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Significance of clinically silent untreated mild acute cellular rejection in lung allograft recipients
Significance of Clinically Silent Untreated Mild Acute Cellular Rejection in Lung Allograft Recipients SAMUEL A, YOUSEM, MD Although acute rejection is a frequent occurrence after transplantation, the clinical behavior and pathological manifestations of untreated mild acute cellular rejection in clinically stable lung allograft recipients is poorly defined. Sixteen patients were identified who had asymptomatic mild acute rejection that was tmtreated but followed by subsequent pulmonary function tests and repeat transbronchial biopsy. Six patients had spontaneous resolution of their infiltrates; the condition of 10 patients worsened as observed from their biopsies or function studies. Those who worsened had more episodes of acute rejection per patient before the A2 biopsy (2.0 vs 1.3), and 50% developed bronchiolitis obliteraus compared with 16% in the spontaneously regressing group. Pathological evaluation
showed that patients with persistent or worsening untreated A2 rejection tended to have more large and small airway inflammation, larger numbers of eosinophils and plasma cells in their biopsies, and airway and airspace granulation tissue. These variables may be used to help determine which low grade lung rejection episodes should receive adjunctive immunosuppressive therapy. HUM PAT8OL 27:269-273. Copyright © 1996 by W.B. Saunders Company Key words: lung transplantation, acute rejection, pathology, histopathology, bronchiolitis obliterans. Abbreviations: ACR, acute cellular rejection; OB, bronchiolitis obliterans.
A c u t e r e j e c t i o n a f t e r l u n g t r a n s p l a n t a t i o n is a c o m m o n p h e n o m e n o n t h a t is a s s o c i a t e d with g r a f t d y s f u n c t i o n , b u t i n f r e q u e n t l y leads to g r a f t loss o r p a t i e n t mortality. ~-4 N o n e t h e l e s s , t h e intensity, persistence, a n d f r e q u e n c y o f a c u t e r e j e c t i o n has b e e n s h o w n to c o r r e l a t e with t h e l o n g - t e r m d e v e l o p m e n t o f b r o n c h i o l i t i s o b l i t e r a n s . 5-s A s i g n i f i c a n t n u m b e r o f t h e s e cases o f a c u t e r e j e c t i o n , p a r t i c u l a r l y lowg r a d e f o r m s , are clinically silent a n d are o n l y r e c o g n i z e d at t h e t i m e o f s u r v e i l l a n c e b r o n c h o s c o p y a n d t r a n s b r o n c h i a l l u n g biopsy. 1'9'~° W h e n i d e n t i f i e d o n p r o t o c o l biopsies, t r e a t m e n t o f t h e a s y m p t o m a t i c rej e c t i o n with h i g h - d o s e s t e r o i d s m u s t be w e i g h e d a g a i n s t t h e risk o f i n f e c t i o n , a m a j o r c a u s e o f early a n d late r e c i p i e n t m o r t a l i t y . In this study, the a u t h o r analyzed the clinical course a n d histopathologic c h a n g e s in clinically asymptomatic l u n g allograft recipients w h o were f o u n d to have mild acute cellular rejection (ACR; A2) o n surveillance transbronchial biopsy. Specific focus was directed at the natural history o f this u n t r e a t e d alloreactive process a n d histological predictors o f s u b s e q u e n t c h r o n i c graft dysfunction.
maintenance immunosuppression. All cases in which the mild rejection episode represented a resolving phase of a previously treated moderate or severe rejection were similarly excluded, as were cases with possible coexistent bacterial, viral, or fungal infection. Pulmonary function tests and chest radiographs in these patients showed no significant change from the most recent evaluation before the time of diagnostic biopsy. Given these rigorous criteria, 16 patients with untreated, clinically silent mild ACR were identified from 2,226 transbronchial biopsy procedures and formed the basis for this study. Repeat transbronchial biopsies were performed at an average of 26 days after the initial biopsy (median, 29 days; range, 7 to 93 days). Lung transplantation was performed for cystic fibrosis (N = 7), centrilobular emphysema (N = 3), al-antitrypsin deficiency (N = 2), plexeginic pulmonary arteriopathy (N = 3), and Eisenmenger's syndrome (N = 1). Clinical variables analyzed included age, sex, race, transplant procedure, postoperative day of biopsy, pulmonary function test results, primary changes in the forced expiratory volumes at 1 second, the development of bronchiolitis obliterans, current status, and immunosuppressive regimen. The latter consisted of either triple drug therapy with cyclosporine, immuran and prednisone, or tacrolimus (FK506) and prednisone. Histopathologic assessment of transbronchial biopsies first required confirmation of the diagnosis of mild (ACR; A2) using criteria defined in the Working Formulation for Lung Allograft Rejection.ll Morphological variables assessed included the following: one or more perivascular infiltrates in the total transbronchial biopsy fragments obtained at one bronchoscopy session, the presence of large or small airway inflammation, endothelialitis, eosinophils, plasma cells, intra-airway and intra-airspace granulation tissue, and alveolar hemosiderosis. All biopsies were evaluated in a blinded fashion without awareness of subsequent clinical course. The Working Formulation u for grading of acute rejection of lung allografts contains five acute rejection grades: A0, none; A1, minimal; A2, mild; A3, moderate; and A4, severe; and is based on the intensity of perivascular mononuclear infiltrates and their extension into alveolar septa. Chronic airway rejection is manifested as bronchiolitis obliterarts. A category of lymphocytic bronchitis and bronchiolitis
MATERIALS AND METHODS The patient records of all lung allograft recipients from January 1990 to January 1995 (246 total lung transplant recipients) were searched for all cases of mild ACR that were not treated with steroid therapy or a change in From the Department of Pathology, Montefiore University Hospital, University of Pittsburgh Medical Center, Pittsburgh, PA. Accepted for publication October 19, 1995. Address correspondence and reprint requests to Samuel A. Yousem, MD, Department of Pathology, Montefiore University Hospital, NW625, University of Pittsburgh Medical Center, 200 Lothrop St, Pittsburgh, PA 15213-2582. Copyright © 1996 by W.B. Saunders Company 0046-8177/96/2703-000755.00/0
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(B) also exists where airway mononuclear inflammation is present without scarring of the airways or perivascular mononuclear cuffs. For the purposes of this study and based on prior studies, 9'~ minimal acute rejection is regarded as a negative or insignificant biopsy finding.
RESULTS Sixteen separate episodes of clinically silent mild ACR were studied in 16 lung allograft recipients (Table 1). In six of these instances, subsequent biopsies showed spontaneous resolution of the infiltrate and pulmonary function studies remained stable (group 1). In 10 patients, subsequent biopsies revealed persistent A2 rejection, worsening rejection, or deteriorated functional studies (group 2). In this latter group of 10 patients, nine received subsequent steroid therapy after repeat biopsies showed persistent or progressive rejection. Group 1
In the clinically stable group (group 1), the initial asymptomatic A2 rejection occurred at a mean of 108 days after transplantation (median, 75 days; range, 30 to 280 days). This group comprised four men and two women, and four had sequential double-lung transplants and two single-lung transplants. Previous acute rejection was observed in nine instances in these six individuals (average, 1.3 episodes per person). One of the six subsequently developed bronchiolitis obliterans, and two died of a cerebrovascular accident and posttransplantation lymphoproliferative disorder, respectively. Of these six patients, the next biopsy performed after the A2 biopsy revealed one A0 and four A1 rejections, whereas the last biopsy showed a mild lymphocytic bronchitis associated with unremarkable pulmonary function studies. Morphological assessment of the initial A2 biopsy showed that more than one perivascular mononuclear infiltrate was observed in four of the six patients in group 1. These infiltrates corresponded to the definition of A2 rejection in the Working Formulation, in that they were readily identified at scanning magnification and usually consisted of a perivascular mononuclear cuff of six to 10 lymphocytes, confined to the perivascular adventifia (Fig 1). The mononuclear infiltrate consisted of small round and angulated lymphocytes with rare transformed cells. No alveolar septal infiltration was observed. Large airway inflammation (four of six), and lymphocytic infiltration of terminal and respiratory bronchioles was seen in three instances. Subendothelial infiltration by lymphocytes was noted in two cases, and eosinophils were present in the infiltrate in two biopsies. Plasma cells were infrequent (one of six), and no granulation tissue was observed in the biopsy specimens. ®roup 2
Ten patients with asymptomatic A2 rejection had subsequent biopsies or pulmonary functions that worsened (group 2). These seven men and three
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women had silent A2 rejection at a mean of 129 days posttransplantation (median, 106 days; range, 13 to 319 days). Six patients had double-lung transplants, three had single-lung procedures, and one had a combined heart-lung transplant. A total of 20 acute rejection episodes preceded the asymptomatic A2 biopsy (average, 2.0 episodes per recipient). Five patients developed bronchiolitis obliterans (OB) in this group, and four of the patients died, three of these patients died of bronchiolitis obliterans. Of this group, the subsequent transbronchial biopsy performed after the untreated A2 rejection showed four patients with persistent A2 rejections, four patients with progressive moderate A3 rejections, one patient with bronchiolitis obliterans, and one patient with severe lymphocytic bronchiolitis, which, in the context of suppressed functional studies, was interpreted as persistent airway rejection and treated with steroids. Histological examination of the group 2 biopsies showed multiple perivascular mononuclear infiltrates in seven patients and a solitary infiltrate in three cases. The composition of the mononuclear infiltrate was similar to that of group 1 in its distribution of small, round, and angulated lymphocytes; and large, transformed cells. Large airway (eight of nine) and small airway (9 of 10) lymphocytic infiltrates were more common than in the spontaneously resolving rejection cases of group I. Eosinophils were also more common (70% vs 33%) as were plasma cells (40% vs 16%). Plugs of loose fibromyxoid connective tissue in airways and airspaces were rare in group 2 (30%) but were not observed at all in naturally regressing A2 cases (Fig 2).
DISCUSSION Acute rejection of the lung allograft occurs early after transplantation in more than 80% of recipients. 13Clinically apparent ACR is characterized by the onset of shortness of breath, fever, hypoxemia, and subtle interstitial infiltrates with effusion on chest radiographs. 3 Such symptomatic ACR has been evaluated by numerous transplant centers, and its intensity, persistence, and frequency correlate with an increased risk for obliterative small airway disease. 2'5'7'9'14 Consequently, histological changes of acute rejection accompanied by functional and clinical disturbances are usually regarded as indications for augmented immunosuppression. 12,15,16 Although symptomatic ACR is regarded as a treatment indication, the same cannot be said for asymptomatic mild acute cellular rejection. Silent A2 rejection has emerged as a therapeutic issue as a result of the widespread use of surveillance transbronchial biopsy as a means of monitoring graft alloreactivity and occult pulmonary infections."9 10 Trulock et al 17 reported A2 rejection in 22% of surveillance biopsies, and DeHoyos et al9 reported occult rejection in 30 of 42 surveillance specimens--"grade 1 in 18 and grade 2 to 3 in 12," indicating a significant rejection rate of 26% (12 of 42) in incidental biopsies. At the University of Pittsburgh Medical Center, surveillance biopsies comprise 31% of
MILD ACUTE REJECTION IN LUNG ALLOGRAFTS (Samuel A. Yousem) TABLE 1. Clinical and Pathological Data on Spontaneously Resolving (Group 1: Patients 1 to 6) and Progressive (Group 2: Patients 7 to 16) Asymptomatic Mild Acute Cellular Rejection in Lung AIIograft Recipients Age Case No.
