JCS：心脏淀粉样变性的诊断和治疗指南（2020）

I. General Remarks on Amyloidosis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1612

1. Concept and Classification of Amyloidosis∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1612 2. AL Amyloidosis∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1614 3. ATTRwt Amyloidosis∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1615 4. ATTRv Amyloidosis∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1616 5. Diagnostic Criteria∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1618

II. Diagnosis of CA ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1621

1. Medical History, Symptoms, and Physical Findings Indicative of CA∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1621 2. Blood Sampling Test∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1624 3. Electrocardiogram (ECG)∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1626 4. Echocardiography∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1627 5. Cardiac Magnetic Resonance Imaging (MRI)/ Computed Tomography (CT)∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1630 6. Nuclear Imaging∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1635 7. Cardiac Catheterization∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1637 8. Biopsy∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1638 9. Pathology/Mass Spectrometry∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1643 10. Genetic Testing and Counseling∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1645 11. Other Items (Aortic Stenosis)∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1646 III. Treatment of Cardiac Amyloidosis (CA) ∙∙∙∙∙∙∙∙∙∙∙1646 1. General Management∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1648 2. Pharmacologic Treatment of Arrhythmia∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1648 3. Device Therapy∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1649 4. Treatment for AL Amyloidosis∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1650 5. Disease-Modifying Therapy for ATTRv Amyloidosis∙∙∙∙1651 6. Treatment for ATTRwt Amyloidosis∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1653 IV. CA Diagnostic Algorithm ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1655 [CQ1] What is the role of 99m Tc-PYP scintigraphy (bone scintigraphy) in clinical practice for CA?∙∙∙∙∙∙∙1655 [CQ2] The role of endomyocardial biopsy. Which site is appropriate for biopsy in cardiac amyloidosis?∙∙∙∙∙∙1655 [CQ3] What patients should receive tafamidis for CA?∙∙∙∙∙1657 [CQ4] What is the role of genetic testing in clinical practice for cardiac amyloidosis?∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1660 References ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1660 Appendix1 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1668 Appendix2 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1669

Abbreviations

|ACEI|angiotensin converting enzyme (ACE) inhibitor| |---|---| |AL|amyloid light-chain| |ANP|atrial natriuretic peptide| |ARB|angiotensinll receptorblocker| |ATTRm|mutant ATTR amyloidosis|

|ATTRv|variant ATTR amyloidosis| |---|---| |ATTRwt|wild-type ATTR amyloidosis| |BNP|B-type (brain) natriuretic peptide| |CA|cardiac amyloidosis| |CMR|cardiac magnetic resonance imaging|

J-STAGE Advance Publication released online August 21, 2020 Mailing address: Scientific Committee of the Japanese Circulation Society, 18F Imperial Hotel Tower, 1-1-1 Uchisaiwai-cho, Chiyoda-ku, Tokyo 100-0011, Japan. E-mail: meeting@j-circ.or.jp This document is an English version of JCS 2020 Guideline on Diagnosis and Treatment of Cardiac Amyloidosis reported at the 84th Annual Scientific Meeting of Japanese Circulation Society performed in 2020 (Website: http://www.j-circ.or.jp/guideline/ pdf/JCS2020_Kitaoka.pdf). Refer to Appendix1 for the details of members. Joint Working Groups: the Japanese Circulation Society, Japanese Society of Amyloidosis, the Japanese Society of Hematology, Japanese Society of Echocardiography, Japanese College of Cardiology, the Japanese Heart Failure Society, Japanese Heart Rhythm Society, the group for surveys and research of amyloidosis in Japan, funded by a grant for the intractable diseases from the Ministry of Health, Labour and Welfare of Japan, group on idiopathic cardiomyopathy supported by the health and labor sciences research grant on intractable diseases. All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: cj@j-circ.or.jp ISSN-1346-9843

|CRT|Cardiac Resynchronization Therapy| |---|---| |ECV|extracellularvolumefraction| |FAP|familial amyloid polyneuropathy| |FLC|free light chain| |GLS|global longitudinal strain| |ICD|implantablecardioverter defibrillator| |LGE|late gadolinium enhancement| |MGUS| monoclonal gammopathy of undetermined significance |

|MRA|mineralocorticoid receptor antagonist| |---|---| |NAG|N-acetyl-β-D-glucosaminidase| |NT-proBNP|N-terminal pro-brain natriuretic peptide| |NYHA|NewYork Heart Asscoiation| |RBP|retinol-binding protein| |SAA|serum amyloid A protein| |SSA| senile systemic amyloidosis | |TTR|transthyretin|

Preamble

It is difficult to say that there is more evidence for cardiac amyloidosis (CA) than for ischemic heart disease. On the other hand, 99m technetium pyrophosphate ( 99m Tc-PYP) scintigraphy has been reported to be useful with high sensitivity and specificity, especially in transthyretin (TTR) amyloidosis (ATTR) 1 , 2 Due to the spread of diagnosis using this method, CA, especially wild-type ATTR (ATTRwt) amyloidosis, which has traditionally been considered a rare disease, is more prevalent than previously assumed, and encountered relatively frequently in daily clinical practice. Furthermore, treatment for not only amyloid light-chain (AL) amyloidosis, but also ATTR, has also progressed rapidly. Tafamidis, a drug that stabilizes the TTR tetramer and suppresses amyloid fibril formation and tissue deposition, was listed and used in Japan in November 2013 for the treatment of peripheral neuropathy in patients with hereditary (variant) ATTR (ATTRv) amyloidosis. In addition, following the results of the 2018 Transthyretin Amyloidosis Cardiomyopathy Clinical Trial (ATTR-ACT), which showed the efficacy of tafamidis for CA, 3 the use of TTR to treat CA was approved in March 2019 in Japan.

Needless to say, amyloidosis is a systemic disease, and in Japan, numerous studies have been conducted and medical treatments devised by the Ministry of Health, Labour and Welfare (MHLW)’s “Research Group on Amyloidosis”, a

research project on intractable disease policy. The present guidelines have been developed in coordination between the MHLW’s “Research Group on Amyloidosis”, the Japanese Circulation Society (JCS) and cardiology-related societies, the Japanese Society of Amyloidosis, and the Japanese Society of Hematology.

Systemic amyloidosis specified by the MHLW as being an incurable disease is classified into the following four types.

1. AL/amyloid heavy-chain amyloidosis (immunoglobulin amyloidosis) 2. ATTRwt amyloidosis (formerly called senile systemic amyloidosis [SSA]) 3. ATTRv amyloidosis (formerly called familial amyloid polyneuropathy [FAP]) 4. Hereditary systemic amyloidosis other than ATTRv amyloidosis

Notations in this guideline are based on the latest classification of the International Society of Amyloidosis. 4 On the other hand, rapid changes in cognition and progress in terms of treatment for CA have caused confusion in the field. We hope that these guidelines will help promote appropriate treatment for patients with CA.

Classes of Recommendations and Levels of Evidence

The recommendations and the levels of evidence in this guideline were basically determined and described in accordance with the guidelines for the diagnosis and treatment of acute and chronic heart failure (JCS 2017/JHFS 2017) 5 and the JCS 2018 guidelines on the diagnosis and

|Table 1. Classes of Recommendations|| |---|---| |ClassI| Evidence and/or general agreement that a given procedure or treatment is useful and effective | |Class II| Conflicting evidence and/or a divergence of opinion about the usefulness/efficacy of the given procedure or treatment | | Class lla| Weight of evidence/opinion is in favor of usefulness/ efficacy | |Class llb| Usefulness/efficacy is less well established by evidence/opinion | |Class Il| Evidence or general agreement that the given procedure or treatment is not useful/effective, and in some cases may be harmful |

treatment of cardiomyopathies. 6 The description of the recommended class and evidence level follow the descriptive method of conventional guidelines ( Tables1 and 2 ). This was determined by the author based on published papers, and was ultimately determined by peer review by team members and external evaluators. In the cardiovascular field in Japan, the conventional recommended classification and level of evidence are widespread, and it is easy to achieve consistency with overseas evidence levels. On the other hand, the medical information service business Minds,

|Table 2. Level of Evidence|| |---|---| |LevelA| Data derived from multiple randomized clinical trials or meta-analyses | |LevelB| Data derived from a single randomized clinical trial or large-scale non-randomized studies | |Level C| Consensus of opinion of the experts and/or small-size clinical studies, retrospective studies, and registries |

|Table 3. MINDS Grades of Recommendations|| |---|---| |GradeA| Strongly recommended and supported by strong evidence | |Grade B| Recommended with moderately strong supporting evidence | |Grade C1| Recommended despite no strong supporting evidence | |Grade C2| Not recommended because of the absence of strong supporting evidence | |Grade D| Not recommended as evidence indicates that the treatment is ineffective or even harmful |

The grade of recommendation is determined based on a comprehensive assessment of the level and quantity of evidence, variation of conclusion, size of effectiveness, applicability to the clinical setting, and evidence on harms and costs. (Adapted from MINDS Treatment Guidelines Selection Committee. 2007 7 )

which is operated by the Japan Agency for Medical Function Assessment, describes different grades and evidence classifications in the “Guide to Creating Minds Medical Care Guidelines 2007” ( Tables3 and 4 ). 7 Therefore, in this guideline, the specific diagnosis and treatment contents are described together as much as possible and displayed in tabular form (recommended class/evidence level and Minds recommended grade/evidence classification).

Whereas the notation of evidence levels in conventional guidelines is based on the belief that the results of a random

| Table 4. MINDS Levels of Evidence (Levels of Evidence in Literature on Treatment) || |---|---| || Systematic review/meta-analysis of randomized controlled trials | || One or more randomized controlled trials| ||Non-randomized controlled trials| |iva|Analytical epidemiological studies (cohort studies)| |b| Analytical epidemiological studies (case-control studies and cross-sectional studies) | ||Descriptive studies (case reports and case series)| || Not based on patient data, or based on opinions from a specialist committee or individual specialists |

(Adapted from MINDS Treatment Guidelines Selection Committee. 2007 7 )

ized interventional trial are more valid than those of a registered study, the Minds evidence classification is based on the study in which the evidence level was based. The Minds classification indicates the type of study, and these notations are not the same. Therefore, the Minds recommended grades and evidence classifications in these guidelines are for reference only.

I. General Remarks on Amyloidosis

1. Concept and Classification of Amyloidosis

▋ ▋ 1.1 Disease Concept and Classification of Amyloidosis

Amyloidoses are diseases in which misfolding proteins form $\beta$ -sheet structured amyloid fibrils, that are deposited in multiple organs throughout the body, leading to organ dysfunction. Amyloidosis is broadly divided into systemic amyloidosis, in which amyloid is deposited in multiple organs, and localized amyloidosis, in which amyloid is localized in specific organs. It is classified further by precursor proteins and their corresponding clinical disease types 4 ( Table5 ).

More than 30 amyloid precursor proteins have been identified. Of these, amyloid fibrils, which are formed by immunoglobulin light chains, TTR, or amyloid A protein (AA) accumulate in the heart and cause cardiac dysfunction ( Table6 ). ATTR can be either ATTRv amyloidosis (formerly called FAP) with a pathogenic mutation in the TTR gene, or systemic ATTRwt amyloidosis without a mutation (formerly called SSA). 8 AA amyloidosis is associated with chronic inflammatory diseases such as rheumatoid arthritis, vasculitis syndrome, and autoimmune diseases, and mainly presents with renal disorders such as proteinuria and renal failure, but rarely with cardiac involvement. 9

▋ ▋ 1.2 Concept of Cardiac Amyloidosis (CA)

CA is a clinical disorder in which the interstitial deposition of amyloid fibrils causes morphological and functional abnor

malities of the heart. As mentioned above, CA is divided into three main types, AL, ATTRv, and ATTRwt amyloidosis, which share common features in terms of clinical signs and laboratory findings. In general, CA presents with heart failure mainly due to diastolic dysfunction with cardiac hypertrophy, in addition to left ventricular (LV) systolic dysfunction, atrioventricular (AV) conduction disorder, atrial fibrillation (AF), and fatal arrhythmias. Prognosis depends on the extent of cardiac involvement and the subtype of amyloid precursor protein. In recent years, effective treatments and drugs have been developed for AL and ATTR. The clinical practical guideline for cardiomyopathy compiled by the JCS/Japan Heart Failure Society also classifies CA as one of the secondary cardiomyopathies clearly associated with a specific cause or systemic disease that needs to be differentiated, and emphasizes the importance of appropriate diagnosis. 6

▋ ▋ 1.2.1 Systemic AL Amyloidosis

AL amyloidosis is a plasma cell dyscrasia characterized by the pathologic production of amyloid fibrils formed by misfolded monoclonal light chains that are deposited in tissues and cause organ dysfunction. Although the major organs involved are the kidney, heart, liver, gastrointestinal tract, and peripheral nerves, any organ throughout the body can be damaged; AL amyloidosis can damage multiple organs, or only a single organ (e.g., heart, kidney, liver). Monoclonal immunoglobulin light chains are mainly produced from plasma cells in the bone marrow. Amyloidosis caused by abnormal production of free light chains from bone marrow plasma cells that does not meet the conditions for multiple myeloma is called “primary AL amyloidosis”,

|Table 5.Classification of Amyloidosis|||| |---|---|---|---| || Fibril protein| Precursor protein|Clnical disease name| |Systemic amyloidosis|||| |Hereditary|ATTRv|Transthyretin, variants|Hereditary transthyretin amyloidosis| |Hereditary|Agel|Gelsolin, variants|Hereditary gelsolin amyloidosis| |Hereditary|AApoAI|Apolipoprotein A-l, variants|Hereditary apolipoprotein A-l amyloidosis| |Hereditary|AApoAll|Apolipoprotein A-ll, variants|Hereditary apolipoprotein A-ll amyloidosis| |Hereditary|AApoCll|Apolipoprotein C-ll, variants | Hereditary apolipoprotein C-ll amyloidosis| |Hereditary|AApoC!ll|Apolipoprotein C-Ill, variants |Hereditary apolipoprotein C-ll amyloidosis| |Hereditary|ALYS|Lysozyme, variants |Hereditary lysozyme amyloidosis| |Hereditary|AFib|Fibrinogen a, variants|Hereditary fibrinogen amyloidosis| |Hereditary|ACys|Cystatin C, variants|Hereditary cystatin C amyloidosis| |Hereditary|AB2M|β2-Microglobulin, variants |Hereditary β2-microgloblin amyloidosis| |Hereditary|APrP|Prion, variants|Prion protein systemic amyloidosis| | Non-hereditary|ATTRwt|Transthyretin, wild type|Wild-type transthyretin amyloidosis| | Non-hereditary|AL|Immunoglobulin light chain|AL amyloidosis| | Non-hereditary|AH|Immunoglobulin heavy chain|AH amyloidosis| | Non-hereditary|AA|Serum amyloid A|AA amyloidosis| | Non-hereditary|AB2M|β2-Microglobulin, wild type| Dialysis-related amyloidosis| |Localized amyloidosis|||| |Brain|AB| Amyloid-β precursor protein (wild-type, variants) | Alzheimer disease, Cerebral amyloid angiopathy (CAA) | |Brain|APrP| Prion protein (wild-type, variants)|Creutzfeldt-Jakob disease| |Brain|ACys|Cystatin C, variants |Hereditary cerebral amyloid angiopathy| |Brain|ABri|ABri precursor protein, variants|Familial British dementia| |Brain|ADan|ADan precursor protein, variants|Familial Danish dementia| | Endocrine|ACal|(Pro)calcitonin|Associated with medullary thyroid cancer| | Endocrine|AIAPP|Amylin|Associated with type II DM| | Endocrine|AANP|ANP|Isolated atrial amyloidosis| | Endocrine|APro|Prolactin| Prolactin-producing tumor| |Cornea|ALac|Lactoferrin| Corneal amyloidosis | |Cornea|Aker|Kerato-epithelin|Corneal amyloidosis| |Others|AMed|Lactadherin|Aortic medial amyloidosis| |Others|AIns|Insulin|Insulin-derived amyloidosis (iatrogenic)| |Others|AL|Immunoglobulin light chain|Localized nodular amyloidosis |

(Modified from Benson MD et al. 2018 4 )

|Table 6. Classification of Cardiac Amyloidosis||||||||| |---|---|---|---|---|---|---|---|---| || Precursor protein | Underlying disorder |Organinvolvement|Organinvolvement|Organinvolvement|Organinvolvement||Treatment| || Precursor protein | Underlying disorder |Heart|Kidneys|Liver|PN(AN)|Other|Treatment| |AL| Monoclonal immunoglobulin light chain | Plasma cell dyscrasia |++++|++++|T+T|+（+)| Soft tissue, gastrointestinal | Chemotherapy or ASCT | |ATTRwt|Wild-type TTR|Aging|++++++|||| Carpal tunnel syndrome |TTR stabilizer| |ATTRv|Mutant TTR| Mutations in TTR gene |TT||| +++++ （+++) | Gastrointestinal, retina | Liver transplant TTR stabilizer Oligonucleotide therapy | |AA|SAA| Inflammatory. disorders (RA, JIA) |一+|+++++|||Gastrointestinal| Suppression of inflammation |

(Modified from Wechalekar AD, et al. 2016 8 )

and amyloidosis associated with B-cell malignancy such as multiple myeloma is called “secondary AL amyloidosis”. In each case, monoclonal immunoglobulin-producing cells are the treatment target.

▋ ▋ 1.2.2 Systemic ATTRwt Amyloidosis (Formerly Called Senile Systemic Amyloidosis [SSA])

ATTRwt amyloidosis is caused by wild-type TTR, which mainly damages heart, tendon, and ligament tissue (e.g., carpal tunnel, ligamentum flavum), the kidney, thyroid,

 Figure1. ( A ) Kaplan-Meier curves for overall survival (OS) from diagnosis among 758 patients based on the new staging system; shaded areas indicate the $95\%$ CI estimates. ( B ) Kaplan-Meier curves for OS from diagnosis among 512 patients based on the staging system, substituting B-type natriuretic peptide for N-terminal pro–B-type natriuretic peptide. (Adapted from Kumar S, et al. 2012 15 )

peripheral nerves, and lungs. It is common in men over 60 years of age. Although aging is thought to be involved in the pathogenesis, the underlying mechanism remains unclear. In Japan, this disease is considered to be misdiagnosed frequently. TTR tetramer stabilizer has been reported to improve the prognosis of cardiac involvement in patients with ATTR. 3

▋ ▋ 1.2.3 Hereditary ATTR (ATTRv Amyloidosis; Formerly Called Familial Amyloid Polyneuropathy [FAP])

ATTRv amyloidosis is a hereditary disease in which TTR forms amyloid that gets deposited in the interstitium of tissues due to a mutation in the TTR gene, causing damage to the nerves, heart, digestive tract, kidneys, and eyes. In Japan, there are large numbers of patients with ATTRv amyloidosis (V30M variant) in Kumamoto and Nagano Prefectures. Although ATTRv amyloidosis is an autosomal dominant disease, about half of the patients in areas other than Kumamoto and Nagano Prefectures show no clear family history. The age of onset (20–70 years or older) and symptoms vary. Early diagnosis is important because the effects of various treatments (e.g., liver transplantation, TTR stabilizers, oligonucleotide therapy) are expected to be seen in the early stage of the disease.

2. AL Amyloidosis

Amyloidoses are protein conformational diseases caused by the misfolding and aggregation of autologous proteins that are deposited in tissues in the form of amyloid fibrils. Amyloid light-chain (AL) amyloidosis is a multi-system disorder caused by a malignant plasma cell clone that results in insoluble fibrillary deposition. AL amyloidosis is seen in 10 to $15\%$ of patients who are diagnosed with multiple myeloma (MM). 10 MM is defined as plasma cells ${\geq}10\%$ with serum monoclonal (M-) protein ${\geq}3\,\mathrm{g}/\mathrm{d}\mathrm{L}$ or 24-h urinary M-protein ${\geq}500\,\mathrm{mg}$ . Patients are diagnosed as having symptomatic MM when they show any of the following myeloma-defining events (MDEs): hypercalcemia, renal

dysfunction, anemia, or bone lesions. AL amyloidosis not associated with MM is called primary AL amyloidosis. The epidemiology and prognosis of primary AL amyloidosis are discussed.

AL amyloidosis is known to be associated with MM, but the characteristics of plasma cells were reported to be different between primary AL amyloidosis and MM. 11 , 12 Translocation t(11;14), which is known to be found in about $15\%$ of MM cases, is found more frequently (about $50\%$ ) in patients with AL amyloidosis. 13 According to these findings, the treatment of AL amyloidosis should be considered differently from that of MM.

Epidemiologic data on primary AL amyloidosis are limited. An analysis in the United States of America between 2007 and 2015 showed that the incidence was 9.7 to 14.0 cases per million person-years, and the prevalence increased significantly from 15.5 in 2007 to 40.5 cases per million in 2015, an annual percentage change (APC) of $12\%$ . 14 It is suspected that more patients can survive with the advances in diagnosis and treatment. In this report, prevalent patients had a mean age of 63 years, and $55\%$ were male. In Japan, the prevalence of patients with amyloidosis (not only AL amyloidosis, but also ATTR amyloidosis) was reported as 6.1 per million persons using the data on intractable disease of the Japanese Ministry of Health and Welfare. There is little information available concerning the status of AL amyloidosis, such as its incidence and the demographic features of AL amyloidosis patients in Japan.

The prognosis of patients with AL amyloidosis is highly dependent on the involved organs and the severity of organ damage. The revised Mayo Clinic staging system is frequently used for predicting prognosis. 15 In this staging system, patients are assigned a score of 1 for each difference in free light chain (FLC; difference between involved and uninvolved FLC) ≥ 18mg/dL (180mg/L), $\mathrm{cTnT}\!\geq\!0.025\mathrm{ng/mL}$ , and NT-proBNP $\geq\!1,\!800\,\mathrm{ps}/\mathrm{mL}$ , creating stages I to IV with scores of 0 to 3 points. The median OS from diagnosis of patients with stages I, II, III, and IV was 94.1, 40.3, 14, and 5.8 months, respectively ( Figure1 ). 15

JCS 2020 Guideline on Diagnosis and Treatment of CA

3. ATTRwt Amyloidosis (Formerly Called SSA)

▋ ▋ 3.1 Concept of ATTRwt Amyloidosis

ATTRwt amyloidosis is caused by the deposition of wildtype TTR-derived amyloid fibrils. TTR is stable in the bloodstream if it forms tetramer, but upon aging, it becomes unstable, develops a misfolded monomer, and is ultimately deposited as amyloid fibrils. Because ATTRwt amyloidosis is often diagnosed in older patients, it has traditionally been known as SSA. However, the term ATTRwt amyloidosis is now recommended because amyloidosis was recently classified according to a precursor protein. 16

▋ ▋ 3.2 Pathophysiology of ATTRwt Amyloidosis

In 1990, TTR was reported as a precursor protein of ATTR. 17 TTR, which is mainly produced and secreted from the liver, binds and transfers thyroid hormone and retinol-binding protein with vitamin A. TTR is stable in circulation as long as it forms a tetramer, but in the case of aging, TTR becomes unstable, develops a misfolded monomer, and becomes a substrate of amyloid fibrils. Although the precise mechanism is unknown, posttranslational biochemical changes in hepatic TTR or chaperone protein are suspected as a cause of instability of the TTR tetramer. 18 , 19

TTR-derived amyloid fibril is deposited in various tissues, such as the heart, lung, kidney, intestinal tract, adipose tissue, joints, and ligaments. However, the joints, ligaments, and heart are the predominant tissues in which symptoms become apparent (e.g., carpal tunnel syndrome, spinal canal stenosis, ventricular hypertrophy, arrhythmia, heart failure). 20 Bilateral carpal tunnel syndrome is one of the earliest symptoms that appears in patients with ATTRwt amyloidosis, usually before cardiac symptoms when they are in their 50s to 70s. In patients undergoing carpal tunnel release surgery (median age, 68 years), $10.2\%$ had a positive biopsy for amyloid and $2\%$ had cardiac involvement. 21 A study that compared 56,032 patients who underwent surgical treatment for carpal tunnel syndrome and a sex- and agematched cohort from the general population demonstrated that carpal tunnel syndrome was associated with a higher incidence of heart failure. 22 It was also reported that the carpal tunnel syndrome symptoms appeared about 7 years before cardiac symptoms. 23 These results suggest that, in contrast to AL amyloidosis, ATTRwt amyloidosis develops over the long term.

▋ ▋ 3.3 Epidemiology of ATTRwt Amyloidosis

The deposition of TTR-derived amyloid fibrils progresses gradually with aging. By analyzing autopsy samples, amyloid deposition was detected in about $25\Bar{\%}$ and $37\%$ of those over 80 and 90 years of age, respectively. 24 , 25 In Japan, wild-type TTR-derived amyloid fibrils were detected in $12\%$ of autopsy samples. 26 If the included patients were limited to those with heart failure with preserved ejection fraction (HFpEF), amyloid fibrils were documented in about $40\%$ of those over 80 years of age. 27 However, these data do not reflect the precise prevalence of ATTRwt amyloidosis because most of these patients had not been diagnosed before death because of the small amounts of deposition and lack of symptoms.

It has been shown that scintigraphy using a tracer with a high affinity for calcium has a high positive predictive value for ATTR based on a comparison of histology samples. 1 Among patients aged over 60 years admitted due to HFpEF and LV hypertrophy $(\geq\!12\,\mathrm{mm})$ , $13.3\%$ showed moderate-to-severe uptake on $^{99\,\mathrm{m}}\mathrm{T}\mathrm{c}{-3,3}$ -diphosphono1,2-propanodicarboxylic acid ( 99m Tc-DPD) scintigraphy. 28 ATTRwt amyloidosis, diagnosed using 99m Tc-PYP scintigraphy, is prevalent in $16\%$ of patients with severe calcific aortic stenosis undergoing transcatheter aortic valve implantation. 29

It has been reported that the prevalence of ATTRwt amyloidosis does not differ by race. 30 On the other hand, a remarkable sex difference has been reported. Significantly higher numbers of male compared with female patients are diagnosed with ATTRwt amyloidosis by histology or 99m TcPYP scintigraphy. 31 – 33 However, the mechanism underlying this sex difference remains unknown.

▋ ▋ 3.4 Diagnosis of ATTRwt Amyloidosis

Conduction system disturbances, such as complete left bundle branch block and AF, are more frequently observed in patients with ATTRwt amyloidosis compared with those with AL amyloidosis. Echocardiography and cardiac magnetic resonance imaging (MRI) have revealed that the extent of cardiac hypertrophy is more robust in patients with ATTRwt amyloidosis. 31 , 34 , 35 The value of high-sensitive troponin T is usually higher in patients with ATTRwt amyloidosis than in those with hypertensive heart disease or hypertrophic cardiomyopathy, but lower than in those with AL amyloidosis. 36

To confirm diagnosis, the detection of TTR-derived amyloid fibrils in biopsy specimens is necessary. In addition, genetic testing is needed to differentiate ATTRwt amyloidosis and ATTRv amyloidosis. Because cardiac amyloid fibril deposition can be detected in patients with ATTRwt cardiomyopathy with high probability, endomyocardial biopsy is regarded as the gold standard for confirming the diagnosis. 37 However, due to the relatively invasive procedure, endomyocardial biopsy cannot be performed for all suspected patients. A diagnosis of ATTRwt amyloidosis is acceptable if the echocardiography and cardiac MRI (CMR) are highly suggestive of ATTRwt amyloidosis and amyloid deposition is detected in other tissues (see “ Chapter I, Section 5. Diagnostic Criteria ”).

