Consistencies and controversies in the application of the International Society for Heart and Lung Transplantation working formulation for heart transplant biopsy specimens. Rapamycin Cardiac Rejection Treatment Trial Pathologists.

BACKGROUND: The International Society for Heart and Lung Transplantation (ISHLT) working formulation was proposed in 1990 to promote standardization in the interpretation of endomyocardial biopsy specimens obtained after heart transplantation, especially in the setting of multicenter clinical trials and for publication purposes. METHODS: To assess uniformity in interpretation, 16 pathologists experienced in posttransplant endomyocardial biopsy specimen interpretation each read independently, in randomized order, an identical series of 23 biopsy specimens representing all ISHLT grades of rejection (n = 12) and other posttransplant biopsy findings (n = 11). The pathologists represented heart transplantation centers participating in the Rapamycin Treatment Trial for Grades 2 and 3A Rejection. The index diagnosis in each case was determined by two pathology consultants who had concurred blindly on 22 of 23 (96%) biopsy specimen evaluations on their first independent reading. Discrepancies that would not affect clinical response (for example grades 0 versus 1A, 1A versus 1B, 3A versus 3B, 3B versus 4) were considered minor; those that could alter therapy were considered major. RESULTS: The 16 trial pathologists were in exact agreement with the index diagnosis in 17 (mean) +/- 3 biopsy specimens (range 10 to 22) and 20 (mean) +/- 2 biopsy specimens (range 16 to 22) if minor discrepancies were excluded. Of 368 diagnoses rendered, 265 agreed exactly with the index diagnosis and 103 differed, of which 50 were minor discrepancies. The 53 major discrepancies included grades 1A/B versus 2, 22 discrepancies; 2 versus 3A, 11; Quilty B versus 2/3A, 10; biopsy site versus 3A, 2; ischemic injury versus 3A/B, 2; Toxoplasma versus 3A, 2; posttransplantation lymphoproliferative disorder versus 3B/4, 3; and Quilty B versus posttransplantation lymphoproliferative disorder, 1. Interobserver agreement assessed by weighted kappa values was 0.67. CONCLUSIONS: First, there was agreement among the trial pathologists and the index diagnosis (excluding minor discrepancies) in 85% of biopsy specimen interpretations. Second, of 53 major discrepancies, 43 (81%) involved grades 1A/B versus 2, 2 versus 3A, and Quilty B versus 2/3A. Third, in 54% of instances in which biopsy findings other than rejection were misdiagnosed as rejection grades, the grade was sufficiently high to have adverse treatment implications. Fourth, the ISHLT working formulation provides for a high degree of diagnostic consistency among experienced observers, and concordance could be further enhanced by clarification of criteria for grade 2 rejection and Quilty B lesions.

Graft arteriosclerosis-induced myocardial pathology in heart transplant recipients: predictive value of endomyocardial biopsy.

BACKGROUND: Ischemic myocardial pathology resulting from graft arteriosclerosis (GA) includes subendocardial myocyte vacuolization (SEMV), indicative of sublethal ischemic injury, and coagulative myocyte necrosis, acute and healing (CMN), indicative of infarction. METHODS: To assess the sensitivity and specificity of myocardial pathology resulting from GA in endomyocardial biopsy specimens, we correlated SEMV and CMN in the final three biopsy specimens (mean interval from last biopsy to death 23 +/- 20 days) with autopsy findings from 30 heart transplant recipients who survived more than 3 months and died of GA (n = 16) or of other causes (n = 14). The two groups were similar in other parameters. RESULTS: Myocardial ischemic injury was present at autopsy in all 16 patients with GA with the following distribution: SEMV (n = 13) nine right ventricle (RV) and left ventricle (LV), four LV only; focal CMN (n = 8) six RV and LV, two LV only; subendocardial infarct (n = 3) three LV only; and transmural infarct (n = 3) one RV, two LV. Ischemic injury was present in the RV of 11 of 16 patients with GA. Of patients without GA, one had SEMV at autopsy; none had infarcts. Ischemic myocardial pathology was present in 10 of 48 biopsy specimens from patients with GA compared with one of 41 biopsy specimens from patients without GA (p < 0.05). The specificity of SEMV on biopsy was 98%, but sensitivity was only 17%. The positive predictive value for ischemic injury was 92%, and negative predictive value was 51%. CONCLUSIONS: Myocardial pathology resulting from ischemia was present at autopsy in all patients dying of GA. Although more prevalent in the LV, 69% of patients had ischemic myocardial pathology in the RV, where it may be accessible to biopsy. Ischemic myocardial pathology in biopsy specimens is highly specific but far less sensitive, for diagnosing GA.

