Intravascular macrophages in cardiac allograft biopsies for diagnosis of early and late antibody-mediated rejection

http://www.jhltonline.org Intravascular macrophages in cardiac allograft biopsies for diagnosis of early and late antibody-mediated rejection Marny Fedrigo, MD, PhD, a Giuseppe Feltrin, MD, PhD, a Francesca Poli, MD, b Anna Chiara Frigo, MSc, a Elena Benazzi, MD, b Antonio Gambino, MD, a Francesco Tona, MD, PhD, a Alida L.P. Caforio, MD, PhD, a Chiara Castellani, PhD, a Giuseppe Toscano, MD, a Gino Gerosa, MD, a Gaetano Thiene, MD, a and Annalisa Angelini, MD a From the a Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Padua; and the b Department of Regenerative Medicine Transplant Immunology, IRCCS OMP MARE, Milan, Italy. KEYWORDS: antibody-mediated rejection; intravascular macrophages; heart transplantation; immunohistochemistry; anti-hla antibodies; C4d BACKGROUND: The aim of our study was to evaluate the role of intravascular macrophages in the diagnosis of early and late antibody-mediated rejection (AMR) on endomyocardial biopsies (EMBs). METHODS: We reviewed 1,420 consecutive EMBs from 131 patients and selected 75 C4d þ EMBs. The C4d þ group was compared with a control group (66 patients) matched for age, gender, date of transplantation, follow-up, immunosuppressive regimen and primary heart disease. A total of 141 EMBs were evaluated. Immunoperoxidase staining for C4d and CD68 were performed. Post-transplant IgG anti-hla reactivity was investigated by Luminex technology. Clinical data were also collected. Fourteen EMBs were available from 11 symptomatic AMR patients. RESULTS: Of the 141 EMBs evaluated, 53 were positive for intravascular macrophages (CD68); among them, 32 were also positive for C4d (32 of 53, 60.4%). Of the 88 CD68 EMBs, 43 were also C4d þ (43 of 88, 48.9%). Of the 53 CD68 þ EMBs, 30 EMBs were within the first year since transplantation (30 of 53, 57.8%), and among these 21 were also positive for C4d (21 of 30, 70.0%). In the late period, among the 23 CD68 þ EMBs (23 of 53, 42.2%) 11 were also positive for C4d (11 of 23, 47.8%). In the early period, intravascular macrophages were more common in symptomatic (3 of 3, 100%) than asymptomatic (3 of 11, 27.3%) patients. Sensitivity and specificity of intravascular macrophages in predicting donor-specific antibodies (DSA) within the first year were 50.0% and 100.0%, respectively. CONCLUSIONS: Intravascular macrophages predict C4d, DSA and symptoms early after transplantation; however, in the late period, they are unable to identify patients with circulating DSA, C4d and/or symptoms. J Heart Lung Transplant 2013;32:404 409 r 2013 International Society for Heart and Lung Transplantation. All rights reserved. The recent worldwide interest in antibody-mediated rejection in heart transplantation highlighted the need for pathologists to define clearly the diagnostic criteria of Reprint requests: Annalisa Angelini, MD, Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Via Gabelli 61, 35121 Padua, Italy. Telephone: þ39-049-8272260. Fax: þ39-049-8272205. E-mail address: annalisa.angelini@unipd.it 1053-2498/$ - see front matter r 2013 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2012.12.017 humoral rejection in monitoring endomyocardial biopsies (EMBs) of heart transplant patients. C4d has been recognized as important biomarker of humoral activation on tissue 1 4 together with intravascular macrophages. Few studies have reported on both technical and interpretative issues for C4d and related C4d positivity to negative outcomes. 5 7 C4d was identified as an important diagnostic biomarker for antibody-mediated rejection and was strongly

Fedrigo et al. Intravascular Macrophages in Early/Late AMR 405 correlated with the presence of circulating anti-hla antibodies. The 2005 ISHLT revised rejection grading system recommended use of C4d and CD68 for immunostaining in the assessment of antibody-mediated rejection (AMR). 8 The lack of experience with CD68 staining patterns, and assessment of the cell threshold required for a positive result indicated that a better definition of CD68 þ criteria is needed. It is unclear at present how many centers are applying CD68 only without accompanying C4d. Those using both C4d and CD68 found that the combination is useful. 8,9 More recently, it has been reported in both kidney and heart transplantation that there is a subset of patients presenting with humoral rejection but with C4d-negative staining. 