▋ ▋ 3.5 Prognosis of ATTRwt Amyloidosis

The prognosis of ATTRwt amyloidosis is better than that of AL amyloidosis, but it is not so good compared with heart failure with other etiologies. The survival of patients with ATTRwt amyloidosis was thought to be over 5 years, 38 , 39 but recent reports suggest that the median survival after diagnosis is 43–47 months. 31 , 33 In 2018, tafamidis, a TTR stabilizer, was reported to be associated with reductions in all-cause mortality and cardiovascular-related hospitalizations, and to reduce the decline in functional capacity and quality of life (QOL) compared with placebo in patients with ATTR cardiomyopathy. 3 Based on these results, tafamidis has been approved for patients with ATTR cardiomyopathy in Japan since 2019. Further evaluation is needed to evaluate the effect of tafamidis on the prognosis of ATTR cardiomyopathy in clinical practice.

Table7. Clinical Features of ATTRv Amyloidosis in Japan

|| Val30Met (p.Val50Met) type (endemic areas) | Val30Met (p.Val50Met) type (non-endemic areas) | Non-Val30Met (p.Val50Met) type (non-endemic areas) | |---|---|---|---| |Onset age|Generally early (20-60 years old)|Generally late (35-90 years old)|| | Family history of the disease|High (80-100%)|Low (about 30%)| Varied (20-85 years old) Low (about 30%) | |Sex|Equal frequency for both sexes|Male-dominant (male: about 80%)| Male-dominant (male: about 60%) About 10 years | |Survival after the onset|About 10 years|About 7 years| Male-dominant (male: about 60%) About 10 years | | Types of ATTR amyloid fibrils deposited in patients |Type B|TypeA|TypeA| | 99mTc PYP scintigraphy|Negative|Positive|Positive| | Initial symptoms|||| |Polyneuropathy|About 60%|About 80%|About30%| | Autonomic symptoms|30-60%|2-10%|About25%| |Cardiac symptoms||2-4%|About35%| |Carpal tunnel syndrome|0-10%|About5%|| | Renal symptoms||0—2%|0o%| | Ocular symptoms||6-13%|About 10%|

4. ATTRv Amyloidosis (Formerly Called TTR-FAP)

▋ ▋ 4.1 Disease Concept and Pathology

ATTRv amyloidosis is the most frequent and representative form of autosomal dominant heritable systemic amyloidosis. 4 , 40 As treatment for ATTRv amyloidosis is different from that for ATTRwt amyloidosis, it is very important to distinguish the two. Furthermore, when a single case of ATTRv amyloidosis is diagnosed, an active search should be considered because there can be multiple carriers of the mutant TTR gene in the patient’s family, and early diagnosis and treatment are possible. “ Chapter II, Section 10. Genetic Testing and Counseling ” details the genetic testing and counselling available for this condition.

TTR, the causative molecule of this disease, is a plasma protein primarily produced in the liver and secreted into the blood. Other known sites of TTR production include the choroid plexus, retinal pigment epithelium of the eyes, and α cells of the pancreatic islets of Langerhans. Although the TTR gene encodes 147 amino acids, the first 20 comprise a signaling peptide that is cut off prior to being expelled from the cell, and the remaining 127 comprise the TTR found in the blood. It is recommended that the mutation sites of the secretory TTR (127 amino acid residues) and the TTR coding sequence (147 amino acid residues) be written together (e.g., Val30Met [p.Val50Met] to describe the TTR mutation that causes this condition. 4 TTR forms a tetramer in the blood and is responsible for the transport of thyroid hormone (thyroxine: T4) and vitamin A (retinol) through the binding of retinol-binding protein (RBP).

Over 150 types of mutants have been reported for the TTR gene, the majority of which are amino acid mutations by means of single residue replacement. 41 The Val30Met (p.Val50Met) mutation, where the 30th amino acid (50th amino acid in the TTR coding sequence) residue, valine, is replaced with a methionine residue, was the first to be reported, and has been observed most frequently. 40 With respect to ATTRv amyloidosis, which is caused by the same Val30Met (p.Val50Met) mutant, the endemic areas in Japan (Kumamoto and Nagano Prefectures) have reported numerous instances of early onset, and the non-endemic areas (other regions) numerous instances of late onset. In

addition to the age at onset, differences have been reported in regard to sex, presence/absence of a family history the disease, and symptoms ( Table7 ). 42 – 44 Many other TTR mutations (non-Val30Met [p.Val50Met]) have been reported, and cardiac symptoms are relatively frequent first symptoms ( Table7 ). 44 Approximately $3.4\%$ of African-Americans are known to have the Val122Ile (p.Val142Ile) TTR variant and be at increased risk of developing TTR amyloid cardiomyopathy (ATTR-CM). 45

The fact that the TTR tetramer destabilizes and dissociates into monomers in the event of a TTR gene mutation is important for the process of amyloid formation. 46 In a basic study involving various TTR mutants, it was reported that the greater the instability of TTR according to the type of mutation, the more common are symptoms such as oculo-leptomeningeal and peripheral nervous system symptoms, while the greater the stability of TTR, the greater is the tendency to experience cardiac symptoms. 47 Biochemical analyses of amyloid fibrils that deposit in the tissues in this condition have shown that there are cases in which both full-length TTR (127 amino acid residues) and C-terminus TTR fragments are detected (Type A), as well as cases in which only full-length TTR is detected (Type B). 48 The relationship between the pathogenesis of this condition and TTR and C-terminus fragmentation has also been discussed.

▋ ▋ 4.2 Epidemiology

Previously, large families of ATTRv amyloidosis (Val30Met [p.Val50Met] type) were thought to exist only in Portugal, Japan (Kumamoto and Nagano Prefectures), and Sweden, but in recent years, improved diagnostic techniques and widespread knowledge about the disease has led to more cases of ATTRv amyloidosis being reported around the world. Globally, it is estimated that approximately 10,000 people have this condition. 49 Although many cases with the Val30Met (p.Val50Met) mutant have been reported in Japan, there have also been numerous reports of other family types of ATTRv amyloidosis caused by other TTR mutants ( Figure2 ). 44 The results of an epidemiological investigation by the MHLW, titled “Research Team on Amyloidosis,” estimated that there were approximately

 Figure2. Distribution of ATTRv amyloidosis and TTR mutants in Japan. (Source: Prepared based on Yamashita T, et al. 2018 44 )

|Major symptoms|| |---|---| |Polyneuropathy|Ala25ser(p.Ala45ser)| |Polyneuropathy| Val28Met(p.Val48Met) Val30Leu(p.Val50Leu) Phe33Val (p.Phe53Val) Asp38Ala(p.Asp58Ala) Glu42Gly (p.Glu62Gly) Phe44Ser (p.Phe64Ser) Gly47Arg (p.Gly67Arg) Gly47Val (p.Gly67Val) Thr49lle (p.Thr69lle) Thr49Ala (p.Thr69Ala) Thr49Ser(p.Thr69Ser) Ser50Arg (p.Ser70Arg) Glu54Lys(p.Glu74Lys) Leu55Pro (p.Leu75Pro) | || Gly47Arg (p.Gly67Arg) Gly47Val (p.Gly67Val) Thr49lle (p.Thr69lle) Thr49Ala (p.Thr69Ala) Thr49Ser(p.Thr69Ser) Ser50Arg (p.Ser70Arg) Glu54Lys(p.Glu74Lys) Leu55Pro (p.Leu75Pro) Glu61Lys(p.Glu81Lys) Val71Ala(p.Val91Ala) Ser77Tyr (p.Ser97Tyr) Lys80Arg (p.Lys100Arg) lle84Asn(p.lle104Asn) Ala97Gly (p.Ala117GIy) Ala109Ser (p.Ala129Ser) Leu58Arg (p.Leu78Arg) Tyr69lle (p.Tyr89lle) lle107Val (p.lle127Val) Tyr114His (p.Tyr134His) | || Leu58Arg (p.Leu78Arg) Tyr69lle (p.Tyr89lle) lle107Val (p.lle127Val) Tyr114His (p.Tyr134His) Ala120Ser (p.Ala140Ser) Ala120Thr (p.Ala140Thr) Asp18Glu (p.Asp38Glu) | | Carpal-tunnel syndrome and polyneuropathy || |Cardiac symptoms| Tyr114His (p.Tyr134His) Ala120Ser (p.Ala140Ser) Ala120Thr (p.Ala140Thr) Asp18Glu (p.Asp38Glu) Pro24Ser (p.Pro44Ser) Ala36Asp (p.Ala56Asp) Ala45Asp (p.Ala65Asp) Ser50lle (p.Ser70lle) Thr59Arg (p.Thr79Arg) Thr60Ala (p.Thr80Ala) Glu89Lys (p.Glu109Lys) Gln92Lys (p.GIn112Lys) Val94Gly (p.Val114Gly) Tyr114Ser (p.Tyr134Ser) Ala25Thr(p.Ala45Thr) Gly53Glu (p.Gly73GIu) | | Oculo-leptomeningeal symptoms | Ala25Thr(p.Ala45Thr) Gly53Glu (p.Gly73GIu) Tyr114Cys (p.Try134Cys) | |Asymptomatic|Arg104His (p.Arg124His)| || Val30Met(p.Val50Met)/ Arg104His (p.Arg124His) Vai30Met(p.Val50Met)/ Lys80Arg (p.Lys100Arg) | |Compound heterozygote||

830 people in Japan who had the condition. 50 As there may also be many undiagnosed cases, we believe that the actual number of such patients is even higher.

▋ ▋ 4.3 Symptoms

ATTRv amyloidosis exhibits various systemic organ symptoms, such as peripheral and autonomic nerve, cardiac, gastrointestinal, kidney, ocular, and central nervous system symptoms. During the advanced stage of the disease, patients tend to present with respiratory muscle paralysis, severe orthostatic hypotension, heart failure, fatal arrhythmia, nephrotic syndrome, kidney failure, protein-losing gastroenteropathy, and severe glaucoma, in addition to paralysis of the extremities. These details are described in “ Chapter II, Section $\mathbf{1.2}^{\ast}$ . It is important to suspect this condition when observing peripheral neuropathy and heart failure, which are difficult to explain from other diseases and pathologies.

▋ ▋ 4.4 Prognosis

When the condition is advanced, it can often lead to death as a result of severe peripheral neuropathy and cardiac, renal, and respiratory disorders. When left untreated, the mean life expectancy from onset for ATTRv amyloidosis is approximately 10 years (early-onset Val30Met [p.Val50Met])

in endemic areas, 51 and approximately 7 years for late-onset disease (late onset Val30Met (p.Val50Met) in non-endemic areas. 43 Even for ATTRv amyloidosis due to non-Val30Met (p.Val50Met) mutants, the mean life expectancy after onset is approximately 10 years if left untreated. 52

Although the mean life expectancy for ATTRv amyloidosis is almost the same in the US and Europe, 53 it is even shorter, at 2–3 years, in cases for which the main symptoms are cardiac symptoms, 54 which may take a long time to be diagnosed because they lack subjective symptoms at the early stages of the disease.

▋ ▋ 4.5 Diagnosis

This condition is diagnosed by identifying the clinical signs specific to the condition, detecting TTR-derived amyloid deposition, and identifying pathogenic mutations in the TTR gene. Many cases of late-onset disease with no family history have been reported in non-endemic areas (other than Kumamoto and Nagano Prefectures). In many such cases, it takes several years or more from the time of onset for the condition to be diagnosed. 44 The items in Chapter II describe in detail the images and biomarkers thought to be useful for diagnosing and evaluating the pathology of amyloid cardiomyopathy. Although nerve conduction tests are often conducted to evaluate the extent of nerve damage, they are not suitable for evaluating small fiber

|Clinical signs|Laboratoryfindings| |---|---| | Symptomsofheart failure |Atrial fibrillation, conduction system disorder (e.g., atrioventricular block, bundle branch block, intraventricular conduction| ||thickening (including right ventricle), atrial septal thickening, ventricular diastolic dysfunction (restrictive), granular| | (e.g., shortness of breath,edema), || |dizziness, and| (apical sparing), elevated B-type natriuretic peptides (BNP) and N-terminal pro-BNP, elevated cardiac troponin T/I, global diffuse myocardial late gadolinium enhancement in the subendocardial layers on cardiac magnetic resonance imaging, elevated native T1 and extracellular volume fraction in T1 mapping | |syncope||

neuropathy. The evaluation of nerve density in the epidermis using skin punch biopsy 55 and of simple sweat function 56 is actively used to evaluate small fiber neuropathy (see “ Chapter I, Section 5. Diagnostic Criteria ”).

5. Diagnostic Criteria

In the diagnosis of amyloidosis, it is important not only to detect amyloid deposition histologically, but also to confirm clinical findings caused by organ damage due to amyloid deposition. Therefore, to diagnose amyloidosis, it is necessary to first suspect this disease from the clinical symptoms caused by organ damage, distinguish it from other diseases based on supporting laboratory findings or imaging tests, and determine the final diagnosis. Especially for the diagnosis of CA, since many nonspecific clinical findings, such as AF, occur in older individuals, regardless of the background of amyloidosis, a comprehensive assessment of clinical and laboratory findings suggesting amyloid deposition in the heart is required. It is necessary to determine amyloid deposition in tissues histologically. Clinical signs or laboratory findings due to each type of amyloidosis are present. Next, using specific antibodies against individual precursor proteins ( κ chain, λ chain, TTR, AA, etc.), immunohistochemical staining is performed to categorize the disease type. For diagnosing AL, it is important to test blood/urine M protein and the free light-chain ratio to confirm the clonal proliferation of plasma cells in bone marrow. In ATTRv amyloidosis, it is necessary to examine TTR gene mutations using genetic testing. In recent years, evidence that 99m Tc-PYP is useful for the diagnosis of ATTR has been accumulated, 57 , 58 and diagnostic criteria using noninvasive imaging without biopsy have been proposed. 1 Because it is sometimes difficult to make a definitive diagnosis and disease typing, each disease type has a diagnostic category of either “definite” or “probable”.

Few official guidelines in Japan or overseas provide clear diagnostic criteria for systemic amyloidosis. For AL amyloidosis, the diagnostic criteria published by the International Myeloma Working Group are widely used as international criteria. 59 In Japan, the “Amyloidosis Clinical Practice Guidelines 2010”, 60 which is published by a research group on amyloidosis that conducts a project for overcoming intractable diseases funded by the MHLW, details the diagnostic criteria for AL, ATTR, AA, A β 2 M, and cerebral amyloidosis. However, given the recent advances in diagnostic technology and changes in the disease concepts, and the launch of new therapeutic agents, the revision of the diagnostic criteria for amyloidosis has become an unmet medical need. Therefore, the Japanese Research Group on Amyloidosis has revised the diagnostic criteria for AL, ATTRwt, and ATTRv amyloidosis based

on a consensus of each society (http://amyloidosisresearch-committee.jp). The following guideline describes the latest diagnostic criteria reflecting these contents.

▋ ▋ 5.1 Common Items of Diagnostic Criteria for Each Type of Amyloidosis

A, B, and C are common to each disease type.

A. Clinical signs and laboratory findings Clinical signs or laboratory findings due to each type of amyloidosis are present. B. Pathohistological findings In biopsied tissue samples, there are amyloid deposits exhibiting positive staining with Congo-red under a light microscope and show apple-green birefringence under a polarizing microscope. C. Amyloid typing Amyloid deposits show positive staining for each type of precursor protein.

Although each item included in “A” is characteristic of each type of disease, it is not a finding specific to amyloidosis, and is considered to be an entry point for diagnosis. The findings of cardiac involvement are common to each disease type, as noted Table8 .

Since the diagnostic criteria are mutually exclusive for each type, differential diagnosis is essential. The diagnosis flowchart is shown in Figure32 (“ Chapter IV. CA Diagnostic Algorithm ”).

▋ ▋ 5.2 Diagnostic Criteria for Systemic AL Amyloidosis

A. Clinical signs and laboratory findings Clinical signs or laboratory findings due to each type of amyloidosis are present ( Table9 ). B. Pathohistological findings In biopsied tissue samples, there are amyloid deposits exhibiting positive staining with Congo-red under a light microscope and showing apple-green birefringence under a polarizing microscope are observed (Note 1). C. Amyloid typing Amyloid deposits show positive staining for immunoglobulin light chains (Note 2). D. M protein M proteins are detected in blood or urine by immunoelectrophoresis, immunofixation, or free light chain (Note 3). E. Differential diagnosis Other conditions that may cause the clinical signs and laboratory findings of “A” are excluded. Especially in the case of CA, since ATTRwt amyloidosis with

|Table 9.Clinical Amyloidosis||| |---|---|---| || Clinical symptoms|Laboratory findings| |Cardiac amyloidosis|SeeTable8|SeeTable8| |Renal amyloidosis|Edema, weight gain| Proteinuria (>0.5 g/day. mainly albumin) Increasedserumcreatinine,decreasedeGFR,increased urinary NAG, increased urinary β2-microglobulin | |Hepatic amyloidosis|Hepatomegaly| Maximum liver longitudinal diameter >15 cm (excluding. heart failure), increased alkaline phosphatase (>1.5 times the upper limit of normal) | | Peripheral nerve amyloidosis | Smallfiber predominant polyneuropathy (a decline mainly in pain and temperature sensations in the extremities) |Nerve conduction abnormalities due to axonal disorders| | Autonomic amyloidosis |Orthostatic hypotension, diarrhea, constipation, dysuria|| | Gastrointestinal amyloidosis | Melena, nausea, loss of appetite, intestinal obstruction, malabsorption syndrome || | Tendon/ligament amyloidosis | Carpal tunnel syndrome (pain and numbness of the hand) | A delay in conduction latencies at wrist in nerve conduction tests | | Joint amyloidosis|Shoulder pad sign, joint swelling|| | Tongue amyloidosis|Macroglossia|| | Cutaneous amyloidosis |Scleroderma-lie thickening, nodules, purpura|| | Amyloidosis of other organs | Hard swelling of the thyroid gland, salivary glands, lymph nodes,etc.,claudication(due to vascular amyloid), myopathy (pseudohypertrophy) | Pattern of diffuse interstitial lung disease on computed tomography |

•B-cell malignancies such as multiple myeloma should be excluded. •The underlined text in the table shows the indicators of major organ damage described in the International Consensus Opinion. 61 In this Consensus Opinion, the index of CA is “thickness of the ventricular septum and posterior wall $(\mathord{>}12\,\mathsf{m m})^{\ast}$ . •AL amyloidosis is often negative on 99m Tc-pyrophosphate scintigraphy. •Monoclonal gammopathy of renal significance (MGRS) is due in part to renal amyloidosis.

monoclonal gammopathy of undetermined significance cannot be ruled out, ATTRwt should be ruled out using the ATTRwt amyloidosis diagnostic criteria/ diagnosis flowchart.

<Diagnosis Category>

Definite: At least one item of “A” and $^{+\mathbf{B}+\mathbf{C}+\mathbf{D}+\mathbf{E}}$ . Or, two or more items of “A” and $_{+\mathrm{B+C+E}}$ . However, when the relevant item of “A” is in the column of heart, kidney, or liver, and $_{\mathrm{B+C+E}}$ is satisfied, the case is diagnosed as systemic, even if only one organ is damaged. Probable: At least one item of “A” and ${\bf+B{+D{+E}}}$ .

\*It is difficult to stain with immunohistochemistry using specific antibodies against κ - or λ -light chain. Therefore, even if no clear result is acquired in immunostaining for immunoglobulin light chain, a “Probable” diagnosis is established assuming a case in which treatment should be started immediately after ruling out ATTR.

(Note1) Biopsy samples can be obtained from the abdominal wall fat, labial salivary glands, digestive tract, bone marrow, etc., and do not necessarily need to be collected from damaged organs. If there are no symptoms with only gastrointestinal lesions, localized gastrointestinal amyloidosis may be present. The occurrence/exacerbation of symptoms such as melena, diarrhea, and constipation needs to be monitored regularly. Localized AL amyloidosis is often asymptomatic or without M protein, so the diagnosis

is confirmed by $_{\mathrm{B+C}}$ .

(Note2) For coexisting cases of monoclonal gammopathy of undetermined significance and ATTRwt amyloidosis, it is necessary to prove the type of immunoglobulin light chain that matches the type of M protein in the Congo red-positive site. AL κ or AL λ $(+)$ , ATTR (−), and AA (−) should be confirmed by immunostaining, or the amyloid precursor protein should be identified using laser microdissection (LMD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in biopsy tissue samples. If it is difficult to perform the test at your own facility, an analysis request can be made to the Research Group on Amyloidosis (http://amyloidosis-research-committee.jp/).

(Note3) For the detection of M protein, it is recommended to examine serum immunofixation, serum free light chain (FLC), and urine immunofixation. Immunofixation is more sensitive than immunoelectrophoresis. Serum FLC should be assessed based on the difference between elevated pathological light chain-type and non-elevated light chaintype (difference FLC: dFLC) rather than by the absolute values of k and l chains or their ratio. Especially when dFLC ${\geq}50\,\mathrm{mg/L}$ , it is useful also as evaluation of treatment effects.

▋ ▋ 5.3 Diagnostic Criteria for Systemic ATTRwt Amyloidosis

Table10. Clinical Symptoms and Laboratory Findings Suggesting ATTRwt Amyloidosis

||Clinical symptoms|Laboratory findings| |---|---|---| |Cardiac amyloidosis|See Table 8|See Table 8| | Peripheral nerve amyloidosis | Small fiber predominant polyneuropathy (a decline mainly in pain and temperature sensations in the extremities) | Decreased intraepidermal nerve fiber density in skin biopsy | | Tendon and ligament amyloidosis | Carpal tunnel syndrome (numbness and pain of the hand), spinal canal stenosis (lumbago, gait disturbance), tendon rupture | A delay in conduction latencies at wrist in nerve conduction tests, spinal MRI |

•If an atypical case is suspected, the above findings are not applicable.

of amyloidosis are present ( Table10 ). B. Pathohistological findings In biopsied myocardium or other tissues, there are amyloid deposits exhibiting positive staining with Congo-red under a light microscope and showing apple-green birefringence under a polarizing microscope are observed (Note 1). C. Amyloid typing Amyloid deposits show positive staining for TTR (Note 2). D. Scintigraphy Intensely diffuse myocardial uptake on 99m Tc-PYP scintigraphy is confirmed (Note 3). E. Genetic testing No pathogenic mutations involve amino acid alterations in the TTR gene. F. M protein is not detected (Note 4). G. Differential diagnosis 1. Localized ATTRwt amyloidosis, which is limited to tendon/ligament tissues, should be excluded. 2. Other diseases that cause clinical signs or laboratory findings of “A” should be excluded. However, it should be noted that ATTRwt amyloidosis can be complicated with other diseases causing cardiac hypertrophy.

<Diagnosis Category> Definite: $_{\mathrm{A+B+C+E+G1}}$ . Probable: $_{\textrm{A+D+E+F+G}2}$ .

\*“Probable” is set for the noninvasive diagnosis of systemic ATTRwt amyloidosis without biopsy using 99m Tc-PYP scintigraphy.

(Note1) In the case of ATTRwt amyloidosis, the detection rate is low for amyloid in abdominal fat pad needle aspiration biopsy, skin biopsy, gastrointestinal biopsy, lip biopsy, etc. Therefore, if amyloid is not detected at these biopsy sites, a myocardial biopsy should be planned. If the disease is strongly suspected due to clinical signs or other laboratory findings, it needs to be detected by repeated biopsy from each tissue site. Amyloid deposition in ATTRwt amyloidosis shows weak staining with Congo-red and weak apple-green birefringence under polarized light. If it is difficult to judge at your own facility, it is recommended to consult with the research group or specialized facilities. (Note2) ATTR $(+)$ , AL κ (−), AL λ $(-)$ , and AA $(-)$ should be confirmed by immunostaining, or the amyloid precursor protein should be identified using LMD and LC-MS/MS in biopsy tissue samples. If it is difficult to

perform the test at your own facility, an analysis request can be made to the Research Group on Amyloidosis (http:// amyloidosis-research-committee.jp/).

(Note3) Visual grading method using frontal planar images taken 3h later (Grade 0: No accumulation in the heart; Grade 1: Mild accumulation in the heart weaker than the ribs; Grade 2: Moderate degree in the heart equivalent to the ribs accumulation; Grade 3: Higher accumulation in the heart than in the ribs; Grade 2 or higher is positive), or a quantitative validation method using planar images taken 1h later (a heart-to-contralateral [H/CL] ratio ${\geq}1.5$ ) should be used for evaluation. (For the details of myocardial scintigraphy, please refer to “ Chapter II, Section 6. Nuclear Medicine Examination ”.)

(Note4) No abnormalities in the immunoglobulin free light-chain $\kappa/\lambda$ ratio. In addition, no M protein in serum or urine immunofixation electrophoresis.

▋ ▋ 5.4 Diagnostic Criteria for Systemic ATTRv Amyloidosis

A. Clinical signs and laboratory findings Clinical signs or laboratory findings due to each type of amyloidosis are present ( Table11 ). B. Pathological examination findings In biopsied tissue, there are amyloid deposits exhibiting positive staining with Congo-red under a light microscope and showing apple-green birefringence under a polarizing microscope are observed (Note 1). C. Amyloid typing Amyloid deposits show positive staining for TTR (Note 2). D. Scintigraphy Intensely diffuse myocardial uptake on 99m Tc-PYP scintigraphy is confirmed (Note 3). E. Genetic testing A pathogenic mutation leading to a change of amino acids in the TTR gene.

<Diagnosis Category> Definite: $_{\mathrm{A+B+C+E}}$ . Probable: $_{\mathrm{A+B+E}}$ or $_\mathrm{A+D+E}$ .

\*Considering FAP patients with young onset in the focus area (Kumamoto and Nagano Prefectures) as a typical clinical picture of ATTRv amyloidosis in Japan, monoclonal gammopathy of undetermined significance (MGUS) is seldom concurrent, and there is little need to check M protein. By contrast, ATTRwt amyloidosis is reportedly frequently associated with MGUS (10–

Table11. Clinical Symptoms and Laboratory Findings Suggesting ATTRv Amyloidosis

|| Clinical symptoms|Laboratory findings| |---|---|---| | Peripheral nerve amyloidosis | Sensory/motor polyneuropathy, small fiber predominant polyneuropathy (a decline mainly in pain and temperature sensations in the extremities, atrophy and weakness of the extremities) | Nerve conduction abnormalities due to axonal disorders,decreased intraepidermal nerve fiber density in skin biopsy, enlargement of dorsal root ganglion and proximal sciatic nerve in MR neurography | |Autonomic amyloidosis| Orthostatic hypotension, vomiting, diarrhea, constipation, dysuria, dysgenesis, dysphoria, etc. | Decreased cardiac uptake in MIBG myocardial scintigraphy, laser Doppler imaging of skin blood flow, sweat function test, R-R interval fluctuation, Schellong test, EGG, etc. | | Tendon/ligament amyloidosis | Carpal tunnel syndrome (numbness and pain of the hand) | A delay in conduction latencies at wrist in nerve conduction tests | |Cardiac amyloidosis|See Table 8|See Table 8| |Renal amyloidosis| Edema, weight gain|Proteinuria, etc.| |Ocular amyloidosis| Dry eyes, vitreous opacity, glaucoma, irregular pupils, etc. |Increased intraocular pressure, etc.| | Central nervous system amyloidosis | Transient focal neurological episodes, disturbance of consciousness, cerebral hemorrhage, etc. | Imaging of meninges in contrast-enhanced head or spine MRl, cerebral hemorrhage including microhemorrhage, etc. | |Other organ amyloidosis|Hypoglycemia, hypothyroidism, etc.||

•Age of onset: Most cases for the Japanese focus area of FAP (Kumamoto and Nagano Prefectures) tend to occur in patients in their 20s to 40s. However, in other areas, onset is more likely after the age of 50 years. About half of patients do not have a clear family history. •Inheritance pattern: Autosomal dominant inheritance, but family history may not be known by interviews alone. •It should be noted that various symptoms (e.g., peripheral nerve type, cardiac type, cerebral meningeal vascular type, ocular type) are exhibited depending on the TTR gene variant. Even within the same family, the age of onset can vary greatly.

$18\%$ ), and $39\%$ of the patients with ATTRwt amyloidosis have abnormal free light-chain values. Therefore, the diagnosis of ATTRwt amyloidosis requires the absence of M protein, but the item related to M protein is excluded from the diagnostic criteria of ATTRv amyloidosis.