Giant cell myocarditis: first report of disease recurrence in the transplanted heart.

A 51-year-old female underwent heart transplantation for endomyocardial biopsy-proved giant cell myocarditis complicated by rapidly progressive congestive heart failure unresponsive to immunosuppression. Preoperatively there was no evidence of an associated extracardiac granulomatous disease. Twenty-one months after heart transplantation, giant cell myocarditis recurred in the allograft associated with sustained ventricular arrhythmias. There remained an absence of concomitant extracardiac granulomatous diseases and infections. Increased corticosteroid therapy cleared myocardial inflammation but did not abolish ventricular arrhythmias, which required pharmacologic intervention and the insertion of an Intertach II antitachycardia pacemaker. Compared with a value of 0.56 obtained 1 year after heart transplantation, left ventricular ejection fraction decreased to 0.29 at the time of diagnosis of giant cell myocarditis and remained subnormal 6 months later. Because giant cell myocarditis can recur in the allograft, the candidacy of patients with this disease for heart transplantation must be carefully assessed.

Prominence of apolipoproteins B, (a), and E in the intimae of coronary arteries in transplanted human hearts: geographic relationship to vessel wall proteoglycans.

BACKGROUND: Transplant arteriopathy (TA) is characterized by vessel wall thickening with prominent glycosaminoglycan and lipid deposits. In this light, we have recently demonstrated the distribution of proteoglycans in allograft coronary arteries. The aim of this study is to examine the distribution of apolipoproteins within allograft coronary arteries and to investigate their localization in relation to proteoglycans. METHOD: Particular transverse sections of left and right epicardial coronary arteries from 46 human cardiac allografts, 11 normal hearts, and 11 hearts with native atherosclerosis were stained immunohistochemically for apolipoprotein B, apolipoprotein (a) (apo[a]), apolipoprotein E (apoE), biglycan, decorin, and versican by use of an automated immunostainer. RESULTS: Apo(a) and apoE immunopositivity in TA was much more intense than that in native atherosclerosis, whereas the reverse was true for apoB. Prominent apoE deposits were evident circumferentially in endothelia and extracellularly in superficial intima of mildly diseased TA, as well as in deeper intima of severely diseased TA. Apo(a) had a staining pattern very similar to apoE except for a patchy deposition also seen in TA media. The intimal areas staining prominently with apoE or apo(a) in TA arteries corresponded very closely to the areas with proteoglycan deposits, especially versican. CONCLUSION: The distinctive patterns of apolipoprotein accumulation in TA and native atherosclefosis appear to reflect different pathogenetic processes in the two conditions. The colocalization of proteoglycans and apolipoproteins in TA intima supports the hypothesis that interactions between proteoglycans and apolipoproteins influence lipid retention and overload in allograft coronary arteries.

Biglycan, decorin, and versican protein expression patterns in coronary arteriopathy of human cardiac allograft: distinctness as compared to native atherosclerosis.