(yr)/ Race, Sex
Group 1 1 23/WF
P
3
5 4 / W M SL
55 82 30 59 58
4
3 5 / W M DL
81 75
5
2 4 / W M DL
6
5 4 / W M SL
2
43/WF
DL
Previous ACR CY/FK POD No. Rx
DL
Group 2 7 4 9 / W M SL
168 280 298 192 202
F / U Biopsy a n d PFTs Results
OB Steroids ( + / - )
Status (d)
2
C
Stable
-
-
Alive: 1,876
0
C
Stable
-
-
Alive: 1,417
0
C
Stable
-
-
D e a d o f stroke: 1,288--no autopsy
1
C
Stable
-
+
D e a d o f PT L D , OB, aspergillosis: 485
3
C
Stable
+
-
Alive: 8 1 6
3
FK
Stable
-
-
Alive: 417
262
1
FK
Func tions stable
+
+
Alive: 1,124
8
4 4 / W M DL
296 122
3
C
D e v e l o p e d OB, f unc tions decreased
+
+
D e a d o f OB: 733
9
4 7 / W M DL
148 13
0
FK
Func tions declined
+
+
D e a d o f OB, herpes pneumonia: 244
10
22/WF
SL
24 94
3
FK
A2 + CMV pneumonitis; f unc tions decreased
+
-
D e a d o f CMV pneumonitis a n d O~ toxicity: 226
11
5 0 / W M DL
111 7
0
C
Func tions decreased
+
-
Alive: 1,215
12
5 1 / W M DL
5
C
Decreased
+
+
Alive: 1,359
13
5 3 / W M SL
414 170 198 70
1
C
Functions unknown
+
-
Alive: 5 7 6
14
22/WF
1] 2 169
3
C
A3 a n d
+
+
D e a d o f OB: 602
DL
Streptococcus pneumoniae,"
PredomBX G r a d e * inant
GranuEndotheP lation LAI SAI lifts EOS Cells Tissue
1 2 1 2 1
A2 A0 A2 A1 A2
Many
+
-
+
Many Many Few
+
+
2 1
A1 A2
Few Many
2 1 2 1 2
A1 A2 B A2 A1
Few Few
+ + N/A
. . +
1
A2
Few
-
2 1
A2 A2
Few Few
2 1
C A2
2 1
+
+
. .
+
+
. +
+
+
+ + . .
.
. .
.
. . .
.
-
. . -
+
+
-
+
+
.
None Many
+
+ +
. +
A2 A2
Many Many
+ +
+ +
2 1
A2 A2
Many Many
+ +
2 1 2 1
A3 A2 B1 A2
Many Few -Many
2 1
A3 A2
-
. .
-
. +
-
.
Few
. .
-
-
.
+
+
-
+
-
. .
.
.
+ .
.
. +
. +
+
+
+ +
+ -
+ +
+ +
-
+ +
+ +
-
+ + + +
+ +
+ -
+ +
+ +
+
-
+ +
Many Many
+ +
+ +
+ -
+ +
+ -
+ -
f unc tions decreased 15
38/WF
DL
218 319
2
FK
Func tions decreased
+
-
Alive: 475
2 1
A3 A2
Many Many
+ N/A
+ +
1 -
+ +
+ -
+ -
3 4 / W M DL
358 70
2
FK
A2 f or 400 d and then -; f unc tions stable
-
-
Alive: 1,841
2 1
A2 A2
Many Many
+ +
+ +
-
16
+ -
-
-
2
A3
Many
+
+
-
+
+
104
Abbreviations: W, white; M, male; F, female; CY, cyclosporine; FIL tacrolimus; Rx, therapy; P, p r o c e d u r e ; DL, d o u b l e - l u n g transplant; SL, single-lung transplant; HL, h e a r t - l u n g transplant; ACR, a c ute c e l l u l a r rejection; F / U , follow-up; P O D , p o s to p e r a tiv e day; P F r , p u l m o n a r y f u n c t i o n tests; LAI, la r g e airway i n f l a m m a t i o n ; SAI, small airway i n f l a m m a t i o n ; EOS, eosinophils; P cells, plasma cells; + , positive cells; - , n e g a t i v e ceils; N / A , n o t available; BX 1, initial biopsy; BX 2, follow-up biopsy; OB, b r o n c h i o l i t i s obliterans; CMV, cytomegalovirus; Ou, oxygen. * G r a d i n g a c c o r d i n g to W o r k i n g F o r m u l a t i o n .
