Chapter 108: Amyloidosis

Amyloidosis is the term for a group of protein misfolding disorders characterized by the extracellular deposition of insoluble polymeric protein fibrils in tissues and organs. A robust cellular machinery exists to chaperone proteins during the process of synthesis and secretion, to ensure that they achieve correct tertiary conformation and function, and to eliminate proteins that misfold. However, genetic mutation, incorrect processing, and other factors may favor misfolding, with consequent loss of normal protein function and intracellular or extracellular aggregation. Many diseases, ranging from cystic fibrosis to Alzheimer’s disease, are now known to involve protein misfolding. In the amyloidoses, the aggregates are typically extracellular, and the misfolded protein subunits assume a common antiparallel, β-pleated sheet–rich structural conformation that leads to the formation of higher-order oligomers and then fibrils with unique staining properties. The term amyloid was coined around 1854 by the pathologist Rudolf Virchow, who thought that these deposits resembled starch (Latin amylum) under the microscope.

Amyloid diseases, defined by the biochemical nature of the protein composing the fibril deposits, are classified according to whether they are systemic or localized, whether they are acquired or inherited, and their clinical patterns (Table 108-1). The standard nomenclature is AX, where A indicates amyloidosis and X represents the protein present in the fibril. This chapter focuses primarily on the systemic forms. AL refers to amyloid composed of immunoglobulin light chains (LCs); this disorder, formerly termed primary systemic amyloidosis, arises from a clonal B cell or plasma cell disorder and can be associated with myeloma or lymphoma. ATTR refers to the most prevalent of the familial amyloidoses, which are most commonly due to mutations in transthyretin (TTR), the transport protein for thyroid hormone and retinol-binding protein. AA amyloid is composed of the acute-phase reactant protein serum amyloid A (SAA) and occurs in the setting of chronic inflammatory or infectious diseases; for this reason, this type was formerly known as secondary amyloidosis. Aβ₂M amyloid results from misfolded β₂-microglobulin, occurring in individuals with long-standing renal disease who have undergone dialysis, typically for years. Aβ, the most common form of localized amyloidosis, is found in the brain of patients with Alzheimer’s disease after abnormal proteolytic processing and aggregation of polypeptides derived from the amyloid precursor protein.

Diagnosis and treatment of the amyloidoses rest upon the histopathologic identification of amyloid deposits and immunohistochemical, biochemical, or genetic determination of amyloid type (Fig. 108-1). In the systemic amyloidoses, the clinically involved organs can be biopsied, but amyloid deposits may be found in any tissue of the body. Historically, blood vessels of the gingiva or rectal mucosa were often examined, but the most easily accessible tissue—positive in more than 80% of patients with systemic amyloidosis—is abdominal fat. After local anesthesia, fat is aspirated from the abdominal pannus with a 16-gauge needle. Fat globules expelled onto a glass slide can be stained, thus avoiding a surgical procedure. If this material is negative, more invasive biopsies of the kidney, heart, liver, or gastrointestinal tract can be considered in patients in whom amyloidosis is suspected. The regular β-sheet structure of amyloid deposits exhibits a unique “apple green” birefringence by polarized light microscopy when stained with Congo red dye; other regular protein structures (e.g., collagen) appear white under these conditions. The 10-nm-diameter fibrils can also be visualized by electron microscopy of paraformaldehyde-fixed tissue. Once amyloid is found, the precursor protein type must be determined by immunohistochemistry, immunoelectron microscopy, or extraction and biochemical analysis employing mass spectrometry; gene sequencing is used to identify mutants causing hereditary amyloidosis. The patient’s history, physical findings, and clinical presentation, including age and ethnic origin, organ system involvement, underlying diseases, and family history, may provide helpful clues as to the type of amyloidosis. However, there can be considerable overlap in clinical presentations, and accurate typing is essential to guide appropriate therapy.

The mechanisms of fibril formation and tissue toxicity remain controversial. The “amyloid hypothesis,” as it is currently understood, proposes that precursor proteins undergo a process of reversible unfolding or misfolding; misfolded proteins form oligomeric aggregates, higher-order polymers, and then fibrils that deposit in tissues. Accumulating evidence suggests that the oligomeric intermediates may constitute the most toxic species. Oligomers are more capable than large fibrils of interacting with cells and inducing formation of reactive oxygen species and stress signaling. Ultimately, the fibrillar tissue deposits are likely to interfere with normal organ function. A more sophisticated understanding of the mechanisms leading to amyloid formation and cell and tissue dysfunction will continue to provide new targets for therapies.

The clinical syndromes of the amyloidoses are associated with relatively nonspecific alterations in routine laboratory tests. Blood counts are usually normal, although the erythrocyte sedimentation rate is frequently elevated. Patients with glomerular kidney involvement generally have proteinuria, often in the nephrotic range, leading to hypoalbuminemia that may be severe; patients with serum albumin levels <2 g/dL generally have pedal edema or anasarca. Amyloid cardiomyopathy is characterized by concentric ventricular hypertrophy and diastolic dysfunction associated with elevation of brain natriuretic peptide or N-terminal pro–brain natriuretic peptide as well as troponin. These cardiac biomarkers can be used for disease staging, prognostication, and disease activity monitoring in patients with AL amyloidosis. Notably, renal insufficiency can falsely elevate levels of these biomarkers. Recently, biomarkers of cardiac remodeling—that is, matrix metalloproteinases and tissue inhibitors of metalloproteinases—have been found to be altered in the serum of patients with amyloid cardiomyopathy. Electrocardiographic and echocardiographic features of amyloid cardiomyopathy are described below. Patients with liver involvement, even when advanced, usually develop cholestasis with an elevated alkaline phosphatase concentration with minimal alteration of the aminotransferases and preservation of synthetic function. In AL amyloidosis, endocrine organs may be infiltrated with fibrils, and hypothyroidism, hypoadrenalism, or even hypopituitarism can occur. