statistically significant when compared among all 4 groups (P = .01), as well as when
Significance of plasma cell infiltrate in ACR
3
Fig. 1 Inflammatory cell infiltrates in portal regions. Hematoxylin and eosin–stained liver biopsy specimens showing inflammatory cell infiltration in the portal areas. A, Typical inflammatory cells in ACR with a mixture of lymphocytes, neutrophils, and eosinophils (original magnification ×200). BD indicates bile duct. B, ACR with prominent plasma cell infiltration—low magnification (original magnification ×20). C, ACR with prominent plasma cell infiltration—intermediate magnification (original magnification ×100). D, ACR with prominent plasma cell infiltration—high magnification (original magnification ×200).
compared between the group with focal mild and mild rejection and the group with moderate and severe rejection (P = .006). The mean percentage of plasma cells in the portal inflammatory infiltrate was progressively higher among groups with higher histologic severity of rejection (Table 1 and Fig. 2). The differences in the percentage of plasma cells among the 4 groups were statistically significant (P = .0014). In addition, the difference in plasma cells between the group with focal mild and mild rejection and the group with moderate and severe rejection was also statistically significant (P = .0002). There was no correlation between percentage of plasma cells and time since transplantation (correlation coefficient, r = 0.15). Plasma cells comprised more than 10% of the inflammatory cells in 5 (9%) of the specimens. Two of the latter specimens, both with 30% plasma cells, had severe rejection, whereas the other 3, with plasma cells between 10% and 30%, had moderate rejection. A total of 7 (12%) mortalities were observed in the cohort, the proximate cause of death in all being infectious
complications or gastrointestinal bleed. A total of 4 (7%) retransplantations were performed—1 for chronic rejection, another for ischemic cholangiopathy, and the other 2 for ACR itself. One of the patients who underwent retransplantation (for chronic rejection) died subsequently (of septicemia). On Cox proportional hazards analysis, there was a statistically significant association between the mean percentage of plasma cells in the portal inflammatory infiltrate and risk of death or retransplantation (hazard ratio, 1.068; 95% confidence interval, 1.0101.128; P = .0201).
4. Discussion In this study, we found that both the presence of plasma cells in the portal inflammatory infiltrate and the intensity of plasma cell infiltration (expressed as a percentage of the total absolute inflammatory cell count) were significantly
4
J. Alexander et al. Table 1
Characteristics of the patients and histologic findings
No. of biopsies Age (y) Sex (male: female) Days since transplantation Etiology Hepatitis B Hepatitis C Alcoholic liver disease NASH Primary biliary cirrhosis Primary sclerosing cholangitis Budd-Chiari syndrome Fulminant hepatic failure Hereditary hemorrhagic telangiectasia Cryptogenic cirrhosis Liver function tests ALT (U/L) AST (U/L) Alkaline phosphatase (U/L) Total bilirubin (mg/dL) INR Biopsies with plasma cells in portal infiltrate Percentage of plasma cells in portal infiltrate Biopsies with no plasma cells
Total
Mild focal (A)
Mild (B)
Moderate (C)
Severe (D)
59 56 (19-69) 37:22 72 (7-3542)
16 52 (23-62) 8:8 42 (9-423)
23 58 (19-69) 12:11 36 (7-3386)
16 56 (35-69) 13:3 106 (8-1507)
4 64 (50-67) 4:0 998 (659-3542)
2 (3%) 20 (34%) 5 (9%) 8 (14%) 9 (15%) 7 (12%) 1 (2%) 2 (3%) 2 (3%)
1 5 0 1 3 3 1 1 1
0 8 3 3 3 3 0 1 1
1 6 2 2 2 1 0 0 0
0 1 0 2 1 0 0 0 0
3 (5%)
0
1
2
0
128 (12-1655) 75 (7-939) 167 (35-1795) 1.6 (0.5-24.4) 1.1 (0.8-1.7) 32 (54%)
82 (12-619) 29 (10-282) 105 (51-471) 1.2 (0.5-12.0) 1.1 (0.9-1.4) 5 (31%)
95 (18-903) 41 (7-654) 239 (35-160) 1.4 (0.5-24.4) 1.0 (0.8-1.6) 11 (48%)
160 (43-1655) 149 (24-662) 234 (84-1795) 2.8 (0.8-22.0) 1.2 (0.8-1.7) 12 (75%)
444 (236-1090) 351 (253-939) 335 (86-1216) 6.1 (1.1-10.0) 1.2 (0.9-1.2) 4 (100%)
0.23 (0.00-30.00)
0.00 (0.00-2.55)
0.00 (0.00-8.48)
0.90 (0.00-25.46)
19.63 (2.02-30.00)
27 (46%)
11 (69%)
12 (52%)
4 (25%)
0
NOTE. Age, days since transplantation, liver function tests, and percentage of plasma cells in the portal infiltrate are expressed as median, with range in parenthesis. All other parameters are expressed as count, with percentage in parenthesis. Abbreviations: NASH, nonalcoholic steatohepatitis; ALT, alanine aminotransferase; AST, aspartate aminotransferase; INR, international normalized ratio.
associated with the histologic severity of ACR in the liver allograft. All specimens with more than 10% plasma cells had either moderate or severe grade of rejection.
