Cardiovascular 21 (2012) 245–274

Original Article 2011 Consensus statement on endomyocardial from the Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology ⁎ ⁎ Ornella Leone a, , John P. Veinot b, , Annalisa Angelini c, Ulrik T. Baandrup d, Cristina Basso c, Gerald Berry e, Patrick Bruneval f, Margaret Burke g, Jagdish Butany h, Fiorella Calabrese c, Giulia d'Amati i, William D. Edwards j, John T. Fallon k, Michael C. Fishbein l, Patrick J. Gallagher m, Marc K. Halushka n, Bruce McManus o, Angela Pucci p, E. René Rodriguez q, Jeffrey E. Saffitz r, Mary N. Sheppard g, Charles Steenbergen n, James R. Stone r, Carmela Tan q, Gaetano Thiene c, Allard C. van der Wal s, Gayle L. Winters r

aBologna, Italy bOttawa, Ontario, Canada cPadua, Italy dHjoerring, Denmark eStanford, CA, USA fParis, France gLondon, UK hToronto, Ontario, Canada iRome, Italy jRochester, MN, USA kNew York, NY, USA lLos Angeles, CA, USA mSouthampton, UK nBaltimore, MD, USA oVancouver, BC, Canada pPisa, Italy qCleveland, OH, USA rBoston, MA, USA sAmsterdam, The Netherlands Received 3 August 2011; received in revised form 28 September 2011; accepted 7 October 2011

Abstract

The Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology have produced this position paper concerning the current role of endomyocardial biopsy (EMB) for the diagnosis of cardiac and its contribution to patient management, focusing on pathological issues, with these aims:

• Determining appropriate EMB use in the context of current diagnostic strategies for cardiac diseases and providing recommendations for its rational utilization

⁎ Corresponding authors. O. Leone is to be contacted at U.O. di Anatomia ed Istologia Patologica, pad. 18, Azienda Ospedaliero-Universitaria S.Orsola-Malpighi, Via Massarenti, 9, 40138 Bologna. Tel.: +39 051 6364511; fax: +39 051 6364571. J.P. Veinot, Department of Pathology and Laboratory Medicine, The Ottawa Hospital, Ottawa, ON, Canada K1H 8L6. Tel.: +1 613 737 8291; fax: +1 613 737 8712. E-mail addresses: [email protected] (O. Leone), [email protected] (J.P. Veinot).

1054-8807/11/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2011.10.001 246 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274

• Providing standard criteria and guidance for appropriate tissue triage and pathological analysis • Promoting a team approach to EMB use, integrating the competences of pathologists, clinicians, and imagers. © 2012 Elsevier Inc. All rights reserved. Keywords: Biopsy; Consensus; ; Molecular biology; Myocardium

1. Introduction standardized diagnostic histopathologic criteria to minimize EMB reporting variability [2,46,47]. Endomyocardial biopsy (EMB) is a commonly performed As EMB is usually a nontargeted biopsy procedure and procedure for the evaluation of cardiac tissue for transplant false-negative results are possible, especially when used monitoring [1,2], [3–5], drug toxicity, cardio- for multifocal or microfocal or localized diseases [36,48,49], myopathy (CMP) [3,6–8], [9–14], and secondary some general rules can optimize diagnostic accuracy: cardiac involvement by systemic diseases [8], and for the diagnosis of cardiac masses [15–21]. Others have high- ■ EMB timing appropriate to specific [24,50,51], lighted EMB utility from prognostic–therapeutic [22–24] ■ adequate bioptic sampling with multiple specimens and pathogenetic view points [25]. from different sites (guided by imaging techniques Pathologists should assume responsibility for providing when indicated) [26,36,52,53]. input concerning the utility of the cardiac biopsy for specific diseases and provide guidance for tissue triage and standards 3. Techniques and risks of morphologic evaluation [26]. This implies considering both the recent developments in myocardial and viral EMB involves percutaneous insertion into the heart of a molecular biology and advances in imaging, electrophysio- fluoroscopically or ultrasound-directed sheath, which allows logy, and genetics, and understanding how these techniques safe and rapid insertion of the and facilitates can complement traditional . The invaluable obtaining multiple specimens. expertise of the pathologist in morphological tissue assess- Bioptic samples can be taken from the right , via ment may be essential in outlining appropriate diagnostic the venous route through jugular, subclavian, or femoral strategies in collaboration with clinicians, imagers, molec- veins, or from the left ventricle with transseptal puncture or ular biologists, and geneticists. by direct access through a peripheral artery, usually the Referral of the tissue to expert pathologists and molecular femoral or brachial artery [5,54,55]. For better anatomic biologists is warranted in many cases, given the complexity definition of the specimen site, noninvasive techniques of of the diagnostic tests. may usefully be associated, such as Moreover, pathologists should endeavor to use uniform gadolinium magnetic resonance imaging (MRI) in targeting reference criteria and language in their reports, including: bioptic specimens in suspected myocarditis [56] and electroanatomic mapping guided techniques in arrhythmo- • degrees of diagnostic certainty: for example, certain– genic right ventricular (ARVC) [10,11,57]. definite, probable, possible, nonspecific; allows accurate intracardiac insertion of • biopsy sample adequacy evaluation: for example, the bioptome without the use of ionizing radiation [58,59] optimal, suboptimal. and may be useful in biopsy of cardiac masses. The principal complications of EMB include hematoma, These guidelines should be viewed as recommendations arteriovenous fistula, vasovagal reaction, , and not as mandatory requirements. They represent an overall consensus of the committee members and the opinions expressed are not necessarily those of all members Table 1 of the Association for European Cardiovascular Pathology Technical procedures for light microscopy examination and/or the Society for Cardiovascular Pathology. Routine procedure Fixation in buffered formalin for 15–30 min Processing in automatic processor overnight Rapid procedure for Microwave fixation 2. Recommendations, limits, and precautions transplant and urgent Processing in microwave or vacuum-prepared diagnostic automatic processor (maximum 30–40 min) The modern view of diagnostic EMB requires the Cutting (routine Serial and numbered sections of paraffin- and rapid) embedded biopsies using at least two thirds of pathologist to have specific professional training [27], use the samples accurate specimen processing [28], and support when Half the cut sections are stained with warranted the traditional histological examination with hematoxylin–eosin; the others are mounted on histochemical, immunohistochemical (IHC), molecular, or sialinized slides (or on slides with polylysine or ultrastructural tests (Tables 1 and 2) [24,29–45] and apply electrostatic charge) for possible further stains. O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 247

Table 2 Stains on paraffin-embedded and frozen tissue Stains on formalin-fixed and paraffin-embedded sections Histomorphological and ▪ Masson or Mallory trichrome, Movat pentachrome, Weigert–Van Gieson for collagen and elastic fibers histochemical stains ▪ PAS with and without diastase for glycogen storage (better on frozen sections) ▪ Congo red, sulfate alcian blue, or S/T thioflavin for amyloid ▪ Perls' iron IHC stains ▪ CD45, CD20, CD3, CD4, CD8, CD68/PGM1, HLA-DR. HLA-ABC for inflammatory and cellular infiltrate assessment ▪ Transthyretin, kappa and lambda chains, apolipoprotein, amyloid A component for amyloid typing ▪ Appropriate antibodies for characterization ▪ Dystrophin, lamin A/C a, desmin, a plakoglobin, a N-cadherin a for some genetic CMPs