BACKGROUND: Histochemical staining has demonstrated previously dramatic deposits of glycosaminoglycans associated with prominent lipid accumulations in thickened vessel walls of allograft coronary arteries. In this study, we characterized the amount, distribution, and types of proteoglycan in the walls of coronary arteries from human cardiac allografts and from native atherosclerotic (NA) controls as part of a strategy to understand the pathogenesis of transplant arteriopathy (TA). METHOD: We used polyclonal rabbit antibodies against human biglycan, decorin, and versican localize the proteoglycan molecules in standardized transverse sections of the proximal left anterior descending and right coronary arteries. Slides were scored in a blinded fashion for intensity of proteoglycan staining (0 to 6+) and for localization in the vessel walls. RESULTS: Unique patterns of proteoglycan distribution were present in TA and NA. Biglycan was particularly prominent in intima and evolving atheromata in severely diseased TA coronary arteries, but not in NA. Decorin was present mainly in adventitia of all vessels and in the intima of NA. Prominent versican accumulation occurred in intima and media of TA coronaries, associated with smooth muscle cells and foam cells. There was a reciprocal pattern of biglycan and decorin staining. Versican colocalized with biglycan. Intimal biglycan and versican deposits were positively correlated to the extent of luminal narrowing in TA. CONCLUSION: The distinctive staining patterns for biglycan, decorin and versican in both native and allograft disease indicate that the synthesis and distribution of these proteoglycans are regulated by different local mechanisms in different atheromatous diseases.

Endocardial infiltrates in the transplanted heart: clinical significance emerging from the analysis of 5026 endomyocardial biopsy specimens.

UNLABELLED: To further elucidate the significance of endocardial infiltrates in heart transplant patients, the presence, frequency, and type of endocardial infiltrates were evaluated in 5026 endomyocardial biopsy specimens obtained from 200 heart transplant patients 0 to 75 months after heart transplantation. The relationship of endocardial infiltrates to immunologic, clinical, and demographic variables was then explored. Endocardial infiltrates were detected in 557 endomyocardial biopsy specimens (11%) from 117 heart transplant patients (58%) at 6.3 +/- 9.4 months (mean +/- SD; range, 0 to 49 months) after heart transplantation. Heart transplant patients with endocardial infiltrates were younger (p = 0.03), had a greater incidence of idiopathic dilated cardiomyopathy before heart transplantation (p = 0.05), and included a greater percentage of females (p < 0.05). Both total and treated rejection rates were significantly higher in patients with endocardial infiltrates versus those without endocardial infiltrates (p = 0.0001). Rejection on the subsequent endomyocardial biopsies was more often present in endocardial biopsy specimens with endocardial infiltrates than in those without endocardial infiltrates, both in the presence (37% versus 24%; p < 0.001) and absence (33% versus 19%; p < 0.0001) of concomitant findings of rejection. No association was identified between endocardial infiltrates and posttransplantation lymphoproliferative disorder, cytomegalovirus infection, Epstein-Barr virus infection, or cardiac allograft vasculopathy. Multivariate regression analysis confirmed that the occurrence of endocardial infiltrates is associated with rejection when adjustment is made for patient's age, gender, heart disease before transplantation, follow-up time, and number of endomyocardial biopsies after heart transplantation (p = 0.0001). CONCLUSIONS: (1) Endocardial infiltrates may occur with or without associated endomyocardial biopsy findings of rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparable proximal and distal severity of intimal thickening and size of epicardial coronary arteries in transplant arteriopathy of human cardiac allografts.

Previous angiographic observations have characterized transplant atherosclerosis as a generally diffuse and more distally severe disease with obliteration of secondary branches. However, it has not been firmly established that the disease is structurally and biologically more severe distally. We evaluated this hypothesis with computer-based digitization of subserial segments of the entire perfusion-fixed left anterior descending coronary artery (100 mm Hg for 1 hour with 10% formaldehyde solution) in 25 allografts at autopsy or explant (19 male and 6 female patients; mean age = 50 years, range 16 to 66; mean implant duration = 490 days, range 3 to 1610). The area, thickness, circumference of the intima and media, and the relative and absolute luminal narrowing were evaluated in a mean of 10 left anterior descending coronary artery sections per allograft. The percentage of luminal narrowing (intimal area/[intimal area + luminal area] x 100) was similar between proximal and distal segments of the left anterior descending coronary artery (45% versus 41%, p > 0.05), and the mean absolute intimal thicknesses (in millimeters) of proximal and distal segments of the left anterior descending coronary artery also were not different (0.32 versus 0.22, p > 0.05). In addition, the 95% confidence intervals for intimal thicknesses of proximal and distal segments were comparable. Because the absolute arterial size of proximal segments is naturally larger than that of distal segments (external diameter 9.37 versus 6.79, p < 0.0001), an appearance of progressive tapering may be visualized angiographically, even though the biologic severity of the disease is geographically uniform. Similarly, observations of obliterated secondary branches in distal segments may result from naturally smaller distal luminal areas which may be occluded by less intimal thickening than would be required proximally. These data emphasize that transplant atherosclerosis is biologically uniform from proximal to distal locations. Etiologic and pathogenetic studies on proximal or distal segments should be equally informative.