(Note2) ATTR $(+)$ , AL κ (−), AL λ $(-)$ , and AA (−) should be confirmed by immunostaining, or the amyloid precursor protein should be identified using LMD and LC-MS/MS in biopsy tissue samples. If it is difficult to perform the test at your own facility, an analysis request can be made to the Research Group on Amyloidosis (http:// amyloidosis-research-committee.jp/).

(Note1) Amyloid can be detected in abdominal fat pad needle aspiration biopsy, skin biopsy, gastrointestinal biopsy, lip biopsy, peripheral nerve biopsy, myocardial biopsy, etc. Since amyloid deposits are often found on the vascular wall of the gastrointestinal submucosa, it is recommended to perform gastrointestinal biopsy down to the submucosal layer. If the disease is strongly suspected due to clinical signs or other laboratory findings, it needs to be detected by repeated biopsy from each tissue site.

(Note3) Visual grading method using frontal planar images taken 3h later (Grade 0: No accumulation in the heart; Grade 1: Mild accumulation in the heart weaker than the ribs; Grade 2: Moderate degree in the heart equivalent to the ribs accumulation; Grade 3: Higher accumulation in the heart than ribs; Grade 2 or higher is positive), or a quantitative validation method using planar images taken 1h later (H/CL ratio: ${\geq}1.5$ ) should be used for evaluation. (For the details of myocardial scintigraphy, refer to “ Chapter II, Section 6. Nuclear Medicine Examination ”.)

II. Diagnosis of CA

1. Medical History, Symptoms, and Physical Findings Indicative of CA

The key to diagnosing CA is to suspect it based on the patient’s medical history, symptoms, and physical findings. Amyloidosis is a systemic disease; therefore, it presents with cardiac and various other findings. These findings must not be overlooked.

▋ ▋ 1.1 AL Amyloidosis

Amyloid deposits can be found in multiple organs, including the heart, kidneys, gastrointestinal system, and nerves. As a result, AL amyloidosis presents with diverse clinical symptoms and must be suspected based on a combination of medical history and physical findings.

Heart failure associated with AL amyloidosis is typically refractory. In many cases, pedal edema and pleural effusion cannot be controlled, even with large doses of diuretics.

 Figure3. Macroglossia in AL amyloidosis. ( A , B ) Tooth pressure marks on the lateral border.

 Figure4. Phalen’s test. The test result is positive if dysesthesia is exacerbated due to increased carpal tunnel pressure after keeping the wrists flexed for one minute.

 Figure5. Tinel’s sign. The test result is positive if tapping the neuropathic site with a hammer causes tingling pain in the innervated area.

Nephrotic syndrome, a complication of AL amyloidosis, results in hypoalbuminemia, which reduces plasma osmolality, attenuates the effects of loop diuretics, and pleural amyloidosis causes pleural effusion in a pathology separate from that of heart failure. 62 Amyloid deposition in the atrial muscle can cause left atrial (LA) thrombus regardless of sinus rhythm, as well as cardiogenic embolism. 63 , 64 Amyloid deposition in arterioles is known to result in jaw claudication, intermittent claudication, and anginal pain. 65 AL amyloidosis can also easily result in His bundle–ventricular block, leading to complete AV block before death in many patients with severe AL amyloidosis and a history of syncope. 66 , 67 Thus, AL amyloidosis may be present in patients with syncope and complete AV block.

Regarding noncardiac physical findings, macroglossia is observed in $10–20\%$ of cases ( Figure3 ). Due to amyloid deposition, the tongue presents as diffuse puffiness or nodular, and tooth pressure marks are seen on the lateral border. Amyloid deposition in vascular walls makes them fragile, heightening bleeding tendency and making patients prone to purpura. A characteristic physical finding is periorbital purpura, colloquially called “raccoon eyes” or “panda eyes.” Amyloid deposition in the gastrointestinal tract causes malabsorption syndrome and diarrhea, leading

to weight loss and general debility. Amyloid deposition in the liver and spleen results in hepatosplenomegaly, which feels harder than typical hepatosplenomegaly associated with right-sided heart failure. Other noncardiac physical findings in AL amyloidosis include the shoulder pad sign, scleroderma-like skin thickening and nodules, and hard swelling in the thyroid gland, salivary glands, and lymph nodes. Peripheral neuropathy manifests as sensory abnormalities, numbness, and muscle weakness. Sensory disturbances are typically symmetrical and often present in the lower extremities. Autonomic neuropathy manifests as dysuria, impotence, and orthostatic hypotension. 60 , 68

▋ ▋ 1.2 Amyloid Transthyretin Amyloidosis (ATTRwt and ATTRv)

ATTRwt amyloidosis occurs most commonly in older men. In many cases, the initial symptoms consist of conduction disturbance or HFpEF, primarily right-sided heart failure (pretibial edema, pleural effusion, etc.). 32 , 69 In addition, a previous study reported that $35\%$ of patients diagnosed with ATTRwt amyloidosis were previously misdiagnosed with another disease, such as hypertrophic cardiomyopathy or hypertensive heart disease. 32 Another problem is that

 Figure6. A perfect O sign for the evaluation of carpal tunnel syndrome. The patient is unable to make a neat circle with the thumb and index finger.

A history of numbness and pain in the median nerve region (radial side of the thumb, index, middle, and ring fingers) is often indicative of carpal tunnel syndrome. Pain in carpal tunnel syndrome is characteristically enhanced at night (particularly at dawn), and is often severe enough to awaken the patient during the night. Carpal tunnel syndrome is also characterized by the patient shaking their hands or changing the positions of their limbs to relieve the pain. A diagnosis of carpal tunnel syndrome is supported by a positive Phalen’s test (exacerbated dysesthesia due to increased carpal tunnel pressure after keeping the wrists flexed for 1min) ( Figure4 ), a positive reverse Phalen’s test (the same sign but with the wrists extended), and a positive Tinel’s sign (tingling pain when the carpal tunnel is tapped with a hammer) ( Figure5 ). 71 The progression of carpal tunnel syndrome can result in atrophy of the thenar eminence, difficulties in fine motor skills (buttoning buttons, etc.), the inability to create a neat circle with the thumb and index finger (perfect O sign) ( Figure6 ). Other comorbidities reported in ATTR amyloidosis include spinal canal stenosis $(14{-}22\%)$ , cubital tunnel syndrome, rotator cuff tear, biceps tendon rupture, and quadriceps tendon rupture. 69 , 72 – 74 Therefore, a history of these conditions should be confirmed for the diagnosis of ATTR amyloidosis.

ATTRwt amyloidosis is not diagnosed until 1–2 years after symptoms manifest. 31 , 70 Thus, many patients receive treatment for an incorrect diagnosis or remain undiagnosed for a long period of time, with ATTRwt amyloidosis not even being suspected.

Many patients with ATTRwt amyloidosis $(40–50\%)$ ) have a history of carpal tunnel syndrome; 31 , 32 , 69 , 70 in fact, comorbid bilateral carpal tunnel syndrome is characteristic of ATTRwt amyloidosis. The mean length of time from the onset of carpal tunnel syndrome to a diagnosis of ATTRwt amyloidosis is reportedly 6.9 years. 23

ATTRv amyloidosis may lead to symptoms, such as peripheral neuropathy, dysautonomia, carpal tunnel syndrome, cardiac conduction defect, heart failure, digestive symptoms, nephropathy and eye symptoms, that affect various organs in the body ( Figure7 ). 60 , 68 , 75

The initial symptoms of peripheral neuropathy begin in the distal part of both lower limbs where the axons are farthest away from the cell bodies and axonopathy is the underlying disease. Generally, the onset of neurological symptoms follows the order of dysautonomia, sensory disorders (thermal hypoalgesia), and movement disorders

 Figure7. Various systemic symptoms in ATTR.

(bilateral peripheral superiority). This is because the disorder progresses from small-diameter unmyelinated fibers to large-diameter myelinated fibers following amyloid deposition. During the initial stage of the illness, while patients retain a normal sense of touch, a dissociated sensory loss is frequently observed, with thermal hypoalgesia starting at the feet or ankles. Thermal hypoalgesia makes it difficult for the patients to recognize burns and injuries to their feet.

Dysautonomia presents with symptoms such as orthostatic hypotension, erectile dysfunction, dysuria, nausea/ vomiting, diarrhea, constipation, dyshidrosis, dry eye, and dry mouth. In cases of juvenile-onset ATTRv amyloidosis (Val30Met (p.Val50Met) mutation), features of dysautonomia are noticeable during the initial stages in areas with a high disease concentration but may be unrecognizable in areas with low disease concentration.

The onset of motor neuron symptoms such as muscular atrophy and muscle weakness is often delayed because of the sensory disorders, but there are also some cases in which there is mild sensory disorder during the initial illness stage with motor neuron disease as the underlying disease. In advanced stages, patients present with sensory disorders, muscle weakness, respiratory muscle paralysis, etc. in the limbs as a result of advanced peripheral neuropathy.

Amyloid deposition may sometimes lead to bilateral carpal tunnel syndrome. In a few cases, amyloid deposition in the cranial meninges and cerebral blood vessels may lead to central nervous system-related symptoms such as disturbance of consciousness and cerebral hemorrhages.

For cases where the family history is not particularly clear, caution is required because many patients are misdiagnosed in the initial stages as having chronic inflammatory demyelinating polyneuropathy (CIDP), diabetic neuropathy, lumbar spinal canal stenosis, etc.

Examples of cardiovascular symptoms include sick sinus syndrome, atrial fibrillation, atrial block due to amyloid deposition in the heart, and early orthostatic hypotension due to dysautonomia. 76 , 77 The onset of ventricular diastolic dysfunction occurs prior to systolic dysfunction due to heart failure caused by amyloid deposition in the myocardium. There are some types of TTR mutations where a heart disorder will be the underlying cause of disease and peripheral neuropathy will not be noticeable. 76 , 77 In particular, it is important to perform careful and frequent evaluations while adequately monitoring the risk of sudden death caused by conduction disorders. In many cases, orthostatic hypotension due to dysautonomia becomes severe as the illness progresses. 78

Examples of digestive symptoms that may occur include gastrointestinal symptoms such as severe alternating diarrhea and constipation and nausea due to dysautonomia. 79 Although severity differs from case to case, the patient’s daily lifestyle may be impaired drastically if symptoms are severe. In particular, persistent watery diarrhea might occur during the endstage of juvenile-onset ATTRv amyloidosis in areas of high disease concentration, leading to malabsorption and protein leakage.

Mutant TTR is produced not only by the liver but also by the retina and is responsible for eye symptoms. Vitreous opacities caused by amyloid deposition are also frequently observed in ATTRv patients and may present as an initial symptom in some cases. Glaucoma is also caused by amyloid deposition in the anterior part of the eye, which, on progression, can become a primary cause of blindness. Lacrimal hyposecretion might lead to dry eye.

Kidney symptoms such as nephrotic syndrome or kidney failure may also be caused by amyloid deposition with severity varying from case to case. Often, kidney symptoms are not noticeable during the initial stage of illness.

Patients may also experience other symptoms such as hypothyroidism, hypoglycemic attacks, and dry cough.

2. Blood Sampling Test

▋ ▋ 2.1 AL Amyloidosis

The most important point in diagnosing AL amyloidosis is to suspect it from clinical symptoms, physical findings, and electrocardiographic, chest X-ray, and echocardiographic findings. When we suspect AL amyloidosis, serum immunoglobulin electrophoresis, serum and urine immunofixation (or electrophoresis), and serum free light chain (FLC) measurements should be performed.

There has been a report on screening panels for detection of monoclonal gammopathies including 581 patients with AL amyloidosis. 80 In the paper, the detection sensitivity for AL amyloidosis was $88.3\%$ by serum FLC, $96.2\%$ by serum FLC+serum immunoglobulin electrophoresis, $97.1\%$ by serum FLC+serum immunoglobulin electrophoresis+serum immunofixation, and $98.1\%$ by serum FLC+serum immunoglobulin electrophoresis+serum and urine immunofixation. 80 Another paper showed that the combination of serum and urine electrophoresis immunofixation with serum FLC had sensitivity of $\bar{1}00\%$ for amyloidogenic light chain detection. 81 Thus, it is not enough to judge by serum FLC alone; at least one additional modality with serum/urine immunofixation is necessary.

To evaluate cardiac findings in AL amyloidosis, plasma natriuretic peptide (BNP or NT-proBNP) and high-sensitivity cardiac troponins are measured. If renal amyloidosis is suspected, urine protein, microalbuminuria, and urinary Bence-Jones protein are measured to evaluate the type and amount of proteinuria. Bone marrow aspiration and biopsy can confirm the presence of clonal plasma cells and evaluate the presence or absence of amyloid deposits in the bone marrow.

Serum FLC measurement is very useful for the diagnosis of AL amyloidosis. Figure8 shows serum FLC values in each disease. 82 In AL amyloidosis, about $80\%$ of cases are of type λ , and the measured values are often distributed in the range of ${50}{-1},000\,\mathrm{mg/L}$ . Serum FLC is elevated due to decreased renal function. Therefore, not the FLC value itself or the $\mathbf{k}/\lambda$ ratio, but the FLC difference (difference FLC: dFLC) is often used. The dFLC is one of the factors associated with a poor prognosis in the Revised Mayo stage 2012, and it is an important index used to determine the efficacy of treatment.

▋ ▋ 2.2 ATTR Amyloidosis

Unlike serum FLC in AL amyloidosis, no biomarkers in ATTR amyloidosis are directly linked to its diagnosis or the assessment of its therapeutic effects. However, the following biomarkers are reportedly useful aids in the diagnosis of ATTR amyloidosis: cardiac troponin, a marker of myocardial damage; B-type natriuretic peptides (BNP) and N-terminal pro-BNP (NT-proBNP), biomarkers of heart failure; and RBP4, a plasma transport protein for vitamin A (retinol). 36 , 83 , 84

 Figure8. Serum free light chain (FLC) in normal sera from healthy donors, patients with multiple myeloma, patients with polyclonal gammopathy, patients with AL amyloidosis, and patients with renal insufficiency. (Adapted from Bradwell AR. 2008 82 )

▋ ▋ 2.2.1 Cardiac Troponin

Elevated cardiac troponin is used to diagnose acute myocardial infarction. However, a persistently elevated cardiac troponin level is a red flag for CA. One study reported that, compared with a group of patients with LV hypertrophy for whom endomyocardial biopsy confirmed the absence of CA (control group), the CA group (including AL and ATTR) demonstrated significantly elevated high-sensitivity cardiac troponin T levels (control group [median]: $0.018\,\mathrm{{ng/mL}}$ ; CA group [median]: $0.048\,\mathrm{ng}/\mathrm{mL}$ ). 36 When the cutoff for high-sensitivity cardiac troponin T levels was set at $0.031\,\mathrm{ng}/\mathrm{mL}$ , the sensitivity and specificity for diagnosing CA based on LV hypertrophy were $74\%$ and $79\%$ , respectively (area under the curve: 0.787).

Another study reported that an elevated high-sensitivity cardiac troponin T level $(>\!0.05\,\mathrm{ng}/\mathrm{mL})$ ) combined with an elevated NT-proBNP level $(>3,000\,\mathrm{pg}/\mathrm{mL})$ is useful for assessing prognosis in ATTRwt CA. In that study, the median survival times for Stage 1 (no elevation in either biomarker), Stage 2 (elevation in one of the two biomarkers), and Stage 3 (elevation in both biomarkers) were 66, 42, and 20 months, respectively. 33

Assessing cardiac troponin requires the consideration of factors that affect its elevation, such as myocardial ischemia, renal dysfunction, pulmonary embolism, and sepsis. Multiple assessments are recommended to confirm persistent elevation. Since cardiac troponin levels are also elevated in acute heart failure, they should be measured in a compensated state. 85 , 86

▋ ▋ 2.2.2 Natriuretic Peptides

BNP and NT-proBNP are widely used to screen for, diagnose, and predict prognosis in heart failure. In CA

(particularly AL amyloidosis), the elevation in NT-proBNP is pronounced compared with heart failure severity and hemodynamics. 87

In ATTRv amyloidosis, since NT-proBNP levels increase before cardiac symptoms manifest, elevated NT-proBNP levels are reportedly useful for detecting cardiac involvement. 83

▋ ▋ 2.2.3 Retinol-Binding Protein 4 (RBP4)

RBP4, a plasma transport protein for vitamin A (retinol), is thought to help stabilize TTR, inhibit misfolding and aggregation, and suppress amyloid fibril formation. A group of patients with ATTRv amyloidosis involving a mutation in the V122I gene reportedly demonstrated significantly lower RBP4 levels than did a group of patients with non-amyloid heart failure, suggesting that RBP4 could be useful in screening for CA. 84 , 88

▋ ▋ 2.2.4 Important Points

In the diagnosis of ATTR, serum FLCs confirm the absence of immunoglobulin abnormalities. Monoclonal gammopathy of undetermined significance, which is assessed to rule out AL amyloidosis, is observed in $5.3\%$ of patients aged ${\geq}70$ years 89 and in $40–50\%$ of those with ATTR. 90 In patients with suspected CA who demonstrate immunoglobulin abnormalities, amyloid precursor protein must be identified in a biopsy specimen to differentiate ATTR from AL amyloidosis.

Recommendations and levels of evidence for blood testing for suspected cardiac amyloidosis are summarized in Table12 .

|Table 12. Recommendations and Levels of Evidence for Blood Testing for Suspected Cardiac Amyloidosis||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | | Measurement of high-sensitivity cardiac troponin to aid the diagnosis of amyloidosis* |na||C1|I4b| | Measurement of BNP/NT-proBNP to aid the diagnosis of amyloidosis |na||C1|I4b| | Measurement of high-sensitivity cardiac troponin to assess prognosis in ATTRwt amyloidosis* |ma||C1|I4b| | Measurement of BNP/NT-proBNP to assess prognosis in ATTRwt amyloidosis |na||C1|Ib| | Measurement of serum immunoglobulin to diagnose AL amyloidosis ||||Ib| | Serum and urine immunofixation (or electrophoresis) to diagnose ALamyloidosis ||||| | Measurement of serum free-light chains to diagnose AL amyloidosis ||||Ib|

\*Not covered by insurance in Japan for diagnosing cardiac amyloidosis. COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS); AL, amyloid light-chain; ATTRwt, wild-type amyloid transthyretin; BNP, b-type natriuretic protein; NT-proBNP, N-terminal pro-BNP.

| Table 13. Rate of Electrocardiogram Findings Among Different Types of Cardiac Amyloidosis||||| |---|---|---|---|---| |Types|Low voltage|Pseudoinfarctpattern|Atrioventricularblock|Atrial fibrillation| |AL|23-64%|15-69%|15-26%|6-32%| |ATTRwt|13-40%|18-71%|11—33%|27-67%| |ATTRv|23-38%|18-69%|25-45%|5-17%|

3. Electrocardiogram (ECG)

Electrocardiogram (ECG) is a noninvasive test often used to screen cardiac diseases. Abnormal ECG findings often lead to a diagnosis of CA. Amyloid deposits in the myocardial interstitium or conduction system may cause abnormal ECG findings such as low voltage, pseudoinfarct pattern, conduction disorders, and AF ( Table13 ). However, most of these ECG findings are not specific to CA, and the diagnostic values of these findings may differ among the types of amyloidosis or by the degree of the disease. Thus, ECG may be normal at the early stage of the disease. As CA is generally a progressive disease, it is important to evaluate chronological changes, even in patients without ECG abnormalities.

▋ ▋ 3.1 Low Voltage and Decreased Voltage/Mass Ratio

Low voltage on ECG despite LV hypertrophy is a classical finding in CA, and relatively common in AL amyloidosis $(23–64\%)$ ; however, it is only observed in $13–40\%$ of patients with ATTRwt CA and $23–38\%$ of those with ATTRv amyloidosis. 31 , 32 , 34 , 54 , 69 , 70 , 91 – 93 Thus, CA cannot be ruled out by the absence of low voltage. Low voltage is often defined as “limb leads $\trianglelefteq0.5\,\mathrm{mV}^{\ast}$ or “precordial leads ${\leq}1.0\,\mathrm{mV^{\ast}}$ . The Sokolow index $\mathrm{SV1}{+}\mathrm{RV}\bar{5}$ or $\mathrm{SV1}{+}\mathrm{RV6}$ ${<}1.5\,\mathrm{mV})$ ) may also be used. Low voltage has been reported as a worse prognostic predictor in studies including both AL and ATTR amyloidosis. 91 , 94 , 95 Furthermore, a decreased voltage/mass ratio (Sokolow index divided by wall thick

ness) has also been reported as a useful finding when diagnosing CA. 54 , 91 , 96

▋ ▋ 3.2 Pseudoinfarct Pattern (Abnormal Q Waves, Poor R Progression)

Abnormal Q waves and poor R progression in a patient without coronary artery disease is called a “pseudoinfarct” or “pseudonecrosis” pattern. The pseudoinfarct pattern is relatively common in all types of CA ( $15–69\%$ of AL, $18–71\%$ of ATTRwt, and $18–69\%$ of ATTRv CA cases). 32 , 34 , 91 , 92 , 94 Rates of the pseudoinfarct pattern are associated with the extent of late gadolinium enhancement (LGE) on CMR, suggesting that an amyloid burden is associated with the occurrence of the pseudoinfarct pattern. 97

▋ ▋ 3.3 Conduction Disturbances (AV Block, Bundle Branch Block, Intraventricular Conduction Disturbance)

Amyloid deposits involving the conduction system may result in AV block, bundle branch block, or intraventricular conduction disturbances. AV block is observed in $15–26\%$ of AL, $11{-}33\%$ of ATTRwt CA, and $25–45\%$ of ATTRv amyloidosis cases. 32 , 34 , 70 , 91 , 98 Right bundle branch block is observed in $3–19\%$ of AL and $\mathrm{{12-16\%}}$ of ATTR amyloidosis cases, and left bundle branch block is observed in $4–6\%$ of AL and $7–40\%$ of ATTR amyloidosis cases. 32 , 34 , 70 , 93 Marume et al. reported that a QRS width ${\geq}120\,\mathrm{ms}$ is significantly associated with positive findings on 99m Tc-PYP

 Figure9. Parasternal long-axis view, parasternal short-axis view and apical 4 chamber view in a patient with cardiac amyloidosis. Thickening of right ventricular free wall, interventricular septum, left ventricular posterior wall, concentric left ventricular hypertrophy, thickening of atrio-ventricular valves, right atrial wall, and pericardial effusion are found.

scintigraphy, suggesting that the presence of conduction disturbances may be useful in diagnosing CA. 99 Conduction disturbances may also reflect disease progression, and intraventricular conduction disturbance in AL or prolonged PQ interval in ATTRv amyloidosis is associated with poor prognosis. 100 , 101

are observed in advanced CA, their clinical significance, as well as the usefulness of implantable cardioverter defibrillators in CA, has not been established (please also refer to CQ3-3 ). Prolonged QT is also a common finding in CA. 32 , 34 , 91 , 93

4. Echocardiography

▋ ▋ 3.4 Atrial Fibrillation (AF)

Although AF is not a specific finding for CA, it is frequently observed in ATTRwt amyloidosis $(27–67\%)$ , whereas it is not as frequent in AL $(6–32\%)$ and ATTRv CA $(5–17\%)$ . 31 – 34 , 54 , 69 , 70 , 91 – 94 , 102 – 104 Thus, patients who have AF and LV hypertrophy should be carefully examined for other findings suggesting CA (especially ATTRwt CA). Previous studies have reported that AF is not associated with survival in CA, 102 , 103 , 105 but is associated with heart failure events. 103

The causes of AF in CA include amyloid deposits in the atrium or increased LA pressure secondary to LV filling pressure and/or LV diastolic dysfunction. Recent studies have also reported that amyloid deposits limited to the atrium, called “isolated atrial amyloidosis”, may be one of the causes of AF. 106 – 109 Rocken et al. reported that amyloid deposits were found in $16\%$ of the right atrial appendages of patients who had undergone cardiac surgery, and that these amyloids were derived from atrial natriuretic peptides (ANPs). 109 It is believed that the excretion of ANPs due to the extension of the atrium or AF itself is associated with this phenomenon; however, the mechanism of ANPs forming amyloid has not been elucidated. 106

▋ ▋ 3.5 Others (Ventricular Arrhythmias, Prolonged QT Interval)

Data regarding the incidence of ventricular arrhythmias in CA are limited; however, ventricular tachycardia or fibrillation may be the first presentation of CA. While non-sustained ventricular tachycardias and sudden deaths

Echocardiography is a noninvasive, reproducible method for assessing cardiac morphology and function in CA, and some echocardiographic indices are prognostic for amyloidosis.

▋ ▋ 4.1 M-Mode Echocardiography

Normal LV diastolic dimensions, increased systolic dimensions, diminished amplitude of excursion, and pericardial effusion are features of CA. 110 The following findings are basic: 1) symmetric LV hypertrophy in the absence of hypertension or aortic valvular disease; 2) hypokinesia and decreased systolic thickening in the interventricular septum and LV posterior wall; and 3) small-to-normal LV cavity size. In addition to the finding of LV hypertrophy, the characteristics of infiltrate cardiomyopathy include a septal/ posterior free wall thickness ratio ${\tt<}1.3$ , increased maximal LA transverse dimension, reduced mitral valve closure (E-F slope), and preserved EF ${>}60\%$ . 111 The right ventricular (RV) anterior wall thickness is reportedly significantly increased in patients with clinically significant amyloid infiltrate cardiomyopathy, and this is consistent with pathologic findings. 112 , 113

▋ ▋ 4.2 Two-Dimensional (2D) Echocardiography ( Figures9 and 10 )

Two-dimensional (2D) echocardiography can reveal additional features such as thickened papillary muscles, thick

 Figure10. Sub-xiphoid view in a patient with cardiac amyloidosis. Thickening of right ventricular free wall, inter-ventricular septum, left ventricular posterior wall, and pericardial effusion are found (allow).

ened valves, better appreciation of the thickened RV wall, and a characteristic “granular sparkling” appearance of the thickened cardiac walls. 114 Although increased echogenicity of the myocardium, particularly with a “granular sparkling” appearance, has been reported in several studies, sensitivity is not high with this pattern, which is seen in about $30\%$ of CA cases. In addition, it should be noted that this granular pattern applies only to standard echocardiographic imaging, without tissue harmonics being applied, as this increases myocardial echogenicity in general. Newer echocardiographic image processing techniques may also reduce the granular appearance. Thus, although increased echogenicity is common in amyloid cardiomyopathies, its usefulness as a discriminating factor remains limited. 115

Mohty et al. examined the prevalence and prognostic impact of left-sided valve thickening in systemic AL amyloidosis. 116 In patients with AL amyloidosis, mitral and/or aortic valve thickening $(\mathord{>}3\,\mathrm{mm})$ was significantly associated with decreased 5-year survival. The electrocardiographic voltage tended to be low and the echocardiographic muscle cross-sectional area tended to be increased. 96 When these two techniques were combined, an inverse correlation was observed between voltage and the muscle cross-sectional area $\mathrm{(r}{=}{-}0.79)$ ; moreover, a marked derangement of the voltage/cross-sectional area relation was associated with clinical symptoms and mortality. The combination of increased LV thickness and a low-voltage electrocardiographic pattern is highly specific for CA, and was found in 3/30 $(10\%)$ and 13/24 $(54\%)$ of secondary and amyloid-light chain (AL) amyloidosis patients, respectively. 117 One model showed that low voltage was present and the interventricular septal thickness was ${\tt>}1.98\,\mathrm{cm}$ , and a diagnosis of CA could be made with a sensitivity of $72\%$ and a specificity of $91\%$ , with positive and negative predictive values of $79\%$ and $88\%$ , respectively. 118 While all patients with amyloidosis show the presence of very bright or highly refractile echoes in the myocardium, this is also observed in patients with uncomplicated ventricular hypertrophy, chronic renal failure, hypertrophic cardiomyopathy, Pompe disease, hemochromatosis, and left heart hypoplastic syndrome, indicating that this finding is nonspecific for CA. 119 The occurrence of clinical congestive heart failure correlated strongly with greater wall thickness and multiple other echocardiographic

abnormalities, and survival was negatively influenced by greater wall thickness and reduced systolic function (fractional shortening), indicating that echocardiographic examinations are an important tool for identifying cardiac amyloid involvement and may be useful in estimating prognosis in systemic amyloidosis. 120 A low voltage pattern on electrocardiography and a reduced LV ejection fraction (LVEF) help identify patients at high risk of death. 94

Extensive CA results in atrial thrombi, even in patients with sinus rhythm. 121 Severe atrial and ventricular infiltration by amyloids may lead to mechanical atrial standstill with resultant thrombus formation. 122 AF, poor LV diastolic function, and lower LA appendage emptying velocity have been shown to be independent risk factors for intracardiac thrombosis, whereas anticoagulation has been found to be associated with a significantly decreased risk.