Stains on frozen sections Histomorphological and Sudan black, oil red 0, PAS with and without diastase histochemical stains Histoenzymatic stains SDH and COX stains Many immunostains listed above, including amyloid subtypes and desmosomal proteins, may be performed on frozen tissue. Frozen tissue may be better than for dystrophin (−COOH and NH2 ends, intermediate domain) and HLA-ABC. PAS = periodic acid-Schiff; SHD = succiante dehydrogenase; COX = cytochrome oxidase. a In the course of validation for diagnostic use. arrhythmia, , , damage, ■ brady- or major ventricular tachyarrhythmias to iden- or systemic embolism during left tify or exclude possible myocardial causes of arrhyth- ventricular biopsy, and cardiac chamber perforation with mias (e.g., myocarditis, ARVC, sarcoidosis, etc.), possible and [60,61]. ■ chronic to evaluate myocardial diseases In skilled hands, EMB is a safe procedure [55]. The with dilated–hypokinetic or hypertrophic–restrictive overall complication rate is low (1%–2%) [5]. In particular, phenotype, the risk of cardiac perforation was estimated at 0.4% in the ■ acute exacerbation of chronic CMP, largest case records in the world published in 1980 [62] and ■ investigation of cardiac masses, and at 0.12% in more recent data regarding a large number of ■ assessment of cardiac transplant biopsy specimens. patients (3048 cases) [63]. 5. Pathological section 4. Patient selection and clinical indications 5.1. General sampling and handling Considering the cost–benefit ratio and possible proce- dural risks, careful patient selection is appropriate [24], using Pathological analysis of EMB is complex and requires these criteria: standard protocols [26,75]. Therefore, it is vital that the pathologist be provided with clinical information as an 1. Clinical setting: EMB should be considered in the aid for diagnosis and to guide the most appropriate context of a sequential diagnostic process, after other technical choices. appropriate basic clinical–instrumental tests have excluded various diseases and focused on the possible 5.1.1. Optimal specimen procurement and triage diagnoses [26,64]; ■ At least three, preferably four, endomyocardial 2. EMB effects on patient clinical management fragments, each 1–2mm[3] in size, should [26,38,65–74]: immediately be fixed in 10% buffered formalin at ■ obtain a definite diagnosis (or exclude diseases) for room temperature for light microscopic examination. therapy, clinical management, and prognosis; In expected focal myocardial lesions, additional ■ monitor the disease clinical course and therapeutic sampling is recommended. efficacy; ■ If indicated, one or two specimens may be snap ■ reduce misdiagnosis and improve risk management. frozen in liquid nitrogen and stored at −80°C for 3. Different diagnostic potential of EMB in various possible molecular tests or specific stains. Otherwise, cardiac diseases [26] (see part on “specific diseases” fragments may be stored in RNA-later (a solution and summary Table 3). preventing RNA degradation) at room temperature. The main clinical scenarios for use of EMB are ■ If indicated, one fragment should be fixed in 2.5% [3,6,8,24,26,73]: glutaraldehyde or Karnovsky solution for ultra- ■ new-onset b6 months unexplained heart failure, structural tests. 248 Table 3 Endomyocardial biopsy diagnostic potential in cardiac diseases with grading of recommendation Clinically suspected EMB diagnostic potential and histological notes Technical aspects and tissue triage Grading of recommendation disease Myocarditis/inflammatory Definite diagnosis: lymphocytic, granulocytic, polymorphous, Recommended: In addition to formalin fixation, two fragments S cardiomyopathy eosinophil, necrotizing eosinophilic, , granulomatous (or one if greater than 3 mm2) could be snap-frozen or myocarditis, with or without associated myocyte damage/ preserved in RNA-later for virus molecular investigation. . One peripheral blood sample (5–10 ml) collected in EDTA or Histological findings alone or together with molecular sodium citrate tubes. techniques may be able to specify the etiology of the NB: Timing and number of EMB and serial histological inflammatory disease. sections are important for sensitive detection of myocarditis. Cardiac sarcoidosis Definite diagnosis: noncaseous granulomatous myocarditis. Possible increased utility with guided biopsy procedure. S Histological findings pathognomonic or highly suggestive of the disease. 245 (2012) 21 Pathology Cardiovascular / al. et Leone O. Myocardial diseases Probable/possible diagnosis: hypersensitivity myocarditis Glutaraldehyde or Karnowsky solution fixed fragment for S: hypersensitivity myocarditis due to drugs and (histological findings suggestive of the disease), toxic damage. electron microscopy may be useful for cardiotoxicity M: cardiotoxicity chemical/“toxic” evaluation of some drugs. substances Peripartum Probable diagnosis: myocarditis/borderline myocarditis. Fresh sample may be useful for virus molecular biology. M cardiomyopathy Histological findings give some indications of probable disease. Important: EMB timing Cardiac Definite diagnosis: amyloid infiltration. Recommended: Congo red, modified sulfated alcian blue, or S Histological findings+histomorphological stains are thioflavin T stains. pathognomonic. IHC, IF, immunoelectron microscopy, protein sequencing, and/or to establish the type of amyloid. Frozen tissue is required for IF and protein sequencing Iron overload Definite diagnosis: iron intracellular deposition. Iron staining is advisable for all diagnostic EMBs from patients S Histological findings+iron staining are pathognomonic. with unexplained DCM. Glycogen storage diseases: Probable diagnosis: diffuse intracellular glycogen storage. Recommended: Fresh sample for histochemical stains and S Type II glycogenosis Histological findings may be suggestive, but not specific, for glutaraldehyde or Karnowsky solution fixed fragment for (Pompe disease) the diagnosis. electron microscopy are useful to assess the nature of – Type III glycogenosis intracellular deposits. 274 (Cori disease) Type V glycogenosis (Andersen disease) Danon disease PRKAG2 mutations Anderson–Fabry disease Probable diagnosis: histology: hypertrophic vacuolated cells with Recommended: Glutaraldehyde or Karnowsky solution fixed S dislocation of contractile elements to the periphery of myocytes. fragment may be useful for electron microscopy to assess Histological findings may be suggestive, but not specific for electron dense concentric lamellar bodies. diagnosis. Desmin cardiomyopathy Definite diagnosis based on ultrastructural findings: abnormal Recommended: Glutaraldehyde or Karnowsky solution fixed S granulofilamentous aggregates of desmin-type intermediate fragment for electron microscopy necessary. filaments in the cytoplasm of cardiomyocytes (interfibrillar Immunoelectron microscopy confirms that these aggregates are areas) and at the Z band level. formed by desmin. Nonspecific light histological findings. Dystrophin Definite diagnosis in Duchenne muscular dystrophy: absence Recommended: dystrophin IHC or IF stain in which dystrophin M cardiomyopathy of dystrophin in myocyte sarcolemma. is markedly diminished or absent in affected individuals. Probable diagnosis in Becker muscular dystrophy: extensive Fresh sample is required for IF and may be required for IHC. irregularities and discontinuities of dystrophin in myocyte sarcolemma. Nonspecific histological findings. Laminopathies Nonspecific diagnosis: interstitial/replacement fibrosis, Data on diagnostic use of IHC staining for lamin A/C in human N (lamin A/C) myocyte and vacuolization, enlarged and EMB are not available. irregular-shaped nuclei. In centers performing IF, fresh samples are required. Nonspecific histological findings. Mitichondrial Probable/possible diagnosis: morphologically altered Recommended: Fresh sample is needed for histoenzymatic M (especially in isolated MIC) mitochondria by electron microscopy stainings. Nonspecific histological findings: enlarged myocytes with Glutaraldehyde or Karnowsky solution fixed fragment is

extensive cytoplasmic vacuolization needed for ultrastructural tests. 245 (2012) 21 Pathology Cardiovascular / al. et Leone O. Arrhythmogenic right Probable diagnosis: fibrous or fibrofatty replacement and IF or IHC for plakoglobin and other cellular junction proteins is S (in selected cases having no clear-cut ventricle myocardial . promising test (being validated). diagnosis with noninvasive and other cardiomyopathy Nonspecific histological findings. EMB from the may not be informative. invasive procedures) Diagnostic accuracy increases if EMB sampling site is guided by imaging (MRI) or electrophysiological (electroanatomic mapping) techniques. Loeffler fibroplastic Definite diagnosis in the acute phase: endomyocardial EMB timing is important as the utility of biopsy probably S in the acute–subacute phase / infiltrates rich in degranulated eosinophils and endocardial decreases with disease time course. endomyocardial fibrosis thrombosis. Specific histological findings. Possible diagnosis in the chronic phase: evidence of endocardial fibrous thickening and subendocardial myocyte abnormalities. Nonspecific histological findings. Cardiac tumors Definite diagnosis IHC useful for tumor typing S Hypertrophic Possible diagnosis: myocyte hypertrophy, interstitial and/or M: The clinical diagnosis of HCM usually relies on cariomyopathy substitutive fibrosis, disarray, and possible small vessel disease. noninvasive diagnostic tools, and EMB is not (sarcomeric mutations) Nonspecific histological findings. recommended in the diagnostic workup. However, in selected cases, EMB may be helpful in excluding – infiltrative or storage diseases with variable 274 diagnostic degrees of certainty. Idiopathic restrictive Possible diagnosis: normal myocardium, and/or fibrosis and/or M:EMBisunabletogiveaspecificdiagnosis. cardiomyopathy disarray. However, in selected cases, EMB may be No other identifiable diseases causing restrictive phenotype. helpful in excluding infiltrative or storage Nonspecific histological findings. diseases. Useful in the workup of constriction versus restriction. Idiopathic dilated Possible diagnosis: myocyte hypertrophy, nuclear alterations, M: EMB is unable to give a diagnostic picture, cardiomyopathy perinuclear halo, with or without fibrosis. but may be helpful in excluding other diseases. Nonspecific histological findings. Definite diagnosis: acute cellular rejection, some infectious Recommended: C4d IHC/IF staining for AMR. S disease, PTLD, and nonrejection posttransplant findings. Fresh sample is required for IF staining. Specific histological findings.

DCM, ; EDTA, ethylenediaminetetraacetic acid; EMB, endomyocardial biopsy; IF, immunofluorescence; IHC, immunohistochemistry; MRI, magnetic resonance imaging; M, mixed; N, not 249 supported; PTLD, posttransplant lymphoproliferative disorders; S, supported. 250 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274

A sample of peripheral blood (5–10 ml) in EDTA or 7. EMB in specific diseases citrate from patients with suspected myocarditis allows for molecular testing for the same viral genomes sought in the 7.1. Myocarditis/inflammatory CMP myocardial tissue, and a sample from patients with genetic CMPs allows for genetic tests. 7.1.1. Evidence of recommendation: S Myocarditis is an inflammatory disease of the myocar- 5.1.2. Pathological analysis dium associated with cardiac dysfunction. Three forms of • Light microscopic examination is routinely performed inflammatory CMP are recognized: infectious, autoimmune, on formalin-fixed and paraffin-embedded tissue and idiopathic. Due to the variable clinical manifestation (Table 1) and includes, when appropriate: from latent to very severe clinical forms, such as acute – • multiple and numbered hematoxylin eosin section congestive heart failure, life-threatening , and examination; sudden death, the prevalence of myocarditis is still • additional histochemical, histomorphologic, and unknown and probably underestimated. In spite of the IHC stains performed in the majority of cases on development of various diagnostic modalities, early and paraffin-embedded fragments or, in limited cases, on definite diagnosis of myocarditis still depends on the frozen sections; detection of inflammatory infiltrates in EMB specimens • morphometric analysis on sections cut for histo- according to the Dallas criteria (Table 4) [46,76–79]. Fig. 1 chemical, histoenzymatic, and IHC tests. shows two cases of viral lymphocytic myocarditis (in Fig. • Molecular tests, such as polymerase chain reaction (PCR), 1A–C, myocarditis is caused by enterovirus; in Fig. 1D–F, quantitative or qualitative, and reverse transcriptase-PCR it is caused by adenovirus). on the fragments frozen in liquid nitrogen or stored in Giant cell myocarditis may present as sudden or RNA-later. chronic heart failure. When it presents as an acute disease, • Transmission electron microscopy examination on EMB may detect it. This is important as the disease is fragments fixed in glutaraldehyde or Karnowsky sensitive to therapy with immunosuppressants [80]. Fig. 2 solution and embedded in resin (Epon). Ultrastructural demonstrates the typical histological findings of giant analysis is preceded by examination of semithin sections cell myocarditis. stained with toluidine blue. In addition, in immune inflammatory diseases, cardiac involvement may occur in the course of systemic connective Table 2 details the stains which may be performed, tissue diseases such as systemic lupus erythematosus (SLE), according to histological picture and clinical information. rheumatoid arthritis, ankylosing spondylitis, dermatopoly- myositis, and scleroderma. Cardiac involvement is more 6. Grading/evidence of recommendation frequent in SLE and in dermatopolymyositis, and includes a