bronchoscopy procedures. Unsuspected rejection (A2 or A3) was found in 12.8%, infection or other treatable abnormalities in 12.2%, and the remainder of biopsies showed no abnormalities. One third of cases of unsuspected ACR were grade A3, and the remainder were grade A2. Although this capture rate of occult rejection
may suggest that silent low grade rejection may contribute to the high rate of development of OB (25% to 40%),11 this group of clinically inapparent A2 rejections has not been systematically studied. Using stringent criteria, the author studied 16 unequivocal cases of occult A2 rejection that were derived
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FIGURE 1, Mild acute cellular rejeclion (A2). At low magnification, easily recognized perivascular mononuclear infiltrates cuffed small pulmonaB/vessels (A). The infillTate was composed primarily of small, round and small, angulated lymphocytes with rare, inconspicuous, large transformed lymphocytes (B). The composition of lymphoid cells was similar in groups 1 and 2.
from 5 years of clinical work and approximately 2,226 transbronchial biopsies in 246 lung allograft recipients. These 16 study cases had no change in their immunosuppression and were followed with repeat transbronchial biopsy and pulmonary function testing. Of these 16 cases, 37.5% (N = 6) had a reduction in the inflammatory infiltrate to a clinically insignificant rejection (ie, A0 and A1 ACR), whereas the remainder persisted with their A2 rejection, or their histological grade
worsened in the next allograft biopsy. When the initial transbronchial biopsies were compared from these two groups, no feature was unequivocal in predicting progression; however, airway inflammation, eosinophils and plasma cells, and intra-airway and airspace granula, tion tissue argue for an aggressive course. These findings are not unexpected. Previous work at the University of Pittsburgh Medical Center has shown that the amount of airway inflammation increases with rejection
FIGURE 2. Mild acute rejection (A2). In the cases of asymptomatic acute rejection with persistent or progressive rejection on a subsequent biopsy, histological features that suggested an aggressive course included large airway inflammation (A; arrows on smooth muscle wall); bronchiolar and alveolar duct infiltrates (B; arrow on muscle and eiastica of alveolar duct); and airway and airspace fibromyxoid tissue (C; arrows).
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MILD ACUTE REJECTION IN LUNG ALLOGRAFTS (Samuel A. Yousem)
grade. 4'1~'~8These study cases may well represent those at the " h i g h " end of the histological spectrum of A2 rejection. Similarly, eosinophils are more common as rejection worsens. ~9 Interestingly, a recent study by Yousem et al 2° suggested that the number of B cells in an acute rejection infiltrate correlates with resistance to interventional steroid therapy. The presence of plasma cells may reflect this humoral component to A2 cases, which makes them especially progressive and resistant to treatment. Finally, prior studies from the author's group and others have shown that intra-airway granulation tissue predisposes to bronchiolitis obliterans and aggressive rejection episodesJ 4'91This fibroproliferative response indicates prior epithelial damage and denudation, with organization. Although the author assumes that the A2 rejection infiltrate is injurious, the presence of granulation tissue indicates a degree of injury to the lung, which, in this study, was present in only the poor prognosis occult A2 rejections. It is also noteworthy that the progressive occult A2 rejection group had more rejection episodes per patient before the documented silent A2 rejection and that 50% (vs 16% of group 1) developed OB. The intensity scores of these episodes had similar distributions in the two groups as well (data not shown). This univariate analysis provides only mild support, however, for the belief that persistence of rejection, even low grade, may impact on long-term graft function. As with any study, this one has its limitations. Despite many surveillance biopsies, most cases of mild ACR are associated with clinical symptoms, and the author was not able to accrue more than 16 bona fide cases. Second, all variables could not be controlled including degrees of immunosuppression, patient compliance with medications, human leukocyte antigen (HLA) cross-matching, cytomegalovirus status, and number of bacterial infections, among others. Despite these restrictions, the overall impression has emerged that a subset of clinically silent mild acute rejection shows a propensity to progress and to be associated with a poor outcome--certainly therapy should be strongly considered and not ignored at this histological diagnosis. Acknowledgment. T h e author acknowledges the contributions of Wayne Grgurich in data collection, correlation, and analysis; Christel Lollo for secretarial assistance; and Linda Shab for p h o t o g r a p h i c aid.