▋ ▋ 4.3 Doppler Echocardiography ( Figure11A , B )

Serial pulsed wave Doppler studies of LV inflow have identified changes from abnormal relaxation or “normal” patterns to restrictive patterns. 123 Doppler-derived LV diastolic filling variables are also important predictors of survival in CA, 124 with patients with a transmitral early filling wave deceleration time of ${\leq}150\mathrm{ms}$ (indicating restrictive pattern) showing poor cardiac outcomes. 125

▋ ▋ 4.4 Pulsed Tissue Doppler Imaging ( Figure11C , D )

CA is characterized by an initial impairment in early cardiac relaxation, whereas congestive heart failure is associated with an impairment of peak systolic wall motion velocities, most prominently seen in the longitudinal axis. 126 Moreover, peak lateral and medial mitral annulus velocities and color M-mode tissue Doppler of the LV posterior wall (for measurements of mean myocardial velocities and myocardial velocity gradient) can differentiate patients with CA and controls with good overall accuracy. 127 The longitudinal myocardial velocity gradients that indicate differences between the basal and mid-myocardial velocities using pulsed tissue Doppler imaging are reportedly significantly impaired in patients with compared with those without congestive heart failure. 128 Furthermore, the LV long-axis function was depressed in all $(100\%)$ patients with CA compared with only $36\%$ of those with idiopathic restrictive cardiomyopathy. 129

▋ ▋ 4.5 Strain Doppler imaging

Longitudinal LV myocardial function as assessed by strain and strain rate tissue Doppler echocardiography can detect early impairments in systolic longitudinal LV function in patients with AL amyloidosis upon fractional shortening, but prior to this, it is not detectable on tissue velocity imaging. 130 , 131 Strain Doppler imaging is a sensitive method that can detect differences in LV function between ATTRv and AL amyloidosis that cannot be distinguished by standard echocardiographic parameters, despite the severity of congestive heart failure and cardiac mortality being much lower in ATTRv amyloidosis. 132 This method can also detect impaired LV systolic function in patients with AL amyloidosis who have no evidence of cardiac involvement upon standard 2D or Doppler echocardiography. 133

Mean LV basal longitudinal strain (LS) was reported to

 Figure11. Transmitral flow, pulmonary venous flow, and pulsed tissue Doppler in mitral annulus in a patient with cardiac amyloidosis. E/A ratio is high, S/D ratio is low, and $\ominus'$ in septum shows low velocity.

be a powerful predictor of clinical outcomes, and found to be superior to standard 2D echocardiographic Doppler flow measurements and simple tissue velocity indices. 134 Additionally, RV function as assessed by Doppler myocardial imaging can also identify early impairment of cardiac function and stratify mortality risk in patients with AL amyloidosis. 135

▋ ▋ 4.6 Speckle Tracking Echocardiography ( Figure12 )

Speckle tracking echocardiography (STE) of myocardial strain and strain rates can discriminate CA from other causes of cardiac hypertrophy. Compared with other causes of LV hypertrophy, cardiac amyloid profoundly alters all strain parameters (longitudinal, circumferential, and radial strain). 136

Endocardial and epicardial longitudinal and circumferential strain and radial strain were found to be significantly lower in patients with hypertrophic cardiomyopathy and ATTR compared with controls. 137 Further, epicardial circumferential strain was significantly lower in patients with ATTR than in those with hypertrophic cardiomyopathy. A systolic septal longitudinal base-to-apex strain gradient (septal apical/basal longitudinal systolic ratio ${>}2.1$ ), combined with a shortened diastolic deceleration time of early filling (deceleration time of early filling ${<}200\,\mathrm{ms}\$ ) aids in differentiating CA from other causes of concentric LV hypertrophy. 138 A higher ratio of septal apical to basal segmental longitudinal peak systolic strain ratio ${>}2.1$ is suggestive of CA (sensitivity: $88\%$ ; specificity: $85\%$ ; positive predictive value: $67\%$ ; negative predictive value: $96\%$ ). 138 When using the strain polar map, the apical sparing of LS visual pattern (a pattern of regional differences in deformation in which LS in the basal and middle segments of the

 Figure12. Parametric display of 2D speckle-tracking longitude strain in a patient with cardiac amyloidosis. Apical sparing pattern is displayed.

left ventricle is more severely impaired than the apical segments) is an easily recognizable and accurate means of differentiating CA from alternative causes of LV hypertrophy. 139 The pathological mechanisms of apical sparing in patients with CA have not been clarified. The echocardiographic imaging parameters, mean tissue Dopplerderived and 2D global LS of the LV cardiac serological biomarkers, and comprehensive clinical characteristics were

prospectively assessed in 206 consecutive patients with biopsy-proven systemic AL amyloidosis. 140 In a multivariate analysis, only diastolic dysfunction and 2D global LS remained independent predictors of survival. In addition, 2D-STE predicted outcomes and provided incrementally better prognostic information compared with existing prognostic staging systems, especially in the group without CA. 141 The EF was preserved while the longitudinal systolic strain was significantly reduced in both compensated and decompensated AL amyloidosis patients. The longitudinal systolic strains were similar in the apical segments and significantly reduced in the basal segments in both groups, and multivariate analysis showed that New York Heart Association (NYHA) functional class and mid-septum systolic LS were independent predictors for survival. 142 Compared with ATTRv and AL amyloidosis, ATTRwt amyloidosis was characterized by greater LV wall thickness and lower EF, and the LV basal LS was more depressed in both ATTRwt and AL amyloidosis. TTR-related causes were favorable predictors of survival, whereas LS and advanced NYHA class were negative predictors. 54 Twentyone consecutive patients with biopsy-proven ATTRwt amyloidosis were compared with 21 patients with ATTRv amyloidosis from the database, matched by age and LV wall thickness. 143 LVEF, LV basal circumferential/radial strain, and mid-radial strain were significantly lower in patients with ATTRwt than in those with ATTRv amyloidosis. No significant differences in the other parameters were observed between the two groups. In the receiveroperating characteristics curve analysis, LVEF and LV basal mean radial strain were the best parameters for distinguishing between the two groups.

LA function used to be a focus in CA. In ATTR cardiomyopathy with increased LV wall thickness, LA myocardial function is abnormal, irrespective of atrial cavity size. Reduced LA myocardial strain rate during atrial systole, irrespective of cavity volume, $\mathrm{E/e}'$ , and LV deformation, is

also a strong predictor of atrial arrhythmic events. 144 LA function was severely impaired and highly correlated with LV deformation in CA. Differences in LA function between amyloid subtypes suggest that amyloid etiology plays a role in the pathophysiology of cardiac dysfunction in CA. 145

Three-dimensional STE (3D-STE) provides new insights into the mechanism of cardiac dysfunction in patients with CA. 3D-STE reveals regional and global biventricular dysfunction (LV basal LS, LV peak basal rotation, RV basal free-wall LS) in CA. The assessment of RV dysfunction has an additive value in differentiating CA from other causes of myocardial wall thickening. 146 Functional LA parameters are progressively altered in patients with AL amyloidosis according to the Mayo Clinic staging system. A decrease in 3D-peak atrial LS is associated with worse outcomes, independent of LA volume ( Table14 ). 147

▋ ▋ 4.7 ATTR in Patients With Aortic Stenosis

See other section.

5. Cardiac Magnetic Resonance Imaging (MRI)/ Computed Tomography (CT)

▋ ▋ 5.1 Cardiac MRI (CMR)

CMR is excellent for diagnosing CA. Although CMR can be used to diagnose advanced CA easily, its ability to diagnose other early diseases and amyloid types is limited. In addition, CA often presents with atypical findings on CMR. Therefore, CMR findings should be assessed in conjunction with the results of other tests and clinical findings. CMR is also useful for differentiating CA from other diseases with similar clinical presentations, such as hypertrophic cardiomyopathy and Anderson-Fabry disease. CA is typically diagnosed with cine CMR and LGE on CMR (LGE imaging). However, myocardial T1 mapping is

|Table 14. Recommendations for Echocardiography in Patients With Cardiac Amyloidosis||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | | M-mode echocardioraphyisrecommended forthe assessment of LVH,LA enlargement, pericaridal effusion, LVEF, and RV anterior wall thickness ||E||| | B-mode echocardioraphy is recommended for the assessment of LVH, LA enlargement, pericaridal effusion, LVEF, thickened pallilary muscle, thickeed RV wall, increased myocardialechogenicity,andvoltage/massratio ||E|E|ivb| | Transesophageal echocardiography is recommended for the detection of intracardiac thrombus ||E|E|iva| | Doppler echocardiography is recommended for the assessment of LV inflow, PV flow, LVOT flow, and/or the prediction of the prognosis ||E|E|iva| | Tissue Doppler echocardiography is recommended for the assessment of LV systolic and diastolic function ||E|B|iva| | Strain Doppler echocardiography is recommended for early detection of systolic dysfunction, and/or the prediction of the prognosis ||E|E|iva| | Speckle tracking enchocardiography is recommended for the differentiation of cardiac amyloidosisfrom other causes of cardiachyperthrophy,and/ortheprediction of the prognosis ||E||iva|

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS); LV (H), left ventricular (hypertrophy); LA, left atrium; EF, ejection fraction; RV, right ventricular; PV, pulmonary vein; LVOT, LV outflow tract.

|Table15.||| |---|---|---| |Imaging| Significance and findings|Notes| |Cine CMR| Significance: Assessment of cardiac morphology and function · Left ventricular hypertrophy predominantly at the basal segments | Caution required in differentiation from hypertro- phic cardiomyopathy Diagnosis is difficult using cine CMR alone | || Symmetrical or asymmetrical left ventricular hypertrophy Thickening of the right ventricular wall and/or atrial septum (≥6mm) · Left ventricular ejection fraction is often preserved || |LGE imaging| Significance: Assessment of amyloid deposition and myocardial fibrosis · Diffuse left ventricular subendocardial LGE . Transmural LGE in advanced stage ·LGE also present in the right ventricular wall, left atrial wall, or atrial septum · Dark blood pools | Atypical LGE patterns are often seen LGE findings change with disease stage Prone to errors in inversion time setting Phase-sensitive inversion recovery sequence is recommended Correlated with prognosis | | T1 mapping| Significance: Assessment of amyloid deposition and myocardial damage || ||. Significantly elevated native T1 and ECV values| Enablesquantitative assessment Reference value for native T1 must be set Measurement should be performed in the inter-Measurement should be performed in the inter- ventricular septum on the left ventricular short-ventricular septum on the left ventricular short- axis images at the basal or middle levelaxis images at the basal or middle level Amyloid type is difficult to diagnose Amyloid type is difficult to diagnose Correlated with severity and outcomesCorrelated with severity and outcomes Applied to monitor progress and assessment of Applied to monitor progress and assessment of therapeutic effects May assist in diagnosis of amyloid type | |||| |T2 mapping| Significance:Assessment of myocardial edema and inflammation . Higher values than healthy controls · Demonstrates especially high values in AL | May assist in diagnosis of amyloid type . Insufficient evidence | |Myocardial strain| Significance: Assessment of myocardial strain :Abnormalities in longitudinal and circumferential strains · Reduced peak strain value (absolute value) . Peak strain time variations |Low disease specificity| ||| Applied in disease state monitoring . Correlated with severity and outcomes |

AL, amyloid light-chain; ECV, extracellular volume fraction; LGE, late gadolinium enhancement; CMR, cardiac magnetic resonance imaging.

|Table 16. Recommendations and Evidence for CMR in Cardiac Amyloidosis||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | |Assessment of cardiac morphology and function with cine CMR||||| |Differentiation from other cardiomyopathies with LGE imaging||C||Ivb| | Differentiation from other cardiomyopathies with T1 mapping||||Ivb| |Differentiation from other cardiomyopathies with T2 mapping|na|C|E|Ib| | Differentiation from other cardiomyopathies with myocardial strain assessment using CMR |ma||E|Ib|

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS); CMR, cardiac magnetic resonance imaging; LGE, late gadolinium enhancement.

reportedly useful for the quantitative assessment of myocardial tissue characterization, and is currently recommended for diagnosing CA. Myocardial T2 mapping and myocardial strain assessment using CMR are also suggested to be effective. CMR assessment that integrates these imaging methods would be useful for diagnosing CA, differentiating it from other diseases, assessing its severity, predicting patient prognosis, and monitoring disease states. Tables15 and 16 show CMR findings in CA and recommendation levels, respectively.

▋ ▋ 5.1.1 Cine CMR

Cine CMR is a basic sequence for assessing cardiac morphology and function. CA often causes LV hypertrophy,

predominantly at the basal segments. Although symmetrical and circumferential hypertrophy has been considered to be typical, a recent study reported that asymmetrical hypertrophy, particularly asymmetrical septal hypertrophy, is often observed 148 ( Figure13 ). The degree of LV hypertrophy tends to be greater in ATTR than in AL amyloidosis. 35 However, it should be noted that $3–8\%$ of CA cases present with a normal geometry. Thickening of the RV wall and atrial septum is frequently observed in CA, with atrial septum thickening ${\geq}6\mathrm{mm}$ considered to be relatively specific to CA. 149 Based on cine CMR functional analysis, LVEF is often preserved, but may decline as the disease progresses. Advanced CA often involves pericardial and pleural effusions. 35 Morphological and functional findings in cine CMR

 Figure13. Cine CMR in cardiac amyloidosis: symmetrical versus asymmetrical cardiac hypertrophy.

are nonspecific, making CA difficult to diagnose using this modality alone.

LGE imaging, the preferred method for diagnosing CA, involves taking images 10min after the injection of a gadolinium contrast agent to visualize myocardial damage (primarily fibrosis). Imaging is generally conducted using inversion recovery sequences. The diagnostic performance of CA with LGE imaging has been reported to have a sensitivity and specificity of $85–90\%$ . 148 , 150 Typical findings of CA in LGE imaging include the following: 1) diffuse LV subendocardial LGE; 2) LGE in the RV wall, LA wall, and atrial septum; and 3) dark blood pools 97 ( Figure14 ). LGE reflects both amyloid deposition in the myocardial interstitium and subendocardial ischemic changes (fibrosis) associated with microangiopathy. 97 Dark blood pools, which originate from the contrast agent kinetics unique to amyloidosis, in which gadolinium is quickly washed away from blood pools, form as a result of the distribution of gadolinium contrast agent in interstitial amyloid depositions throughout the body. Patterns of LGE tend to be more prominent in ATTR than in AL amyloidosis, and frequently develop into transmural LGE. 35 LGE mostly occurs in the basal segments. 151 The presence of LGE is considered a powerful prognostic predictor of CA. 152 Recent findings indicate that LGE transitions from subendocardial to transmural as CA progresses, and that CA presents with various atypical LGE patterns (patchy, subepicardial, and diffuse transmural LGE) according to disease stage. 153 No more than $50\%$ of all patients with CA present with typical LGE (subendocardial LGE). 153

LGE imaging based on inversion recovery was developed to detect local myocardial damage such as myocardial infarction. Diffuse myocardial diseases, especially CA, are prone to inadequate contrast enhancement due to technical imaging errors, which can cause LGE underestimations. The use of phase-sensitive inversion recovery sequences has recently been recommended as a way to avoid such imaging errors. 153

 Figure14. LGE imaging in cardiac amyloidosis: Typical patterns include diffuse left ventricular subenodcardial LGE; right ventricular wall, left atrial wall, and atrial septal LGE; and dark blood pools.

Although LGE imaging is effective for diagnosing CA, the following limitations and disadvantages must be recognized: 1) LGE imaging cannot detect early-stage lesions; 2) CA often presents with atypical LGE; 3) LGE patterns are underestimated in diffuse myocardial damage; 4) imaging errors are likely to occur; 5) quantitative assessment is difficult; and 6) gadolinium contrast agent cannot be used in patients with advanced renal dysfunction or those on dialysis. T1 mapping, discussed below, is an important method that complements the disadvantages of LGE imaging.

▋ ▋ 5.1.3 T1 Mapping

T1 mapping is an imaging method that quantitatively measures myocardial T1 values (T1 relaxation times [ms]). Due to the ability of T1 mapping to assess myocardial damage quantitatively, it is expected to be applied for the detection of early-stage lesions, the assessment of severity and outcomes, and the evaluation of therapeutic effects. There are two parameters in T1 mapping: native T1, which does not use a contrast agent, and extracellular volume fraction (ECV), which involves the use of a contrast agent. ECV is calculated based on the myocardial and blood T1 values of pre- and post-contrast agent injection using the following formula and the most recent hematocrit (Hct) result within the last $24\,\mathrm{h}$ :

$$\begin{array}{r}{\mathrm{ECV\,(\%)}\,{=}\,(1\mathrm{\,-\,}\mathrm{Hct})\times(1\mathrm{\,/\,}\mathrm{T\,}1_{\mathrm{mo\,\,post}}\mathrm{\,-\,}1\mathrm{\,/\,}\mathrm{T\,}1_{\mathrm{mo\,\,pre}})\mathrm{\,/\,}}\\ {(1\mathrm{\,/\,}\mathrm{T\,}1_{\mathrm{bl o o d\,\,post}}\mathrm{\,-\,}1\mathrm{\,/\,}\mathrm{T\,}1_{\mathrm{bl o o d\,\,pre}})\times100\qquad\qquad\qquad}\end{array}$$ where native T1 includes both intracellular and extracellular information, and ECV reflects changes in the extracellular space. Native T1 is easily affected by the magnetic field strength of the systems ( $1.5\,\mathrm{T}$ or 3.0 T scanners) and imaging sequences, thereby requiring the establishment of reference values for each system and imaging sequence. 154 By contrast, ECV, which shows the percentage of extracellular space in which the contrast agent is distributed, is mostly unaffected by magnetic field strength or imaging sequences. Therefore, ECV enables the stable assessment of myocardial properties. The reference value for ECV is $23–28\%$ . Imaging and measurements should be performed in the interventricular septum on the LV short-axis images at the basal or middle level. 154

In CA, native T1 and ECV frequently yield extremely abnormally high values 155 ( Figure15 ). Native T1 and ECV values in CA are reported to be significantly higher than

 Figure15. T1 mapping in cardiac amyloidosis: Native T1 and ECV demonstrate abnormally high values in cardiac amyloidosis compared to hypertensive heart disease.

those in morphologically similar hypertrophic cardiomyopathy and aortic stenosis. For native T1 alone, the diagnostic sensitivity and specificity of CA are $80–92\%$ and $56–91\%$ , respectively. For ECV alone, the diagnostic sensitivity and specificity are $93\%$ and $82\%$ , respectively. 155 – 157 While native T1 and ECV demonstrate excellent diagnostic capacities, combining them with other parameters in a comprehensive assessment may further improve their diagnostic capacity. In CA, ECV is reportedly $44–61\%$ . 153 , 157 – 159 Thus, CA must be strongly suspected when ECV is ${\geq}40\%$ . 160 Although T1 mapping findings differ by the type of CA (AL, ATTRwt, or ATTRv amyloidosis), there is a great deal of overlap, which hinders the diagnosis of CA type. 159 T1 mapping detects lesions with greater sensitivity than does LGE imaging; therefore, its use may aid the early diagnosis of CA. 161 , 162 In addition, native T1 and ECV demonstrate correlations with CA severity and outcomes, creating anticipation for their application in the assessment of risk and therapeutic effects, progress monitoring, and other aspects of clinical management. 153 , 158 , 159 , 162

▋ ▋ 5.1.4 T2 Mapping

T2 mapping is an imaging method that measures myocardial T2 values (T2 relaxation times [ms]). T2 values reflect water in the tissue; thus, they are prolonged with the occurrence of myocardial edema or inflammation. Normal myocardial T2 values are reportedly 40–50ms. However, as with T1 mapping, reference values must be established for each system and imaging sequence. 154 According to recent studies, myocardial T2 values are significantly higher in patients with CA than in healthy controls, and values are particularly high in AL amyloidosis. Reported T2 values (with a 1.5 T MRI system) for AL amyloidosis, ATTR amyloidosis, and healthy controls were 56.6–63.2ms, 54.2– 56.2ms, and 48.9–51.1ms, respectively. 163 , 164 These findings suggest that a degree of myocardial edema occurs in CA,

 Figure16. T2 mapping in cardiac amyloidosis: Myocardial T2 is significantly higher in AL than in ATTR.

 Figure17. Myocardial strain assessment using CMR in cardiac amyloidosis. Peak circumferential strain is significantly reduced, and peak times in the myocardial segment vary.

particularly in AL amyloidosis ( Figure16 ). In addition, myocardial T2 is a significant predictor of prognosis in AL amyloidosis. 163

▋ ▋ 5.1.5 Myocardial Strain Using CMR

Analysis of myocardial strain with 2D-STE has been established as an effective method for diagnosing CA. Myocardial strain can also be analyzed in a similar fashion in CMR. Imaging methods include the tagging and feature tracking techniques. As in echocardiography, LS has also been shown to be effective in CMR. Circumferential strain is also considered highly useful in CMR. In CA, peak circumferential strain is significantly reduced, and peak times in the myocardial segment vary ( Figure17 ). These findings are reportedly correlated with severity and associated with prognosis. 151 , 165 , 166 Myocardial strain assessment using CMR may also be effective for detecting early-stage lesions and disease state monitoring. 165

▋ ▋ 5.2 Cardiac CT

Cardiac CT can be used as an alternative to CMR for assessing myocardial late enhancement and ECV. The use of low tube voltage techniques and dual-energy CT-based virtual monochromatic imaging enables the assessment of myocardial late enhancement equivalent to that of CMR. 167 In addition, ECV can be calculated based on myocardial contrast enhancement or iodine density. 167 , 168 CT can be performed safely in patients with heart failure or in whom implantable devices have been inserted, i.e., cases in which CMR would be difficult to perform, as well as in patients undergoing hemodialysis, in whom gadolinium contrast

Table17. Cardiac CT Findings in Cardiac Amyloidosis

|Imaging| Significance and findings|Notes| |---|---|---| |Late enhancement|| Enables three-dimensional assessment | || Significance:Assessment of myocardial fibrosis . Diffuse left ventricular subendocardial late enhancement | . lodinated contrast agents can be used in patients undergoing hemodialysis | |||.Contrast resolution is inferior to that of CMR| |CT-ECV| Significance:Assessment of amyloid deposits and myocardial damage | Calculated with a subtraction method in conventional single-energy CT systemssingle-energy CT systems . Can be calculated with an iodine method in dual-energy CT systems | ||· Significantly elevated ECV value||

CT, computed tomography; ECV, extracellular volume fraction; CMR, cardiac magnetic resonance imaging.

|Table 18. Recommendations and Evidence for Cardiac CT in Cardiac Amyloidosis||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | | Myocardial late enhancement/ECV assessment with cardiac CT as an alternative toCMR |na||E||

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS); CT, computed tomography; ECV, extracellular volume fraction; CMR, cardiac magnetic resonance imaging.

 Figure18. Late enhancement CT and CT-ECV in cardiac amyloidosis. Diffuse subendocardial late enhancement is visible and the ECV is significantly elevated.

agents are contraindicated. Thus, CT is highly practical in real-world clinical settings. Tables17 and 18 show cardiac CT findings in CA and recommendation levels, respectively.

▋ ▋ 5.2.1 Late Enhancement CT

The iodinated contrast agents used in contrast-enhanced CT demonstrate pharmacokinetics similar to those of gadolinium contrast agents in MRI. Therefore, myocardial late enhancement assessment is also theoretically possible in contrast-enhanced CT. However, the contrast resolution of CT is inferior to that of CMR. As a result, the use of late enhancement CT has been limited in clinical settings. Recently, low tube voltage techniques and dual-energy CT-based virtual monochromatic imaging have greatly improved the image quality of late enhancement CT. Although late enhancement CT may be capable of yielding the same findings as CMR for the diagnosis of CA, 169 relevant evidence is currently insufficient ( Figure18 ).

▋ ▋ 5.2.2 CT-ECV

In late enhancement CT, myocardial contrast enhancement or iodine density values can be used to calculate ECV and assess it in a manner comparable to CMR T1 mapping. 167 There are two methods for calculating ECV in CT, both of which are considered to yield comparable results: 1) a subtraction method based on myocardial contrast enhancement that can be obtained using a conventional CT system (single-energy CT system); and 2) an iodine method based on myocardial iodine density values using dual-energy CT systems. 170 Similar to CMR, CT-ECV demonstrated significantly higher values in CA; thus, it appears useful for diagnosis 169 , 171 , 172 ( Figure18 ). The typical CT imaging protocol is as follows: prospective ECG gating mode, CTDI vol , 11–25mGy, 500–600mgI/kg iodine contrast agent, iterative image reconstruction, and 7–10min of scan delay time.

Table19. Nuclear Imaging Findings of Cardiac Amyloidosis

|ldosls|ldosls|ldosls| |---|---|---| |Imaging| Significance and findings|Notes| | 99mTc-PYP scintigraphy | Significance:Diagnosis of ATTR cardiac amyloidosis .Grade 2 or 3 in visual assessment (3-h imaging) · H/CL ratio > 1.5 (1-h imaging) . H/CL ratio > 1.3 (3-h imaging) | : Grade 2 may also be observed in AL cardiac amyloidosis . False positives may occur due to physiological accumulation in blood pools ·Combination with SPECT is useful .Useful for assessing prognosis | | 123|-MIBG scintigraphy | Significance: Assessment of myocardial sympathetic nerve function . H/M ratio ||

AL, amyloid light-chain; ATTR, amyloid transthyretin; H/CL, heart/contralateral; H/M, heart/mediastinum; MIBG, metaiodobenzylguanidine; PET, positron emission tomography; PYP, pyrophosphate; SPECT, single-photon emission computed tomography.

|Table 20. Recommendations and Evidence for Nuclear Imaging for Cardiac Amyloidosis||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | | Diagnosis of ATTR cardiac amyloidosis with 99mTc-PYP scintigraphy ||||| | Assessmentofmyocardialsympatheticnervefunctionwith 1231-MIBGscintigraphy |ila||E|Ib| |Diagnosis of cardiac amyloidosis using amyloid PET|||C1||

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS); ATTR, amyloid transthyretin; MIBG, metaiodobenzylguanidine; PET, positron emission tomography; PYP, pyrophosphate.

6. Nuclear Imaging

Nuclear imaging plays an important role in diagnosing CA. 99m Tc-PYP scintigraphy, a type of bone scintigraphy, is highly capable of diagnosing ATTR amyloidosis. 123 I-metaiodobenzylguanidine ( 123 I-MIBG) scintigraphy, a type of myocardial sympathetic innervation imaging used in the noninvasive diagnosis of disease stage, is able to detect denervation in CA and assess the state of heart failure. In addition, preclinical research has recently begun on amyloid positron emission tomography (PET), which uses amyloid-specific tracers. Tables19 and 20 show the nuclear imaging findings of CA and recommendation levels, respectively.