Existing recommendations face limitations since, at present, they are not supported by controlled and randomized clinical studies [26,64].Onlycontrol–case record series; Table 4 registries; single-center case records, often limited in number; EMB diagnosis of myocarditis: the Dallas criteria [77] and expert opinions are available, and moreover, the majority Definition of idiopathic myocarditis: “an inflammatory infiltrate of the of publications are pathological diagnosis focused. myocardium with necrosis and/or degeneration of adjacent myocytes not ” A cardiologist should not perform EMB “in the dark,” but typical of the ischemic damage associated with only after consideration of the clinical presentation suggests Classification a possible diagnosis and restricts the field of possible First biopsy • Myocarditis with/without fibrosis diseases. We strongly suggest using the following recom- • Borderline myocarditis (repeat biopsy may be indicated) mendation grading, based on published peer-reviewed • No myocarditis evidence on various expected diseases. Subsequent biopsy • ■ S: published peer-reviewed evidence SUPPORTS Ongoing (persistent) myocarditis with or without fibrosis • Resolving (healing) myocarditis with or without fibrosis the utility of the test. • Resolved (healed) myocarditis with or without fibrosis ■ M: published peer-reviewed evidence is MIXED concerning the utility of the test. Descriptors ■ N: there is NO published peer-reviewed evidence Inflammatory infiltrate Fibrosis assessing the utility of the test. Distribution Focal, confluent, diffuse Endocardial, interstitial Extent Mild, moderate, severe Mild, moderate, severe Table 3 summarizes EMB diagnostic potential and Type Lymphocytic, eosinophilic, Perivascular, replacement grading of recommendation in cardiac diseases, detailing granulomatous, giant cell, the histological picture and special technical notes. neutrophilic, mixed O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 251

Fig. 1. (A–C) Viral active lymphocytic myocarditis in 35-year-old male. (A–B) EMB showing a multifocal moderate lymphomonocytic inflammatory infiltrate (A, hematoxylin–eosin, 25×); at higher magnification, myocyte damage (arrow) associated with inflammatory infiltrate is evident (B, hematoxylin–eosin, 200×). (C) PCR gel electrophoresis for enterovirus. Line 1: DNA marker (VIII factor); line 2: positive control for enterovirus (180 bp); line 3: EMB positive for enterovirus; line 4: negative control (reagents without DNA). (D–F) Viral active lymphocytic myocarditis in 5-year-old female. (D–E) EMB showing a diffuse lymphomonocytic infiltrate (D, hematoxylin–eosin, 100×) associated with myocyte damage (arrows) (E, hematoxylin–eosin, 200×). No viral cytopathic effects are present. (F) PCR gel electrophoresis. Line 1: DNA marker (VIII factor); line 2: adenovirus positive control (308 bp); line 3: EMB positive for adenovirus; line 4: beta-globin amplification (housekeeping gene, 269 bp); line 5: negative control (reagents without DNA). range of clinical expressions, from conduction diseases to bowel disease. Such examples further emphasize the progressive cardiac dysfunction with the clinical features relevance of receiving updated clinical information. Cardiac of dilated cardiomyopathy (DCM). The EMB may be helpful histopathology in these immune diseases can be polymor- in distinguishing between SLE-related myocarditis and phous depending on the type or the evolutive phase of the cardiotoxic effects of chloroquine, a drug for treatment of disease; however, EMB frequently reveals a myocardial the disease. inflammatory picture and/or vasculitis indicating heart Furthermore, the pathologic involvement of the heart can involvement [82,83]. occur in many other immunologic systemic diseases Myocarditis is found in about 10% of cases of clinical including Churg–Strauss disease, Wegener granulomatosis, DCM, so may play a role in the of the DCM sarcoidosis, Takayasu arteritis, and idiopathic inflammatory group [84,85]. Myocarditis may evolve into DCM as the

Fig. 2. (A) Giant cell myocarditis in 43-year-old female. Histology: EMB shows a severe polymorphous inflammatory infiltrate (lymphocytes, macro- phages, eosinophil granulocytes, multinucleated giant cells) associated with diffuse myocyte damage (necrosis and degeneration) (hematoxylin–eosin, original magnification 100×). (B) Detail of picture (A) showing multinucleated giant cells at higher magnification (arrows) (hematoxylin–eosin, 400×). From Leone et al., modified [81]. 252 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 initial viral infection may lead to myocyte destruction, principal antibodies used for immunophenotype characteri- followed by a chronic immunological attack of the zation are CD45, CD45RO, CD3, CD20, CD4, CD8, and myocytes with autoantibodies and involvement of CD68/PGM1. Additional stains relevant to immune activa- the cellular immune system. tion include HLA-ABC and HLA-DR.

7.1.2. EMB diagnostic potential 7.1.4.1. Molecular analysis for viral detection. The EMB is a key diagnostic tool [86,87]. Imaging tech- introduction of new molecular techniques, particularly niques (echocardiography, nuclear imaging, and magnetic amplification methods like PCR or nested-PCR, allows the resonance) can be useful for determining the best place to detection of low-copy viral genomes from even an biopsy, but these cannot replace EMB for a definitive diagnosis extremely small amount of tissue such as in an EMB – of myocarditis. specimen [88 91]. EMB can provide: Molecular techniques may be helpful as an ancillary diagnostic tool only with other mandatory investigations ■ Definitive diagnosis of inflammatory myocarditis (clinical and morphological) and should be performed by ■ Identification of the likely etiology (both infective those with this specific expertise. and immunological) Molecular testing may be done in a special laboratory ■ Monitoring of inflammatory CMP separate from the histology laboratory. Peripheral blood ■ Confirmation of cardiac involvement (myocarditis or analysis should accompany the molecular tissue analysis vasculitis) in the context of systemic immune so that the results may be interpreted in the correct con- diseases text. Knowledge of the viral findings of the patient's blood and of the incidence of the virus in the patient's community To assess immune etiology, cardiac autoantibodies in the is an important datum to be aware of before interpretation of blood should also be investigated. the myocardial specimen molecular results. Appropriate At histology, routine (hematoxylin and eosin) stained quality assurance and control tissues for molecular biology serial sections should be evaluated for the presence of: are important. The pathologist's role is to gather the available histo- ■ the inflammatory infiltrate: immunotype and semi- logical and molecular data and provide an interpretation. quantitative or quantitative analysis The principal viruses to be considered are adenovirus, – ■ myocyte damage type: myocytolysis, , or cytomegalovirus, enterovirus, Epstein Barr virus, hepa- other myocyte alteration titis C virus, herpes simplex virus 1 and 2, human herpes ■ fibrosis: type and semiquantitative or morphometric virus 6, influenza viruses A and B, parvovirus B19, and analysis; special stains may be helpful rhinovirus [35,36]. The Dallas criteria have proven useful in the histolo- Immunotyping and quantitation of the and gical assessment of myocarditis, but some investigators fibrosis are controversial, with strong opinions regarding the feel that these criteria have some limitations including utility of these. In view of this, both Pathologist Groups lack of definition and quantification of myocyte damage, support quantification, as a research tool, to obtain data to lack of quantification of the inflammatory infiltrate, lack assess its utility. of characterization (site/topography) and quantification of fibrosis, and lack of an etiological characterization [76]. These criticisms are variably accepted. The diagnosis of 7.1.3. Specific technical aspects myocarditis by EMB is thus in evolution, as every medical If viral genome detection is being pursued, it is test and procedure go through. Data are limited and recommended to have: conflicting, so the acquisition of reliable data is essential to determine the optimal means for diagnosis. ■ 2 two fragments (or one if greater than 3 mm ), snap- From this standpoint, we suggest that pathological frozen or preserved in RNA-later, for molecular diagnosis of myocarditis may report histological find- investigation ings including the cell type, immunophenotypic charac- ■ – one peripheral blood sample (5 10 ml) collected in terization of the inflammatory infiltrate, and molecular EDTA or sodium citrate tubes viral data, if these are available. An EMB registry might be useful. NB: Timing and number of EMB specimens are If EMB for suspected myocarditis were to be routinely important for sensitive detection of myocarditis. performed following a standardized protocol, important and extremely helpful information could well be obtained on 7.1.4. IHC and molecular aspects this complex and varied disease, including epidemiology Immunohistochemistry is a useful corollary to identify as well as etiology and prevalence, since this latter is very and characterize inflammatory infiltrates in EMB. The likely underestimated. O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 253

7.2. Idiopathic/primary DCM 7.3. Dystrophin CMP

7.2.1. Evidence of recommendation: M 7.3.1. Evidence of recommendation: M DCM is a disease of the myocardium characterized by Mutations in the dystrophin gene can result in DCM. The dilatation and impaired systolic contraction of one or both phenotype may be exclusively progressive cardiac failure (X- ventricles [92]. DCM is a common CMP and may affect linked DCM), or more commonly, it also involves skeletal patients of any age. The wall thickness may be reduced muscle, causing concurrent Duchenne or Becker muscular due to stretching of the myocytes as the ventricle dilates. dystrophies. Becker-type dystrophy can be associated with There may be atrial or ventricular thrombus in all mild functional skeletal involvement and more profound chambers and on both sides. DCM may be complicated myocardial involvement. Dystrophin gene, found on the X by biventricular congestive heart failure; arrhythmias, both chromosome (Xp21), is maternally inherited, and the supraventricular and ventricular; thromboemboli; and phenotype is only seen in males. Dystrophin mutations have sudden death. been found in up to 7% of males with DCM, representing a To reach a diagnosis of idiopathic/primary DCM, minor but not insignificant cause of “idiopathic” DCM [41]. clinical information is very helpful, including knowledge Diagnosing dystrophin-induced DCM is complex. An of other illnesses and systemic diseases, and exclusion of elevated serum creatinine phosphokinase has been reported significant , coronary artery disease, in N80% of affected men, suggesting that this serum test may and systemic arterial hypertension. Although the histolog- be useful as an initial screening test. ical findings are nonspecific, EMB diagnosis may be valuable in the workup of heart failure (active myocarditis 7.3.2. EMB diagnostic potential and IHC aspects versus acute exacerbation of a chronic disease) or Routine light microscopy may identify nonspecific arrhythmias to exclude specific diseases. findings including interstitial/replacement fibrosis, fibrofatty replacement, myocyte hypertrophy, vacuolization, and 7.2.2. EMB diagnostic potential and IHC aspects myocytolysis (Fig. 3A). The most useful analysis is a Nonspecific histology includes fibrosis, myocyte nuclear dystrophin IHC stain in which dystrophin is markedly enlargement, and often sarcoplasmic degenerative changes diminished or absent in affected individuals (Fig. 3B). such as a perinuclear halo, and myocyte clearing or However, this is best done in skeletal muscle biopsies along vacuolization. Myofiber attenuation and stretching may be with IHC for spectrin, a positive control for membrane marked. Nuclei may be irregular in shape and hyper- staining. By ultrastructural examination, membrane irregu- chromatic. Fibrosis, most commonly pericellular, endocar- larities including discontinuation of the sarcolemma have dial and substitutive, may also be perivascular in location. been described, although this is not a specific finding. Immunohistochemistry for muscle fiber abnormality Ultimately, genetic mutation screening is necessary to make may be attempted in CMPs associated with skeletal myo- a definitive diagnosis and should be considered the primary pathy. However, skeletal muscle biopsy may be easier in diagnostic tool. such patients. 7.3.3. Genetic aspects 7.2.3. Genetic aspects We suggest genetic testing as a first-line diagnostic tool Familial DCM represents approximately 30% of cases when dystrophin CMP is strongly suspected based on of DCM [93–95]. Nonfamilial and familial forms of clinical findings and mode of inheritance. However, in cases DCM cannot be distinguished by routine histology or of “idiopathic” DCM, dystrophin IHC and serum creatinine imaging [96]. phosphokinase can be used to entertain this diagnosis and

Fig. 3. A 36-year-old male patient with Becker muscular dystrophy. (A) Histology: marked interstitial fibrosis, myocyte sarcoplasmic vacuolization, hypertrophy, and focal atrophy (Mallory trichrome, 200×). (B) Immunohistochemistry shows loss or discontinuity of sarcolemmal stain for dystrophin molecule (200×). 254 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 focus genetic testing. In Becker disease with solely or predominantly cardiac involvement and in X-linked CMP, EMB may be the only diagnostic tool able to raise the question of a dystrophin-gene-related disease.

7.4. Mitochondrial cardiomyopathies (MICs)

7.4.1. Evidence of recommendation: M (especially in isolated forms) MICs can occur both in the context of multiple organ syndromes and as the sole or predominant feature of respiratory chain dysfunction. MICs may occur at any age, and their frequency in patients with mitochondrial disease is high. Because of the dual genetic control of respiratory chain, MICs can be caused by mutations in either the mitochondrial or the nuclear genome. Thus, MICs can be maternally inherited, autosomal dominant or recessive, X- linked, or sporadic. The phenotype is usually hypertrophic, nonobstructive type. Biventricular dilation and congestive heart failure also can occur in the natural history of the disease [97,98].

7.4.1.1. EMB diagnostic potential. When MICs are associated with multisystemic disease, the diagnosis of mitochondrial etiology relies upon skeletal muscle biopsy, biochemical analysis, and genetic tests. However, in isolated MICs, when the skeletal muscle biopsy is unremarkable, EMB can be useful. At histology, myocytes are enlarged, with extensive cytoplasmic vacuolization (Fig. 4A). His- toenzymatic stains on frozen sections for succinate dehy- drogenase (SDH) and cytochrome oxidase (COX) are required for the diagnosis. The presence of COX-negative myocytes and an increased staining for SDH demonstrate the respiratory chain defect and mitochondrial prolifera- tion (Fig. 4B). At ultrastructure, there is diffuse mito- chondrial proliferation (Fig. 4C), often with giant mitochondria, mitochondrial inclusions, abundant cytoplas- mic glycogen, lipid droplets, and contractile myofibril loss.

7.4.2. Molecular aspects and genetics Fig. 4. Maternally inherited MIC due to a point mutation in the gene In isolated MICs, we recommend storing frozen myocar- encoding for isoleucine tRNA. (A) Histology: extensive myocyte vacuoliza- tion (hematoxylin–eosin, 40×). (B) Combined histoenzymatic stain for dial tissue to check for mutations, deletions, or depletion of nuclear-encoded SDH (in blue) and mitochondrial-encoded COX (brown). mitochondrial DNA in homogenate tissue, which may pass Several myocytes are stained in blue, indicating proliferation of mitochon- unnoticed when using DNA extracted from peripheral blood. dria lacking COX activity (100×). (C) Ultrastructural view of interfibrillar Mutations of most genes coding for structural and functional mitochondria proliferation (5000×). components of mitochondria can be appropriately assessed using peripheral blood.

7.5. Lamin A/C-related CMP lamins B1 and B2, respectively. Mutations in the LMNA 7.5.1. Evidence of recommendation: N gene produce clinical disease with several distinct syn- Lamins are intermediate filaments that play a structural dromes such as Charcot–Marie–Tooth disease type 2, role in the formation of the nuclear lamina that lines the inner mandibuloacral , familial partial lipodystrophy- core of the nuclear envelope of cells. The proteins lamin A Dunnigan type, restrictive dermopathy, and premature aging and lamin C are the products of alternative splicing of the syndromes (Hutchinson–Gilford progeria and atypical gene LMNA. The genes LMNB1 and LMNB2 encode Werner syndrome) as well as CMP. O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 255

Fig. 5. A 32-year-old female patient with lamin AC deficiency DCM. (A–B) EMB showing interstitial/replacement fibrosis, myocyte hypertrophy and vacuolization, and enlarged and irregular-shaped nuclei. (A: Mallory trichrome 250×; B: hematoxylin–eosin 400×). (C) Immunohistochemical staining for lamin A/C shows discontinuity or loss in perinuclear staining (400×). (D) Normal IHC control with regular and strong perinuclear staining (400×).

Cardiovascular manifestations in patients with mutations 7.6. Arrhythmogenic right ventricle cardiomyopathy in lamin A/C include conduction system abnormalities early in the disease with pacemaker requirement in up to 44% of 7.6.1. Evidence of recommendation: S (in selected cases the patients over 30 years of age. These patients may show having no clear-cut diagnosis with noninvasive and other evidence of delayed intracardiac conduction. Their electro- invasive procedures) cardiograms show low-amplitude P waves and prolonged PR ARVC is a rare primary heart muscle disease with a interval with normal QRS complex. Atrial 1:2000–1:5000 prevalence in the general population. appears later with thromboembolic complications. Prognosis Genetic defects have been identified in genes mostly is poor, with congestive heart failure as the most common encoding for desmosomal proteins. The clinical presentation clinical evolution [99]. includes ventricular arrhythmias [103]; sudden death, particularly in the young and athletes; and also, less 7.5.2. EMB diagnostic potential commonly, mechanical dysfunction. With the latter presen- Pathologic examination shows nonspecific features of tation, such systolic dysfunction may mimic DCM [104]. DCM: interstitial/replacement fibrosis, myocyte hypertrophy From the pathology viewpoint, ARVC is characterized by and vacuolization, and enlarged irregularly shaped nuclei progressive atrophy of the ventricular myocardium with (Fig. 5A, B) [100,101]. fibrofatty replacement. The septum is rarely involved. The clinical diagnosis is a major challenge since there is no single 7.5.3. IHC and molecular aspects gold standard and it requires fulfilment of major and minor Data on diagnostic use of IHC staining for lamin A/C in diagnostic criteria (scoring system) [9]. human EMBs are not available, although promising research studies are ongoing (Fig. 5C). Ultrastructural 7.6.2. EMB diagnostic potential nuclear changes have been reported including large nuclear If noninvasive and other invasive procedures do not allow blebs (not folds) [102]. These reports consist mostly of a clear-cut diagnosis of ARVC, right ventricle EMB may nonspecific features of hypertrophy, nuclear contour serve as a complementary diagnostic tool (tissue character- irregularity, and degeneration of the cardiac myocytes. At ization diagnostic criterion). Moreover, in sporadic forms this juncture, the diagnosis should be established by (not familial), EMB can be useful to exclude phenocopies genetic testing. (i.e., myocarditis, sarcoid, etc.). 256 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274

Fig. 6. EMB findings in a proband affected by a diffuse form of ARVC. The three biopsy samples are from different regions of the right ventricle free wall.In each, there is extensive fibrofatty tissue replacement with myocardial atrophy, which is a major criterion (i.e., residual myocytes b60% by morphometric analysis or b50% if estimated) (Mallory trichrome, 60×). From Marcus et al., modified [105].

Histological examination (on hematoxylin–eosin- and The clinical diagnosis is based upon instrumental connective-tissue-stained slides) shows fibrous or fibrofatty demonstration of left (electrocardio- replacement and myocardial atrophy (Fig. 6), which are not gram and echo) and requires exclusion of other causes of specific (see, for example, muscular dystrophies). Fatty ventricular hypertrophy. HCM is most commonly due to a tissue alone is not considered diagnostic for ARVC since fat mutation in one of the genes encoding sarcomeric proteins. is normally found in the myocardium, particularly at the From the pathology viewpoint, HCM is characterized by anterolateral apical right ventricular free wall. Myocyte increased wall thickness/mass with variable distribution, hypertrophy and vacuolization as well as inflammatory most commonly with asymmetric hypertrophy, involving the infiltrates can be present. basal interventricular septum. Histological features include Histomorphometry evaluation of right ventricle EMB is myocyte hypertrophy, interstitial and/or replacement fibro- mainly supported as a research tool to gather data. However, sis, myocardial disarray, and thickening of the walls of the according to the updated diagnostic criteria, fibrous or small mural vessels. fibrofatty replacement with b60% residual myocardium in at least one EMB sample is a major criterion, and 60%–75% residual myocardium is a minor criterion for ARVC [12,105]. 7.7.2. EMB diagnostic potential The clinical diagnosis of HCM usually relies on noninvasive diagnostic tools, and EMB is not recommended 7.6.3. Specific technical aspects in the diagnostic workup. However, in selected cases, EMB EMB samples from the septum may not be that helpful. If may be helpful in excluding infiltrative or storage diseases possible, consideration to biopsying the affected area, (such as Fabry and PRKAG disease) with variable diagnostic including the right ventricle free wall, may be considered. degrees of certainty [109]. Diagnostic accuracy increases if the EMB sampling site is guided by imaging (MRI) or electrophysiologic (electro- anatomic mapping) techniques. 7.8. Idiopathic/primary restrictive cardiomyopathy (RC)

7.6.4. Notes on IHC and molecular aspects 7.8.1. Evidence of recommendation: M Immunofluorescence for plakoglobin and other cellular RC is a primary heart muscle disease characterized by junction proteins is promising as it may decrease the need impaired filling of the ventricles in the setting of normal for free wall biopsy since it works on morphologically wall thickness and systolic function. Formerly believed to preserved myocardium and thus on septal bioptic samples. be of idiopathic nature, once inflammatory, infiltrative, or Attention to specific antibody dilutions and technique is systemic diseases were excluded, it is now known to be important. However, this test needs validation before associated with mutations in sarcomeric contractile protein entering routine practice and cannot at present replace genes, like HCM. Patients may develop severe symptoms morphologic diagnosis [106]. of heart failure over a short period of time, even requiring heart transplantation. The clinical diagnosis is based on 7.7. Sarcomeric hypertrophic cardiomyopathy (HCM) clinicoinstrumental demonstration of restriction and re- quires exclusion of other causes of constriction and res- 7.7.1. Evidence of recommendation: M triction. EMB may be of utility in the distinction between HCM is a primary heart muscle disease with a 1:500 restrictive myocardial disease and constriction from peri- prevalence in the general population [107,108]. cardial disease [110]. O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 257

Fig. 7. Eosinophilic disease. (A) A 44-year-old man affected by Loeffler disease in the subacute thrombotic stage. EMB shows endocardial thrombus and endomyocardial infiltrates rich in eosinophils. (A) Fresh thrombus (hematoxylin–eosin, 200×); (B) organizing thrombus (hematoxylin–eosin, 100×); (C–D) typical eosinophil-rich endomyocardial inflammation (hematoxylin–eosin, C, 400×; D, 250×).

7.8.2. EMB diagnostic potential scar (Fig. 7). Three stages can be distinguished along the EMB is unable to give a diagnostic picture as histological time course: a necrotic (rarely symptomatic) stage, a findings (i.e., interstitial fibrosis, myocardial disarray, or thrombotic stage (with risk of thromboembolism), and a even normal myocardium) are not specific. However, in fibrotic stage with cardiac and atrioventricular valvular selected cases, EMB may be helpful in excluding infiltrative dysfunction [112,113]. or storage diseases [110,111]. Tropical (Davies) endomyocardial fibrosis is typical in the tropical areas, with a male/female ratio of 1:1, usually 7.9. Loeffler fibroplastic endocarditis/endomyocardial occurring in children and young adults. Eosinophilia is fibrosis—also known as “obliterative cardiomyopathy” infrequent and reflects a parasitic infection. Tropical endomyocardial fibrosis is commonly associated with 7.9.1. Evidence of recommendation: S (in the nutritional deficiency, hypersensitivity to malaria, viral and acute–subacute phase) parasitic , magnesium deficiency, and high cerium This section addresses heart diseases with thickening of intake. Pathologically, there is fibrotic endocardial thicken- lining the heart cavities and progressive ing at the apex and inflow of right or both ventricle, with obliteration of the ventricular cavities. atrioventricular valve apparatus entrapment, endocardial Loeffler endocarditis (also known as eosinophilic calcification, and mural thrombosis, as well as variable endomyocardial disease or fibroplastic parietal endocardi- degrees of subendocardial necrosis or degeneration of the tis) is typical in the temperate zone, with a male/female underlying myocardium [114–116]. ratio of 9:1, and usually manifests in the fourth decade of life. It appears to be a subcategory of the hypereosi- 7.9.2. EMB diagnostic potential nophilic syndrome in which the heart is predominantly EMB can allow a “definite” diagnosis only in the acute involved. It is an uncommon myocardial disease, thought phase, with the demonstration of endomyocardial infiltrates to be secondary to damage from eosinophils. At gross rich in degranulated eosinophils and eosinophil cationic examination, there is diffuse deposition of mural thrombus protein, and endocardial thrombosis. A “probable/possible” over the ventricular endocardium incorporated into a diagnosis can be reached in the chronic phase of endomyo- whitish fibrous plaque. cardial fibrosis, with evidence of endocardial fibrous On histological examination, there may be eosinophilic thickening and subendocardial myocyte abnormalities. myocarditis, endocardial damage, thrombotic deposition Clinical features and laboratory evidence of past/present infiltrated by eosinophils, and dense myocardial fibrous hypereosinophilia are useful for final diagnosis. 258 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274

Fig. 8. Cardiac sarcoidosis in a 53-year-old female. (A) Histology: a giant cell granulomatous inflammatory infiltrate is visible with$2 absence of . In the cytoplasm of a giant cell, an asteroid body (arrow) should be noted (hematoxylin–eosin, 200×). (B) Immunohistochemistry: the and the multinucleate giant cells are positive with the CD68/PGM1 antibody (200×). (C) Subsequent control EMB showing the persistence of nodular polymorphous inflammatory infiltrate (hematoxylin–eosin, 100×). (D) Subsequent chronic evolution with interstitial fibrosis and sparse inflammatory infiltrate (Masson trichrome, 100×). From Leone et al. [26].

7.10. Cardiac sarcoidosis 7.11. Peripartum cardiomyopathy (PPCM)

7.10.1. Evidence of recommendations: S 7.11.1. Evidence of recommendations: M PPCM is often defined as the onset of unexplained left Sarcoidosis is a systemic granulomatous disease of ventricular dysfunction in the last trimester of pregnancy or unclear etiology (Fig. 8). Assessment of cardiac involvement within 5 months postpartum. The etiology of this rare is important as the cause of death in half of sarcoidosis disorder remains unclear. Borderline myocarditis and active patients is cardiac in origin. In some cases, an unexpected myocarditis, as well as healing myocarditis, have all been diagnosis of sarcoidosis is made on biopsy of patients with demonstrated in EMB from some of these women, with a ventricular , suspected ARVC, or RC. Sarcoid prevalence of myocarditis on EMB ranging from 8.8% to enters the differential in the investigation of unexplained arrhythmias [117–119]. 62%, depending upon the diagnostic criteria used and the timing of biopsy with respect to onset of symptoms.

7.10.2. EMB diagnostic potential 7.11.2. EMB diagnostic potential Due to the focal nature of the infiltrates, EMB has The role of EMB in the diagnosis of PPCM is controversial been reported to have poor sensitivity in systemic since there may be no difference in clinical outcome between sarcoidosis with presumed cardiac involvement, being those with and without myocarditis on biopsy [122]. Spon- positive in 19%–25% of cases [120,121]. A positive taneous resolution of myocarditis has been observed in some biopsy, however, may be reflective of more extensive patients, while others require immunosuppressive therapy or disease and is associated with worse survival [121]. More progress to DCM. importantly, a positive biopsy guides therapeutic manage- ment of these patients with corticosteroids and potential 7.11.3. Molecular aspects prophylactic use of a defibrillator. To increase the Investigations into the presence of viral genomes have sensitivity of the procedure, electrophysiologic (electro- yielded inconsistent results [123]. anatomic mapping) or image-guided biopsy procedures have been proposed. 7.12.

7.12.1. Evidence of recommendations: S 7.10.3. Special technical aspects The amyloidoses comprise a group of disorders charac- Special stains for microorganisms are important to rule terized by the deposition of abnormally folded protein with out infectious granulomatous diseases. an extended beta-sheet conformation. O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 259

Table 5 chemistry [125], immunofluorescence (Fig. 9C, F) [124], Summary of the main forms of amyloidosis that affect the heart immunoelectron microscopy [126], protein sequencing Nomenclature Precursor of amyloid fibril Systemic (S) or [127], and mass spectrometry [128]. Thus, it is recom- localized (L) mended that efforts be made to subtype amyloid deposits AL Immunoglobulin light chain S in EMBs. Subclassification of amyloid deposits is nontri- ATTR Familial (mutant) and senile S vial, and the misclassification of amyloid in pathologic systemic (wild type) transthyretin specimens has been reported (reviewed by Collins et al. AApoA1 Mutant apolipoprotein S/L AA Serum amyloid A S [124]). Mistyping of amyloid can have catastrophic effects, AANP Atrial natriuretic peptide L as the prognosis is very different and treatment options are specific for the type of amyloid present [70]. Amyloid subtyping should be performed in a center with expe- 7.12.2. EMB diagnostic potential rience using techniques that have been validated to be EMB is well recognized as an important modality in the reliable in that center. workup of patients with cardiac amyloidosis. At least 11 types of amyloidosis may involve the heart [124]. Table 5 summarizes the main forms of amyloidosis that affect the 7.13. Iron overload heart [70]. In hematoxylin–eosin-stained sections, the amyloid appears as a homogeneous, slightly eosinophilic, 7.13.1. Evidence of recommendations: S amorphous, and nonstructured substance (Fig. 9A, B, D). It The heart normally contains no iron, and therefore, is recommended that nontransplant biopsies be stained for finding stainable iron in the cardiomyocytes indicates the the presence of amyloid using Congo red (deposits appear presence of pathology. Heart failure induced by iron with a characteristic apple-green birefringence under polar- deposition is a leading cause of death in patients with ized light; Fig. 9 E), modified sulfated alcian blue, or transfusion-dependent anaemia, notably beta-thalassemia thioflavin T. Frozen tissue is necessary if the amyloid is to be [129]. Cardiac iron deposition is also a feature of hereditary typed by immunofluorescence. hemochromatosis, but is seldom a direct cause of death [130]. Beta-thalassemia CMP may have a dilated phenotype 7.12.3. IHC and molecular aspects with impaired contractility or a restrictive pattern with EMB has been proven useful for establishing the type of pulmonary hypertension and right heart failure [129].In cardiac amyloid using techniques such as immunohisto- cardiac biopsy studies, there is a good correlation between

Fig. 9. (A–C) Late-onset systemic familial transthyretin-related amyloidosis (mutation: Ile68Leu) in a 62-year-old male. The EMB (A) shows diffuse interstitial deposits of amyloid (25×). (B) In the hematoxylin–eosin routine section (400×), amyloid appears as a homogeneous, amorphous, and eosinophilic substance. (C) Immunohistochemistry: amyloid deposits show a diffuse and intense brown positivity for the anti-transthyretin antibody (400×). (D–F) Right ventricular EMB from an 84-year-old man. Hematoxylin–eosin (D) and Congo red stain (E) show interstitial amyloid deposition. Subtyping of the amyloid by immunofluorescence (F) shows specific staining for transthyretin consistent with senile systemic amyloidosis (wild-type transthyretin). (D–F) All images are at 400× magnification. 260 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 serum ferritin levels and the degree of iron deposition [131]. diagnosis, but in selected cases with no clear-cut diagnosis, Recently, MRI has proven to be effective in quantifying iron EMB is recommended. EMB from patients with unexplained in the heart and liver [132]. In a comparative study of EMB DCM should be assessed for the presence of iron, and an and MRI, patients with stainable iron in more than 50% of iron stain should be performed when necessary. Biopsy myofibers were shown to have lower T2 relaxation times maybealsousefulinmonitoringofpatientstoverify than those with less iron on biopsy [133]. Iron tends to therapeutic results. Genetic testing on blood is commonly initially deposit subepicardially and may not always be utilized to make the diagnosis of hereditary hemochromatosis. found in the right ventricle septal region; thus, the possibility of sampling error exists [134]. 7.14. Desmin-related cardiomyopathy (DRM)

7.13.2. EMB diagnostic potential 7.14.1. Evidence of recommendations: S The histological appearance of iron storage on hematox- DRM is a clinically heterogeneous group of diseases ylin–eosin-stained sections is that of intramyocytic granular with progressive skeletal and/or cardiac myopathy, which brownish deposits (Fig. 10A), which are blue on a pink field results from mutations in the intermediate filament by blue Prussian (Perls) staining (Fig. 10B). It is not possible desmin (DES) gene [45,135,136]. Cardiac phenotypes to histologically distinguish primary from secondary iron related to DRM include dilated, restrictive, and occasionally overload. Magnetic resonance imaging may contribute to ARVC-like [137].

