Heart Transplant Rejection Pathology

Rejection refers to the organ recipient's immune system recognizing a transplanted organ as foreign and mounting a response to it via cellular and/or humoral (antibody-mediated) mechanisms. Routine endomyocardial biopsy remains the criterion standard for monitoring for such rejection. ^[1] This process involves sampling of the right ventricle using a bioptome, and a pathologist evaluating the tissue. The concept of rejection is not unique to cardiac allograft transplantation and, to a varying extent, occurs in all forms of solid organ transplantation.

Cardiac transplant rejection manifests in one or more of the following three ways: acute cell-mediated rejection, antibody-mediated rejection (AMR), and allograft vasculopathy.

Acute cell-mediated rejection is primarily a host T-lymphocyte-mediated response mounted against the allograft tissue.

AMR refers to allograft injury resulting from activation of the complement system, typically by recipient-generated antibodies directed against the allograft tissue. ^[2]  Although AMR most commonly occurs months to years following transplantation, a rare subtype, hyperacute rejection, can occur within minutes to hours after transplantation.

Cardiac allograft vasculopathy (CAV), also called accelerated graft arteriosclerosis, generally refers to concentric luminal narrowing of the epicardial and intramural coronary arteries. This process generally occurs months to years following transplantation and is currently the limiting factor in long-term allograft survival. This is also thought to represent a manifestation of an antibody-mediated response.

Further details (ie, clinical, gross, microscopic findings) of these various types of rejection are detailed later in this article, although the primary focus of this article is the pathology of acute cellular rejection in heart transplantation.

The first two images below show normal transplant endomyocardial specimens. Contrast these with the third image which demonstrates severe acute cell-mediated rejection.

Heart Transplant Rejection Pathology. Transplant endomyocardial biopsy specimen from the right ventricle. This photomicrograph is of a biopsy specimen with no evidence of rejection (hematoxylin-eosin, 40x).

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Heart Transplant Rejection Pathology. Transplant endomyocardial biopsy specimen from the right ventricle. This photomicrograph is also of a biopsy specimen with no evidence of rejection (hematoxylin-eosin, 40x).

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Heart Transplant Rejection Pathology. Severe acute cellular rejection. This photomicrograph exhibits diffuse myocardial inflammation with associated myocyte damage (International Society for Heart Lung Transplantation [ISHLT] 1990 grade 3B or ISHLT 2004 grade 3R) (hematoxylin-eosin, 100x).

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The following should be considered in the differential diagnosis of heart transplant rejection:

• Infection
• Posttransplantation lymphoproliferative disorder
• Previous biopsy site
• Quilty lesion

Risk factors for developing antibody-mediated rejection (AMR) include pregnancy, previous transplantation, blood transfusions, sensitization by OKT3 induction therapy, and use of ventricular assist devices. ^[3]

Cardiac allograft vasculopathy (CAV) inevitably occurs in most heart transplant recipients over a variable time course. More than half of adult heart transplant recipients exhibit angiographically detectable CAV by 10 years posttransplantation. ^[4] Identified risk factors for developing CAV include donor hypertension, cellular rejection during the first year following transplantation, and being a recipient of a heart from a male donor. ^[5]

Generally speaking, solid organ rejection is mediated via host T lymphocytes that recognize donor major histocompatibility complexes (MHC) I and II. The endothelium, being the primary interaction site between graft and host, is where rejection is usually first seen. Antibodies generated by these antigenic differences between donor and recipient as well as direct cytotoxic effects of the donor's immune system are responsible for the pathology seen in transplant rejection.

A more detailed discussion of the basic immunologic mechanisms behind transplant rejection is beyond the scope of this review. For a detailed review of these processes, please see Coico R, Sunshine G. Immunology: A Short Course. 6th ed. Hoboken, NJ: Wiley-Blackwell; 2009.