Cytomegalovirus and other herpesviruses: do they have a role in the development of accelerated coronary arterial disease in human heart allografts?

In conclusion, a great deal of indirect and inferential data point to herpesviruses as having a role in atherogenesis. It has been shown that the herpesviruses are able to remain within vascular tissue in a latent state, allowing for reactivation to occur with subsequent sequelae of an active infection. Herpesviruses affect the cellular metabolic activity of cells, induce the accumulation of lipids, and inhibit the production of matrix proteins. They have the ability to inhibit endothelial cell binding to the basement membrane. It is also known that the herpesviruses, particularly CMV, can initiate a variety of immunologic responses that may contribute to endothelial damage, precipitating atherogenesis. We are only beginning to understand how CMV may participate in ACAD. Greater attention must be focused on the exact cause-and-effect relationship between CMV infection and ACAD. Even the presence of CMV genomes in arterial walls of allografts must be viewed conservatively in the knowledge of CMV ubiquity and other probable contributions to ACAD. If CMV is involved in the development of ACAD, as an active or latent infection, directly or indirectly, it probably involves numerous coexistent mechanisms (Figure 5).

The international society for heart and lung transplantation working formulation for heart transplant rejection: Is it making the grade?

The cardinal criterion of a classification is utility. It provides one means in an attempt to give order to what would otherwise be a hopeless jumble of disconnected facts. As such it is of the first importance in any field of scholarly endeavor.

Endomyocardial biopsy findings after photopheresis treatment of cardiac transplant rejection.

Photopheresis is a potential therapy for allograft rejection in which reinfusion of mononuclear cells exposed to ultraviolet-A irradiation after pretreatment with 8-methoxypsoralen may initiate immunosuppressive responses. Endomyocardial biopsies (EMBs) of cardiac transplant recipients with moderate acute rejection (IHSLT grades 2 and 3) treated with photopheresis (7 patients/9 treatments) and followed for six months or more were evaluated and compared with biopsies of patients treated with corticosteroids (7 patients/8 treatments) and followed for a similar time period. The first posttreatment EMB showed improvement in 100% of corticosteroid-treated patients, compared with 56% of photopheresis-treated patients (p < 0.005). Interstitial infiltrates of >90% T-lymphocytes were present in a greater percentage of photopheresis-treated patients than in corticosteroid-treated patients on the first five posttreatment EMBs (p < 0.005) as follows: EMB 1, 90% vs. 25%; EMB 2, 90% vs. 25%; EMB 3, 78% vs. 0%; EMB 4, 56% vs. 0%, EMB 5, 56% vs. 0%. Postphotopheresis EMBs also showed giant cell reaction in 1 patient and extensive band-like infiltrates in 3 patients. Our results suggest that interstitial T-cell infiltrates are more prevalent and persist longer after photopheresis than after corticosteroid treatment of heart allograft rejection. Whether these T-lymphocytes are alloreactive or mediate immunosuppressive signals is unknown. The use of new immunosuppressive therapies may modify endomyocardial biopsy findings, requiring adjustment of the diagnostic criteria for assessing and grading allograft rejection.

The pathology of heart allograft rejection.