10 This group of patients (donor-specific antibody positive [DSA þ ], C4d ) could be identified by CD68 þ staining. Cardiac AMR has generally been considered an early phenomenon, although many patients produce de novo donor-specific antibodies in a time-dependent manner after transplantation. In heart transplantation, AMR can develop late or very late after transplantation. 11 Some investigators have suggested that early and late AMR could be characterized by different features. 12 The aim of the present study was to evaluate the roles of: (1) intravascular macrophages in the diagnosis of humoral rejection on EMBs; (2) intravascular macrophages in asymptomatic and symptomatic patients; and (3) intravascular macrophages in early and late AMR. Methods We analyzed 1,420 consecutive EMBs from 131 patients (age at transplant: mean 48.9 years, standard deviation 18.4 years). Donor mean age was 41.6 15.2 years, and 15% were 460 years old. Since 2004, at our institution we have routinely applied C4d staining of EMBs. Eighty-one EMBs stained positive for C4d from 65 patients and, from these, we excluded 6 EMBs from 4 patients with acute cellular rejection Z2R, resulting in 75 index EMBs from 61 index patients. Sixty-six control patients were closely matched for age, gender and time after transplantation. All control patients were hemodynamically stable; had few to no episodes of prior acute cellular rejection; and never had a clinical, pathologic or serologic (presence of circulating DSA) AMR. Among the 75 C4dpositive EMBs, 14 (18.7%) were diagnosed with symptomatic AMR on the basis of circulating DSA and graft dysfunction and 15 were asymptomatic (AMR without graft dysfunction). For followup after transplantation we subdivided the EMBs in 2 periods: early, r1 year after transplantation; and late, 41 year. We found that 79 of 141 (56.0%) of the EMB specimen were collected within 1 year after heart transplantation. A separate evaluation was carried out on 26 EMBs from 13 patients who had repeated C4d capillary positivity on EMBs at intervals of 42 weeks. New C4d-positive events were considered separately when positivity appeared at intervals of 42 weeks. The patients clinical data collected during the follow-up were reviewed. Cardiac allograft dysfunction was defined either as the finding of left ventricular ejection fraction o50%, as measured by transthoracic echocardiography, and signs and symptoms of heart failure. Figure 1 Definition of CD68 negativity and positivity. (A) CD68 negativity, with absence of intravascular macrophages or aggregates (original magnification 20); (B) CD68 positivity, with many intravascular macrophages, sometimes in aggregate (410% capillaries involved) (original magnification 20). Immunohistochemistry staining technique Histopathologic evaluation and C4d staining were performed on formalin-fixed, paraffin-embedded sections. Immunostaining for C4d was performed using affinity-purified anti-human C4d rabbit polyclonal antibody (Cat. No. BI-RC4; Biomedica Gruppe, Vienna). Prior to immunostaining, sections were retrieved using an ethylene-diamine tetraacetic acid (EDTA) solution on pressure-cook (Pascal; DakoCytomatic). These were incubated with anti-c4d antibody at a 1/50 dilution at room temperature for 1 hour, with anti-rabbit EnVision (Dako Corporation, Hamburg, Germany) for 30 minutes and, finally, with peroxidase diaminobenzidine (DAB) for 5 minutes. Sections were counterstained with Mayer s hematoxylin for 1 minute, dehydrated in alcohol, and mounted with medium mounting (Eukitt; Bioptica). The C4d staining intensities and patterns were scored in a semiquantitative manner using grades ranging from 0 to 3 þ8. We considered C4d positivity in the setting of multifocal or diffuse (450%) of capillaries involvement with a faint or strong intensity. The C4d prevalence was 6%. Immunohistochemical (IHC) staining of CD68, a monocyte/ macrophage marker, was performed routinely and retrospectively on the paraffin sections of each biopsy, all evaluated by light microscopy. We considered CD68 þ when we found intravascular macrophage aggregates with 410% capillary involvement (Figure 1) according to the new ISHLT classification. 8