REFERENCES 1. CooperJD, Patterson GA, Trulock EP, et al: Results of single and bilateral lung transplantation in 131 consecutive recipients. J Thorac Cardiovasc Surg 107:460-471, 1994
2. HutterJA, Despins P, Higenbottam T, et al: Heart-lung transplantation: Better use of resources. A m J Med 85:4-11, 1988 3. Otulana BA, Higenbottam TW, Scott J, et al: Lung function associated with histologically diagnosed acute lung rejection and pulmonary infection in heart-lung transplant patients. Am Rev Respir Dis 142:329-332, 1990 4. Yousem SA, Burke CM, Billingham ME: Pathologic pulmonary alterations in long-term human heart-lung transplantation. HUM PATHOL 16:911-923, 1985 5. Clelland CA, Higenbottam T, Otulana B, et al: Histologic prognostic indicators for the lung allografts of heart-lung transplants. J Heart Transplant 9:177-186, 1990 6. Scott JP, Higenbottam TW, Sharples L, et al: Risk factors for obliterative bronchiolitis in heartqung transplant recipients. Transplantation 51:813-817, 1991 7. Sharples LD, ScottJP, Dennis C, et ah Risk factors for survival following combined heart-lung transplantation. Transplantation 57:218-223, 1994 8. Starnes VA, Theodore J, Oyer PE, et al: Evaluation of heartlung transplant recipients with prospective, serial transbronchial biopsies and pulmonary function studies. J Thorac Cardiovasc Surg 98:683-690, 1989 9. DeHoyosA, Chamberlain D, Schvartzman R, et ah Prospective assessment of a standardized pathologic grading system for acute rejection in lung transplantation. Chest 103:1813-1818, 1993 10. Starnes VA, Theodore J, Oyer PE, et al: Pulmonary infiltrates after heart-lung transplantation: Evaluation by serial transbronchial biopsies. J Thorac Cardiovasc Surg 98:945-950, 1989 11. Yousem SA, Berry G, Brunt E, et al: A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection. J Heart Transplant 9:593-601, 1990 12. Clelland CA, Higenbottam TW, Stewart S, et al: The histological changes in transbronchial biopsy after treatment of acute lung rejection in heart-lung transplants. J Pathol 161:105-112, 1990 13. Trulock EP. Management of lung transplant rejection. Chest 103:1566-1576, 1993 14. Scott JP, Fradet G, Smyth RL, et al: Prospective study of transbronchial biopsies in the management of heart-lung and single lung transplant patients. J Heart Lung Transplant 10:626-637, 1991 15. Hurter JA, Stewart S, Higenbottam T, et al: Histologic changes in heart-lung transplant recipients during rejection episodes and at routine biopsy. J Heart Transplant 7:440-444, 1988 16. Yousem SA, DauberJA, Keenan R, et al: Does histologic acute rejection in lung allografts predict the development of bronchiolitis obliterans? Transplantation 52:306-309, 1991 17. Trulock EP, Ettinger NA, Brunt EM, et al: The role of transbronchial lung biopsy in the treatment of lung transplant recipients: An analysis of 200 consecutive procedures. Chest 102:1049-1054, 1992 18. Yousem SA, Paradis IL, DauberJH, et al: Efficacy oftransbronchial lung biopsy in the diagnosis of bronchiolitis obliterans in heart-lung transplant recipients. Transplantation 47:893-895, 1989 19. Yousem SA: Graft eosinophilia in lung transplantation. HUM PATHOL 23:1172-1177, 1992 20. Yousem SA, Martin T, Paradis IL, et ah Can immunohistologic analysis of transbronchial biopsies predict responder status in early acute rejection of lung allografts? HUM PATHOL 25:525-529, 1994 21. Yousem SA, Duncan SR, Griffith BP: Interstitial and airspace granulation tissue reactions in lung transplant recipients. A m J Surg Pathol 16:877-884, 1992
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showed that patients with persistent or worsening untreated A2 rejection tended to have more large and small airway inflammation, larger numbers of eosinophils and plasma cells in their biopsies, and airway and airspace granulation tissue. These variables may be used to help determine which low grade lung rejection episodes should receive adjunctive immunosuppressive therapy. HUM PAT8OL 27:269-273. Copyright © 1996 by W.B. Saunders Company Key words: lung transplantation, acute rejection, pathology, histopathology, bronchiolitis obliterans. Abbreviations: ACR, acute cellular rejection; OB, bronchiolitis obliterans.
A c u t e r e j e c t i o n a f t e r l u n g t r a n s p l a n t a t i o n is a c o m m o n p h e n o m e n o n t h a t is a s s o c i a t e d with g r a f t d y s f u n c t i o n , b u t i n f r e q u e n t l y leads to g r a f t loss o r p a t i e n t mortality. ~-4 N o n e t h e l e s s , t h e intensity, persistence, a n d f r e q u e n c y o f a c u t e r e j e c t i o n has b e e n s h o w n to c o r r e l a t e with t h e l o n g - t e r m d e v e l o p m e n t o f b r o n c h i o l i t i s o b l i t e r a n s . 5-s A s i g n i f i c a n t n u m b e r o f t h e s e cases o f a c u t e r e j e c t i o n , p a r t i c u l a r l y lowg r a d e f o r m s , are clinically silent a n d are o n l y r e c o g n i z e d at t h e t i m e o f s u r v e i l l a n c e b r o n c h o s c o p y a n d t r a n s b r o n c h i a l l u n g biopsy. 1'9'~° W h e n i d e n t i f i e d o n p r o t o c o l biopsies, t r e a t m e n t o f t h e a s y m p t o m a t i c rej e c t i o n with h i g h - d o s e s t e r o i d s m u s t be w e i g h e d a g a i n s t t h e risk o f i n f e c t i o n , a m a j o r c a u s e o f early a n d late r e c i p i e n t m o r t a l i t y . In this study, the a u t h o r analyzed the clinical course a n d histopathologic c h a n g e s in clinically asymptomatic l u n g allograft recipients w h o were f o u n d to have mild acute cellular rejection (ACR; A2) o n surveillance transbronchial biopsy. Specific focus was directed at the natural history o f this u n t r e a t e d alloreactive process a n d histological predictors o f s u b s e q u e n t c h r o n i c graft dysfunction.