Fig. 2 Scattergram showing percentage of plasma cells in portal inflammatory infiltrate in each grade of severity of acute rejection.
ACR, reported to occur in 50% to 75% of the allografts, is one of the most common complications after liver transplantation [2-4]. Although ACR, irrespective of organ type, is predominantly a T-cell–mediated immunologic process, recent studies have identified evidence that humoral immunity is important in certain cases of acute rejection in renal allografts. Interstitial/peritubular infiltrates containing more than 10% mature plasma cells are present in approximately 5% of acute renal allograft rejections and are associated with a poor response to therapy with pulse steroids and immunosuppressants and an increased risk of graft failure and death [6,7,9]. Graft loss associated with plasma cell–rich acute rejection is significantly higher (52% versus 20%) and earlier (median time to graft loss, 6.3 months versus 21.5 months) than in classic acute rejection [7,9]. As in the renal transplant setting, infiltration of B lymphocytes, macrophages, and plasma cells, as well as portal capillary deposition of C4d, has been demonstrated in acute liver allograft rejection [13,17]. In addition, the presence of a relatively high proportion of plasma cells has recently been described in inflammatory cell infiltrates in liver allografts without evidence of acute or chronic rejection
Significance of plasma cell infiltrate in ACR and found to be associated worse outcomes in this setting [18,19]. However, there have been no previous reports of a similar phenomenon in liver allografts with histologic evidence of rejection, nor has there been any study on the association between the presence or intensity of plasma cell infiltration and the histologic severity of rejection. Our finding of plasma cell infiltration in liver allografts with ACR is complementary to the concept of PCAR in renal allografts, as well as to the previously reported association of plasma cell infiltration with worse outcomes after liver transplantation. As can be seen in Table 1, ACR with moderate to severe plasma cell infiltrates were noted to occur later in the posttransplant course in patients in our series. This finding is considered analogous to the plasma cell–rich rejection in kidney allografts, which occurs late after transplantation [6,10]. Taken together, these observations raise the interesting and novel possibility that plasma cells could have a significant pathogenic role in the allograft liver, both in presence of and in the absence of evidence of rejection. Distinguishing PCAR from de novo autoimmune hepatitis is often a vexing diagnostic problem because of the similarity in the histologic features between the 2 entities [20]. The term plasma cell hepatitis encompasses both PCAR and de novo autoimmune hepatitis and is commonly used when the distinction between the 2 is difficult. There is no single diagnostic test or histologic feature that has been found to consistently discriminate between these 2 conditions. However, the relative prominence of bile duct damage and venous endotheliitis and lack of marked interface hepatitis or lobular inflammation in ACR is useful in distinguishing the 2 conditions. Both bile duct damage and venous endotheliitis are characteristic of ACR and can be quite prominent, whereas these findings are minimal or absent in autoimmune hepatitis. On the other hand, interface hepatitis and lobular inflammation are prominent features of autoimmune hepatitis, but quite mild in ACR [15]. The current practice is to make a diagnosis of ACR when typical laboratory and histologic features of autoimmune hepatitis are absent, and plasma cell infiltrates are present in association with other features of ACR such as prominent bile duct damage and endotheliitis [19]. We have adhered to this convention and restricted this study to patients who had unequivocal evidence of ACR, without any features suggestive of de novo autoimmune hepatitis. We acknowledge that the current study has some limitations. This is a retrospective study with a mediumsized number of patients and did not have sufficient power to detect the association of plasma cell infiltration with clinical outcomes. Lack of a control group without ACR may be considered as a limitation, although the groups with different degrees of plasma cell infiltration served as internal controls. Not using specialized stains for plasma cells could also be considered a limitation, but the appearance of the plasma cells on the routine hematoxy-
5 lin-eosin staining is distinctive enough that this is unlikely to have affected the diagnostic accuracy. In conclusion, both the presence of plasma cells in the portal inflammatory infiltrate and the intensity of plasma cell infiltration are significantly associated with the histologic severity of ACR in the liver allograft. These findings suggest a potential role for plasma cells in the pathophysiology of ACR in the liver allograft and define a robust additional histologic marker of liver allografts with or at risk for highgrade rejection. Further prospective studies to confirm this observation and to evaluate whether plasma cell infiltration could be predictive of clinical outcomes such as response to therapy, graft failure, and patient survival are warranted.
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