▋ ▋ 6.1 99m Tc-PYP Scintigraphy (Bone Scintigraphy) (see CQ1 )

Pyrophosphate, which possesses an affinity for calcium, has long been used to assess bone disease and acute myocardial infarction, and is generally known as a tracer in bone scintigraphy. 99m Tc-PYP scintigraphy has recently attracted attention due to the discovery that it is extremely effective for detecting ATTR (ATTRv and ATTRwt amyloidosis) 1 , 2 , 173 ( Figure19 ). Planar imaging is acquired 1 or 3h after the injection of 99m Tc-PYP. The 1-h imaging results in improved detection sensitivity, while the 3-h imaging results in improved detection specificity. 2 The diagnosis of ATTR

 Figure19. 99m Tc-PYP scintigraphy in cardiac amyloidosis. Significant myocardial uptake is observed in ATTR amyloidosis.

amyloidosis using 99m Tc-PYP scintigraphy has a sensitivity of $58–99\%$ and a specificity of $79–100\%$ . 1 , 2 Most false positives are AL CA; however, if these cases are excluded, 99m Tc-PYP scintigraphy is considered to have a diagnostic specificity and positive predictive value of $100\%$ . 1 There are two methods of assessing 99m Tc-PYP scintigraphy: visual and quantitative assessment. Both of these methods are based on anterior planar images ( Table21 , Figure20 ). 160 As the visual assessment has been established in the 3-h imaging, the same standards should not be applied in the 1-h imaging, which easily occurs high uptake in blood pools. A visual assessment of Grades 0, 1, and 2 requires differentiation of AL and/or physiological uptake in blood pools. Adding single-photon emission CT (SPECT) along with planar imaging is recommended at facilities when possible because it enables a more accurate assessment of

|Table 21. 99mTc-PYP Scintigraphy Assessment Methods|| |---|---| |Visual assessment|| |Grade0|No cardiac uptake| |Grade1|Mild cardiac uptake, less than rib uptake| |Grade2|Moderate cardiac uptake,equal to ribuptake| |Grade3|High cardiac uptake, greater than rib uptake| ||| |Grades 2 and 3 are classified as positive.|| |(Note) Visual assessment should be performed using 3-h|| |image.|| |Quantitative assessment|| | H/CL ratio > 1.5 (1-h imaging) H/CL ratio > 1.3 (3-h imaging) || ||| |H/CL:heart-to-contralateral|| |The H/CL ratio is calculated by marking a region of interest|| |in the site corresponding to the heart (H) on an anterior|| | planar image and another region of interest of the same size in the contralateral chest, lower lung field (CL). ||

(Source: Prepared based on Dorbala S, et al. 2019 160 )

 Figure20. 99m Tc-PYP scintigraphy (3-h imaging): visual assessment and quantitative assessment. H/CL, heart-tocontralateral ratio.

uptake in the myocardium and blood pools. 160 SPECT/CT fusion imaging is also effective as an aid in visual diagnosis. The cardiac uptake of 99m Tc-PYP has been demonstrated to be associated with prognosis. An H/CL ratio ${>}1.6$ is considered to be an independent predictor of poor prognosis. 2 Although much remains unknown regarding the mechanism of uptake of pyrophosphate and other bone tracers in ATTR amyloidosis, it is suspected to be calcium-mediated. 99m Tc-hydroxymethylene diphosphonate ( 99m Tc-HMDP) and 99m Tc-DPD are other bone scintigraphy tracers considered to have diagnostic accuracy comparable to that of 99m Tc-PYP. 1 , 174 , 175

▋ ▋ 6.2 123 I-MIBG Scintigraphy

123 I-MIBG is a myocardial sympathetic nerve function imaging agent with a structure similar to that of the neurotransmitter norepinephrine. Myocardial uptake of 123 I-MIBG reflects cardiac sympathetic innervation and can be used to assess myocardial denervation and other forms of sympathetic dysfunction. In addition, clearance of 123 I-MIBG from the myocardium is thought to reflect sympathetic nerve activity. Imaging typically consists of anterior planar and SPECT imaging, which include early-and late-phase images taken 15min and 3–4h, respectively, after the injection of ${}^{123}\mathrm{I}.$ -MIBG. These anterior planar images are used to calculate the heart/mediastinum (H/M) and washout ratios ( Figure21 ). 123 I-MIBG can be used to assess myocardial denervation in CA. In ATTRv amyloidosis, a reduced late H/M ratio has been demonstrated to be associated with prognosis. A late H/M ratio ${\tt<}1.6$ is considered an indicator of poor prognosis, with reported 5-year mortality rates for an H/M ratio ${\tt<}1.6$ and ${\geq}1.6$ of $42\%$ and $7\%$ , respectively. 176 In another study, an H/M ratio ${\tt<}1.6$ in ATTRv amyloidosis was significantly associated with all-cause mortality, hospitalization for heart failure, pacemaker implantation, and cardiac resynchronization therapy. 101

 Figure21. 123 I-MIBG scintigraphy in cardiac amyloidosis: Late H/M ratio is significantly reduced. H/M, heart/mediastinum; MIBG, metaiodobenzylguanidine.

 Figure22. Measurement of pulmonary arterial wedge pressure. ( A ) Mean pulmonary arterial wedge pressure during a whole cardiac cycle. ( B ) End-diastolic pulmonary arterial wedge pressure calculated from the mean of a wave. ( C ) End-diastolic pulmonary arterial wedge pressure calculated using the QRS-gated method.

▋ ▋ 6.3 Amyloid PET

The application of amyloid PET, which uses amyloidspecific tracers, in CA has been the subject of preclinical research reports. Myocardial uptake of $^{11}\mathrm{C}$ -Pittsburgh compound B, 177 18 F-florbetapir, 178 and $^{18}\mathrm{F}.$ -florbetaben 179 has been confirmed in AL and ATTR amyloidosis, and has been shown to be significantly higher in such patients compared with controls. The cardiac uptake of $^{18}\mathrm{F}$ -florbetapir and 18 F-florbetaben tends to be higher in AL than in ATTR amyloidosis. Unlike the other modalities discussed here, amyloid PET can be used to assess amyloid deposition not only in the heart, but also throughout the body. Thus, amyloid PET may be capable of quantifying amyloid deposition throughout the body.

7. Cardiac Catheterization

Cardiac amyloidosis refers to a secondary form of cardiomyopathy caused by the extracellular and vascular deposition of amyloid fibrils presenting with cardiac dysfunction. 27 It is characterized by left ventricular (LV) diastolic dysfunction. LV systolic dysfunction also appears at an advanced stage leading to arrhythmias, conduction disturbance, and heart failure. To make a definitive diagnosis, pathological evidence is required. On cardiac catheterization, endomyocardial biopsy (EMB) is primarily performed in patients with suspected cardiac amyloidosis. In addition, coronary angiography is performed to assess the presence of coronary stenosis if coronary artery disease is suspected. Left ventriculography can provide dynamic, volumetric, and morphologic assessments, and assess the severity of mitral valve regurgitation. In addition, LV pressure and the intraventricular pressure gradient can be measured. In patients with clinically suggested aortic valve stenosis, aortic valve

area and the LV-aortic pressure gradient are measured. Pulmonary hemodynamics are assessed by right heart catheterization if needed. Electrophysiological examination is performed in patients with symptomatic bradycardia or tachyarrhythmia when considering a pacemaker or catheter ablation treatment.

▋ ▋ 7.1 Right Heart Catheterization

The importance of right heart catheterization has not been confirmed in patients with cardiac amyloidosis. The procedure is performed when it is difficult to evaluate hemodynamics on echocardiography in patients with pulmonary hypertension (PH) or refractory heart failure. PH is frequently observed in left heart disease (LHD). In fact, the number of patients with PH due to LHD is the largest within the clinical classification of PH. The pathogenesis of PH-LHD involves an increase in the pulmonary venous pressure related to increases in end-diastolic LV and left atrial pressures. In the guidelines for the classification of PH, PH-LHD is defined as group 2 PH. It is defined as post-capillary PH by an increase in mean pulmonary arterial pressure (mPAP) ${\geq}25\,\mathrm{mmHg}$ and a pulmonary arterial wedge pressure (PAWP) >15mmHg. 180 To obtain accurate resting PAWP measurement, the 6th World Symposium on Pulmonary Hypertension in 2018 recommended that it could be measured at end-expiration and end-diastole 181 ( Figure22 ). In sinus rhythm, this corresponds to the mean of the a-wave (B). In atrial fibrillation, it is appropriate to measure PAWP 130–160ms after the onset of the QRS and before the v-wave (C) ( Figure22 ). Caution is needed in mitral valve regurgitation or stiff left atrial syndrome with a large v-wave, because PAWP during a whole cardiac cycle (A) can be overestimated.

▋ ▋ 7.2 Left Heart Catheterization

LV pressure can be measured directly by inserting a catheter into the LV, and LV compliance can be estimated from the LV end-diastolic pressure. Furthermore, if echocardiography suggests the presence of ventricular obstruction, the intraventricular pressure gradient can be accurately measured. In patients with clinically suggested aortic valve stenosis, the indication for surgical valve replacement or transcatheter aortic valve implantation can be assessed by directly measuring the intracardiac pressure.

▋ ▋ 7.3 Coronary Angiography

In cardiac amyloidosis, coronary angiography is indicated for patients with angina symptoms in whom revascularization may reduce myocardial ischemia and for those with suspected silent myocardial ischemia. Exercise testing, coronary computed tomography (CT), and/or myocardial perfusion scintigraphy may be considered in patients without symptoms and electrocardiographic or echocardiographic evidence of myocardial ischemia. If there is a low pre-test probability of coronary artery disease, coronary CT can evaluate coronary artery stenosis. 182 Coronary angiography is recommended for symptomatic patients with a history of percutaneous coronary intervention or coronary artery bypass graft, a high pre-test probability, cardiopulmonary resuscitation for cardiac arrest, and fatal ventricular tachycardias. In patients with amyloidosis, myocardial ischemia might be present because of vascular compression by hypertrophic myocardium, and amyloid infiltration of the small intramyocardial vessels can induce angina. In such patients, the assessment of coronary lesions is limited using coronary angiography. 183 – 185

▋ ▋ 7.4 Left Ventriculography

In patients with cardiac amyloidosis, the morphological findings are shared with many other cardiomyopathies. Misdiagnosis can occur frequently in cases with asymmetric LV hypertrophy. 148 In patients with hypertrophic myocardium, dynamic, volumetric, and morphologic assessments are commonly evaluated by echocardiography or cardiac magnetic resonance imaging (MRI). 186 Left ventriculography may be performed for assessment of LV characteristics in unevaluable patients using such modalities and in patients with a non-MRI-conditional pacemaker, implantable cardioverter-defibrillator, or cardiac resynchronization device. Some patients with cardiac amyloidosis present with normal cardiac contraction, and others have impaired contraction. In patients with cardiac amyloidosis, left ventriculography frequently indicates wall motion abnormalities that are mismatched to the coronary artery dominant areas and longitudinal dysfunction in mid and basal

LV segments. In addition, left ventriculography can demonstrate the severity of mitral valve regurgitation.

▋ ▋ 7.5 Endomyocardial Biopsy (see also CQ2 )

Despite imaging advances, EMB remains the gold standard technique to obtain pathological information on the myocardium, excluding surgical biopsy. To establish the diagnosis of cardiac amyloidosis, EMB is appropriate to differentiate from other cardiomyopathies, including hypertrophic cardiomyopathy (HCM). 6 , 187 The incidence of serious complications related to EMB is reportedly less than $1\%$ . 188 , 189 However, the procedure is difficult in patients with s conduction disturbance and advanced age. For EMB, specimens are commonly collected from the right ventricular (RV) septum, although they are collected from the LV in some cases. It remains to be clarified which site of collection is better with respect to obtaining useful histological information or the incidence of complications. 189 Because cardiac amyloid lesions are diffusely present through the LV and RV, there are few sampling errors in EMB. 190

According to international guidelines, the indication for EMB is classified as class IIa in patients with heart failure associated with unknown restrictive cardiomyopathy such as cardiac amyloidosis and Fabry’s disease. 191 , 192 As a common disease in cardiology, HCM is often diagnosed by familial inheritance, electrocardiography, echocardiography, and MRI without pathological evaluation by EMB. One to nine percent of suspected HCM patients were reported to have been diagnosed with cardiac amyloidosis based on the results of genetic testing, serum or urine protein electrophoresis, FLC testing, and bone scintigraphy. 193 – 195 There is a report of the incidental diagnosis of amyloidosis in patients with HCM undergoing septal myectomy for LV outflow tract obstruction. 196 Therefore, the diagnosis of patients with hypertrophic myocardium must be carefully performed, including information from EMB. The prognosis of patients with cardiac amyloidosis is markedly inferior to that of patients with HCM. EMB plays a crucial role in determining treatment strategies. The recommendations for EMB in cardiac amyloidosis and evidence levels are presented in Table22 .

8. Biopsy (see also CQ2 )

▋ ▋ 8.1 Biopsy From Organs Other Than the Heart

In the pathological diagnosis of systemic amyloidosis, a high detection rate of amyloid in the organs or tissues exhibiting symptoms can be achieved. As an alternative to organs highly invasive for biopsy, such as the heart, nerves, and kidney, relatively less invasive biopsy sites, such as the abdominal wall fat, skin, digestive tract, and lips, can be used to detect amyloid deposition. If the biopsy shows

|Table 2. Recommendation of Endomyocardial Biopsy for Cardiac Amyloidosis and Evidence Levels||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | | Suspected cardiomyopathy with hypertrophic myocardium undiagnosed byothermodalities |na||E||

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS).

 Amyloid deposition of abdominal fat pad in ATTRv amyloidosis. Congo red staining ( Left : bright field, Right : under $\wp\!=\!100\,\mu\mathsf{m}$ .

Figure23.

amyloid deposits, a definitive diagnosis can be obtained by typing the amyloid precursor protein by immunohistochemical staining and mass spectrometry. In the pathological diagnosis of CA, the detection rate of amyloid deposits by myocardial biopsy is almost $100\%$ , which is considered the gold standard for diagnosing this disorder. However, in some cases, especially in older patients, performing a myocardial biopsy involves an increased risk for complications. In CA, it is necessary to distinguish between the three typical types of amyloidosis, namely AL, ATTRwt, and ATTRv amyloidosis, and to introduce treatment specific to each disease at an early stage. To establish the diagnosis of AL amyloidosis, it is important to confirm the presence of amyloid deposits derived from immunoglobulin light chain in any organ. On the other hand, in the early stages of ATTRwt amyloidosis, amyloid deposits are difficult to detect in biopsies at sites other than the heart; thus, clinical diagnostic methods using diagnostic imaging such as pyrophosphate scintigraphy have been proposed in Europe and the US. 1 , 160

▋ ▋ 8.1.1 Abdominal Fat Pad Biopsy

Abdominal fat pad biopsy is a safe and simple procedure; hence, it can be repeated over time for outpatient screening and follow-up. After the induction of local anesthesia in the abdominal fat pad around the umbilicus, the subcutaneous tissue is punctured using an 18G needle, which is reinserted several times while applying negative pressure to the syringe. When the needle is removed with the application of negative pressure, a small amount of adipose tissue is aspirated into the syringe. The collected tissue is crushed between two glass slides to prepare a smear or a formalin-fixed paraffin section, and pathological evaluation is performed by Congo red staining. 197 The amyloid deposits around the adipocytes and small blood vessels contained in the adipose tissue ( Figure23 ). Abdominal fat pad biopsy can produce falsenegative results because of the small amount of sample collected, but multiple biopsies may reduce this risk. The amount of tissue around the adipose tissue is usually small, and it is difficult to perform typing of the amyloid precursor proteins by immunohistochemical staining or mass spectrometry. The amyloid detection rate of abdominal liposuction biopsy is reported to be $79–84\%$ for systemic AL amyloidosis, 198 – 200 45–67% for ATTRv amyloidosis, 190 , 200 and $14{-}15\%$ for ATTRwt amyloidosis. 190 , 200 In ATTRwt amyloidosis, the amount of amyloid deposited in adipose

tissue is small and often sparse. If amyloid deposits are not detected using this method, deep and large skin tissue may be collected by surgical skin incision, as described below. 201

▋ ▋ 8.1.2 Skin Biopsy

Skin biopsy by surgical incision or skin punch biopsy using a biopsy trepan is slightly more invasive than abdominal fat biopsy, but can preserve tissue morphology and collect a larger amount of tissue. Furthermore, in skin biopsy, the amyloid precursor protein can be identified by immunohistochemistry (IHC); hence, the possibility of false-negative results due to sampling errors can be reduced. After the induction of local anesthesia, the skin is cut into a spindle shape using a scalpel, and cross-sectional pathological images of the epidermis, dermis, and subcutaneous adipose tissue are obtained. In a punch biopsy, the epidermis, dermis, and subcutaneous adipose tissue are collected in a cylindrical shape using a biopsy trepan (inner diameter, $3–6\,\mathrm{mm})$ ). Although the amount of sample collected is limited by the size of the trepan in skin biopsy, it is considered a simpler procedure that results in fewer scars than does abdominal fat pad biopsy. In ATTRwt amyloidosis, the utility of abdominal fat pad needle biopsy is limited because the amount of amyloid deposits is limited in the adipose tissue. Conversely, it has been reported that detection sensitivity can be improved by obtaining deep and large skin tissue via surgical incision. 201 , 202 Moreover, in AL and ATTRv amyloidosis, the amyloid detection rate by skin biopsy is higher than that by abdominal fat pad biopsy. 55 , 202

▋ ▋ 8.1.3 Lip Salivary Gland Biopsy

Lip salivary gland biopsy does not require sedation and is associated with few serious bleeding complications. Moreover, in lip salivary gland biopsy, the submucosal tissue can be reliably collected. As a general method, the mucosa side of the lower lip is locally anesthetized and incised, and a small amount of salivary gland tissue is collected. Next, after achieving hemostasis, suturing is performed. Amyloid deposits are frequently detected in small blood vessels and around the salivary glands, and salivary gland biopsy can be performed to obtain the amount of amyloid deposits required to identify the amyloid precursor protein by immunohistochemical staining. 203 The detection rate of lip salivary gland biopsy is $81–89\%$ for systemic AL amyloidosis 204 , 205 and $91–100\%$ for ATTRv amyloidosis. 206 , 207 However, no studies evaluating ATTRwt amyloidosis

 Duodenal amyloid deposition in ATTRwt amyloidosis. Congo red staining ( Left : bright field, Right : under polarized

Figure24.

light), Bars=100 μ m.

using lip salivary gland biopsy have been conducted.

▋ ▋ 8.1.4 Gastrointestinal Biopsy

Gastric/duodenal biopsy using an endoscope is frequently performed to establish a diagnosis of systemic amyloidosis. In the gastrointestinal tract, amyloid deposits are detected on the muscularis mucosa and submucosal blood vessels. Collecting amyloid deposits on only the mucosal surface does not allow the presence of amyloid deposits in the submucosal blood vessels to be evaluated, which produces false-negative results. Therefore, it is necessary to collect amyloid deposits in the submucosa thoroughly while paying careful attention to gastrointestinal perforation and bleeding. Specifically, in ATTRwt amyloidosis, the amyloid deposits, which are nondiffusely distributed, are frequently detected in the submucosal vascular wall only ( Figure24 ); hence, biopsies at multiple sites are required. In the upper gastrointestinal tract, the detection rate of amyloid is higher in the order of the duodenum (part 2, bulbs) and stomach (vestibule). 208 , 209 Using gastrointestinal biopsy tissue, the amyloid precursor protein can be identified by immunohistochemical staining or mass spectrometry. The amyloid detection rate in gastrointestinal tract biopsy is reported to be $72–95\%$ for systemic AL amyloidosis 210 , 211 and $\mathrm{\bar{8}6–100\%}$ for ATTRv amyloidosis. 212 , 213 In a study comprising a few cases of ATTRwt amyloidosis, amyloid was detected in three of the eight cases $(38\%)$ . 26

▋ ▋ 8.1.5 Bone Marrow Biopsy

In AL amyloidosis, bone marrow biopsy is performed to evaluate the presence of abnormal plasma cells. In AL amyloidosis, bone marrow biopsy can detect $57–60\%$ of amyloid deposits; 214 , 215 therefore, bone marrow biopsy with Congo red staining is recommended when assessing the bone marrow. Since the amount of amyloid deposits in bone marrow biopsy tissues is often significantly small, it is frequently difficult to identify the amyloid precursor protein by IHC or mass spectrometry. Positive rates of amyloid deposition in ATTRv and ATTRwt amyloidosis in bone marrow biopsies are reported to be $41\%$ and $30\%$ , respectively. 190

In patients experiencing significant renal failure, such as proteinuria and nephrotic syndrome, renal biopsy frequently leads to the establishment of a diagnosis, especially in AL

and AA amyloidosis. Amyloid deposits are usually detected in the glomeruli, interstitium, and vascular wall. Since renal biopsy is invasive, amyloid deposition can be detected in other biopsy sites, including the abdominal fat pad, skin, lip salivary gland, and gastrointestinal tract, in patients suspected to have systemic amyloidosis. ATTRv amyloidosis also presents with renal failure due to renal amyloidosis and nephrotic syndrome at the end stage, but renal biopsy is rarely performed because diagnosis has already been established by biopsy from other sites. In ATTRwt amyloidosis, no or only a small amount of amyloid is deposited in the kidney.

▋ ▋ 8.1.7 Nerve Biopsy

Nerve biopsy is performed to identify the cause of peripheral neuropathy of unknown cause. Regarding the biopsy site, it is common to select a sural nerve that does not contain a motor nerve component. ATTRv amyloidosis frequently develops with peripheral neuropathy, and the detection of amyloid deposits on nerve biopsy occasionally leads to the diagnosis of this disease. Amyloid deposits are predominantly proximal in peripheral nerves such as the dorsal root ganglia and autonomic ganglia; 216 therefore, sural nerve biopsy might fail to detect amyloid deposits in the early stage of the disease. 217 Although AL amyloidosis frequently presents with a variety of sensory, motor, and autonomic dysfunctions, 218 invasive nerve biopsy is rarely performed if the disease is suspected.

▋ ▋ 8.1.8 Tendon/ligament Tissue Biopsy

In ATTR amyloidosis, amyloids are frequently found in various tendon and ligament tissues, including the lateral carpal ligament and the ligamentum flavum. 20 , 22 , 219 – 221 Specifically, in bilateral carpal tunnel syndrome in older individuals, the rate of detection of TTR amyloid deposition is high when biopsy is performed during lateral carpal ligament release surgery. 20 , 219 TTR amyloid deposition in the tendons and ligaments is considered to occur prior to cardiac lesions; therefore, if TTR amyloid deposition is observed in these tissues, it is necessary to monitor the appearance of the cardiac lesions carefully. On the other hand, in the early stages of ATTRwt amyloidosis, TTR amyloid deposition is frequently localized to the tendon and ligament tissues; therefore, it should be noted that tendon and ligament amyloid deposition cannot necessarily establish the pathological diagnosis of CA.

JCS 2020 Guideline on Diagnosis and Treatment of CA

|Table 23. Detection Rate of Amyloid at Screening Biopsy Sites in CA|||| |---|---|---|---| ||Amyloiddetectionrate|Amyloiddetectionrate|Amyloiddetectionrate| ||AL|ATTRV|ATTRwt| |Abdominal fat pad|79-84%198-200|45-67%190.200|14-15%190.200| |Skin|100%202|94%55|63-73%201*,202| | Lip salivary gland|81-89%204,205|91-100%206,207|No data| |Stomach/duodenum|72-95%210.211|86-100%212,213|38%26**|

\*When performing deeper and wider biopsies than usual. \*\*Examination in a few cases.

|Table 24. Recommendations and Evidence for Biopsy (Other Than the Heart) in Cardiac Amyloidosis||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | | Perform abdominal fat pad biopsy as a means of detecting amyloid other than myocardial biopsy when cardiac amyloidosis is suspected |||E|ya| | Perform lip salivary gland biopsy*, skin biopsy*, and gastrointestinal tract biopsy* as a means of detecting and typing other than myocardiaibiopsywhencardiacamyloidosisissuspected |||E|Iva*| | Perform lip salivary gland biopsy*, skin biopsy*, and gastrointestinal tract biopsy* as a means of detecting and typing other than myocardiaibiopsywhencardiacamyloidosisissuspected |||E|Ivb**| | Repeat biopsies from other sites(abdominal fat, skin,digestive tract, labial salivary glands) when single biopsy failed to detect amyloid deposits |na||E|ya| | Perform abdominal fat pad biopsy, skin biopsy, gastrointestinal biopsy, and labial salivary gland biopsy as a means of assessing theseverity and prognosis of cardiac amyloidosis |||E||

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS).

▋ ▋ 8.1.9 Other Organs

High levels of ALP or liver damage due to amyloid deposition may be observed in AL amyloidosis, but liver biopsy for the diagnosis of this disease is considered to result in a high risk of bleeding, and thus, is not generally recommended. Table23 shows detection rate of amyloid at screening biopsy sites other than the heart in CA. Table24 shows the recommendations and evidence for biopsy other than the heart in CA.

▋ ▋ 8.1.10 Important Points in Biopsy From Organs Other Than the Heart

Abdominal fat pad biopsy is considered to be the simplest procedure, and to be beneficial as a screening test because it is minimally invasive and has a relatively high amyloid detection rate. When CA is clinically suspected, abdominal fat pad biopsy, along with M protein, urinary Bence Jones protein, free light-chain protein, and pyrophosphate scintigraphy are considered beneficial. On the other hand, typing the amyloid precursor protein by abdominal fat pad biopsy is considered difficult because of the small amount of tissue collected. The detection rates of lip salivary gland, stomach/duodenum, and skin biopsy are sufficiently high in AL and ATTRv amyloidosis, and these biopsy sites are considered beneficial when amyloid precursor protein typing (immunohistochemical staining and tissue mass spectrometry) is required. It should be noted that the amyloid detection rate in screening biopsies is not necessarily $100\%$ at any site, and if biopsy from one site fails to detect amyloid deposition, then biopsy from another site or repeat biopsy is recommended. Conversely, in ATTRwt

amyloidosis, amyloid detection rates at biopsy sites other than the heart are relatively insufficient. Therefore, biopsies from multiple sites are frequently required for the histological detection of amyloid, and in some cases, the detection of amyloid deposits in biopsy specimens from sites other than the heart can be difficult.

▋ ▋ 8.2 Endomyocardial Biopsy

CA is essentially diagnosed based on the pathological findings that amyloid deposits are recognized in the myocardial tissues on autopsy or EMB. Amyloid is pathologically defined as substances that turn red with Congo-red staining and show green birefringence through the polarized light microscope, known as the apple green sign. Amyloid deposits can be detected by EMB in almost all patients with CA, leading to the idea that “sampling error” need not be a concern during the process of investigating amyloid by EMB.

▋ ▋ 8.2.1 Light Microscopic Findings

It is important to suspect CA on investigating endomyocardial tissues conventionally stained by hematoxylin-eosin and Masson-trichrome. It may be difficult to differentiate amyloid from interstitial fibrosis by hematoxylin-eosin staining because both tissues can be observed as pale-pink vitreous amorphous ( Figure25A ). On the other hand, Masson-trichrome staining can easily lead to precise differentiation: amyloid seems to be bluish-grey, whereas fibrosis looks clear blue ( Figure25B ). Although direct fast scarlet 4BS or Dylon staining can be used in clinical prac

 Figure25. Light microscopic findings. ( A ) Hematoxylin-eosin staining. ( B ) Masson-trichrome staining. ( C ) Congo-red staining. ( D ) the polarized light microscope. ( E ) imunohistochemical analysis.

tice, in principle, amyloid is defined using Congo-red staining ( Figure25C ) and the polarized light microscope ( Figure25D ). 222 It is useful to ensure no co-staining of the internal elastic membrane to avoid false-positive results during these diagnostic processes.