Fig. 10. Storage diseases. (A–B) Hemochromatosis in a 60-year-old male with sudden onset of refractory cardiac failure. (A) The EMB shows numerous intracytoplasmic granules in the cardiomyocytes (hematoxylin–eosin: 400×). (B) Specific staining for iron highlights the intracytoplasmic granules in blue (Perls blue, 100×). (C–D) Fabry disease. (C) Hematoxylin–eosin-stained section (200×) showing hypertrophied vacuolated myocytes. (D) Transmission electron micrograph (10.000×) showing lamellar bodies within the cytoplasm of cardiac myocytes. (E–F) Glycogenosis type II (Pompe's disease). (E) PAS-stained section (400×) showing cytoplasmic glycogen deposits. (F) Transmission electron micrograph (27,500×) showing lysosomal bound glycogen surrounded by mitochondria; some extralysosomal glycogen is also present. O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 261

7.14.2. EMB diagnostic potential cient glycoprotein syndromes, and Sandhoff's disease). All EMB may be helpful to provide a definitive diagnosis in of these diseases may lead to cardiac hypertrophy. suspected cases, confirm cardiac involvement in patients with positive genetic testing, and rule out other processes. 7.16.2. EMB diagnostic potential The histological picture is nonspecific (fibrosis and some- Microscopically, there are hypertrophied vacuolated times a small increment in myofiber size). Electron myocytes on EMB [140,141]. Special stains (periodic acid- microscopy shows abnormal aggregates of desmin-type Schiff [PAS], alcian blue) may be used to assess the nature of intermediate filaments (granulofilamentous aggregates) in the storage products, preferably on frozen sections (as the cytoplasm of cardiomyocytes (interfibrillar areas and glycogen may wash out with routine processing for paraffin at Z band level). sections). Electron microscopy is used to assess the nature and location of the intracellular deposits. Enzyme replace- 7.14.3. IHC aspects ment therapy is utilized for lysosomal storage diseases and Immunoelectron microscopy may confirm that these mucopolysaccharidoses; thus, the use of EMB in monitoring aggregates are formed by desmin. IHC for desmin on may be applicable, but skeletal muscle biopsy is more often histological sections shows abnormal distribution and, employed [142,143]. in advanced cases, large aggregates of desmin fila- ments, although these IHC findings are not entirely spe- 7.17. Cardiac tumors cific for DRM. 7.17.1. Evidence of recommendation: S 7.15. Anderson–Fabry disease 7.17.1.1. Primary cardiac tumors. Most primary cardiac 7.15.1. Evidence of recommendations: S tumors (90% in surgical series) are benign [144–147].In Anderson–Fabry disease is an X-linked inborn metabolic series, the frequency of primary cardiac tumors was defect caused by a deficiency of alpha-galactosidase A, 1/1000, whereas cardiac metastases (mainly from lung, resulting in the accumulation of glycosphingolipid, which breast, or kidney, or from malignant melanoma) were far may cause conduction disturbances, valve disease, and left more prevalent (1/100) [147]. ventricular hypertrophy [138]. The most common primary cardiac neoplasm is the Long-term treatment with recombinant human alpha- cardiac myxoma, a benign tumor that has no microscopic galactosidase A may halt the progression of microvascular counterpart in other organs and tissues [148,149], whereas pathology and prevent the clinical manifestations [139]. the most frequent malignant tumors are represented by EMB may be very informative for the assessment of sarcomas, which have the same classification as soft tissue unexplained left ventricular hypertrophy, particularly in sarcomas [150]. According to the World Health Organiza- carrier women that develop heart disease later in life and in tion classification, primary cardiac tumors are categorized patients that have mutations associated with residual enzyme activity and a milder disease phenotype.

7.15.2. EMB diagnostic potential EMB may be a useful guide for clinicians and pathologists who diagnose and follow these patients [139]. At histology, hypertrophic vacuolated cells are evident, with dislocation of contractile elements to the periphery of myocytes (Fig. 10C). Electron microscopy reveals electron dense concentric lamellar bodies, which are typical, although not exclusive to the disease (Fig. 10D).

7.16. Other inherited storage diseases

7.16.1. Evidence of recommendations: S Cardiac manifestation of systemic metabolic diseases caused by deficiencies of various enzymes in metabolic pathways includes glycogen storage diseases [e.g., glyco- Fig. 11. Angiosarcoma: biopsy of a right tumor. At low genosis type II (Pompe's disease, Fig. 10E–F), glycogenosis magnification, spindle cell tumor with hemorrhage and thrombus (hema- – – type III, Danon disease, and PRKAG2 mutations] and toxylin eosin 50×). At higher magnification (insert A, hematoxylin eosin – 400×), pleomorphic and atypical nuclei are conspicuous, and some tumor lysosomal storage diseases (e.g., Niemann Pick disease, cells show intracytoplasmic vacuoles containing red blood cells (arrows). Gaucher disease, I-cell disease, mucopolysaccharidoses, Immunohistochemistry (insert B, 400×) showing strong positivity for factor- GM1 gangliosidosis, galactosialidosis, carbohydrate-defi- VIII-related antigen. 262 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274

Fig. 12. Right atrium fibrosarcoma. (A) Transvenous endocardial biopsy specimen of mass (hematoxylin–eosin stain, 120×). (B) At higher magnification, histologic examination of mass shows spindle-shaped cells arranged in fascicles at acute angles with frequent mitoses (hematoxylin–eosin stain, 480×). (C) Diffuse positivity for vimentin at immunohistochemistry (insert, 480×) and negative immunostaining with other tested antibodies. From Basso et al., modified [16].

as benign tumors and tumor-like lesions, malignant tumors, EMB is a valuable tool for preoperative diagnosis of and pericardial tumors [151]. intracardiac masses. It is mainly indicated for the investiga- For more detailed information and diagnostic criteria, tion of right-sided masses showing an infiltrative or extensive literature is available [146,148,152–154]. Tumors obstructive growth pattern and for the differential diagnosis reported in the literature that have been diagnosed by EMB of sarcomas, lymphomas, and metastatic tumors. Unresect- are numerous, and mainly malignant or metastatic able cardiac masses may benefit from EMB to help plan [8,21,155–170]. Figs. 11 and 12 show two examples of therapy or palliation [15,16,21,172,173]. Table 6 lists biopsied cardiac primary tumors, and Fig. 13 shows two some biopsied malignant and metastatic that cases of biopsied cardiac metastases. have been reported.

7.17.2. Indications for EMB 7.17.2.2. Limits. False-negative results are possible due to sampling error but can be minimized by procurement of 7.17.2.1. Indications. Clinical diagnosis of cardiac masses is multiple specimens guided by echocardiography. Inspecting mainly done by echocardiography, computerized tomogra- the biopsy specimens for a pale color may also be useful. phy, and/or MRI. Histology remains crucial for differenti- Left-sided EMB is possible but sometimes avoided because ating neoplastic from nonneoplastic masses and benign from of the potential for systemic embolism. malignant, and for subtyping the neoplasms. It therefore provides critical information for treatment and prognosis, 7.17.3. Specific technical aspects which are largely dependent upon tumor histotype and At least three to five formalin-fixed and paraffin- biological behavior [171]. embedded biopsy samples (2–3mm2 each) are advisable.

Fig. 13. Anaplastic large cell lymphoma and carcinoma cardiac metastasis. (A) Transvenous EMB specimen taken from the right ventricle showing a diffuse neoplastic myocardial infiltration by atypical lymphoid cells (hematoxylin–eosin 100×). (B) CD20 IHC staining reveals a B-cell lymphoma infiltrate (100×). (C) Transvenous right ventricular EMB showing a cardiac breast carcinoma metastasis (hematoxylin–eosin 200×). O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 263

Table 6 7.18. Hypersensitivity reactions and cardiac toxicity due to Malignant primary or metastatic cardiac tumors reported in the literature as drugs and chemical/toxic substances diagnosed by EMB Malignant primary tumors Metastatic tumors 7.18.1. Evidence of recommendation: hypersensitivity Angiosarcoma Adenocarcinoma myocarditis: S; toxic cardiopathies: M (depending on Fibrosarcoma Cervical carcinoma the drug) Granulocytic sarcoma (chloroma) Endometrial carcinoma Drug-related cardiac toxicity and hypersensitivity re- Leiomyosarcoma Lymphoma Lymphoma Melanoma actions represent, for physicians, the most worrying aspect of Malignant fibrous histiocytoma Squamous cell carcinoma the wide panel of substances capable of injuring the heart Rhabdomyosarcoma since many prescribed drugs can be associated with cardiac Sarcoma NOS toxicity or hypersensitivity reactions [176–178].Itis Synovial sarcoma expressed in two clearly distinct entities: hypersensitivity myocarditis and toxic “damage.”

7.18.2. Hypersensitivity myocarditis This is probably the most common form of drug-induced One to two additional snap-frozen samples for molecu- cardiac dysfunction and may occur with many usual lar biology may be useful for diagnosis of sarcomas therapeutic agents from many classes (Table 7). It is and lymphomas. unpredictable; is independent of drug dosage; and, as with “direct toxicity,” is likely due to sensitization after previous 7.17.4. Immunohistochemistry safe use. The most common pattern of hypersensitivity The type of antibodies to be utilized depends upon tumor myocarditis is pancarditis with a perivascular mixed type. An initial useful suggested panel includes vimentin, inflammatory infiltrate that is rich in eosinophils. Lympho- factor-VIII-related antigen, CD31, CD34, epithelial mem- cytes, macrophages, giant cells, and neutrophils may be brane antigen, wide-spectrum cytokeratins, S-100 protein, present in variable numbers, and the process may be smooth muscle actin, desmin, myogenin, HMB-45 molecule, associated with the development of small . The CD45, CD20, and CD3. For detailed diagnosis of lympho- inflammation has a tendency for an interstitial or perivas- mas, a wide panel of lymphocytic antibodies is usually cular pattern of distribution, with little in the way of myocyte required [174]. injury (Fig. 14).