maintenance immunosuppression. All cases in which the mild rejection episode represented a resolving phase of a previously treated moderate or severe rejection were similarly excluded, as were cases with possible coexistent bacterial, viral, or fungal infection. Pulmonary function tests and chest radiographs in these patients showed no significant change from the most recent evaluation before the time of diagnostic biopsy. Given these rigorous criteria, 16 patients with untreated, clinically silent mild ACR were identified from 2,226 transbronchial biopsy procedures and formed the basis for this study. Repeat transbronchial biopsies were performed at an average of 26 days after the initial biopsy (median, 29 days; range, 7 to 93 days). Lung transplantation was performed for cystic fibrosis (N = 7), centrilobular emphysema (N = 3), al-antitrypsin deficiency (N = 2), plexeginic pulmonary arteriopathy (N = 3), and Eisenmenger's syndrome (N = 1). Clinical variables analyzed included age, sex, race, transplant procedure, postoperative day of biopsy, pulmonary function test results, primary changes in the forced expiratory volumes at 1 second, the development of bronchiolitis obliterans, current status, and immunosuppressive regimen. The latter consisted of either triple drug therapy with cyclosporine, immuran and prednisone, or tacrolimus (FK506) and prednisone. Histopathologic assessment of transbronchial biopsies first required confirmation of the diagnosis of mild (ACR; A2) using criteria defined in the Working Formulation for Lung Allograft Rejection.ll Morphological variables assessed included the following: one or more perivascular infiltrates in the total transbronchial biopsy fragments obtained at one bronchoscopy session, the presence of large or small airway inflammation, endothelialitis, eosinophils, plasma cells, intra-airway and intra-airspace granulation tissue, and alveolar hemosiderosis. All biopsies were evaluated in a blinded fashion without awareness of subsequent clinical course. The Working Formulation u for grading of acute rejection of lung allografts contains five acute rejection grades: A0, none; A1, minimal; A2, mild; A3, moderate; and A4, severe; and is based on the intensity of perivascular mononuclear infiltrates and their extension into alveolar septa. Chronic airway rejection is manifested as bronchiolitis obliterarts. A category of lymphocytic bronchitis and bronchiolitis
MATERIALS AND METHODS The patient records of all lung allograft recipients from January 1990 to January 1995 (246 total lung transplant recipients) were searched for all cases of mild ACR that were not treated with steroid therapy or a change in From the Department of Pathology, Montefiore University Hospital, University of Pittsburgh Medical Center, Pittsburgh, PA. Accepted for publication October 19, 1995. Address correspondence and reprint requests to Samuel A. Yousem, MD, Department of Pathology, Montefiore University Hospital, NW625, University of Pittsburgh Medical Center, 200 Lothrop St, Pittsburgh, PA 15213-2582. Copyright © 1996 by W.B. Saunders Company 0046-8177/96/2703-000755.00/0
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Volume 27, No. 3 (March 1996)
(B) also exists where airway mononuclear inflammation is present without scarring of the airways or perivascular mononuclear cuffs. For the purposes of this study and based on prior studies, 9'~ minimal acute rejection is regarded as a negative or insignificant biopsy finding.
RESULTS Sixteen separate episodes of clinically silent mild ACR were studied in 16 lung allograft recipients (Table 1). In six of these instances, subsequent biopsies showed spontaneous resolution of the infiltrate and pulmonary function studies remained stable (group 1). In 10 patients, subsequent biopsies revealed persistent A2 rejection, worsening rejection, or deteriorated functional studies (group 2). In this latter group of 10 patients, nine received subsequent steroid therapy after repeat biopsies showed persistent or progressive rejection. Group 1
In the clinically stable group (group 1), the initial asymptomatic A2 rejection occurred at a mean of 108 days after transplantation (median, 75 days; range, 30 to 280 days). This group comprised four men and two women, and four had sequential double-lung transplants and two single-lung transplants. Previous acute rejection was observed in nine instances in these six individuals (average, 1.3 episodes per person). One of the six subsequently developed bronchiolitis obliterans, and two died of a cerebrovascular accident and posttransplantation lymphoproliferative disorder, respectively. Of these six patients, the next biopsy performed after the A2 biopsy revealed one A0 and four A1 rejections, whereas the last biopsy showed a mild lymphocytic bronchitis associated with unremarkable pulmonary function studies. Morphological assessment of the initial A2 biopsy showed that more than one perivascular mononuclear infiltrate was observed in four of the six patients in group 1. These infiltrates corresponded to the definition of A2 rejection in the Working Formulation, in that they were readily identified at scanning magnification and usually consisted of a perivascular mononuclear cuff of six to 10 lymphocytes, confined to the perivascular adventifia (Fig 1). The mononuclear infiltrate consisted of small round and angulated lymphocytes with rare transformed cells. No alveolar septal infiltration was observed. Large airway inflammation (four of six), and lymphocytic infiltration of terminal and respiratory bronchioles was seen in three instances. Subendothelial infiltration by lymphocytes was noted in two cases, and eosinophils were present in the infiltrate in two biopsies. Plasma cells were infrequent (one of six), and no granulation tissue was observed in the biopsy specimens. ®roup 2
Ten patients with asymptomatic A2 rejection had subsequent biopsies or pulmonary functions that worsened (group 2). These seven men and three
270
women had silent A2 rejection at a mean of 129 days posttransplantation (median, 106 days; range, 13 to 319 days). Six patients had double-lung transplants, three had single-lung procedures, and one had a combined heart-lung transplant. A total of 20 acute rejection episodes preceded the asymptomatic A2 biopsy (average, 2.0 episodes per recipient). Five patients developed bronchiolitis obliterans (OB) in this group, and four of the patients died, three of these patients died of bronchiolitis obliterans. Of this group, the subsequent transbronchial biopsy performed after the untreated A2 rejection showed four patients with persistent A2 rejections, four patients with progressive moderate A3 rejections, one patient with bronchiolitis obliterans, and one patient with severe lymphocytic bronchiolitis, which, in the context of suppressed functional studies, was interpreted as persistent airway rejection and treated with steroids. Histological examination of the group 2 biopsies showed multiple perivascular mononuclear infiltrates in seven patients and a solitary infiltrate in three cases. The composition of the mononuclear infiltrate was similar to that of group 1 in its distribution of small, round, and angulated lymphocytes; and large, transformed cells. Large airway (eight of nine) and small airway (9 of 10) lymphocytic infiltrates were more common than in the spontaneously resolving rejection cases of group I. Eosinophils were also more common (70% vs 33%) as were plasma cells (40% vs 16%). Plugs of loose fibromyxoid connective tissue in airways and airspaces were rare in group 2 (30%) but were not observed at all in naturally regressing A2 cases (Fig 2).