The clinical manifestations of cardiac transplant rejection are variable and may or may not correlate with the severity of rejection. During an episode of rejection, the clinical presentation can range from asymptomatic to profound heart failure. ^[6] Arrhythmias and sudden death have also been reported following heart transplantation. ^{[7, 8]} Ischemia may be produced secondary to cardiac allograft vasculopathy (CAV); however, typical angina is usually absent, owing to the fact that the donor heart is not innervated by the host. ^[3]

The gross findings of transplant rejection are seen in one of two settings: the explant of a previously transplanted failing heart so that the recipient can receive another transplant or in the autopsy setting. The gross findings of these hearts are largely dependent on the type of underlying rejection.

In hyperacute antibody-mediated rejection (AMR), the heart is generally swollen, with a dusky appearance to the myocardium. ^[3] Acute cell-mediated rejection can produce a spectrum of findings ranging from a grossly normal appearance to ischemic changes similar to that seen in hyperacute rejection.

Perhaps the most common gross finding in transplanted hearts is that of cardiac allograft vasculopathy (CAV). This process manifests grossly as varying degrees of diffuse concentric luminal narrowing involving the entire length of the transplanted arterial vessels. This process, if severe enough, is often accompanied by chronic ischemic changes (ie, fibrosis) in the distribution of the involved vessels.

Regarding transplant endomyocardial biopsy specimens, the most important gross finding involves the assessment of specimen adequacy. Adequate sampling of the myocardium is essential to providing accurate information on the status of the transplanted heart to the clinical team. The International Society for Heart and Lung Transplantation working formulation (ISHLT 2004) of cardiac allograft pathology recommended an absolute minimum of three pieces (each of which contain >50% myocardium) of tissue be taken, primarily because of the focal and patchy distribution of acute cellular rejection. ^{[9, 10]} The current recommendation is evaluation of 4-6 pieces of transplant endomyocardial biopsy specimens when screening for rejection.

Endomyocardial biopsy is the gold standard for differentiating between types of immunologic injury-acute versus antibody-mediated rejection (AMR).

The histologic findings of acute cellular rejection, AMR, and cardiac allograft vasculopathy (CAV) are reviewed in this section. Note that contraction bands are very common artifactual findings in endomyocardial biopsy specimens and should not be used as the sole criteria for a diagnosis of ischemia or myocardial tissue injury.

Acute cell-mediated rejection

Acute cell-mediated rejection is typified by a lymphocytic infiltrate that begins in a perivenular distribution and then progresses into the cardiac interstitium. The degree to which this infiltrate is present in these spaces is the primary basis for the grading of cardiac transplant rejection (see Tumor Spread and Staging). The lymphocytic infiltrate seen in rejection is primarily composed of T lymphocytes. This feature of rejection is useful when differentiating rejection from the so-called Quilty lesion (see the following image), which will contain both B lymphocytes and T lymphocytes, with the latter typically being more prominent.

Heart Transplant Rejection Pathology. Quilty lesion. This photomicrograph shows a subendocardial Quilty lesion. The location, in concert with the interspersed capillaries and collagen among the lymphocytes, are classic features of a Quilty lesion (hematoxylin-eosin, 200x).

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Quilty lesions are an entity unique to cardiac allograft specimens and are found in 10-20% of transplant endomyocardial biopsies. ^[11] They are defined as dense inflammatory foci that are generally seen in the endocardium of transplanted hearts. They may be large and sometimes extend deep into the myocardium, making them difficult to distinguish from rejection.

Helpful distinguishing features of Quilty lesions are that they generally contain both B lymphocytes and T lymphocytes, they often have collagen between the lymphocytes, they often have capillaries near the middle of the infiltrate, and they can usually be found to connect up with the endocardium (see the image above). The etiology of this phenomenon is not known; however, some authors have suggested that it is related to posttransplant immunosuppression with cyclosporine A.

More severe rejection is associated with a more pronounced infiltrate (which can include neutrophils and eosinophils), myocyte injury, hemorrhage, and/or vasculitis (see the images below). Although "myocyte injury" has not been precisely defined, histologically, some features should suggest such injury to the pathologist. They include sarcoplasmic scalloping, myocyte disruption, hypereosinophilia, and nuclear pyknosis.