The pathologist plays an important role in the care of cardiac transplant recipients. Day-to-day management of immunosuppression is largely dependent on the diagnosis and grading of acute rejection. While noninvasive methods have been tried experimentally, the endomyocardial biopsy remains the gold standard in monitoring the rejection status of the heart allograft. The diagnosis of rejection, however, is complicated by a variety of other histologic findings, which may be procedural or processing related, due to sampling, or specifically related to transplantation. Failed allografts, whether from autopsy or explantation, provide the opportunity to study short- and long-term changes in the transplanted heart. Allograft arteriopathy, or graft atherosclerosis, is the major limiting factor in long-term recipient survival. While the morphological features of graft arteriopathy have been well described, the mechanism and factors contributing to its development remain unclear.

The challenge of endomyocardial biopsy interpretation in assessing cardiac allograft rejection.

The endomyocardial biopsy has long been the preferred technique for monitoring the rejection status of the cardiac allograft. During the past year, published reports have addressed important issues concerning the endomyocardial biopsy, including the reliability of the International Society for Heart and Lung Transplantation grading system; problem areas in posttransplantation biopsy interpretation, including grade 2 rejection and myocyte injury, Quilty lesions, and ischemic injury; the natural history of grade 2 rejection; the necessity of surveillance biopsies late in the posttransplantation course; and the efficacy of numerous noninvasive techniques in diagnosing or predicting rejection. No technique developed to date has been shown to have the sensitivity or specificity needed to replace the endomyocardial biopsy as a diagnostic tool. In addition, studies of endomyocardial biopsy specimens have furthered our understanding of the pathobiology of rejection and other transplant-related conditions.

Pathological findings in 2300 consecutive endomyocardial biopsies.

Endomyocardial biopsy (EMB) is a valuable diagnostic procedure for rejection surveillance in heart allograft recipients and is widely used for evaluation of native heart disease. However, the spectrum and incidence of diagnoses encountered on a heart failure/cardiac transplant service deserve clarification. Of 2300 consecutive EMBs performed during a 2.5-yr period, 79.9% had been performed for rejection surveillance in heart allograft recipients. Of these, 1281 (69.7%) were negative for rejection; 536 (29.1%) were positive (18.9% mild, 9.7% moderate, 0.5% severe); 21 (1.1%) were not interpretable due to insufficient samples. Endocardial lymphocytic infiltrates ("Quilty" effect) were present in 86 (4.7%), ischemia in 12 (0.7%), myocardial calcification in five (0.3%), foreign body giant cells in two (0.1%), valvular tissue in two (0.1%), and liver tissue in one (0.05%). Of the 20.1% of EMBs performed in patients with native heart disease, 298 (64.5%) were abnormal. A total of 239 (51.7%) had myocyte hypertrophy and/or fibrosis, while 37 (8.0%) had active or ongoing myocarditis, two of which were of the giant cell type. Other diagnoses included anthracycline cardiotoxicity in 11 (2.4%), amyloidosis in five (1.1%), hemochromatosis in two (0.4%), healed infarct in two (0.4%), scleroderma in one (0.2%), and foreign body granuloma in one (0.2%). A total of 159 (34.4%) samples had no diagnostic abnormalities; five (1.1%) were insufficient samples. As the number of EMBs performed grows, pathologists must develop expertise in the detection of morphological features pertaining to various cardiac conditions which may have similar clinical presentations.

Natural history of focal moderate cardiac allograft rejection. Is treatment warranted?