DISCUSSION Acute rejection of the lung allograft occurs early after transplantation in more than 80% of recipients. 13Clinically apparent ACR is characterized by the onset of shortness of breath, fever, hypoxemia, and subtle interstitial infiltrates with effusion on chest radiographs. 3 Such symptomatic ACR has been evaluated by numerous transplant centers, and its intensity, persistence, and frequency correlate with an increased risk for obliterative small airway disease. 2'5'7'9'14 Consequently, histological changes of acute rejection accompanied by functional and clinical disturbances are usually regarded as indications for augmented immunosuppression. 12,15,16 Although symptomatic ACR is regarded as a treatment indication, the same cannot be said for asymptomatic mild acute cellular rejection. Silent A2 rejection has emerged as a therapeutic issue as a result of the widespread use of surveillance transbronchial biopsy as a means of monitoring graft alloreactivity and occult pulmonary infections."9 10 Trulock et al 17 reported A2 rejection in 22% of surveillance biopsies, and DeHoyos et al9 reported occult rejection in 30 of 42 surveillance specimens--"grade 1 in 18 and grade 2 to 3 in 12," indicating a significant rejection rate of 26% (12 of 42) in incidental biopsies. At the University of Pittsburgh Medical Center, surveillance biopsies comprise 31% of
MILD ACUTE REJECTION IN LUNG ALLOGRAFTS (Samuel A. Yousem) TABLE 1. Clinical and Pathological Data on Spontaneously Resolving (Group 1: Patients 1 to 6) and Progressive (Group 2: Patients 7 to 16) Asymptomatic Mild Acute Cellular Rejection in Lung AIIograft Recipients Age Case No.
(yr)/ Race, Sex
Group 1 1 23/WF
P
3
5 4 / W M SL
55 82 30 59 58
4
3 5 / W M DL
81 75
5
2 4 / W M DL
6
5 4 / W M SL
2
43/WF
DL
Previous ACR CY/FK POD No. Rx
DL
Group 2 7 4 9 / W M SL
168 280 298 192 202
F / U Biopsy a n d PFTs Results
OB Steroids ( + / - )
Status (d)
2
C
Stable
-
-
Alive: 1,876
0
C
Stable
-
-
Alive: 1,417
0
C
Stable
-
-
D e a d o f stroke: 1,288--no autopsy
1
C
Stable
-
+
D e a d o f PT L D , OB, aspergillosis: 485
3
C
Stable
+
-
Alive: 8 1 6
3
FK
Stable
-
-
Alive: 417
262
1
FK
Func tions stable
+
+
Alive: 1,124
8
4 4 / W M DL
296 122
3
C
D e v e l o p e d OB, f unc tions decreased
+
+
D e a d o f OB: 733
9
4 7 / W M DL
148 13
0
FK
Func tions declined
+
+
D e a d o f OB, herpes pneumonia: 244
10
22/WF
SL
24 94
3
FK
A2 + CMV pneumonitis; f unc tions decreased
+
-
D e a d o f CMV pneumonitis a n d O~ toxicity: 226
11
5 0 / W M DL
111 7
0
C
Func tions decreased
+
-
Alive: 1,215
12
5 1 / W M DL
5
C
Decreased
+
+
Alive: 1,359
13
5 3 / W M SL
414 170 198 70
1
C
Functions unknown
+
-
Alive: 5 7 6
14
22/WF
1] 2 169
3
C
A3 a n d
+
+
D e a d o f OB: 602
DL
Streptococcus pneumoniae,"
PredomBX G r a d e * inant
GranuEndotheP lation LAI SAI lifts EOS Cells Tissue
1 2 1 2 1
A2 A0 A2 A1 A2
Many
+
-
+
Many Many Few
+
+
2 1
A1 A2
Few Many
2 1 2 1 2
A1 A2 B A2 A1
Few Few
+ + N/A
. . +
1
A2
Few
-
2 1
A2 A2
Few Few
2 1
C A2
2 1
+
+
. .
+
+
. +
+
+
+ + . .
.
. .
.
. . .
.
-
. . -
+
+
-
+
+
.
None Many
+
+ +
. +
A2 A2
Many Many
+ +
+ +
2 1
A2 A2
Many Many
+ +
2 1 2 1
A3 A2 B1 A2
Many Few -Many
2 1
A3 A2
-
. .
-
. +
-
.
Few
. .
-
-
.
+
+
-
+
-
. .
.
.
+ .
.