▋ ▋ 8.2.2 Electron Microscopy Findings

Amyloid fibrils are observed as felt-like unbranched structures, $8–12\,\mathrm{nm}$ in width, in a disarrayed configuration ( Figure26 ). Even when amyloid deposits are not seen on

light microscopy, electron microscopy can rarely show them. When evaluated by electron microscopy, histological tissues from EMB must be fixed by glutaraldehyde. When considering CA clinically as one of the differential diagnoses, part of the biopsied samples should be fixed immediately for electron microscopy. 223

▋ ▋ 8.2.3 Immunohistochemical Analysis

Following the detection of amyloid, immunohistochemical analysis should be performed using specific antibodies to

 Figure26. Electromicroscopic findings.

identify amyloid subtype. 224 Above all, subtyping for transthyretin ( Figure25E ), $\kappa$ chain, or λ -chain is especially important in CA. However, specific antibodies have not been fully developed and widely spread. So it is not rare to get definite subtyping by the stained findings currently.

9. Pathology/Mass Spectrometry

▋ ▋ 9.1 Histopathological Differential Diagnosis of CA

Amyloid typing is essential in determining the treatment plan in patients with CA. After confirming amyloid deposition by Congo red staining, IHC is usually recommended. Since AL, AA, ATTR, and $\mathrm{A}\beta2$ -microglobulin amyloidosis are common in Japan, IHC is performed as the first step using antibodies recognizing these amyloid proteins (Class I, Grade A). However, amyloid proteins in AL amyloidosis may often be unrecognized by commercially available antibodies against $\kappa-$ or λ -light chain because they are mainly composed of variable region fragments of immunoglobulin light chain. 224 Therefore, it is recommended to use antibodies developed for diagnosis in AL amyloidosis 225 (Class IIa, Grade B). At present, amyloidosis diagnostic services using those special antibodies are provided by the Amyloid Research Committee of the MHLW in Japan (http:// amyloidosis-research-committee.jp/consultation/).

In ATTR amyloidosis, it is impossible to distinguish ATTRwt from ATTRv amyloidosis using only IHC; therefore, TTR gene analysis is necessary. In Japan, the most common TTR variant is V30M (p.V50M), which causes severe peripheral and autonomic neuropathy as well as cardiac involvement. However, some TTR variants, such as V122I and T60A, which cause severe CA, have also been detected in Japanese patients. 226 , 227 In patients with these variants, the clinical features typically include lateonset cardiac failure, and the neuropathic symptoms, except for carpal tunnel syndrome, are very mild, which is similar to ATTRwt amyloidosis.

▋ ▋ 9.2 Amyloid Typing Using Mass Spectrometry

▋ ▋ 9.2.1 Detection of Serum Variant TTR

As the majority of TTR variants are one amino acid substitutions, such as V30M, the presence of variant TTR in serum can be confirmed by the detection of the difference of molecular weight in one amino acid substitution using mass spectrometry. 228 In healthy controls and patients with ATTRwt amyloidosis, only a wild-type TTR peak is seen on mass spectrometry. On the other hand, both wild-type and variant TTR peaks are detected in patients with ATTRv amyloidosis. However, the peak of variant TTR becomes undetectable in the case of small differences in the molecular weight, such as in E61K and E89Q.

▋ ▋ 9.2.2 Amyloid Typing Using Mass Spectrometry

When the type of amyloid protein is not confirmed by IHC, an investigation of amyloid protein is needed. With recent advances in mass spectrometry, the investigation of quite small amounts of amyloid protein is possible, 229 , 230 and tiny biopsied samples, such as cardiac biopsy, can be analyzed. Even in formalin-fixed, paraffin-embedded samples, amyloid typing can be performed using LMDLC-MS/MS. 231 Protein analysis using LMD-LC-MS/MS is currently one of the most useful diagnostic procedures, and shows higher diagnostic accuracy than IHC. 232 In LMD, deposited amyloid can be selectively isolated with a laser cutter ( Figure27A – F ). Amyloid is composed of amyloid fibril protein and various amyloid-associated proteins, such as serum amyloid-P component; therefore, which protein is a main body of amyloid should be determined carefully. In Japan, amyloid protein typing using LMD is mainly carried out at Kumamoto University and Shinshu University, and diagnostic services can also be ordered online through the Amyloid Research Committee of the MHLW in Japan (http://amyloidosis-research-committee. jp/consultation/) ( Table25 ).

 Figure27. Extraction of deposited amyloid by laser microdissection (LMD). ( A , B , C ): Extraction of amyloid from nerve tissues. Arrows denote deposited amyloid on the endoneurium ( A ) and those amyloid deposits were isolated by LMD ( B ). ( C ) shows isolated amyloid (arrows). ( D , E , F ): Histopathology of localized atrial amyloidosis diagnosed by LMD. Amyloid deposits were detected in the atrium and it was conformed using LMD and LC-MS/MS that amyloid protein is derived from atrial natriuretic peptides (ANP). ( D , E ): Congo red staining ( E , polarized condition). ( F ): IHC using anti-ANP (performed by Dr. Yoshinobu Hoshii, Yamaguchi University, Ube, Japan).

|Table 25. Levels of Recommendation and Evidence of Histopathological Diagnosis in Cardiac Amyloidosis ||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | |IHC after detecting amyloid deposits||E||Ib| |Massspectrometryforcasesundiagnosed byIHC||EB||Ivb|

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS).

| Table 26. Recommendations and Evidence Levels of Genetic Testing for the Definitive Diagnosis of ATTR-Type Cardiac Amyloidosis ||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | |Detection of TTR variant|||||

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS).

| Table 27. Difference Between "Genetic Testing for the Diagnoses of Patients Who Have Developed a Disease" and "Genetic Testing for Presymptomatic Diagnosis" |||| |---|---|---|---| ||Purpose of the test| Physicianandmedical staff in charge of the test |Cost of the test| | Genetictestingforpatients who have already developed a disease | Establishment of diagnosis of ATTRv and ATTRwt amyloidosis | Attending doctor (e.g., Cardiologist) | Covered by public health insurance including genetic counseling fee | | Genetic testing for presymptomatic diagnosis | Prediction of disease development of ATTRv amyloidosis beforehand | Genetic counseling team · Clinical geneticists ·Genetic counselor · Clinical psychologist | Not covered by public health insurance | | Genetic testing for presymptomatic diagnosis ||||

10. Genetic Testing and Counseling

▋ ▋ 10.1 Role of Genetic Testing for the Diagnosis of CA

Genetic testing in patients with CA is necessary for the definitive diagnosis of ATTRv and ATTRwt amyloidosis ( Table26 ). Genetic testing may also be performed for the purpose of presymptomatic genetic testing in at-risk relatives. The target gene for testing is TTR .

TTR is composed of four exons and encodes 147 amino acids. Mature TTR protein consists of 127 amino-acids, as codons 1–20 encode a 20-amino-acid signal sequence. The most common pathogenic variant in Japan is $\mathrm{c.148G{>}A}$ (p.V50M). Historic protein numbering was based on the mature protein after cleavage of a 20-amino-acid signal sequence (e.g., p.Val50Met would be referred to as Val30Met). In this guideline, both historic and standard protein numbering as recommended by the Human Genomes Variation Society are employed.

In Japan, genetic testing should be performed in accordance with the “Guidelines for Genetic Tests and Diagnoses in Medical Practice” approved by the Japanese Association of Medical Sciences, 233 and “genetic testing for the diagnoses of patients who have developed a disease” and “genetic testing for presymptomatic diagnosis” should be distinguished ( Table27 ).

▋ ▋ 10.2 Genetic Testing for the Diagnoses of Patients Who Have Developed a Disease

Definitive diagnosis of patients with CA is essential to choose the optimal therapeutic strategy, and thus, genetic testing should be performed for patients who are suspected as having ATTR amyloidosis. However, sufficient consideration is necessary, as variants in the TTR gene have a profound impact on relatives. In particular, patients should be informed about the purpose and implication of the test, as well as the mode of inheritance, clinical course, prognosis, and therapy for the disease prior to testing. In addition, possible conditions after the results are obtained should be indicated. If necessary, the attending physician should

arrange genetic counseling by experts so that patients can receive support for autonomous decision-making.

V122I (p.V142I) is a representative variant that shows a cardiac-dominant phenotype, and is found in $3.9\%$ of the African-American population. 234 Recently, a patient with this variant was also reported in Japan. 226 In addition, several variants, including T60A (p.T80A), D38A (p.D58A), and S50I (p.S70I), have been associated with the cardiacdominant phenotype in Japan. It is not rare that late-onset V30M patients show the cardiac-dominant phenotype. A considerable number of patients with late-onset ATTRv amyloidosis do not have a familial history of amyloidosis. 42 Therefore, genetic testing is needed to differentiate ATTRwt from ATTRv amyloidosis, even in sporadic older patients.

In Japan, TTR gene analysis is covered by public health insurance (medical remuneration points: 3,880) in patients who are suspected as having ATTRv amyloidosis. A genetic counseling fee (medical remuneration points: 1,000) can be added when the results of genetic testing are disclosed at genetic counseling.

▋ ▋ 10.3 Genetic Testing for Presymptomatic Diagnosis

Patients with hereditary disorders often do not wish to disclose information about the disease to at-risk relatives. However, with early diagnosis, ATTRv amyloidosis is a treatable disease, and thus, the provision of information to at-risk relatives is highly important in terms of health management. 235 Actually, it has been reported that the percentage of presymptomatic testing usage is higher in ATTRv amyloidosis than in untreatable diseases. 236 , 237 It is desirable that the provision of information to at-risk relatives and presymptomatic testing be managed by the genetic counseling department. Presymptomatic testing of hereditary disorders should be managed in accordance with the “Guidelines for Genetic Tests and Diagnoses in Medical Practice” approved by the Japanese Association of Medical Sciences, 233 and performed by an adequate genetic counseling team. As ATTRv amyloidosis is an adult-onset disease, it is necessary to confirm that 1) the client is an adult, 2) the client is willing to take the test, and 3) the client

| Table 28. Recommendations and Evidence Levels of Genetic Counseling for Patients and Relatives of ATTRvAmyloidosis ||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | |Genetic counseling for patients with ATTRv amyloidosis|||C1|| | GeneticcounselingforasymptomaticrelativesofATTRv amyloidosispatients |||C1||

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS).

has sufficiently understood the information concerning the mode and risk of inheritance, clinical course, prognosis, and available therapeutic strategies of the disease, purposes and implications of the test, and possible conditions after the result is obtained.

ATTRv amyloidosis is an autosomal-dominant genetic disorder, and patients are usually heterozygous for a TTR pathogenic variant. Therefore, each asymptomatic child of a patient has a $50\%$ risk of inheriting the TTR pathogenic variant. 235 In early-onset families with the V30M variant in endemic areas, the penetration of the disease is approximately $100\%$ . However, the penetration of the disease is not so high in late-onset families with the V30M variant in non-endemic areas, especially female carriers, who sometimes do not develop the disease throughout their lifetime. As the male:female ratio is approximately 4:1 in late-onset V30M patients in non-endemic areas, 42 penetration is considered to be low in female patients. A limited number of patients with biallelic TTR pathogenic variants have been reported. In such a situation, all offspring of a proband will inherit the variant gene. 235

When presymptomatic tests identify an asymptomatic variant carrier, the carrier should be followed on a periodic basis by the genetic counseling and clinical departments (e.g., Department of Cardiology, Department of Neurology), and psychological support and screening tests for the onset of amyloidosis should be provided. 238 It should also be noted that presymptomatic testing is not covered by public health insurance in Japan.

Recommendations and evidence levels of genetic counseling for relatives of and patients with ATTRv amyloidosis are summarized in Table28 .

11. Other Items (Aortic Stenosis)

Aortic stenosis combined with ATTR cardiac amyloidosis has attracted attention in recent years. 239 , 240 Myocardial amyloid deposits are observed in $6\%$ of patients aged ${\geq}65$ years with advanced aortic stenosis who have undergone aortic valve replacement. 241 Another study reported that $99\,\mathrm{mTc}$ pyrophosphate scintigraphy detected myocardial amyloid deposits in $16\%$ of patients who underwent trans

catheter aortic valve implantation (TAVI) (mean age, 84 ± 6 years, $68\%$ male). 29 The patients with positive results in $\mathrm{99\,mTc}$ pyrophosphate scintigraphy were of advanced age (mean age, $86.3{\pm}5.7\$ years). Positive results were more common among men than women ( $22\%$ vs. $4\%$ , respectively). Many patients with positive results demonstrated prolonged QRS duration; right bundle branch block; and low-flow low-gradient aortic stenosis. In addition, one study observed occult cardiac amyloidosis in $13.9\%$ of advanced aortic stenosis patients prior to TAVI. 242

In advanced aortic stenosis combined with ATTR cardiac amyloidosis, therapeutic intervention involves high risk. Some studies report that symptoms improve little despite treatment and prognosis is poor. 241 , 243 – 245 However, occult amyloid deposition is commonly observed in elderly people. 25 Different severities of cardiac amyloidosis may affect prognosis differently. In the examination of therapeutic interventions for aortic stenosis in the elderly, the coexistence of ATTR cardiac amyloidosis must always be considered. Treatment suitability should be determined on a case-bycase basis based on an accurate assessment of the severities of ATTR cardiac amyloidosis and aortic stenosis.

Both ATTR cardiac amyloidosis and advanced aortic stenosis result in left ventricular hypertrophy, hindering the suspicion of coexistent ATTR cardiac amyloidosis. However, ATTR cardiac amyloidosis can be suspected based on various red flags (carpal tunnel syndrome, etc.) or late enhancement or high extracellular volume in contrast-enhanced CT prior to TAVI. 246

The causal relationship between aortic stenosis and ATTR cardiac amyloidosis is not well understood. In cases of surgically removed heart valves, amyloid in aortic stenosis was not only observed frequently $(74\%)$ , it was observed more frequently than in aortic regurgitation or mitral valve stenosis. 247 Aortic stenosis-induced valve degeneration, inflammation-induced atherosclerotic changes, and high shear stress are suggested to potentially result in the progression of aortic stenosis by promoting amyloid deposition in the aortic valve. However, cardiac load associated with aortic stenosis may lead to left ventricular remodeling and promote TTR amyloid deposition, resulting in ATTR cardiac amyloidosis. 240

III. Treatment of Cardiac Amyloidosis (CA)

In general, heart diseases are roughly classified into pump failure and rhythm abnormality. Figure28 shows the overview of treatment for cardiac amyloidosis by stage of heart

failure. The recommendations and evidence levels for drug therapy in patients with cardiac amyloidosis are presented in the following Table29 .

 Figure28. Overview of treatment for cardiac amyloidosis by stage. ACCF, American College of cardiology Foundation; AHA, American Heart Association; HF, heart failure; HFpEF, heart failure with preserved ejection fraction; EFrEF, heart failure with reduced ejection fraction.

|Table 29. Recommendations and Levels of Evidence for Drug Therapy in Patients With CA||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | |Diuretics for fluid retention|||C1|| | Nitrates or carperitide for pulmonary congestion in acute settings|ma||C1|| | Catecholamines or phosphodiesterase inhibitors in pump failure with low output |ma||C1|| | Tolerated dosing of ACE inhibitors, β-blockers and aldosterone antagonists to reduce clinical events |||C1|| |DOACfor AF|||E|| |DOACforATwithLVdysfunction|ma||C1|| |β-blockerfor AF tachycardia| (case by case) ||C1|| | NonDPH Ca antagonist for ATTR amyloidosis with preserved systolic function | (case by case) ||02|| | NonDPH Ca antagonist for ATTR amyloidosis with impaired systolic function |||02|| |NonDPH Ca antagonist for AL amyroidosis||C|02|| |Digitalis for AF|||02||

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS).

|Table 30. Recommendations and Levels of Evidence for Palliative Care for Patients With CA||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | | Conduct advance care planning (ACP) in which physicians share decisions with the patient and his/her family members about treatment and care before the patient becomes difficult to make his or her own decisions |||E|| | Continued treatment to manage heart failure and complications and relieve symptoms |||E|| | Multidisciplinary team-based frequent assessment for the patient's physical,mental and spiritual needs |||C1||

(Adapted from Japanese Circulation Society and the Japanese Heart Failure Society Joint Working Group. 2018 5 ) COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS).

1. General Management

▋ ▋ 1.1 Basic Policies for CA Treatment

As a clinical phenotype in CA patients, cardiologists encounter heart failure (HF) more often than arrhythmias. $^{69}\mathrm{F}$ treatment generally consists of two categories: one is to prolong lives through the prevention of disease progression, and the other is to relieve signs and symptoms while reducing the risk of death through improving hemodynamic deterioration. Furthermore, the former consists of two components: one is the intervention for long-lasting disease progression commonly seen in chronic HF, and the other is to treat the cause of HF. The basic policies for CA management are to deal with symptomatic complaints based on HF, together with the consideration of two therapeutic possibilities: general HF pharmacotherapy and causal treatment for CA.

▋ ▋ 1.2 Treatment to Prevent HF Progression

Few clinical studies have focused on HF management in CA patients. HF patients with left ventricular systolic dysfunction should receive pharmacological treatment generally recommended based on the clinical evidence, such as ACE inhibitors, angiotensin II receptor blockers, $\beta$ -blockers, and aldosterone antagonists. 5 However, there have been no trials so far to show their prognostic benefits in HF with preserved ejection fraction resembling the clinical phenotypes in most CA cases. 5 Moreover, low cardiac output can be easily seen through the restrictive compromise in CA. The neurohumoral modulator agents described above not only have uncertain properties for improving the prognosis in diastolic dysfunction, but they may also induce adverse effects such as hypotension and renal impairment. 249 Accordingly, causative therapy for amyloidosis has to play a major role to prevent disease progression in CA cases. It may be a wise policy to use neurohumoral modulators carefully in order to relieve HF symptoms partly through vasodilation and diuresis, as long as critical complications are not induced.

▋ ▋ 1.3 Treatment to Ameliorate HF Signs and Symptoms

Additional drugs should be given to patients with signs of

congestion and low cardiac output. Although vasodilators and diuretics are generally used for the treatment of congestion, vasodilators can play a limited role because of the potential risk of hypotension occasionally seen in CA. 248 Diuretic agents including loop diuretics have frequently been used to relieve congestion. 249 However, intravascular volume depletion may cause hypotension in cases of restrictive hemodynamics, leading to diuretic resistance. Tolvaptan may be a therapeutic option in cases with loop-diuretic resistance presumably through aggressive fluid removal with preservation of intravascular volume by refilling from the extravascular space, 250 even in cases with renal dysfunction and hyponatremia.

Inotropic drugs such as catecholamines and phosphodiesterase inhibitors are used for the treatment of low perfusion. These drugs have mainly been used temporarily in the acute phase, but oral inotropic agents may be unavoidable in cases with persistent low output during the chronic phase. Although not often recommended, oral inotropics may occasionally lead to relief of HF symptoms or avoid subsequent clinical events. Especially in patients in sinus rhythm, one must be careful not to reduce the heart rate excessively, because an increased heart rate has a compensatory role in a restrictive heart with low stroke volume. 248

▋ ▋ 1.4 Palliative Care

Even with advances in therapy aimed at the cause, end-stage CA will continue to result in life-threatening conditions and a poor prognosis. Advance care planning is an important step to help individual patients to prepare for future changes in their disease condition. In addition, many CA patients suffering from general pain should be supported by healthcare professionals with multidisciplinary approaches. 5 CA is a representative disease for considering palliative care ( Table30 ).

2. Pharmacologic Treatment of Arrhythmia

Patients with CA have various supraventricular (e.g., AF, atrial flutter, atrial tachycardia, conduction disturbance) and/or ventricular arrhythmia (e.g., ventricular tachycardia, ventricular fibrillation, pulseless electrical activity [PEA]). However, no evidence regarding the relationship between the prognosis of CA and these arrhythmias has been estab

lished because no international cohort or interventional studies have been conducted. Although a guideline is already available from the JCS, physicians should choose the therapy individually according of the characteristics of CA (e.g., etiology, general status, stage of amyloidosis, effectiveness of treatments, prognosis). Therefore, in this guideline, we have provided notes in regard to the use of previous guidelines for patients with CA.

▋ ▋ 2.1 Atrial Fibrillation, Atrial Flutter, and Atrial Tachycardia

In patients with CA, amyloid fibers deposit in the myocardial interstitial matrix. Diastolic dysfunction due to LV hypertrophy increases the LA pressure. The deposition of amyloid fibers in the atrium also results in conduction disturbances. These phenomena involve AF and/or other atrial arrhythmias. Sanchis et al. reported that $44\%$ of 238 patients with CA already had AF at diagnosis (persistent AF: $60\%$ ; nonpersistent AF: $40\%$ ). AF was found in $26\%$ of patients with AL and $60\%$ with ATTR amyloidosis. Overall, in the observation period, AF was found in $64\%$ of patients with CA. 102 Pinney et al. also reported that $11\%$ of patients with AL and $46\%$ of those with ATTR amyloidosis had AF at diagnosis. 70 The prognosis of patients with AF was worse than that of patients without AF. 251 The prognosis with permanent AF did not differ from nonpermanent AF. 102

▋ ▋ 2.2 Anticoagulant Therapy for AF

Edward et al. reported that transesophageal echocardiography revealed spontaneous echocardiogram contrast and atrial thrombi in $\cdot67\%$ and $28\%$ of patients with CA accompanied by AF, respectively; however, no differences were found between AL and other CAs. 252 Furthermore, intracardiac mural thrombi were found in $35–50\%$ of patients with CA without AF. AL amyloidosis, AF, LV diastolic dysfunction, tachycardia, and RV hypertrophy were independent risk factors for thoromboembolism. 63 , 122 Therefore, anticoagulant therapy should be prescribed for all patients with CA accompanied by AF after an evaluation of hemorrhage risk. Patients with atrial tachycardia and systolic/diastolic dysfunction should also receive anticoagulant therapy. In patients who require tooth extraction or a surgical procedure, anticoagulant therapy should be discontinued in accordance with the 2020 JCS guideline. 253

▋ ▋ 2.3 Heart Rate Control

Tachycardia due to AF $(\mathord{>}130/\operatorname*{min})$ can result in congestive heart failure, even if patients have no organic cardiac disorders. Therefore, heart failure occurs more frequently in patients with CA with diastolic dysfunction due to LV hypertrophy. Generally, the Race II Trial recommended mild heart rate control $(<\!110/\mathrm{min})$ during AF to prevent heart failure and other cardiac events. 254 Similarly, mild heart rate control was recommended for patients with CA. However, in patients with conduction disturbance, the dosage of medications to avoid AV block must be chosen carefully. Heart rate control is not needed in anemic patients. First of all, treatment for anemia should take precedence over heart rate control. In the general population, digitalis, $\beta\cdot$ -blockers, verapamil, diltiazem, and amiodarone are available for heart rate control. However,

digitalis is contraindicated in CA because the usual daily dose could cause life-threatening arrhythmia. Intravenous injection of landiolol hydrochloride $(1{-}10\,\mu\mathrm{g/kg/min})$ can reduce heart rate safely in patients with LV dysfunction (LVEF $25–50\%$ ). However, there is little evidence of its safety and effectiveness in patients with CA. Non-dihydropyridiane Ca blockers (verapamil and diltiazeme) have negative inotropic effects. These Ca blockers are contraindicated in patients with amyloidosis (especially AL amyloidosis) with LV dysfunction (Class III, level C). Bisoprolol fumarate and carvedilol can be prescribed to reduced heart rate in patients with heart failure; however, careful attention is needed when prescribing carvedilol to patients with autonomic dysfunction. Carvedilol containing α blockade could worsen postural hypotension.

▋ ▋ 2.4 Pulmonary Vein Isolation for AF

The indication of pulmonary vein isolation (PVI) for AF should be decided individually according to the 2018 JCS/ JHRS Guideline on Non-Pharmacotherapy of Cardiac Arrhythmias. 255 The etiology of CA, LA dilatation, LV hypertrophy, and other general statuses are important factors affecting prognosis after PVI. Recently, PVI for AF has been established as non-pharmacotherapy in the general population. The success rate in patients with paroxysmal AF is $80–90\%$ . However, the effectiveness of PVI in patients with CA has not been thoroughly evaluated. Tan et al. reported that the success rate in patients with CA was lower than that in the general population ( $75\%$ at 1 year and $60\%$ at 3 years). 256 Therefore, patients with paroxysmal AF without LA dilatation or LV hypertrophy may be candidates for PVI (Class IIb, level C). However, PVI is contraindicated for patients with AL amyloidosis, poor prognosis and severe LA dilatation, and LV hypertrophy (Class III, level C).

3. Device Therapy

▋ ▋ 3.1 AV Block

Patients with CA frequently have some sort of ECG abnormality. 257 Penney et al. reported that AV conduction abnormalities were recognized in $43\%$ of patients with AL and $58\%$ of patients with ATTR amyloidosis. 70 Pacemaker implantation is indicated for $20\%$ of patients with CA. The indication of an implantable pacemaker for AV block can be discussed according to the 2018 JCS/JHRS Guideline on Non-Pharmacotherapy of Cardiac Arrhythmias. 255 Amyloidosis is generally a progressive disease. Therefore, the time courses until advanced or complete AV block differ among patients. Moreover, some patients are accompanied by life-threatening ventricular arrhythmias. Thus, scheduled ECG or Holter ECG and implantable cardiac monitoring (ICM) should be performed for the early detection of these arrhythmias. Vincent et al. demonstrated that pacemaker implantation was indicated within 45 months in patients with CA with the following findings: first-degree AV block, Wenckbach rate ${<}100\,\mathrm{bpm}$ , AH interval ${>}70\mathrm{ms}$ , HV interval ${>}55\,\mathrm{ms}$ , and bundle block). In addition, the preventive implantation of a pacemaker for such high risk patients has been shown to be effective for avoiding cardiac events. 258 However, the usefulness of preventive pacemaker implantation has not been established (Class IIb, level C).

▋ ▋ 3.2 Sick Sinus Syndrome and AF Bradycardia

The indication for an implantable pacemaker for sick sinus syndrome and AF bradycardia can be discussed according to 2018 JCS/JHRS Guideline on Non-Pharmacotherapy of Cardiac Arrhythmias. 255 AF is more frequently accompanied by AV block in patients with CA than in the general population. Asymptomatic AF bradycardia is not recommended for pacemaker implantation; however, AF bradycardia accompanied by heart failure could be a candidate for pacemaker implantation. Regardless of the presence of symptoms (e.g., syncope, convulsion, dizziness, dyspnea, easy fatigability) in patients with AF bradycardia, the relationship between the presence of symptoms and bradycardia need to be verified by scheduled ECG or Holter ECG and ICM.

▋ ▋ 3.3 Cardiac Resynchronized Therapy (CRT)

Generally, cardiac resynchronized therapy (CRT) for patients with heart failure has been recognized as an effective therapy for LV dysfunction associated with intraventricular conduction disturbance, especially left bundle branch block (LBBB). However, no evidence has been established for CA. To date, only case reports or small cohort studies have been published. Therefore, the evidence level is not available. The indication for CRT should be discussed individually. Candidate patients can be discussed according to the 2018 JCS/JHRS Guideline on NonPharmacotherapy of Cardiac Arrhythmias. 255 CRT can be indicated for patients with LBBB and a relatively good prognosis $^{(>1}$ year) (Class IIb, level C). CRT is contraindicated for patients with a poor prognosis ( ${<}1$ year), a QRS interval less than 150ms, and conduction disturbances other than LBBB (Class III, level C).