Heart Transplant Rejection Pathology. Severe acute cellular rejection. This photomicrograph exhibits diffuse myocardial inflammation with associated myocyte damage (International Society for Heart Lung Transplantation [ISHLT] 1990 grade 3B or ISHLT 2004 grade 3R) (hematoxylin-eosin, 200x).

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Heart Transplant Rejection Pathology. Severe acute cellular rejection. This photomicrograph exhibits diffuse myocardial inflammation with associated myocyte damage (International Society for Heart Lung Transplantation [ISHLT] 1990 grade 3B or ISHLT 2004 grade 3R). Scalloping of the sarcolemma is readily apparent (hematoxylin-eosin, 400x).

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Antibody-mediated rejection

Initially, AMR was defined as the presence of complement and immunoglobulins in myocardial capillaries, in the absence of a cellular infiltrate. Inflammatory reactions, especially intravascular macrophages, endothelial cell swelling, and tissue edema has been appreciated as features of AMR. ^[12]

The International Society for Heart and Lung Transplantation (ISHLT) revised the reporting structure for AMR in 2011. ^[13]  The pathologic diagnosis of AMR is now divided into those with histopathologic findings only (pAMR 1 (H+)), immunopathologic findings alone (pAMR 1 (I+)), both histopathologic and immunopathologic findings (pAMR 2), and severe findings of hemorrhage, capillary injury, and/or marked edema (pAMR 3).

Cardiac allograft vasculopathy

The luminal narrowing seen in cardiac graft vasculopathy is the result of concentric intimal hyperplasia within the epicardial coronary arteries and medial disease in the microvasculature. ^[14] CAV has also been shown to have an association with intravascular thrombosis and may be a form of healed vasculitis.

Immunoperoxidase studies for acute cellular rejection are useful in the identification of Quilty lesions and are critical in the diagnosis of antibody-mediated rejection (AMR). CD20 is helpful in identifying B lymphocytes in Quilty lesions that may be mistaken for cellular rejection, if their endocardial location is obscure. Cellular rejection is typically a T-lymphocyte-mediated response, whereas Quilty lesions often contain numerous B lymphocytes as well as T lymphocytes, with a few macrophages.

Immunochemistry for CD68 is helpful in diagnosing AMR, which is characterized by intraluminal macrophages that may be difficult to identify on routine stains.

Xu et al found that in the first year after a heart transplant, C4d(+) occurred early in children and young adults with pretransplant donor-specific antibody or with clinical suspicion of AMR. Additional research is needed to determine appropriate posttransplant review. ^[15]

In relatively recent years, gene expression profiling (GEP) has been mentioned in the literature as a noninvasive means of monitoring for cardiac transplant rejection. ^{[16, 17, 18]} This method involves obtaining a blood sample and, then, peripheral blood mononuclear cells are screened for gene expression patterns commonly seen in varying degrees of acute rejection. Despite the reported 84% sensitivity of this methodology, the specificity was 38%. ^[16] Head-to-head studies with endomyocardial biopsies have yet to be performed.

Other potential noninvasive methods for detecting heart transplant rejection include the use of biomarkers such as donor-derived cell-free DNA, soluble cardiac protein biomarkers, mircro-mRNAs, and circulating donor heart exosomes. ^{[18, 19]} Further investigation is needed.

The International Society for Heart Lung Transplantation (ISHLT) published the first consensus statement on grading rejection in transplant endomyocardial biopsy specimens in 1990. It described the various histologic patterns of inflammation in biopsy specimens and is summarized in the image below.

Heart Transplant Rejection Pathology. International Society for Heart Lung Transplantation (ISHLT) 1990 classification of acute cellular rejection in transplant endomyocardial biopsy specimens.