. +
. +
+
+
+ +
+ -
+ +
+ +
-
+ +
+ +
-
+ + + +
+ +
+ -
+ +
+ +
+
-
+ +
Many Many
+ +
+ +
+ -
+ +
+ -
+ -
f unc tions decreased 15
38/WF
DL
218 319
2
FK
Func tions decreased
+
-
Alive: 475
2 1
A3 A2
Many Many
+ N/A
+ +
1 -
+ +
+ -
+ -
3 4 / W M DL
358 70
2
FK
A2 f or 400 d and then -; f unc tions stable
-
-
Alive: 1,841
2 1
A2 A2
Many Many
+ +
+ +
-
16
+ -
-
-
2
A3
Many
+
+
-
+
+
104
Abbreviations: W, white; M, male; F, female; CY, cyclosporine; FIL tacrolimus; Rx, therapy; P, p r o c e d u r e ; DL, d o u b l e - l u n g transplant; SL, single-lung transplant; HL, h e a r t - l u n g transplant; ACR, a c ute c e l l u l a r rejection; F / U , follow-up; P O D , p o s to p e r a tiv e day; P F r , p u l m o n a r y f u n c t i o n tests; LAI, la r g e airway i n f l a m m a t i o n ; SAI, small airway i n f l a m m a t i o n ; EOS, eosinophils; P cells, plasma cells; + , positive cells; - , n e g a t i v e ceils; N / A , n o t available; BX 1, initial biopsy; BX 2, follow-up biopsy; OB, b r o n c h i o l i t i s obliterans; CMV, cytomegalovirus; Ou, oxygen. * G r a d i n g a c c o r d i n g to W o r k i n g F o r m u l a t i o n .
bronchoscopy procedures. Unsuspected rejection (A2 or A3) was found in 12.8%, infection or other treatable abnormalities in 12.2%, and the remainder of biopsies showed no abnormalities. One third of cases of unsuspected ACR were grade A3, and the remainder were grade A2. Although this capture rate of occult rejection
may suggest that silent low grade rejection may contribute to the high rate of development of OB (25% to 40%),11 this group of clinically inapparent A2 rejections has not been systematically studied. Using stringent criteria, the author studied 16 unequivocal cases of occult A2 rejection that were derived
271
HUMAN PATHOLOGY
Volume 27, No. 3 (March 1996)
FIGURE 1, Mild acute cellular rejeclion (A2). At low magnification, easily recognized perivascular mononuclear infiltrates cuffed small pulmonaB/vessels (A). The infillTate was composed primarily of small, round and small, angulated lymphocytes with rare, inconspicuous, large transformed lymphocytes (B). The composition of lymphoid cells was similar in groups 1 and 2.
from 5 years of clinical work and approximately 2,226 transbronchial biopsies in 246 lung allograft recipients. These 16 study cases had no change in their immunosuppression and were followed with repeat transbronchial biopsy and pulmonary function testing. Of these 16 cases, 37.5% (N = 6) had a reduction in the inflammatory infiltrate to a clinically insignificant rejection (ie, A0 and A1 ACR), whereas the remainder persisted with their A2 rejection, or their histological grade
worsened in the next allograft biopsy. When the initial transbronchial biopsies were compared from these two groups, no feature was unequivocal in predicting progression; however, airway inflammation, eosinophils and plasma cells, and intra-airway and airspace granula, tion tissue argue for an aggressive course. These findings are not unexpected. Previous work at the University of Pittsburgh Medical Center has shown that the amount of airway inflammation increases with rejection
FIGURE 2. Mild acute rejection (A2). In the cases of asymptomatic acute rejection with persistent or progressive rejection on a subsequent biopsy, histological features that suggested an aggressive course included large airway inflammation (A; arrows on smooth muscle wall); bronchiolar and alveolar duct infiltrates (B; arrow on muscle and eiastica of alveolar duct); and airway and airspace fibromyxoid tissue (C; arrows).
272
MILD ACUTE REJECTION IN LUNG ALLOGRAFTS (Samuel A. Yousem)
grade. 4'1~'~8These study cases may well represent those at the " h i g h " end of the histological spectrum of A2 rejection. Similarly, eosinophils are more common as rejection worsens. ~9 Interestingly, a recent study by Yousem et al 2° suggested that the number of B cells in an acute rejection infiltrate correlates with resistance to interventional steroid therapy. The presence of plasma cells may reflect this humoral component to A2 cases, which makes them especially progressive and resistant to treatment. Finally, prior studies from the author's group and others have shown that intra-airway granulation tissue predisposes to bronchiolitis obliterans and aggressive rejection episodesJ 4'91This fibroproliferative response indicates prior epithelial damage and denudation, with organization. Although the author assumes that the A2 rejection infiltrate is injurious, the presence of granulation tissue indicates a degree of injury to the lung, which, in this study, was present in only the poor prognosis occult A2 rejections. It is also noteworthy that the progressive occult A2 rejection group had more rejection episodes per patient before the documented silent A2 rejection and that 50% (vs 16% of group 1) developed OB. The intensity scores of these episodes had similar distributions in the two groups as well (data not shown). This univariate analysis provides only mild support, however, for the belief that persistence of rejection, even low grade, may impact on long-term graft function. As with any study, this one has its limitations. Despite many surveillance biopsies, most cases of mild ACR are associated with clinical symptoms, and the author was not able to accrue more than 16 bona fide cases. Second, all variables could not be controlled including degrees of immunosuppression, patient compliance with medications, human leukocyte antigen (HLA) cross-matching, cytomegalovirus status, and number of bacterial infections, among others. Despite these restrictions, the overall impression has emerged that a subset of clinically silent mild acute rejection shows a propensity to progress and to be associated with a poor outcome--certainly therapy should be strongly considered and not ignored at this histological diagnosis. Acknowledgment. T h e author acknowledges the contributions of Wayne Grgurich in data collection, correlation, and analysis; Christel Lollo for secretarial assistance; and Linda Shab for p h o t o g r a p h i c aid.
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