▋ ▋ 3.4 Implantable Cardioverter-Defibrillators (ICDs)

Previous reports have demonstrated the occurrence of sudden cardiac death in $10–30\%$ of patients with CA. 259,260 Especially in AL amyloidosis, the prognosis is extremely poor and PEA is occasionally documented. Therefore, reviews regarding the efficacy of ICDs do not recommend ICD implantation for patients with amyloidosis. 261 , 262 In a Japanese cohort study (Nippon Storm), few cases of implanted ICDs for CA were reported. 263 Kristen reported that many patients with CA eventually died as a result of ventricular fibrillation or PEA, and no significant difference in prognosis was found between patients with and without an implanted ICD. 261 On the other hand, favorable results for ICD implantation were reported in patients with preserved cardiac function who received effective therapy. 264 Therefore, indications for ICDs must be carefully discussed. The 2018 JCS/JHRS Guideline on Non-Pharmacotherapy of Cardiac Arrhythmias 255 is limited as a reference regarding the indication for ICD in patients with CA. ICD implantation is not strongly recommended for prevention against sudden cardiac death in any patients (so called Class I indication). ICD implantation for patients with a poor prognosis ( $\mathord{\left<1\right>}$ year) is contraindicated (Class III, level C). A limited number of patients (e.g., those with mild hypertrophy and preserved systolic/diastolic function, those with a good prognosis after adequate therapy) could be candidates for ICD implantation (Class IIb, level C). In the future, the indications for ICD implantation may expand

| Table 31. Guidelines for Patient Selection for Stem Cell Transplantation (Mayo Clinic) || |---|---| |Suggested requrement|| |“Physiologic" age ||

(Adapted from Sher T, et al. 2016 276 ) ©2016 American Society for Blood and Marrow Transplantation. Licensed under the CC BY-NC-ND 4.0.

if cardiac involvement can be eliminated after early diagnosis and adequate therapy can be provided. These issues require further discussion.

4. Treatment for AL Amyloidosis

The aim of treatment for AL amyloidosis is to decrease FLC, which is a major cause of toxic amyloid fibrils. The National Comprehensive Cancer Network (NCCN) guideline recommends the combination of cyclophosphamide (CPA), bortezomib (BOR), and dexamethasone (DEX) (CyBorD) as treatment for both transplant-eligible and transplant-ineligible patients. 265 This combination is also recommended in the United Kingdom (UK) guideline. 266 Unfortunately, BOR has not been approved for primary AL amyloidosis in Japan (as of March 2020). The UK guideline notes that treatment-related toxicity is increased when BOR is used for patients with AL amyloidosis as compared with MM, and dose reduction is recommended. No randomized, clinical trial has shown the efficacy and safety of BOR-including regimens to date.

In Japan, standard treatment for AL amyloidosis is the combination of oral melphalan (MEL) and dexamethasone (DEX) (MEL/DEX), and high-dose melphalan and stemcell transplantation (HDM/ASCT) may lead to a higher response rate and long-term survival for eligible patients. 267 – 273 There are few data on the superiority of HDM/ASCT compared with other treatment modalities. A randomized trial reported in 2007 failed to show the superiority of HDM/ASCT over oral MEL/DEX. 272 The lack of evaluation of cardiac biomarkers may lead to high treatmentrelated mortality (TRM) in patients receiving HDM/ASCT.

The indications for HDM/ASCT have been reported from the Mayo Clinic 274 and the UK amyloidosis treatment trial. 275 A recent review from the Mayo Clinic provides simple criteria 276 ( Table31 ).

MEL/DEX seems to lack power and have low efficacy, but it is recommended for HDM/ASCT-ineligible patients because of low treatment toxicity. Palladini reported that better survival is expected in hematological responders. 277

Although the efficacy of BOR and thalidomide (THAL) has been reported from the USA and Europe, these drugs are not approved in Japan as of March 2020. The combination of CPA, BOR, and DEX is reported as CyBorD from the Mayo Clinic 278 and as CVD from the National Amyloidosis Center, London. 279 Both reports showed efficacy and safety, and such combination treatment will be a powerful strategy if eligible patients are carefully chosen. Venner reported the result of a matched pair analysis

JCS 2020 Guideline on Diagnosis and Treatment of CA

 Process of ATTR amyloid fibril formation and action mechanism of disease modifying therapies approved in Japan.

Figure29.

showing better response with CVD than CTD. 280 Approval of BOR and THAL for primary AL amyloidosis is expected.

5. Disease-Modifying Therapy for ATTRv Amyloidosis (see to CQ3 )

The clinical efficacy of disease-modifying therapies including liver transplantation, transthyretin (TTR) tetramer stabilizers, and gene therapies has been proven for hereditary ATTR (ATTRv) amyloidosis. Figure29 shows the process of ATTR amyloid fibril formation and the mechanism of action of disease-modifying therapies approved in Japan.

▋ ▋ 5.1 Liver Transplantation

Since most serum TTR is produced by the liver, liver transplantation was first performed in a patient with ATTRv amyloidosis in 1990. After liver transplantation, the serum concentration of TTR decreased rapidly 281 , 282 ( Figure29 ). Since then, liver transplantation has been performed for this disease at many hospitals worldwide, and useful clinical data are accumulating. According to data from the Familial Amyloidotic Polyneuropathy World Transplant Registry and Domino Liver Transplant Registry, 283 2,236 liver transplantations have been performed to date with 5-year and 20-year survival rates of $\bar{7}7\%284$ and $55\%$ , 285 respectively. Prognosis after liver transplantation is affected by TTR genotype, and it was reported that the 5-year survival rate was $82\%$ in V30M (p.V50M) patients, whereas that in non-V30M patients was $59\%$ . 286 Among V30M patients, the prognosis of early-onset patients is excellent, 51 although that of elderly male patients is relatively poor. 287 , 288 The main cause of death after liver transplantation is cardiacrelated death. 283 , 284

Liver transplantation decreases the amount of amyloid deposition in abdominal fat tissue 289 and improves periph

eral and autonomic nerve function in early-onset V30M patients. 290 However, turnover of amyloid is different among organs, and progression of amyloid deposition is often observed in the heart after liver transplantation. 291 – 293 In addition, it should be noted that ocular amyloidosis and leptomeningeal/cerebral vessel amyloidosis are not relieved by liver transplantation due to variant TTR synthesis by the retinal pigment epithelium and choroid plexus. 294 – 296

The situation surrounding the use of liver transplantation for ATTRv amyloidosis in Japan is different from that in other areas of the world. Cadaveric liver tissue is mainly used for transplantation outside Japan, whereas liver from live donors is mainly used in Japan due to a lack of brain dead donors. It takes considerable time to obtain live donor liver transplantation after registration with the Japan Organ Transplant Network because the urgency of transplantation for ATTRv amyloidosis is relatively low compared with other diseases. Requisites for live donors include healthy adults ≤ 65 years, relatives ≤ sixth-degree or spouse’s relatives ≤ third-degree, blood type possible for blood transfusion, and not having a pathogenic TTR variant.

▋ ▋ 5.2 TTR Tetramer Stabilizer

TTR exists as a tetramer in the human body, but dissociation of the tetramer to a monomer is necessary to form amyloid fibrils ( Figure29 ). It has been shown that the TTR tetramer structure is destabilized in ATTRv patients due to TTR gene variation. 47 Based on these findings, research on small molecules that bind to and stabilize TTR tetramer advanced, and tafamidis was developed ( Figure29 ). Tafamidis has been shown to inhibit progression of peripheral neuropathy and decrease all-cause death and cardiovascular-related hospitalizations.

The first randomized, controlled trial of tafamidis (Fx-005 study) 297 enrolled 125 ATTRv patients with V30M variant and administered tafamidis (tafamidis meglumine 20mg/day)

| Table 32. Recommendations and Evidence Levels of Disease Modifying Therapies for ATTRv Neuropathy and Cardiomyopathy* ||||| |---|---|---|---|---| ||COR|LOE| GOR (MINDS) | LOE (MINDS) | |Liver transplantation|||E|Ivb| |Tafamidis for neuropathy**||E|E|| |Patisiran for neuropathy|||E|| | Tafamidsis for patients with NYHA Class I/ll and satisfy patient requirementsin thestatement of theJapaneseCirculationSociety* |na|EB|E|| | Tafamidsisforpatients withNYHA Class Ill and satisfy patient requirements in the statement of the Japanese CirculationSociety** ||EB|E||

Tafamidis should be administered with a dosage of 20mg for neuropathy and 80mg for cardiomyopathy (dosage of tafamidis can be reduced in the case of intolerance to $80\,\mathsf{m g}$ ). \*Refer to Table33 for therapeutic options in each patient. \*\*Refer to section of “ ATTRwt amyloidosis ” concerning indication of tafamidis for ATTRv cardiac amyloidosis. COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS).

or placebo for 18 months. The primary endpoints were treatment response (with response defined as ${<}2$ -point increase in the Neuropathy Impairment Score in the Lower Limbs [NIS-LL]) and change from baseline in total quality of life as measured by the Norfolk Quality of Life–Diabetic Neuropathy total score. Fx-005 study demonstrated that significantly more tafamidis patients than placebo patients were NIS-LL responders ( $60.0\%$ vs. $38.1\%$ , respectively), and tafamidis patients had better preserved total quality of life (0.1 vs. 8.9, respectively) in the efficacy-evaluable (EE) population. Based on these findings, tafamidis was approved in 2013 in Japan for the inhibition of peripheral neuropathy in ATTRv amyloidosis. Concerning ATTR-type cardiac amyloidosis (ATTRv and ATTRwt), a second randomized, controlled trial of tafamidis (ATTR-ACT study) 3 was conducted and demonstrated that tafamidis significantly inhibited the primary endpoints (i.e., all-cause death and cardiovascular-related hospitalizations) compared with placebo. Based on these findings, an additional indication for tafamidis, ATTR-type cardiac amyloidosis (both ATTRwt and ATTRv), was approved in March, 2013 in Japan (refer to “ Chapter III, 6. Treatment for ATTRwt Amyloidosis ”).

Similar to tafamidis, diflunisal, a well-known nonsteroidal anti-inflammatory drug, also has a TTR tetramer stabilization effect and was proven to inhibit progression of peripheral neuropathy in a randomized, controlled trial. 298 However, diflunisal is not currently available in Japan and is not approved for the treatment of ATTRv amyloidosis.

▋ ▋ 5.3 Nucleic Acid Medicine

ATTRv amyloidosis is considered to be a good target for gene silencing therapy because ATTRv amyloidosis is a typical gain of toxic function disease, and TTR knockout mice do not show any abnormal phenotype. 299 In addition, delivery of a nucleic acid drug is relatively easy, since most TTR is produced by the liver.

Under these circumstances, patisiran, a small interfering RNA (siRNA) therapeutic agent targeting TTR mRNA, was developed, and the result of a randomized, controlled trial (APOLLO study) was reported in 2018. 300 The primary endpoint of the APOLLO study was change from baseline

of the modified Neuropathy Impairment Score $+7$ (mNIS+7), and 225 ATTRv amyloidosis patients were randomly assigned to receive patisiran $0.3\,\mathrm{mg/kg}$ or placebo by intravenous injection every 3 weeks for 18 months. Patisiran reduced the serum TTR level by approximately $80\%$ from baseline, and significantly improved the $\mathrm{mNIS+7}$ compared with placebo (changes from baseline in the patisiran group and in the placebo group were $-6.0$ and 28.0, respectively). In addition, the analysis of the cardiac subpopulation, patients with left ventricular wall thickness ${\geq}13\,\mathrm{mm}$ and no history of hypertension or aortic valve disease, showed that patisiran significantly inhibited deterioration of left ventricular wall thickness, global longitudinal strain, cardiac output, end-diastolic volume, and NT-proBNP. 300 – 302 Regarding the safety, although the incidence of infusionrelated reaction was observed in about $20\%$ of the patients with patisiran, this treatment was well tolerated.Based on the result of the APOLLO study, patisiran was approved and became available in September, 2019 in Japan for TTR-type amyloid neuropathy (indication described in the package insert).

Inotersen, an antisense oligonucleotide therapeutic agent targeting TTR mRNA, was also proven to inhibit mNIS+7 and improve the Norfolk Quality of Life-Diabetic Neuropathy score compared with placebo in a randomized, controlled trial of ATTRv amyloidosis. 303 However, serious adverse events including glomerulonephritis and thrombocytopenia were reported in the inotersen group, and all patients need to receive enhanced monitoring for these events. Due to the serious adverse events described above, a phase III clinical trial of inotersen was cancelled in Japan; therefore, inotersen is not approved in Japan ( Table32 ).

▋ ▋ 5.4 Therapeutic Options in ATTRv Amyloidosis

ATTRv amyloidosis is a lethal disease if disease-modifying therapies are not given. Therefore, either liver transplantation or pharmacotherapy (tafamidis or patisiran) should be performed. Therapeutic options in each patient should be decided based on characteristics of each therapy ( Table33 ) and the patient’s condition, including TTR genotype, severity of amyloidosis, general condition, and social environment, such as occupation.

|| Table 33. Characteristics of Disease Modifying Therapies for ATTRv Amyloidosis Disease modifying Merits (efficacy, safety, etc.) therapy || |---|---|---| || Table 33. Characteristics of Disease Modifying Therapies for ATTRv Amyloidosis Disease modifying Merits (efficacy, safety, etc.) therapy |Points of attention| | Liver transplantation ||| | Liver transplantation | Lon-term evidenceofefficacyinpatientswithearly- onset V30M variants (peripheral nerve damage is stopped or slightly improved if transplantation is performed in early stage of the disease) || | Liver transplantation | Lon-term evidenceofefficacyinpatientswithearly- onset V30M variants (peripheral nerve damage is stopped or slightly improved if transplantation is performed in early stage of the disease) || | Liver transplantation || Invasiveness Shortage of donor Requirement for immunosuppressive drugs after transplantation Transplant-related complications (biliary obstruction, infection, rejection, etc.) Limitation of efficacyin non-V30Mpatients Progression of cardiac amyloidosis after transplantation due to wild-type TTR deposition Progression of ocular and leptomeningeal/cerebral vessel amyloidosis after transplantation Can not stop progression of pheripheral neuropathy and cardiacfailurecompletely | |||| |||| |Tafamidis| V30M patients Reduce frequency of all cause of death and cardiovascular-related hospitalizations Inhibition of deterioration of cardiac failure symptoms Excellent safety profile Good compliance as being a once a day oral drug | No evidence concerning inhibition of progression of peripheral neuropathy in non-V30Mpatients (tafamidis covered by public health insurance for both V30M and non-V30M patients in Japan) No evidence concerning ocular and leptomeningeal/ cerebral vessel amyloidosis High medical cost | |||| |Patisiran| Arrest or slight improvement of peripheral neuropathy Inhibition of deterioration of echocardiographic parameters and NT-proBNP | Lack of long term evidence (efficacy, safety) Intravenous injection every 3 weeks (require approximately 3h including premedications) No evidenceconcerning ocular and leptomeningeal/ cerebral vesselamyloidosis High medical cost |

Liver transplantation was the only therapeutic option for ATTRv amyloidosis for a long time and was still a firstline therapy for early-onset V30M patients even after approval of tafamidis. 75 However, pharmacotherapy (tafamidis or patisiran) has become first-line therapy for all ATTRv amyloidosis patients because patisiran showed an excellent therapeutic effect on peripheral neuropathy, comparable with liver transplantation in the APOLLO study ( Table33 ). In particular, the indications for liver transplantation are limited because cardiac amyloidosis progresses even after liver transplantation. Since a limited amount of evidence has been accumulated to date, combination therapy with patisiran and tafamidis should be considered only in patients who do not respond to monotherapy with each drug.

6. Treatment for ATTRwt Amyloidosis (see also CQ3 )

▋ ▋ 6.1 TTR Stabilizers

The tetrameric structure of TTR is thought to be destabilized by gene mutations or aging, and dissociates into monomers, causing misfolding and polymerization. TTR stabilizers suppress amyloid fibril formation by binding at the thyroxine-binding site of TTR to stabilize the tetrameric structure.

In a phase 2/3 study in 2012, 297 tafamidis was well tolerated in patients with ATTRv amyloidosis with peripheral neuropathy for 18 months, and it suppressed the progression of peripheral nerve symptoms. In 2013, approval for tafamidis was granted in Japan.

The ATTR-ACT, 3 a phase III international, multicenter, double-blind, randomized clinical study, was conducted to investigate the efficacy, safety, and tolerability of an oral daily dose of $20\,\mathrm{mg}$ or 80mg tafamidis meglumine compared with placebo over a 30-month period in 441 patients with

ATTR-CM (including both ATTRwt and ATTRv amyloidosis). In the primary analysis, a significant reduction in the hierarchical combination of both all-cause mortality and the frequency of cardiovascular-related hospitalizations was observed in the tafamidis groups (264 cases) compared with the placebo group (177 cases). Tafamidis significantly reduced all-cause mortality (tafamidis group: $29.{\bar{5}}\%$ [78/264 cases]; placebo group: $42.9\%$ [76/177 cases]) and cardiovascular event-related hospitalization frequency compared with the placebo group (0.48/year in the tafamidis group; 0.70/year in the placebo group) ( Figure30 ). 3

In the secondary study end points, a decline in functional capacity as measured by 6-min walking test distance was alleviated in the tafamidis group, and a decline in QOL as evaluated by the Kansas City Cardiomyopathy Questionnaire-Overall Summary score was also improved compared with the placebo group at 30 months. Tafamidis was also well tolerated, with an observed safety profile comparable to that of placebo.

In prespecified subgroups, tafamidis was effective for both ATTRwt and ATTRv amyloidosis. No significant difference in the primary end point between the high- and low-dose groups was found, but all-cause mortality was significantly higher in the high-dose group ( Figure31 ). 3 However, it took more than 1.5 years from the start of treatment to observe a decrease in mortality due to tafamidis, and it could not be confirmed to be effective by subgroup analysis in patients with NYHA class III at baseline with cardiovascular-related hospitalization, suggesting the importance of early administration.

Following the results of the ATTR-ACT, ATTR-CM (including both ATTRwt and ATTRv amyloidosis) was added to the indications for tafamidis in March 2019. Although the efficacy of tafamidis has been demonstrated in a patient group enrolled in the ATTR-ACT, the efficacy in those excluded from this study ( Table34 ), such as those with NYHA class IV, remains unknown. The MHLW has

 All-cause mortality in ATTR-ACT study. (Adapted from Maurer MS, et al. 2018 3 ) Copyright $\circledcirc$ 2018 Massachusetts Medical Society. All rights reserved.

Figure30.

 Figure31. Subgroup analysis in ATTR-ACT study. (Adapted from Maurer MS, et al. 2018 3 ) Copyright $\circledcirc$ 2018 Massachusetts Medical Society. All rights reserved.

set the requirements for prescribing tafamidis, and the JCS has established the certification for prescribing facilities and physicians to promote the appropriate use of this drug. Table35 shows the recommendations and levels of evidence for tafamidis for ATTRwt CA.

▋ ▋ 6.2 Nucleic Acid Medicine

Regarding the neurological symptoms of patients with ATTRv amyloidosis who have peripheral neuropathy, the efficacy of two oligonucleotide-based drugs that knock

down the expression of TTR — patisiran, a siRNA drug, and inotersen, an antisense oligo drug — have been confirmed in phase III trials. 300 , 303 Patisiran was approved in Japan in June 2019 (for details, see “ Chapter III, Section 5. Treatment for ATTRv Amyloidosis ”). Since the efficacy of these nucleic acid drugs for ATTR has not been sufficiently evaluated, a phase III trial of patisiran, APOLLO-B, and a clinical trial of the next-generation inotersen, AKTEATTR-LRX, are underway for ATTR, including ATTRwt amyloidosis.

JCS 2020 Guideline on Diagnosis and Treatment of CA

Table34. Main Patient Groups Excluded in ATTR-ACT Study

|.Heart failure not caused by ATTR-CM| |---| |.NYHAClassIV| |·AL amyloidosis| |·Implanted with any cardiac device| |· Estimated glomerular filtration rate (eGFR) ≤25 mL/min/1.73 m2| |·Liver transaminase more than twice the upper normal limit| |. Notable undernutrition patients: Modified body mass index (serum albumin (g/L)xBMl) |

(Source: Prepared based on Maurer MS, et al. 2018 3 )

| Table 35. Classes of Recommendation and Levels of Evidence for Tafamidis in the Treatment of ATTRwt CardiacAmyloidosis ||||| |---|---|---|---|---| ||COR*|LOE| GOR (MINDS) | LOE (MINDS) | | Tafamidis for ATTRwt cardiac amyloidosis patients with NYHA Class I/Il who meet patient requirements in the JCS statement |iia|E|E|| | Tafamidis for ATTRwt cardiac amyloidosis patients with NYHA Class Ill whomeet patient requirements in the JCSstatement ||E|E||

COR, class of recommendation; LOE, level of evidence; GOR (MINDS), grade of recommendation (MINDS); LOE (MINDS), level of evidence (MINDS). \*The JCS statement is subject to change, so always refer to the latest information (see CQ3–1 ). \*Only the ATTR-ACT has been reported as a randomized controlled trial for ATTR. Therefore, the recommendations and evidence levels associated with tafamidis greatly depend on the results of this trial, and the following points need to be noted. · NYHA Class IV patients were excluded from the study, and thus, the efficacy and safety of tafamidis in such patients have not been confirmed. · The efficacy and safety of tafamidis in the population excluded from the ATTR-ACT ( Table34 ) have not been confirmed. · In the ATTR-ACT trial, it took >1 year after administration to confirm the effectiveness of tafamidis. Therefore, in cases with a life expectancy not expected to be ${>}1$ year, the administration of tafamidis should be carefully considered.

IV. CA Diagnostic Algorithm

The diagnostic algorithm for CA is shown in Figure32 . 1 , 75 , 304 – 309

[CQ1] What is the role of 99m Tc-PYP scintigraphy (bone scintigraphy) in clinical practice for CA?

[Response]

99m Tc-PYP scintigraphy is fairly effective for detecting both ATTRv and ATTRwt CA 1 , 2 , 173 (see “ Chapter II, Section 6. Nuclear Imaging ”). When 99m Tc-PYP scintigraphy yields a positive result, ATTRv and ATTRwt CA must be differentiated with genetic testing.

In the visual assessment of 99m Tc-PYP scintigraphy, AL amyloidosis sometimes presents as Grade 2, thereby requiring hematology testing to rule out monoclonal immunoglobulin (M protein). If 99m Tc-PYP scintigraphy yields a positive result and AL amyloidosis is excluded, the diagnostic specificity and positive predictive value for ATTR are reportedly $100\%$ . 1 Physiological uptake into blood pools may result in false-positive results. Therefore, SPECT should be performed to assess myocardial uptake accurately.

An important consideration in the use of 99m Tc-PYP scintigraphy is selecting suitable cases (see in Chapters II and IV ). 99m Tc-PYP scintigraphy positivity can be assessed

using the Kumamoto criteria: 99 high-sensitivity cardiac troponin T ${\geq}0.03\,\mathrm{ng}/\mathrm{mL}$ , LV posterior wall thickness ${\geq}13.6\,\mathrm{mm}$ , and QRS width ${\geq}120\,\mathrm{ms}$ . For patients who meet two and three of these criteria, 99m Tc-PYP scintigraphy positivity rates are $63\%$ and $96\%$ , respectively. Thus, the Kumamoto criteria may be useful when selecting patients to undergo 99m Tc-PYP scintigraphy.

In real-world clinical practice, the suitability of 99m TcPYP scintigraphy must be considered on a case-by-case basis. Table36 shows the clinical scenarios and recommendation levels for 99m Tc-PYP scintigraphy based on existing academic evidence. 160 , 308 , 310

[CQ2] The role of endomyocardial biopsy. Which site is appropriate for biopsy in cardiac amyloidosis?

1. Biopsy From Other Than the Heart

Generally, pathological information can be obtained from the tissues or organs related to symptoms in patients with systemic amyloidosis. Abdominal wall fat, skin, salivary gland of the lip, and the intestinal tract are selected as noninvasive biopsy sites. 26 , 190 , 198 – 201 , 204 , 205 , 207 – 215 The detection

 Figure32. Algorithm for diagnosis of cardiac amyloidosis.

|Table 36. Clinical Scenarios and Recommendation Levels for 99mTc-PYP Scintigraphy|| |---|---| |Clinical scenario| Recommendation level | |Suspicion of ATTR cardiac amyloidosis|Recommended| |Cardiac hypertrophy and previous bilateral carpal tunnel syndrome| Recommended| |Cardiac amyloidosis suspected in echocardiography, CMR, or cardiac CT|Recommended| |Low-flow, low-gradient aortic stenosis in a patient aged ≥60 years|Recommended| | Persistently mildly elevated high-sensitivity cardiac troponin T in a patient aged ≥70 years |Considered| | Heart failure of unidentified cause with left ventricular hypertrophy in a patient aged ≥60 years |Considered| | Atrioventricular block, bundle branch block, or atrial fibrillation with cardiac hypertrophy in a patient aged ≥60 years |Considered| |Symptoms of polyneuropathy or autonomic neuropathy|Considered| |Monitoring of progress for a patient diagnosed with ATTR cardiac amyloidosis|Not recommended| |Suspicion of AL amyloidosis|Not recommended|

AL, amyloid light-chain; ATTR, amyloid transthyretin; CMR, cardiac magnetic resonance imaging; CT, computed tomography.

rate of amyloid protein from various tissues/organs varies depending on the type of amyloid precursor protein. If amyloid protein is detected from non-cardiac organs in suspected amyloidosis patients with clinical and imaging findings, it is acceptable to make a diagnosis of cardiac amyloidosis.

1.1 Biopsy From Other Than the Heart in AL Amyloidosis In patients with systemic AL amyloidosis, abdominal fat pad, lip salivary gland, and bone marrow biopsies are commonly performed. The amyloid protein detection rate at each biopsy site is shown in Table37 . In suspected AL amyloidosis with evidence of monoclonal proteins, bone marrow biopsy is often performed to search for abnormal plasma cells. Bone marrow biopsy is not the first choice for screening, because the detection rate of amyloid deposits by bone marrow biopsy is relatively low in AL amyloidosis. It is recommended that bone marrow specimens be assessed by Congo red staining for detection of amyloid protein. Although the amyloid detection rate of liver and spleen biopsies is high, they should be avoided because they are highly invasive procedures inducing bleeding complications.

1.2 Biopsy From Other Than the Heart in ATTR

In transthyretin-related (ATTR) amyloidosis, biopsies are often performed from the abdominal fat pad, labial salivary gland, and gastrointestinal tract. As shown in Table37 , the amyloid protein detection rates of biopsies in patients with wild-type transthyretin-related (ATTRwt) amyloidosis were relatively low, such as $14{-}15\%$ for the abdominal fat pad 200 , 214 and $38\%$ for the intestinal tract. 26 It has been demonstrated that the detection rate of abdominal wall skin biopsy was $73\%$ . 201 Skin biopsy can yield improvement of the sensitivity for diagnosis of ATTRwt amyloidosis. Since there are some localized cases of ATTR amyloidosis with limited amyloid deposition in tendon and ligament tissues, diagnosis of systemic ATTR amyloidosis based on pathological findings of tendon and ligament tissues alone

should be avoided.

\*Usefulness of bone scintigraphy and endomyocardial biopsy in ATTR

It has been reported that the detection rate of amyloid protein by EMB was almost $100\%$ in patients with ATTR amyloidosis. 190 The frequency of sampling errors from myocardium is extremely low. In particular, the amyloid protein detection rate of biopsies other than the heart is not high at the initial stage of ATTRwt amyloidosis. Bone scintigraphy leads to the diagnosis of ATTRwt cardiomyopathy being made reliably with EMB. 1 , 160

2. Endomyocardial Biopsy

EMB can show amyloid fibrils in clinically suspected patients with hypertensive heart disease or HCM on echocardiography. Therefore, it is important to pathologically differentiate cardiac amyloidosis from other hypertrophic cardiomyopathies. In high-risk patients with conduction disturbances and advanced age, biopsy is performed to detect amyloid protein from the abdominal fat pad, skin, lip salivary gland, and intestinal tract. A non-invasive abdominal fat biopsy is often performed in such cases. However, the amyloid detection rate of abdominal liposuction biopsy is not high in patients with ATTRwt amyloidosis. 200 , 214 Other biopsy sites, especially myocardium in reference to bone scintigraphy, are selected. Patients with myocardial uptake on bone scintigraphy and no FLC and confirmation of pathological amyloid protein cannot be definitely diagnosed as having ATTR amyloidosis, but such cases are regarded as probable ATTR amyloidosis (see “ Chapter I, Section 5. Diagnostic Criteria” ).