406 The Journal of Heart and Lung Transplantation, Vol 32, No 4, April 2013 Table 1a Global Relation of Intravascular Macrophages and C4d Intravascular macrophages Monoclonal mouse CD68, Clone PGM1 (dilution 1/100; Dako), was retrieved with a citrate solution using a microwave oven. Staining for CD34 pre-diluted antibody (Immunotech) was performed to determine whether CD68 (macrophages) was intraand/or extracapillary. Assessment of donor-specific antibodies IgG anti-human leukocyte antigen reactivity in the sera, obtained before transplantation and at the time of C4d þ detection on EMBs, was analyzed using Luminex bead based screening assays. Thirtyfour DSA tests at the time of C4d þ EMBs specimens from 25 index patients and 9 tests at the time of C4d EMBs from 9 controls were assessed for a total of 43 determinations. Eighteen DSA tests were performed within the first year after transplantation and 25 DSA tests in the late period. Antibodies were considered positive if the median fluorescence intensity (MFI) was 41,000. Informed consent All patients provided a consent statement regarding the research we performed on their surveillance EMBs and they were informed about how their clinical data would be utilized. Use of these sensitive data followed in accordance with Italian Law No. 196, June 30, 2006. Statistical analysis C4d þ C4d Total CD68 þ 32 (42.7) 21 (31.8) 53 CD68 43 (57.3) 45 (58.2) 88 Total 75 66 141 Data are expressed as mean and standard deviation for quantitative variables and as number and percent in each category for categorical variables. Sensitivity, specificity, positive and negative predictive value and positive and negative likelihood ratio are presented with 95% confidence interval (95% CI). The 95% CI was calculated with the bimodal proportion exact method for sensitivity, specificity and predictive values, and according to Simel et al 13 for likelihood ratios. The predictive values were calculated considering that, at our center, C4d positivity has a prevalence of 6%. Likelihood ratios are alternative statistics for summarizing diagnostic accuracy that are independent from the p 0.18 Table 1c Distribution of Intravascular Macrophages (CD68 þ ) During HTx Follow-up prevalence of the condition analyzed and summaries of how many times more (or less) likely patients with the condition are to have that particular test result than patients without the condition. The larger the positive likelihood ratio, the greater the likelihood of disease; the smaller the negative likelihood ratio, the lower the likelihood of disease. Positive likelihood ratios 410 and negative likelihood ratios o0.1 are considered strong evidence for ruling-in or ruling-out diagnoses, respectively, in most circumstances. The statistical significance of the association of macrophages with C4d and symptoms was evaluated with chi-square test conducted at the 5% level. Results r1 year / 0.4 0.3 year (range 0.1 1.0 year) 41 year / 7.6 6.7 years (range 1.0 22.1 years) C4d þ C4d p C4d þ C4d p CD68 þ 21 (70.0) 9 (30.0) 0.04 11 (47.8) 12 (52.2) CD68 23 26 20 19 (46.9) (53.1) (51.3) (48.7) Total 44 35 31 31 0.79 Intravascular macrophages could be detected in 53 of 141 (37.6%) EMBs analyzed. Intravascular macrophage positivity was similarly detected in early (30 of 79, 38.0%) and late follow up (23 of 62, 37.1%). Intravascular macrophages were present in 32 of 75 (42.7%) C4d þ EMBs and in 21 of 66 (31.8%) control group EMBs (p ¼ 0.1845) (Table 1a). The distribution of intravascular macrophages in relation to 4 grades of C4d showed that there was no relation between intravascular CD68 and severity of C4d grade (Table 1b). Sensitivity and specificity of intravascular macrophages to identify humoral activation assessed as C4d positivity were 42.7% (95% CI 31.1% to 54.6%) and 68.2% (95% CI 55.6% to 79.1%), respectively. At our center, the C4d prevalence was 6%. The positive and negative predictive value of intravascular macrophages to predict humoral activation was 7.9% (95% CI 5.5% to 10.9%) and 94.9% (95% CI 93.2% to 96.3%), respectively. Table 1b Distribution of Intravascular Macrophages (CD68 þ ) in Relation to 4 Grades of C4d (0 3) C4d grade CD68 þ CD68 p 0 8 (26) 23 (74) 1 13 (37) 22 (63) 2 10 (38) 16 (62) 3 22 (45) 27 (55) Total 53 88 0.40 Table 2 Intravascular Macrophages in Repeated C4d Positivity During Follow-up in EMBs Intravascular macrophage r1 year 41 year CD68 þ 11 (68.8) 3 (30.0) 14 CD68 5 (31.2) 7 (70.0) 12 Total 16 10 26 As a whole