7.18.3. Toxic damage 7.17.5. Molecular biology This is due to substances usually known for their potential Fluorescence in situ hybridization and PCR tests to cause direct cardiac toxicity (Table 7). However, the are commonly used, at least in most referral centers, for development of new drugs with undetermined capacities for the evaluation of sarcomas and lymphomas [e.g., reciprocal cardiotoxicity can occur. Cardiac toxicity is dose dependent, translocation t(x;18)(p11.2;q11.2) in synovial sarcoma possibly has a cumulative effect, and is often potentiated by and t(2;13)(q35;q14) in rhabdomyosarcoma] [175]. other antineoplastic therapies such as radiation therapy.

Table 7 Selected drugs and other chemical substances associated with cardiac toxicity and their most frequent histological picture Histological picture Drugs Hypersensitivity Miscellaneous Acetazolamide, amitriptyline, aminophylline, amphotericin B, ampicillin, azathioprine, benzodiazepines, myocarditis bumetanide, captopril, carbamazepine, cephalosporins, chloramphenicol, chlorthalidone, clozapine, cyclosporine, digoxin, dobutamine, furosemide, heparin, hydralazine, hydrochlorothiazide, indomethacin, interleukin-2, isoniazid, isosorbide dinitrate, methyldopa, metolazone, nitroprusside, oxyphenbutazone, para-aminosalicylic acid, penicillin, phenindione, phenylbutazone, phenytoin, spironolactone, streptomycin, sulfadiazine, sulfamethoxypyridazine, sulfisoxazole, sulfonylureas, tetracyclines, triazolam, tricyclic antidepressants. Toxic damage Antineoplastic drugs Amsacrine, cyclophosphamide, anthracyclines (doxorubicin, daunorubicin, epirubicin), 5-fluorouracil, interferon-alpha, interleukin-2, mitomycin C, mitoxantrone, tyrosine kinase inhibitors (trastuzumab anti- HER2/neu, cetuximab and erlotinib anti-EGFR, bevacizumab and sunitinib anti-VEGF, imatinib anti-cKIT) Toxic damage Miscellaneous Amphetamine-derived compounds (benfluorex, type 2 diabetes modulator), antimony compounds (antileishmaniasis treatment), antiretroviral agents, catecholamines, chloroquine, hydroxychloroquine, dopamine agonists (Parkinson's disease), emetine (antiamoebiasis treatment), lithium, phenothiazines, -derived compounds (fenfluramine/phentermine, appetite suppressants) Toxic damage Drugs of abuse Alcohol, amphetamine and related compounds (methamphetamines, ecstasy), anabolic steroids, cocaine Toxic damage Poisoning Arsenicals, cobalt, lead 264 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274

Fig. 14. (A) EMB showing eosinophilic myocarditis from patient with history of allergies, who was on multiple medications (hematoxylin–eosin 400×). (B) Drug-induced hypersensitivity myocarditis. EMB in a heart transplant patient treated with horse antilymphocyte serum for rejection in the early 80s. He developed serum sickness with cutaneous rash, pulmonary infiltrate, and high eosinophil count at bronchoalveolar lavage, and a typical pattern of eosinophilic myocarditis. All the signs resolved with steroids (hematoxylin–eosin 400×).

Cardiotoxicity may be reversible after discontinuation of chloroquine toxicity, the myocytes may have a the medication, allowing for its reintroduction later [179] or vacuolated appearance, and lamellar lysosomal in- for avoidance of drug-related heart failure [180–182]. clusions may be identified by electron microscopy. Recently, new drugs have revealed cardiac toxicity, In other cases, such as with Adriamycin toxicity, including new antineoplastic drugs [179,180,183–185], dilation of the sarcoplasmic reticulum may be seen appetite suppressants [186], and drugs used in type 2 on electron microscopy. diabetes [187] or in Parkinson's disease (Table 7) [188]. Currently, cases of cardiac toxicity due to street drugs or 7.18.4. EMB diagnostic potential illicit substances and alcohol, and industrial or environmen- Drug-induced hypersensitivity myocarditis may be sug- tal chemicals have been overshadowed by the frequency of gested using EMB when myocarditis is clinically suspected. – cocaine cardiotoxicity (Table 7) [189 191]. As with all forms of myocarditis, sampling error may lead to The pathology of cardiac toxicity [177,178] is quite variable false-negative diagnosis. and often lacks specificity so that the implication of a drug can In toxic CMPs, the role of EMB is variable depending be difficult. This is compounded by the fact that many patients upon the specific drug. For drugs with a well-established are often on a polypharmacy of drugs with potential cardio- cardiac toxicity, for example, anthracyclines (including toxicity. The gross appearance of the heart varies from no Adriamycin) [193], EMB has been replaced by noninvasive “ ” structural changes to a pattern of dilated cardiomyopathy in techniques such as stress or strain echocardiography, the later stages of some cumulative drug exposures. , MRI, and troponin I level Histology can show three main general histological patterns: monitoring [176,191]. On the other hand, in recently marketed drugs, EMB ■ Myocardial necrosis usually without inflammatory is important in order to document cardiac toxicity, to infiltrate: patchy eosinophilic changes in myofibers, study the mechanisms of cardiac toxicity, and to establish focal myofibril degeneration or focal coagulative the imputability of the drug. Even with the use of the EMB, necrosis, and prominent contraction bands which it may still be difficult to pinpoint one specific drug as may be associated with fibrin deposition, interstitial the culprit. , and hemorrhagic extravasation. ■ “Toxic myocarditis,” characterized by multiple areas 7.18.5. Specific technical aspects of of different ages, surrounded by a mixed For toxic cardiopathies, transmission electron microscopy inflammatory infiltrate of macrophages, neutrophil may sometimes be performed, implying special fixation of granulocytes, and a small number of lymphocytes. EMB samples in glutaraldehyde. Interstitial edema and hemorrhagic extravasation and myocyte may be present to a variable degree. Cyclophosphamide-induced cardiac 7.19. Transplantation toxicity targets endothelial cells and results in microthrombi, hemorrhagic edema, and myocyte 7.19.1. Evidence of recommendation: S necrosis [192]. ■ DCM in the chronic phase, with nonspecific 7.19.1.1. Acute cellular rejection. In 1990, an international histological alterations (interstitial fibrosis and grading system for cardiac allograft biopsies was adopted by myofiber alterations). In some cases, such as with the International Society for Heart and Lung Transplantation O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 265

Table 8 1990 and 2004 ISHLT grading systems for cellular and antibody-mediated rejections (AMR) [2] 2004 ISHLT revised standardized cardiac biopsy grading 1990 ISHLT standardized cardiac biopsy grading Grading of acute cellular rejection Grade 0 No rejection Grade 0 Grade 1R mild Interstitial and/or perivascular inflammatory infiltrate with up to Grades 1A, 1B, 2 one focus of myocyte damage Grade 2R moderate Two or more foci of inflammatory infiltrates with associated myocyte damage Grade 3A Grade 3R severe Diffuse inflammatory infiltrates with multifocal myocyte Grades 3B and 4 damage±edema±hemorrhage±vasculitis

Grading of acute AMR Grade 0 Negative for acute AMR No histologic or immunopathologic features of AMR Grade 1 Positive for AMR Histologic features of AMR: myocardial capillary injury (endothelial swelling, intravascular macrophages), interstitial edema and hemorrhage, neutrophils in and around capillaries, intravascular thrombi, myocyte necrosis Positive immunofluorescence or immunoperoxidase staining for AMR: immunoglobulin (IgG, IgM, and/or IgA) and complement (C3d, C4d, and/or C1q) deposition in capillaries by immunofluorescence on frozen sections; macrophages (CD68) in capillaries (CD31 or CD34) and complement (C4d) deposition on capillary endothelium by paraffin immunohistochemistry

(ISHLT) [1]. The grading system was widely adopted and between transplant centers, but most agree that the incidence served the transplant community well for over a decade. In is low (b5%), especially since high-risk patients may be 2004, under the direction of the ISHLT, a multidisciplinary treated prophylactically before transplantation. review of the grading system was undertaken to address The histological features of AMR include interstitial challenges and inconsistencies in its use, to consider changes edema; endothelial cell swelling; intravascular macrophages; in rejection incidence and treatment practices, and to and, less frequently, interstitial hemorrhage, mixed inflam- incorporate advances in medical knowledge [194–206]. matory infiltrates, capillary fragmentation, and endothelial The outcome of the consensus conference was revision of cell (Fig. 16A). There should be immunopheno- the 1990 working formulation for cellular rejection as typic evidence of immunoglobulin and complement depo- outlined below with “R” after the rejection grade indicating sition, especially C4d (and perhaps C3d), in capillaries the 2004 revision (Table 8) [2]. (Fig. 16B). However, several studies since 2005 have raised In Fig. 15, 2004 ISHLT cellular rejection grades are the question of asymptomatic AMR [213], questioned the shown. sensitivity and specificity of histological criteria for screening biopsies (as stated in the 2004 grading system 7.19.1.2. Acute antibody-mediated rejection (AMR). AMR is revision) [214,215], shown positive histology (in the kidney) an evolving issue in cardiac transplantation [207–210]. for AMR without C4d capillary deposition [216], and shown While mentioned only briefly in the 1990 grading system, C4d capillary deposition without positive histology or the 2004 revision (Table 8) recommends reporting whether intravascular macrophages [217,218]. This suggests that AMR is absent (AMR 0) or present (AMR 1). AMR has been there is a morphologic and immunophenotypic spectrum of recognized as a clinicopathologic entity that has been AMR-related changes, which should be regarded as a characterized by (a) cardiac dysfunction in the absence of clinical–pathologic continuum that starts with a latent cellular rejection or ischemic injury, (b) characteristic humoral response of circulating antibodies alone and histological features on EMB, (c) positive immunostaining progresses through a silent phase of circulating antibodies of capillaries for the complement component C4d, and (d) with C4d deposition, without histological or clinical presence of circulating donor-specific antibodies (DSAs). alterations; to a subclinical stage with circulating antibodies, AMR often occurs in sensitized patients (including those and histological and immunopathologic findings; and to with previous transplantation, transfusion or pregnancy, and symptomatic AMR with clinical manifestations. previous ventricular assist device placement) and is Furthermore, although C4d is the most widely used associated with a worse graft survival [210,211]. Increas- immunohistologic marker for AMR, there is no uniformity of ingly AMR is being detected late after transplantation both in approach to frequency of testing or to what constitutes a presensitized patients and in patients developing de novo positive result in terms of distribution and intensity of DSAs [212]. The incidence of AMR is highly variable staining [219–222]. Lastly, ACR and AMR may coexist in 266 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274