[CQ3] What patients should receive tafamidis for CA?

|Table 37. Biopsy Sites in Amyloidosis|||| |---|---|---|---| | Author (reference number) |Subjects| Biopsy site| Detection rate| |Garcia Y198| 97 suspected amyloidosis undergoing abdominal fat pad excisional biopsy | Abdominal fat excisional tissue |AL 79%, ATTR 12%| |van Gameren I1199|120 amyloidosis (38 AA, 70 AL, 12 ATTR)| Abdominal fat aspiration tissue |AA 92%,AL 94%,ATTR 83%| |Quarta CC200| 600 cardiac amyloidosis (216 AL, 113 ATTRv, 271ATTRwt) | Abdominal fat aspiration tissue | AL 84%,ATTRv 45%,ATTRwt 15% | |Suzuki T204| 34 patients with evidence of monoclonal proteins and symptoms related amyloidosis | Skin, bone marrow, and Labial salivary gland | Skin 72%, Bone marrow 77%, Labial salivary gland 89% | |Hachulla E205| 59 cases undergoing labial salivary gland biopsy (30 patients with AA or AL amyloidosis) | Labial salivary gland|Labial salivary gland 86%| |SwanN214|100 AL amyloidosis|Bone marrow| Bone marrow 60%| |Kimmich C215|168 AL amyloidosis| Bone marrow, abdominal fat aspiration tissue | Bone marrow 57%, abdominal fat96% | |Fine NM190| 286 ATTR amyloidosis with positive extra- cardiac biopsy (186 ATTRv and 100 ATTRwt) | Myocardium, abdominal fat aspiration tissue | Myocardium 100%, abdominal fat (ATTRv 67%,ATTRwt 14%) | |Lechapt-Zalcman E207|32 polyneuropathy with unknown etiology|Labial salivary gland|ATTRv 100%| |Ikeda S201|11ATTRwt|Abdominal wall skin|Skin73%|

1. Statement by the Japanese Circulation Society (JCS)

Tafamidis (Vyndaqel®) received approval in Japan for suppressing the progression of peripheral neuropathy caused by ATTRv amyloidosis in September 2013. Following the results of the ATTR-ACT, 3 in March 2019, ATTR CA was added to the indications for this drug.

According to the precautions related to the efficacy of tafamidis, the Health Care Division of the MHLW states, “When applying this drug, refer to the latest guidelines and confirm that the diagnosis of ATTR has been determined.” It was recommended that tafamidis should be used in patients judged to be eligible by a physician skilled in diagnosing and treating ATTR.

In response to the recommendations of the MHLW, the JCS has begun to prepare guidelines for CA, and has issued a statement that stipulates the requirements of facilities and physicians for the introduction of tafamidis (shown below), in addition to the patient requirements from the MHWL ( Table38 ). 311 , 312

2. What patients with ATTR amyloidosis should take tafamidis?

A: ATTR patients with NYHA functional class I or II are eligible for active treatment using tafamidis. In addition, it is necessary to determine the indication for treatment in consideration of general conditions such as comorbidities and frailty.

Class of Recommendation: IIa Level of Evidence: B Grade of Recommendation (MINDS): B Level of Evidence (MINDS): II

Commentary

A tafamidis phase III trial (ATTR-ACT) showed that patients with ATTR in the tafamidis oral administration group had better survival compared with controls. 3 In particular, patients with NYHA functional class I or II showed significant improvements in both all-cause mortality and cardiovascular-related hospitalizations.

ATTR amyloidosis is a progressive disease that shows

poor prognosis, although the prognosis of the ATTR type is not as bad as that of the AL type. 313 , 314 In patients with ATTR amyloidosis, there are also several reports that there is no difference between ATTRwt and ATTRv amyloidosis in terms of prognosis. 77 , 313 In ATTRv amyloidosis, it is necessary to consider the patient’s general condition because of neuropathy. In ATTRwt amyloidosis, cardiac events are the most common cause of death. 31 , 315

There are several reports, mainly from overseas, on the prognostic factors for patients with ATTR, but in those reports, the number of target patients is only about 100. An accurate prognostic prediction method has not yet been established. The following prognostic factors have been raised from previous reports: age, NYHA functional class, biomarkers (e.g., BNP/NT-proBNP, troponin, renal function, uric acid), echocardiographic indices (e.g., LVEF, relative wall thickness, strain), and CMR (e.g., LGE, ECV). 31 , 33 , 54 , 70 , 92 , 93 , 148 , 153 , 157 , 314 , 316 – 319 In ATTRv amyloidosis, MIBG scintigraphy was reported to be useful for prediction. 176 AF, a common electrocardiographic finding in patients with ATTR amyloidosis, has been reported to be associated with the onset of heart failure, but not clearly associated with survival. 102 , 103 , 105 There are few prognostic data for Japanese patients with ATTR, and further large scale investigations are needed. 101 , 320

With regard to predicting survival in heart failure (not limited to CA), many risk prediction models using multiple clinical indicators have been reported. 321 – 325 However, whether these models are useful for predicting events in patients with ATTR amyloidosis remains unclear. In general, for patients with heart failure, the most consistently reported predictors were age, NYHA functional class, renal function, low blood pressure, low body mass index, malnutrition, and exercise capacity. 326 , 327 In addition, in aged patients with heart failure, the presence of comorbidities is often the primary determinant of prognosis, rather than heart failure itself. Although it is reported that cardiac events are the most frequent mode of death in patients with ATTR, the indication for treatment using determined in comorbidities (e.g., renal dysfunction, cere

Table38. A Statement for the Appropriate Administration of Tafamidis in Patients With Transthyretin Cardiac Amyloidosis

1) Patient requirements

$\textcircled{1}$ ATTRwt cardiac amyloidosis

a. A medical history of hospitalization for heart failure or a heart failure condition requiring treatment with a diuretic for improvement b. Presence of amyloid deposits in biopsy tissue (cardiac or non-cardiac) c. Transthyretin precursor protein identification by immunohistochemistry d. Evidence of cardiac involvement on echocardiography with an end-diastolic interventricular septal wall thickness of $_{>12\,\mathsf{m m}}$

$\circledcirc$ ATTRv amyloidosis

a. A cardiomyopathy symptom and a TTR gene mutation associated with cardiomyopathy b. A medical history of hospitalization for heart failure or a heart failure condition requiring treatment with a diuretic for improvement c. Evidence of cardiac involvement on echocardiography with an end-diastolic interventricular septal wall thickness of $_{>12\,\mathsf{m m}}$ d. Presence of amyloid deposits in biopsy tissue (cardiac or noncardiac)

2) Facility requirements

As a general rule, only facilities that meet all of the following requirements $\textcircled{1}$ to $\textcircled{6}$ are permitted to start administration of tafamidis meglumine:

$\textcircled{1}$ Cardiologist training facility certified by the JCS $\circledcirc$ Radiologist general training institute certified by the Japan Radiological Society $\textcircled{3}$ Pathologist training facility certified by the Japan Society of Pathology $\textcircled{4}$ Hematologist Training Facility certified by the Japanese Society of Hematology $\textcircled{5}$ Neurologist educational facilities certified by the Japanese Society of Neurology $\textcircled{6}$ Institution that treats more than 15 cases of myocardial biopsy annually

3) Physician requirements

Physicians who fulfill the following requirements $\textcircled{1}$ or $\circledcirc$ and commit to $\textcircled{3}$ :

$\textcircled{1}$ Physicians who have used tafamidis meglumine for ATTRv amyloidosis before the approval date of the indication expansion for ATTR-CM $\circledcirc$ Physicians who have identified at least three cases of ATTR by identifying the TTR precursor protein by immunohistological staining or mass spectrometry from a biopsy tissue sample obtained at their own institution or by asking another institution for identification $\textcircled{3}$ Registration of all administration cases Continuous therapy for patients who have been introduced to the treatment can be prescribed at a cooperating hospital, considering the convenience of patients, but the facilities that, and physicians who, have introduced tafamidis meglumine are responsible for maintaining follow-up of the clinical course.

(Excerpted from Endo J, et al. 2019 311 )

brovascular disorder, COPD, anemia), general condition, and nutritional status, including frailty and sarcopenia.

3. What are echocardiographic findings to recommend the use of tafamidis?

• There are no data about echocardiographic findings to determine whether tafamidis should be used.

• The effects of tafamidis are expected in patients with ATTR amyloidosis in the early stage. Therefore, it is important to diagnose ATTR amyloidosis at an earlier stage using specific cardiac echocardiographic findings.

A placebo-controlled randomized trial revealed that tafamidis improved prognosis (all-cause mortality and cardiovascular events) in patients with ATTR amyloidosis and inhibited the reduction of frailty and QOL. 3 However, the survival rate (end point: all-cause mortality) at 1.5 years after administration was almost the same between patients who received tafamidis and those who received placebo, and gradually became different after 1.5 years. Therefore, although tafamidis appears to inhibit the progression of the disease stage, it does not seem to improve the pathology of ATTR amyloidosis. In patients with NYHA I or II, the risk of all-cause mortality and/or hospitalization due to cardiovascular events was reduced by tafamidis, although it did not improve in patients with NYHA III. Judging from these results, tafamidis appears to be effective for inhibiting disease progression in patients

with ATTR amyloidosis with NYHA I or II. Therefore, ATTR amyloidosis needs to be diagnosed and treatment with tafamidis needs to be started before patients show obvious symptoms of heart failure with more than NYHA III.

To our knowledge, no studies have investigated echocardiographic findings for predicting prognostic improvement with tafamidis. Therefore, we conclude that there are no data about echocardiographic findings to determine whether tafamidis should be used. However, considering the effects of tafamidis described above, it is important to identify patients with ATTR amyloidosis at an early stage by echocardiography. To confirm these findings, we reviewed previous studies about echocardiographic findings as prognostic predictors and specific findings on echocardiography seen at an early stage of ATTR amyloidosis.

Regarding echocardiographic findings as prognostic predictors in patients with ATTR amyloidosis, LVEF was the most reported. Grogan et al. 33 reported that LVEF ${<}50\%$ was an independent prognostic predictor in patients with ATTRwt CA (hazard ratio [HR]: 1.85, $95\%$ confidence interval [CI]: 1.12–3.06). Moreover, in patients with ATTRwt CA, Connors et al. 31 and Hanson et al. 319 reported that reduced LVEF was a prognostic predictor. LVEF has ever been the most evidence-available echocardiographic finding as a prognostic predictor. Therefore, it is important to diagnose ATTR amyloidosis and start tafamidis before LVEF begins to reduce. On the other hand, previous studies have reported that myocardial

contraction fraction (MCF), which is the ratio of LV mass to LV stroke volume, is useful for predicting prognosis in patients with ATTR amyloidosis. Rubin et al. 318 reported that MCF ${<}25\%$ (HR: 8.5, $95\%$ CI: 4.8–14.9) is more useful as a prognostic predictor than LVEF ${<}50\%$ (HR: 2.8, $95\%$ CI: 1.8–4.4) in patients with ATTR amyloidosis. The usefulness of MCF was also shown in patients with AL amyloidosis; 328 thus, MCF as well as LVEF should be considered as prognostic predictors for patients with amyloidosis.

Moreover, some studies have reported that tricuspid annular plane systolic excursion (TAPSE) and mitral annular plane systolic excursion (MAPSE) are more useful prognostic predictors than LVEF, MCF, and LV global strain. 93 , 329 TAPSE was significantly lower in patients with than in those without RV LGE on CMR (RV LGEpositive: TAPSE 12 [9–15] mm, RV LGE-negative: TAPSE 20 [18–25] mm). RV LGE reflected RV fibrosis in patients with CA and was related to prognosis. 329

Tafamidis should be started before these echocardiographic findings are detected. This treatment strategy could contribute to improved prognosis in patients with ATTR amyloidosis.

As clues for the early diagnosis of ATTR amyloidosis, apical sparing (apical-to-basal strain ration ${>}2{:}1$ ), in which LV motion at the apex is well maintained compared with basal motion, and relatively high LVEF values (EF-tostrain ratio ${>}4$ ) were specific findings in CA. 309 , 330 Moreover, it has been reported that differences in strain values between LV apex and basal increased by using exercise stress echocardiography, and this difference in strain values was related to symptoms in patients with CA (e.g., NYHA class, metabolic equivalents at peak exercise, maximum V O ). 331 2

Apical sparing is observed both in patients with ATTR amyloidosis and in those with AL amyloidosis. However,

the strain value at the LV apex in patients with ATTR amyloidosis has been shown to be lower than that in patients with AL amyloidosis. 332 The earlier diagnosis of ATTR amyloidosis by using these echocardiographic findings may lead to earlier administration of tafamidis.

[CQ4] What is the role of genetic testing in clinical practice for cardiac amyloidosis?

In clinical practice, genetic testing is essential for the definitive diagnosis of ATTRv and ATTRwt amyloidosis in patients with cardiac amyloidosis. Genetic testing may be performed for the purpose of presymptomatic testing of relatives. The target gene for the test is TTR . If a pathogenic variant is not identified, ATTRv amyloidosis can be completely ruled out. Situations necessary for the TTR gene test are as follows:

1. Differential diagnosis of ATTRv and ATTRwt amyloidosis in a patient diagnosed as having ATTR amyloidosis by biopsy or complementary tests such as 99m Tc-pyrophosphate scintigraphy. 2. Definitive or differential diagnosis of ATTRv amyloidosis in a cardiomyopathy patient with neuropathy and/or a family history (genetic test may be performed before biopsy in this situation). 3. Definitive diagnosis of ATTRv amyloidosis in symptomatic relatives of an ATTRv amyloidosis patient. 4. Presymptomatic diagnosis in asymptomatic relatives of an ATTRv amyloidosis patient (genetic counseling by specialists is necessary before testing in presymptomatic testing; presymptomatic testing is not covered by public health insurance in Japan).

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Appendix1 Details of Members

Collaborators

• Hiroaki Kitaoka, Department of Cardiology and Geriatrics, Kochi Medical School, Kochi University

Members

• Takayuki Inomata, Department of Cardiovascular Medicine, Kitasato University Kitasato Institute Hospital • Chisato Izumi, Department of Cardiovascular Medicine, National Cerebral and Cardiovascular Center • Yasuhiro Izumiya, Department of Cardiovascular Medicine, Osaka City University, Graduate School of Medicine • Jun Koyama, Department of Internal Medicine, Maruko Central Hospital • Toru Kubo, Department of Cardiology and Geriatrics, Kochi Medical School, Kochi University • Motoaki Sano, Department of Cardiology, Keio University School of Medicine • Yoshiki Sekijima, Department of Medicine (Neurology and Rheumatology), Shinshu University • Nobuhiro Tahara, Division of Cardiovascular Medicine, Department of Medicine, Kurume University School of Medicine • Takeshi Tomita, Department of Cardiovascular Medicine, North Alps Medical Center Azumi Hospital • Kenichi Tsujita, Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University • Nobuhiro Tsukada, Department of Haematology, Japanese Red Cross Medical Center • Hiroyuki Tsutsui, Department of Cardiovascular Medicine, Kyushu University • Mitsuharu Ueda, Department of Neurology, Kumamoto University • Masashi Amano, Department of Cardiovascular Medicine, National Cerebral and Cardiovascular Center • Yuichi Baba, Department of Cardiology and Geriatrics, Kochi Medical School, Kochi University • Jin Endo, Department of Cardiology, Keio University School of Medicine • Seitaro Oda, Department of Diagnostic Radiology, Faculty of Life Sciences, Kumamoto University • Atsushi Okada, Department of Cardiovascular Medicine, National Cerebral and Cardiovascular Center • Yohei Misumi, Department of Neurology, Kumamoto University Graduate School of Medical Sciences • Seiji Takashio, Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University • Masahide Yazaki, Institute for Biomedical Sciences, Shinshu University

External Evaluation Committee Members

• Yukio Ando, Department of Amyloidosis Research, Nagasaki International University • Toshihisa Anzai, Department of Cardiovascular Medicine, Hokkaido University • Keiichi Fukuda, Department of Cardiology, Keio University School of Medicine • Mitsuaki Isobe, Sakakibara Heart Institute • Takeshi Kimura, Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine (The affiliations of the members are as of January 2020)

Appendix2 Disclosure of Potential Conflicts of Interest (COI): JCS 2020 Guideline on Diagnosis and Treatment of Cardiac Amyloidosis

|Author|Potential COIof the participant| Potential COI of the marital partner, first- degree family | PotentialCOIf thehead oftheorganizat departetwhchparalo (when the participant isin the positonf cooperativerserchihtheheadofth organization/department) |||||||||| |---|---|---|---|---|---|---|---|---|---|---|---|---| |Author| Employer/ leadership position (private company) |Stakeholder|royalty'|Honorarium| Payment for manuscripts |Research grant| Scholarship (educational) grant |Endowed chair| Other rewards | thosewho share income and property |Research grant| Scholarship (educational) grant | | Chair: Hiroaki Kitaoka |||| Daichi Sankyo Company, Limited Takeda Pharmaceutical Company Limited Mitsubishi Tanabe Pharma Corporation Bayer Yakubin, Ltd. ||| OnoPharma Co., Ltd. Bayer Yakuhin, Ltd. |||||| | Members: Chisato Izumi |||| Daichi Sankyo Company, Limited Otsuka Pharmaceutical Co. Lt. || Daichi Sankyo Company,Limited ||||| Daiichi Sankyo Company, Limited || | Members: Yasuhiro Izumiya |||| Sanofi K.K. Pfizer Japan Inc. Daichi Sankyo Company, Limited || Daiichi Sankyo Company, Limited ||||||| | Members: Takayuki Inomata |||| Bayer Yakuhin, Ltd. Pfizer Japan Ine. Bristol-Myers Squibb OtsukaPhamaceuticalCo Daichi Sankyo Company, Limited Nipon BohringerIngelhem Co.,Ltd. Medtronic Japan Co. Ltd. | MedtronicJapan Co., Ltd. |||||||| | Members: Mitsuharu Ueda ||||Alnylam Pharmaceuticals Inc.|| Takeda Science Foundation Prothena Corporation Public Limited Company ||||||| | Members: Toru Kubo |||| Sumitomo Dainippon Pharma Co., Ltd. Pfizer Japan Inc. ||||||||| | Members: Jun Koyama ||||Pfizer Japan Inc.||||||||| | Members: Motoaki Sano |||| MSD K.K. Astellas Pharma Inc. AstraZeneca K.K. Novartis Pharma K.K. Boehringer Ingelheim GmbH Kowa Company, Ltd. Ono Pharmaceutical Co. Lt. Taisho Pharmaceutical Co., Ltd. TAIYONIPPON SANSO CORPORATION Daichi Sankyo Company, Limited Mitsubishi Tanabe Pharma Corporation || TAIYO NIPPON SANSO CORPORATION | TAIYONIPPON SANSO CORPORATION ||||| Takeda Pharmaceutical Company Limited | | Members: Yoshiki Sekiima |||| Pfizer Japan Inc. Alnylam Pharmaceuticals Inc. |||Pfizer Japan Inc.|||||| | Members: Nobuhiro Tahara |||| Pfizer Japan Inc. ActelionPharmaceuticals Japan Ltd_ ||||||||| | Members: Nobuhiro Tsukada ||||Company Limited||||||||| | Members: Kenichi Tsujita |||| MSD K.K. Amgen Astellas BioPharma K.K... Sanofi K.K. Bayer Yakuhin, Ltd. Pfizer Japan Inc. Bristol-MyersSquibb KowaPharmaceutical CoLtd Otsuka Pharmaceutical Co. Ltd. Daichi Sankyo Company, Limited Takeda Pharmaceutical Company Limited || AstraZeneca K.K. Bristol-Myers Squibb Linical Co.,Ltd. KowaCompany. Ltd.. Sugi bee garden TEJINHOME HEALTHCARE LIMITED. Japan Medical Device Technology Co., Ltd. | MSD K.K. ITICO.,LTD. Astellas Pharma Inc. AbbottVascular Japan Co., Ltd. AbbottMedical Japan L.L.C Eisai Co., Ltd. Cardinal Health Japan KANEKAMEDIX CORP. Goodman Co.,LTD. Sanofi K.K. GM MEDICAL CO.,LTD.. St. Jude Medical Japan Co., Ltd. TERUMO CORPORATION Nipro Corporation Novartis Pharma K.K. Bayer Yakuhin, Ltd. BIOTRONIK Japan, Inc. Pfizer Japan Inc. Fides-one,Inc. Fukuda Denshi Co., Ltd Bristol-Myers Squibb Boston Scientific CCorporation Shionogi Co., Ltd. ||||||

|Author|entialCOIo|entialCOIo|entialCOIo|entialCOIo|entialCOIo|entialCOIo|entialCOIo|entialCOIo|entialCOIo| Potential COI of the marital partner, first- degree family | PotentialCOIof theheadoftheorganization department to which the participant belongs whenthepra tpsof cooperatereachwihthhadofte organization/department) || |---|---|---|---|---|---|---|---|---|---|---|---|---| |Author| Employer/ leadership position (private company) |Stakeholder|royalty|Honorarium| Payment for manuscripts |Research grant| Scholarship (educational) grant |Endowed chair| Other rewards | members, or those who share income and property |Research grant| Scholarship (educational) grant | | Members: Kenichi Tsujita ||||||| Otsuka Pharm utical Co., Ltd. Daiichi Sankyo Company, Limited Chugai Pharmaceutical Co.,Ltd. Mitsubishi Tanabe Pharma Corporation NipponBoehringer Ingelheim Co., Ltd. Medtronic Japan Co., Ltd. Japan Lifeline Co.,Ltd. Nihon Kohden TaseR Pharmaceutical Company Limited |||||| | Members: Hiroyuki Tsutsui |||| MSD K.K. Novartis Pharma K.K. Bayer Yakuhin, Ltd. Pfizer Japan Inc. Bristol-MyersSquibb Otsuka Pharmaceutical Co., Ltd. Daichi Sankyo Company. Limited Teijin Pharma Limited Mitsubishi Tanabe Pharma Corporation Nippon BoehringerIngelheim Co., Ltd. Takeda Pharmaceutical Company Limited | Nipponrinshos Co., Ltd. | Actelion Pharmaceuticals Japan Ltd. IQVIA Services Japan K.K. OMRON HEALTHCARE Co., Ltd. Daiichi Sankyo Company, Limited Mitsubishi Tanabe Pharma Corporation Japan Tobacco Inc. Nippon Boehringer Ingelheim Co., Ltd. | MSDK.K. Daiichi Sankyo PCompany,Limited Teijin Pharma Limited Mitsubishi Tanabe Pharma Corporation |||||| | Members: Takeshi Tomita |||| MSD K.K. Amgen Astells BioPharma K-K. AstraZeneca K.K. Novartis Pharma K.K. Bayer Yakuhin, Ltd. BIOTRONIK Japan, Inc. Boehringer Ingelheim GmbH Mylan EPD G.K. Kowa Pharmaceutical Co., Ltd. Ono Pharmaceutical Co., Ltd Otsuka Pharmaceutical Co., Ltd. Daichi Sankyo Company, Limited Mitsubishi Tanabe Pharma Corporation Nippon Boehringer Ingelheim Co., Ltd. Medtronic Japan Co., Ltd. TakedaPharmaceutical Company Limited | Goodman Co.,LTD. |||||||| | Collaborators Jin Endo ||||Pfizer Japan Inc.|||| Co., Ltd. Taisho Toyama Pharmaceutical Co., Ltd. Daiichi Sankyo Company, Limited ||||| | Collaborators Seitaro Oda |||| Eisai Co., Ltd. Guerbet Japan K.K. Bayer Yakuhin, Ltd. Philips Japan, Ltd. Daichi Sankyo Company, Limited | Guerbet Japan K.K. |Ziosoft, Inc.||Philips Japan, Ltd.||| Philips Japan, Ltd. CANON MEDICAL SYSTEMS CORPORATION | Philips Japan, Ltd. Guerbet Japan K.K. CANON MEDICAL SYSTEMS CORPORATION | | Collaborators: Seuii Takashio ||||||||||| TEIJIN HOME HEALTHCARE LIMITED | Abbott Vascular Japan Co., Ltd. TERUMO CORPORATION MedtronicJapan ICo., Ltd. Nihon Kohden Corp. | | Collaborators Masahide Yazaki ||||||| MSD K.K. Astellas Pharma Inc. Daiichi Sankyo Company,Limited JapanBlood Products Organization ||||||

(Appendix2 continued the next page.)

|Author|Potential COIof the participant| Potential COI of the marital partner, first- degree family | Potential COIof thehead oftheorganization/ department t whichthe partipant belons (whenthe participantis in the position of cooperativeresearch with thehead of the organization/department) |||||||||| |---|---|---|---|---|---|---|---|---|---|---|---|---| |Author| Employer/ leadership position (private company) |Stakeholder| Patent royalty |Honorarium| Payment for manuscripts |Research grant| Scholarship (educational) grant |Endowed chair| Other rewards | members, or thosewho share income and property |Research grant| Scholarship (educational) grant | | Extermal Evaluation Committee Members: Toshihisa Anzai |||| Bayer Yakuhin, Ltd. Bristol-Myers Squibb Ono Pharmaceutical Co., Ltd. Otsuka Pharmaceutical Co., Ltd. Daichi Sankyo Company, Limited Mitsubishi Tanabe Pharma Corporation Nippon Boehringer Ingelheim Co., Ltd. Takeda Pharmaceutical Company Limited ||| Bayer Yakuhin, Ltd. Boehringer Ingelheim GmbH Daiichi Sankyo Company, Limited | WIN INTERNATIONAL CO.,LTD. BIOTRONIK Japan, Inc- HOKUYAKU TAKEYAMA Holdings,Inc. MEDICAL SYSTEM NETWORK Co., Itd. Medtronic Japan Co., Ltd. ||||| | External Evaluation Committee Members: Mitsuaki Isobe |||| Daichi Sankyo Company, Limited Chugai Pharmaceutical Co.,Ltd. Otsuka Pharmaceutical Co., Ltd. Pfizer Japan Inc. ||||||||| | Extermal Evaluation Committee Members: Takeshi Kimura |||| Amgen Astellas BioPharma K.K. Abbott Vascular Japan Co., Ltd. Sanofi K.K. Bristol-MyersSquibb Boston Scientific Corporation Kowa Pharmaceutical Co., Ltd. Daichi Sankyo Company. Limited Nippon BoehringerIngelheim Co, Ltd. || Nipro Corporation EP-CRSU Co., Ltd. Edwards Lifesciences Corporation Daiichi Sankyo Company, Limited Pfizer Japan Inc. | Astellas Pharma Inc. Otsuka Pharmaceutical Co., Ltd. Daiichi Sankyo Company,Limited Mitsubishi Tanabe Pharma Corporation Nippon Boehringer Ingelheim Co., Ltd. Takeda Pharmaceutical Company Limited |||||| | External Evaluation Committee Members: Keiichi Fukuda | Heartseed Inc- | Heartseed InC- ||||Heartseed Inc.| Mitsubishi Tanabe Pharma Corporation Otsuka Pharmaceutical Co., Ltd. Sumitomo Dainippon Pharma Co., Ltd. Bayer Yakuhin, Ltd. Takeda Pharmaceutical Company Limited | Kowa Pharmaceutical Co., Ltd. Taisho Toyama Pharmaceutical Co., Ltd. Medtronic Japan Co., Ltd. BIOTRONIKJapan, Inc- Nippon Shinyaku Co., Ltd. Bridgestone Corporation |||||

Notation of corporation is omitted. No potential COI for the following members. Collaborators: Masashi Amano, Absent Collaborators: Atsushi Okada, Absent Collaborators: Yuichi Baba, Absent Collaborators: Yohei Misumi, Absent External Evaluation Committee Members: Yukio Ando, Absent