Fedrigo et al. Intravascular Macrophages in Early/Late AMR 407 Table 3a Sensitivity and Specificity of CD68 and C4d to Predict Circulating DSA in Early AMR (r1 year) % Sensitivity (95% CI) % Specificity (95% CI) CD68 þ 50.0 (24.7 73.4) 100.0 (15.8 100.0) C4d þ 93.8 100.0 (69.8 99.8) (15.8 100.0) CD68 þ 100.0 100.0 or C4d þ (79.4 100.0) (15.8 100.0) CD68 þ and C4d þ 43.8 100.0 (19.8 70.1) (15.8 100.0) þlr LR (95% CI) (95% CI) 0.500 (0.306 0.816) 0.063 (0.009 0.417) 0.563 (0.365 0.867) Intravascular macrophages (CD68 þ ) and C4d in early and late follow-up after heart transplantation There was a statistically significant association between presence of CD68 þ and C4d þ in the first year after transplantation (p ¼ 0.04), whereas this relation decreased over time (Table 1c). The presence of intravascular macrophages seems to be time-related. Patients with repeated episodes of IHC C4d positivity were evaluated separately for CD68 þ (Table 2).The results showed a similar trend over time for IHC C4d þ and CD68 compared with patients at the first episode. Intravascular macrophages (CD68 þ ) and circulating anti-hla antibodies in early and late follow-up The sensitivity and specificity of intravascular macrophages in predicting DSA was 50.0% (95% CI 24.7% to 73.4%) and 100.0% (95% CI 15.8% to 100.0%), respectively, in the early follow-up period, and 16.7% (95% CI 3.6% to 41.4%) and 42.9% (95% CI 9.9% to 81.6%), respectively, in the late follow-up period. Considering the two most important immunohistochemical markers for pamr1(i) separately, the sensitivity was 100.0% (95% CI 79.4% to 100.0%) and the specificity was 100.0% (95% CI 15.8% to 100.0%) (Table 3a). Conversely, in the late period, the sensitivity and specificity of CD68 and/or C4d positivity to predict circulating DSA dropped dramatically (Table 3b). The sensitivity of CD68 and/or C4d was the same as for C4d positivity alone, with no contribution of CD68 to diagnose the presence of circulating DSA. Within the first year, 16 of 18 (88.9%) EMBs presented with concomitant circulating DSA; among these, C4d was positive in 15 (15 of 16, 93.8%) with 7 also CD68 þ (7 of 15, 47.7%). The only one DSA þ and C4d (6.3%) was conversely CD68 þ.the2dsa were also C4d and CD68. In the late period, 18 of 25 (72.0%) EMBs presented with concomitant DSA and, among these, C4d was positive in 15 (15 of 18, 83.3%), with 3 also positive for CD68 (3 of 15, 20.0%). Of the 7 DSA, 4 were C4d þ (57.1%) and among these 3 were also positive for CD68 (75.0%). Symptomatic and asymptomatic AMR In the group of asymptomatic patients with C4d positivity and circulating DSA, intravascular macrophages were present in only 4 of 15 (26.7%), and in 5 of 14 (35.7%) of the symptomatic patients (Figure 2). Within the first year, intravascular macrophages were constantly present in symptomatic patients (p ¼ 0.05), whereas the same was not true in the late period for symptomatic patients (Table 4). Discussion The main result of our study is that intravascular macrophages evaluated at immunohistochemistry on EMBs are an important diagnostic marker of AMR in the first year after heart transplantation and correlate well with C4d and DSA. The detection of intravascular macrophages is recognized as a major pathologic criterion both on hematoxylin eosin (H&E) [pamr1(h)] and on immunoperoxidase staining [pamr1(i)]. CD68 has been recognized as an important antibody for staining, able to identify activated intravascular macrophages. The consensus study on AMR by Kobashigawa et al 14 confirmed the key role of macrophages in the humoral activation pathway acting as an antigen-presenting cell to endothelial cells, initiating the complement cascade with C4d and C3d cleavage and deposition in the tissue. This has also been endorsed in the recent update of the ISHLT classification, which recognizes CD68 among the key pathologic criteria for diagnosis of AMR. 8 Our result is also in keeping with previous observations by Fishbein et al, 15 who used CD68 immunohistochemical staining for the characterization of intravascular cells, even before the introduction of C4d in clinical practice. Fishbein et al suggested that if immunofluorescence is not available, the finding of numerous CD68-positive macrophages within capillaries in the absence of T-cell infiltrates would be considered diagnostic of humoral rejection. They demonstrated that the majority of swollen cells within the microvasculature in AMR were actually macrophages and not endothelial cells. 9 Table 3b Sensitivity and Specificity of CD68 and C4d to Predict Circulating DSA in Late AMR (41 Year) % Sensitivity (95% CI) % Specificity (95% CI) þlr (95% CI) LR (95% CI) CD68 þ 16.7 (3.6 41.4) 42.9 (9.9 81.6) 0.292 (0.086 0.984) 1.944 (0.807 4.688) C4d þ 83.3 (58.6 96.4) 42.9 (9.9 81.6) 1.458 (0.743 2.861) 0.389 (0.102 1.487) CD68 þ or C4d þ 83.3 (58.6 96.4) 28.6 (3.7 80.0) 1.167 (0.699 1.947) 0.583 (0.122 2.781) CD68 þ and C4d þ 16.7 (3.6 41.4) 57.1 (18.4 90.1) 0.389 (0.102 1.487) 1.458 (0.743 2.861)

408 The Journal of Heart and Lung Transplantation, Vol 32, No 4, April 2013 Figure 2 Relation between intravascular macrophages (CD68) and symptomatic and asymptomatic AMR. According to our results, however, intravascular macrophages cannot be considered a diagnostic biomarker for AMR in late follow-up, suggesting a low sensitivity for C4d. It was recently hypothesized that there are 2 different phenotypic patterns of AMR occurring in a time-dependent manner: early AMR (r1 year), relating more to presensitized patients (crossmatch-positive) and to drug therapy; and a late or very late AMR, related more to triggers such as acute cellular rejection episodes and/or infections. 16,17 Hodges proposed that histopathologic and immunopathologic features were not sensitive markers of AMR in a late follow-up group of patients (45 years since transplantation), suggesting a need to identify specific biopsy criteria for the late cardiac AMR. Consequently, clinically and pathogenetically early AMR and late AMR could be considered 2 distinct entities. It is now well accepted that there are 4 stages of humoral response to an organ allograft, based on circulating DSA, pathologic substrates of tissue and clinical dysfunction, characterized by asymptomatic (latent and silent and subclinical) and symptomatic phases 18 with different pathologic features. Even the early asymptomatic phases (latent and silent) carry poorer outcomes 5,19 ; in the heart, this was true both in terms of mortality and cardiac allograft vasculopathy (CAV) development. Our results show that, overall, intravascular macrophages had a low sensitivity and specificity to predict the presence of circulating HLA antibodies (DSA). This low sensitivity/specificity was also found if we considered symptomatic and asymptomatic patients. One could expect that asymptomatic patients would be characterized by mild morphologic tissue involvement, whereas symptomatic patients would have a more severe and advanced AMR morphologic injury. Our previous experience on morphologic criteria of AMR with H&E showed that there were no detectable differences on EMBs between symptomatic and asymptomatic patients. 20 Even with IHC, sensitivity/specificity of intravascular macrophages in relation to symptoms was low. C4d retained a higher sensitivity/specificity to predict DSA compared with CD68 both in early and late follow-up. In early AMR, CD68 had good sensitivity and very high specificity (50% and 100%, respectively); when considering the presence of at least 1 of the 2 most important diagnostic criteria of AMR we observed increased sensitivity/specificity for predicting donor-specific antibody positivity. The use of CD68 in the early period after HTx could identify some patients as C4d, which may have been missed otherwise by using C4d alone. On the contrary, in the late follow-up period, the use of 2 IHC markers did not increase sensitivity and specificity, suggesting that the high sensitivity/specificity was due to C4d positivity and that CD68, when present, was associated with C4d positivity. Our data show that, in the early follow-up period, intravascular macrophages (CD68 þ ) were present in a few C4d EMBs (see Table 3) presenting with circulating DSA. This entity, proposed by Sis and colleagues, first in kidney and then also in heart, 10,21 using endothelial-associated transcripts to define molecular gene expression for AMR, 22 is challenging from a pathologic diagnostic point of view and its presence should be carefully acknowledged. Intravascular macrophages as a sign of microvascular inflammation represent the earliest evidence of antibodymediated allograft injury and, if recognized early before complement activation deposition on tissue, could represent a potential therapeutic target for prevention of consequent graft dysfunction and failure. Study limitations There are some limitations to this study, mainly with regard to its retrospective nature and to the relatively small number of patients and EMBs with CD68 and C4d positivity. Also, DSA assessment was done in only a subgroup of patients. In conclusion, intravascular macrophages predict C4d and DSA positivity and symptoms early after transplantation. However, in the late period, intravascular macrophages are unable to identify patients with circulating DSA, C4d positivity and/or symptoms. Intravascular macrophages alone do not appear to be an adequate marker for pathologic diagnosis of late AMR. Table 4 Intravascular Macrophages vs Symptoms of AMR During Follow-up r1 year / 0.4 0.3 years (range 0.1 1.0 year) 41 year / 7.6 6.7 years (range 1.0 22.1 years) Symptomatic Asymptomatic p Symptomatic Asymptomatic p CD68 þ 3 (100.0) 3 (27.3) 0.05 2 (18.2) 1 (25.0) 1.00 CD68 0 (0.0) 8 (72.7) 9 (81.8) 3 (75.0) Total 3 11 11 4

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