BACKGROUND: The rate of progression and potential long-term consequences of isolated foci of moderate acute rejection (FMR) on endomyocardial biopsy (EMB) have not been defined; therefore, whether FMR necessitates augmented immunosuppression remains controversial. METHODS AND RESULTS: At our institution, recipients with EMBs having FMR, defined as one or two isolated foci of cellular infiltrates with associated myocyte damage (International Society for Heart and Lung Transplantation [ISHLT] grade 2 and a subset of grade 3A), do not routinely receive intensified immunosuppression. Accordingly, to determine the outcome of untreated FMR, we reviewed 4398 EMBs (mean, 4.4 samples each) obtained after orthotopic heart transplantation in 208 consecutive recipients maintained on triple immunosuppressive therapy. The incidence of progression versus resolution of FMR, the time interval after transplantation when FMR was detected, and the relation of untreated FMR to recipient survival were analyzed. FMR categorized as one (n = 312) or two (n = 89) foci was present in 401 EMBs (9% of total) obtained 10 days to 7.5 years after transplantation from 149 recipients (72%). EMBs with FMR resolved without treatment in 341 of 401 (85%), and only 60 of 401 (15%) progressed to higher grade rejection. EMBs that progressed occurred 7.5 +/- 7.9 months (mean +/- SD) after transplantation compared with 14.0 +/- 16.5 months after transplantation for those that resolved (P < .005). Of the 60 EMBs that progressed, 55% occurred within the first 6 months, 78% within the first year, and 97% within the first 2 years after transplantation. EMBs with two foci of FMR were no more likely to progress than those with one focus. Thirty-nine recipients experienced one (n = 25), two (n = 9), three (n = 3), or four (n = 2) episodes of FMR that progressed. One or more episodes of FMR that did not progress occurred in 110 recipients. By Kaplan-Meier analysis, survival at 1 and 5 years was similar in recipients with and those without FMR progression. CONCLUSIONS: First, untreated FMR consisting of either one or two foci has a low rate of progression. Second, progression of FMR decreases with increasing postoperative interval and becomes rare after 2 years. Last, FMR progression did not identify recipients with decreased survival.

Metastasizing mixed tumor of the parotid gland: a rare tumor with unusually rapid progression in a cardiac transplant recipient.

We report the case of a 69-year-old man who had a mixed tumor (pleomorphic adenoma) removed from his parotid gland 3 years after orthotopic heart transplantation. Two years later, he presented with widely metastatic mixed tumor, which resulted in his death within 6 months. Metastatic mixed tumor is histologically identical to a benign mixed tumor, but it inexplicably metastasizes. Such tumors are rare and have not been reported to date in a transplant recipient. This case illustrates the rapid and aggressive course that malignancies can follow in an immunosuppressed population. Mixed tumors are common salivary neoplasms, so transplant recipients should be carefully followed after resection for evidence of metastatic spread.

Enhanced lymphocyte longevity and absence of proliferation and lymphocyte apoptosis in Quilty effects of human heart allografts.

"Quilty effect" (QE) is a common and problematic observation in endomyocardial biopsy specimens from patients after cardiac transplantation. The origin, fate, and significance of QE cellular elements are unknown. Twenty-six paraffin-embedded endomyocardial biopsy specimens with QE (five QE As and twenty-one QE Bs) from twenty-two cardiac allografts were studied by immunohistochemistry for expression of Bcl-2, Fas antigen, proliferating cell nuclear antigen (PCNA), perforin, T cells (UCHL-1), macrophages (CD68), and apoptosis by in situ terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Approximately 50% of the lymphocytes present, mainly in the deeper region of 20 of 21 QE Bs and all 5 QE As, expressed Bcl-2 in a pseudo-nodular pattern surrounding high endothelial venules. Fas expression was detected in lymphocytes in 20 of 21 QE Bs and 5 QE As in a similar pattern to Bcl-2. However, endothelial cells and macrophages were Bcl-2 negative, whereas both cell types were Fas positive. Perforin was negative in nearly all lymphocytes. TUNEL staining revealed that lymphocytes in QEs did not undergo apoptosis; however, TUNEL positivity was observed in approximately 70% of endothelial cells and macrophages and certain adjacent cardiac myocytes in 20 of 21 QE Bs and 5 QE As. One large QE B with a germinal center was noted. Germinal center cells expressed PCNA intensely but were negative for Bcl-2, Fas, and TUNEL. Cells surrounding the germinal center expressed abundant Bcl-2. The following conclusions were drawn. 1) Apoptosis does not occur in lymphocytes in QE where enhanced Bcl-2 (apoptosis inhibitor) and Fas antigen (apoptosis inducer) are expressed. 2) PCNA negativity indicates that QE lymphocytes may not proliferate, and perforin negativity indicates that they may not exhibit perforin-based cytotoxicity. We propose that there may be a relationship between the longevity of lymphocytes in QE and the absence of apoptosis.