in 2011 focused solely on AMR specifically to address (a) appropriate testing and interpretation of histological findings and immunohistochemistry (at present, C4d immunofluo- rescence and immunoperoxidase detection seem to be more or less equivalent) [223,224]; (b) use of serologic data, specifically donor specific antibodies; and (c) correlation of allograft dysfunction and histopathologic/immunophenoty- pic/serologic findings. In both consensus conferences, it was agreed that the diagnosis of AMR should be made according to specific pathologic features. The combination of histopathologic and immunopathologic findings should be reported as the “pathological diagnosis of AMR” and should be designated by pAMR (Table 9). Descriptors for immunologic and clinical presentation, such as DSA and cardiac dysfunction, would assist in diagnosis and management [223,224]. Uncertainty still remains as to the best treatment for both symptomatic AMR, currently defined as positive histology and C4d staining on EMB and positive serum antibodies in a hemodynamically compromised patient [225], and asymp- tomatic AMR in a patient who is clinically doing well with negative histology yet has positive C4d staining on EMB with or without positive DSA [213].

7.19.1.3. Nonrejection posttransplant biopsy findings. Early perioperative ischemic injury is an extremely common finding up to 6 weeks (or more) posttransplant, characterized by myocyte necrosis which often extends to the endocardial surface. As healing ensues, a mixed inflammatory infiltrate including neutrophils as well as lymphocytes, macrophages, and eosinophils must be distinguished from acute rejection (Fig. 17A) [226,227]. Late ischemic injury resulting from allograft coronary disease may be detected by secondary myocardial changes since vessels large enough to demon- strate this process are rarely present in biopsy material. Myocyte vacuolization and/or microinfarcts are characte- ristic of this process and may be helpful in determining the cause of late cardiac failure or, more importantly, ruling out other potentially treatable etiologies [228,229]. Quilty effect (nodular endocardial infiltrates) (Fig. 17B) may be Fig. 15. (A) Cellular rejection, ISHLTR 1R. Perivascular and/or interstitial confined to the endocardium (1990 ISHLT Quilty A) or lymphocytic infiltrate with no more than one focus of myocyte damage extend into the underlying myocardium where asso- (hematoxylin–eosin, 400×). (B) Cellular rejection, ISHLTR 2R. Two or more foci of lymphocytic infiltrates with associated myocyte damage ciated myocyte damage may be present (ISHLT 1990 (hematoxylin–eosin, 200×). (C) Cellular rejection, ISHLTR 3R. Diffuse Quilty B) [230–234]. However, because the subtyping of infiltrate with multifocal myocyte damage and edema. In some cases, Quilty A and B was never shown to have clinical hemorrhage and vasculitis may be present (hematoxylin–eosin, 200×). significance, the designations “A” and “B” were eliminated in the 2004 revision. the same biopsy, but given the above challenges we face 7.19.1.4. EMB diagnostic potential. Despite many advances in diagnosing AMR, its frequency and clinical significance in immunosuppression therapy, acute rejection still occurs, are unknown. most commonly during the first posttransplant year [235]. Due to the many unanswered questions regarding AMR, The EMB has been the “gold standard” for the diagnosis of the heart transplantation section of the Tenth Banff allograft rejection, and its routine use has been credited as a Conference on Allograft Pathology in August 2009 and factor in improved recipient survival [236]. No established two Consensus Conferences held in conjunction with the noninvasive marker of rejection is available, and no indicator ISHLT Annual Meeting in Chicago in 2010 and San Diego of heart allograft rejection has been proven more sensitive, O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 267

Fig. 16. Antibody-mediated rejection (AMR). (A) Histology of AMR: endothelial cell swelling with intravascular macrophages and perivascular edema (hematoxylin–eosin 400×). (B) Immunohistochemical staining for C4d complement factor is diffuse and strong in capillary endothelial cells (100×).

Table 9 2011 ISHLT preliminary nomenclature scheme for pathologic AMR [223] pAMR 0: Negative for pathologic AMR: Both histologic and immunopathologic studies are negative. pAMR 1 (H+): Histopathologic AMR alone: Histological findings are present, and immunopathological findings are negative. pAMR1 (I+): Immunopathologic AMR alone: Histological findings are negative, and immunopathological findings are positive. pAMR 2: Pathologic AMR: Both histologic and immunopathologic findings are present. pAMR 3: Severe pathologic AMR: This category recognizes the rare cases of severe AMR with histopathologic findings of interstitial hemorrhage, capillary fragmentation, mixed inflammatory infiltrates, endothelial cell pyknosis and/or , and marked edema. The reported experience of the group was that these cases are associated with profound allograft dysfunction and poor clinical outcomes.

Fig. 17. (A) Ischemic injury: coagulative myocyte necrosis with mixed inflammatory infiltrate representing early healing (hematoxylin–eosin, 200×). Note that, in contrast to cellular rejection, the degree of myocyte damage is more extensive than the cellular infiltrate. (B) Quilty effect: endocardial infiltrate, often with prominent vascularity, which may extend into the underlying myocardium (hematoxylin–eosin 100×). specific, or temporally useful. The sensitivity of the EMB is detected), confirming that, to date, the EMB remains the high (generally 85%–100%), and the complication rate is “gold standard” for diagnosis of acute allograft rejection low [237–239]. The use of gene expression profiling in [240,241]. Today, new knowledge regarding AMR and its which peripheral blood specimens are analyzed is the most more accurate pathologic diagnostic criteria also confirms recent challenge to the EMB for rejection surveillance. this role. However, the recent Invasive Monitoring Attenuation EMB is useful in differential diagnosis between acute through Gene Expression trial was limited to recipients cellular rejection and myocardial posttransplant infections more than 6 months posttransplant (when rejection frequen- (e.g., Cytomegalovirus, Toxoplasma gondii) and may play cy is lower) with an end point of no increased risk of serious an important role in the diagnosis of posttransplant clinical outcome (not the number of rejection episodes lymphoproliferative disorders (Fig. 18) [242,243]. 268 O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274

Fig. 18. Three years posttransplant EMB from a 34-year-old woman affected by a posttransplant lymphoproliferative disorder. (A) Hematoxylin–eosin stain with the diffuse inflammatory cell infiltration (200×). (B) Immunohistochemistry for antibody against B lymphocytes (CD20) (200×). (C) PCR amplification of variable diversity joining (VDJ) regions of heavy chain immunoglobulins showing the monoclonality for B cells.

7.19.1.5. Technical considerations. Routine diagnosis and Given the complexity of EMB protocols, it should be grading of cardiac allograft cellular rejection are performed performed only in centers which are fully equipped for on formalin-fixed tissue prepared for light microscopy pathological workup and where specifically trained and examination [2]. Staining with hematoxylin and eosin are experienced pathologists are available and can apply sufficient for diagnosis of ACR. A connective tissue stain standardized diagnostic histopathologic criteria to minimize such as Masson's trichrome may be helpful for assessing EMB reporting variability. myocyte damage, interstitial fibrosis, and Quilty lesions. In the perspective of personalized medicine, pathologists, Assessment of AMR or mixed ACR and AMR may be with their expertise in morphological tissue assessment, are performed by immunohistochemistry on paraffin-embedded central to the establishment of diagnosis of myocardial tissue, which enables assessment of staining localization and diseases and in outlining appropriate diagnostic strategies in distribution on a deeper level of all pieces of submitted collaboration with clinicians, imagers, molecular biologists, endomyocardial tissue. An alternative approach utilizes and geneticists. immunofluorescence on fresh/frozen tissue. While this Given the current key role of translational medicine, technique allows assessment of staining intensity and pathologists can make a significant contribution in transfer- comparison with previous studies, it is usually performed ring the new knowledge from basic research to applied on only one fragment of frozen tissue. clinical research, and promoting the institution of shared databases, using digital slide technology, especially for rare diseases or controversial diagnostic criteria. EMB remains a 8. Conclusion valuable tool for both diagnosis and research. EMB is a common procedure for the evaluation of cardiac tissue for transplant monitoring, myocarditis, drug toxicity, References CMPs, arrhythmia, secondary cardiac involvement by [1] Billingham ME, Cary NR, Hammond ME, et al. A working systemic diseases, and cardiac masses. It is the gold standard formulation for the standardization of nomenclature in the diagnosis for the diagnosis of cardiac rejection, myocarditis, and of heart and lung rejection: Heart Rejection Study Group. The infiltrative/storage disorders and can be crucial for differen- International Society for Heart Transplantation. J Heart Transplant tial diagnosis in heart failure and ventricular arrhythmias. Its 1990;9:587–93. role in differential diagnosis, i.e., in excluding other diseases, [2] Stewart S, Winters GL, Fishbein MC, et al. Revision of the 1990 working formulation for the standardization of nomenclature in should be stressed. the diagnosis of heart rejection. J Heart Lung Transplant 2005;24: EMB can also be strategic, both in unexplained patho- 1710–20. logical conditions to focus diagnosis and the subsequent [3] Parrillo JE, Aretz HT, Palacios I, et al. The results of transvenous diagnostic workup and in pathological conditions where, endomyocardial biopsy can frequently be used to diagnose despite a clinical diagnostic orientation, a definite diagnosis myocardial diseases in patients with idiopathic heart failure. Endomyocardial biopsies in 100 consecutive patients revealed a and specific etiological data for therapy, clinical manage- substantial incidence of myocarditis. Circulation 1984;69:93–101. ment, and prognosis (e.g., amyloidosis, hemochromatosis, [4] Arbustini E, Gavazzi A, Dal Bello B, et al. Ten-year experience with Anderson–Fabry disease, myocarditis, etc.) are required. endomyocardial biopsy in myocarditis presenting with congestive O. Leone et al. / Cardiovascular Pathology 21 (2012) 245–274 269

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