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The following were the histologic descriptors for various grades of acute cellular rejection:

• Grade 1A: Focal, mild acute rejection

• Grade 1B: Diffuse, mild acute rejection

• Grade 2: Focal, moderate acute rejection

• Grade 3A: Multifocal, moderate rejection

• Grade 3B: Diffuse, borderline severe acute rejection

• Grade 4: Severe acute rejection

Over the ensuing years, various issues with the 1990 classification scheme arose. Along with the advancement of immunosuppressive regimens and a drop in the incidence of acute cellular rejection, evidence grew that inconsistencies existed in the usage of the ISHLT 1990 grading scheme. ^{[9, 11]} In 2004, task forces were gathered by the ISHLT to address these matters, and a revised consensus statement was published in early 2005.

The ISHLT 2004 standardized cardiac biopsy grading for acute cellular rejection (see the summary in the first image below) modified the 1990 criteria in three major ways. First, ISHLT 1990 grades 1A, 1B, and 2 were merged into the revised ISHLT 2004 grade 1R (mild, acute cellular rejection). Second, ISHLT 1990 grade 3A was reclassified as ISHTL 2004 grade 2R (moderate, acute cellular rejection). Finally, ISHLT 1990 grades 3B and 4 were combined into ISHLT 2004 grade 3R (severe, acute cellular rejection).

Nearly 20 years after the adoption of the first standardized criteria, variability still exists in the way that rejection is reported. ^[18] Although the ISHLT 2004 criteria is the most updated, its widespread adoption has yet to have occurred, with many institutions still reporting rejection using the ISHLT 1990 criteria. Some institutions have made the transition to using the ISHLT 2004 criteria, whereas others use a hybrid system, reporting both. The reason for this variability is likely due to the comfort level and familiarity of both the reporting pathologist and treating clinician with the different grading schemes.

Antibody-mediated rejection

The ISHLT now recommends routine evaluation for antibody-mediated rejection (AMR), beginning at least 2 weeks after graft placement. Likewise, the reporting scheme has been made formal with a detailed and descriptive grading rubric. Evaluating for the presence of AMR both histopathologically as well as with the assistance of immunohistochemistry is recommended.

Histopathologic criteria for AMR includes, activated intravascular mononuclear cells within capillaries and venules, swelling of endothelial cells, and interstitial edema. Findings of hemorrhage, myocyte necrosis, capillary fragmentation, or intravascular thrombi may constitute a diagnosis of severe AMR. Immunopathologic surveillance for AMR is most commonly accomplished with antibodies directed against C4d and CD68. Strong reactivity of C4d with capillary endothelial cells in more than 50% of observed capillaries constitutes a positive C4d stain. See the image below.

Heart Transplant Rejection Pathology. International Society for Heart Lung Transplantation (ISHLT) 2013 grading of cardiac transplant biopsies for antibody-mediated rejection (AMR).

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The prognosis of patients undergoing cardiac transplantation has improved significantly over the last few decades. In 1987, 1-year survival following transplantation was 79.5%; in 2005, this statistic increased to 87.3%, likely owing to advancements in surgical technique and postoperative (immunosuppressive) care. ^[20]

Although the overall prognosis following heart transplantation has markedly improved, a number of factors have been identified as portending a worse prognosis. People older than 55 years seem to do worse than younger people, ^[21] as do patients with diabetes. ^[22] Underlying cardiac disease necessitating transplantation is also a factor that affects graft survival both in the long and short term.

The role of race in adjusted graft survival is complicated. Black individuals (92.3%) have a comparable graft survival rate to White persons (91.6%) at 3 months following transplantation. However, the graft survival in Black persons drops more drastically to 66.4% at 5 years posttransplantation, whereas graft survival in White individuals drops to 73.2%. ^[20] The reason for this difference is not known; however, it may be related to comorbid conditions or socioeconomic variables. ^{[23, 24]}

The prognosis of heart transplant recipients is driven by a number of factors including postoperative course and transplant rejection. Individuals who develop antibody-mediated rejection (AMR) tend to do poorly, and the severity of AMR seems to correlate with cardiovascular mortality; however, a severity scale has yet to be officially adopted by the International Society for Heart Lung Transplantation (ISHLT). ^{[25, 26]}