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THE SAGA OF ANTIBODY-MEDIATED REJECTION:
THE PATHOLOGY CHALLENGE

Patrick Bruneval, MD
Pathology Council Chair
University Paris-Descartes and Hôpital Européen Georges-Pompidou, Paris, France


patrick brunevalWhere we were:
Antibody-mediated rejection (AMR) was initially recognized in severe conditions challenging the cardiac graft function and frequently presenting as a life-threatening episode of rejection. Full-blown pathology was described from endomyocardial biopsies and autopsies, including some myocyte necrosis, interstitial edema, plump endothelial cells and microthrombi in capillaries. Rapidly, the deposits of the complement fraction C4d on the capillary walls supplanted any other immunofluorescence tests. Therefore, diffuse and strong deposits of C4d, initially by immunofluorescence on frozen tissues, then by immunohistochemistry on paraffin sections, became the gold standard for the diagnosis of AMR on endomyocardial biopsies (Figure 1).

aug linksMeanwhile the presence of intravascular mononuclear cells, so-called "activated macrophages," "CD68-positive cells" retained the attention and also became a marker of cardiac AMR detectable by plain histology and/or by immunohistochemistry.

ISHLT and input from its Pathology Council allowed: i) to better characterize cardiac AMR opening this entity to conditions where no cardiac dysfunction was present at the time of biopsy diagnosis; ii) to grade AMR on endomyocardial biopsies as follows:

pAMR 0: both histological and immunopathological studies are negative
pAMR 1 (H+): histopathological alone
pAMR 1 (I+): immunopathological alone
pAMR 2: both histological and immunopathological findings are positive
pAMR 3: severe AMR

(from Berry et al. J Heart Lung Transplant 2011)

Where we are:
The 2012 meeting of ISHLT in Prague showed that our knowledge of the pathology of AMR is still progressing, benefited in part from the results of a transatlantic multicenter survey based on the analysis of 25 digitalized endomyocardial biopsies by a panel of 15 skilled pathologists. The results were presented and discussed in a pre-meeting workshop held on April 17th (manuscript under preparation), clearly showing that the 2011 working formulation is still valid and displays pathology-based criteria for the diagnosis of AMR on endomyocardial biopsies. The grading system is correlated to the certainty of AMR. pAMR3 corresponding to severe cases is seldom observed nowadays. The 2012 AMR workshop in Prague pointed at some questions:

  • aug linksHow to consider "focal strong" C4d labeling (Figure 2)?
    So far the dogma is and remains that positivity requires diffuse labeling i.e. above 50% of capillaries; lower grades labeling, <10% and focal between 10 and 50% are still considered as below the threshold of positivity. However the panel of pathologists recommended to notify when focal labeling is strong and to correlate it with DSA or other biopsy findings such as intravascular macrophages. How to explain that strong focal pattern? Since the panel ruled out technical problems considering that now immunohistochemistry and immunofluorescence are standardized, several speculative explanations were proposed: i) DSA at low levels or DSA species poorly activators of complement; ii) capillary loss due to previous AMR damage to microcirculation.
  • What is the threshold for positivity of intravascular macrophages? The panel of pathologists rejected a grading system similar to C4d labeling interpretation and proposed a threshold above 10% for positivity of intravascular macrophages.
  • Is still C4d positivity mandatory for the diagnosis of AMR? Most of the pathologists attending the workshop agreed that they have cases of C4d-negative AMR. However it is too early to change the working formulation for diagnosis and grading AMR. Further studies are needed.

Where we're going:
Finally after the pathologists did their own introspection from 2010-present to characterize the histological and immunohistochemical markers of cardiac AMR, clearly the time is now to correlate our data with those of other specialists dealing with AMR, mainly immunologists. Sophisticated analysis of implicated antibodies [titers of donor specific antibodies (DSA), complement-fixing DSA, non HLA antibodies...] and sequential detection of DSA are mandatory to support new concepts in the pathology of AMR. A multidisciplinary approach is necessary to tackle the following problems:

  • aug linksC4d-negative biopsies in AMR are challenging the C4d detection as the marker of AMR;
  • Subclinical cardiac AMR: We are not often far from the situations of an acute episode of AMR immediately threatening the graft function. Now AMR presents usually as an ongoing phenomenon with fluctuating levels of DSA and variable damages on biopsy.
  • Several studies pointed out that subclinical AMR is harmful for the coronary arteries and is an important factor for cardiac allograft vasculopathy (CAV) assessed by coronary angiography or IVUS. A challenge for the pathologist would be to predict the risk of CAV from biopsy analysis considering that a common denominator between coronary arteries and the myocardium sampled by biopsies is the endothelium lining. In this respect looking for markers of endothelial cell activation/damage on endomyocardial biopsies beyond C4d deposition should draw our attention using transcriptome analysis, immunohistochemical detection (Figure 3).
  • Characterization of intravascular cells: the dogma that intravascular cells are macrophages is questionable given the frequent inadequacy between the numbers of intravascular cells seen by H&E and those detected by CD68-immunohistochemistry. Phenotype characterization of intravascular cells should provide new insights in the diagnosis and the natural history of AMR.

Many studies remain to be done, some of them should benefit from a multicenter approach and for that the Pathology Council of ISHLT should ease the task.

Disclosure Statement: The author received travel and research grants from Astellas in 2009 and 2010, research grant from Novartis in 2012.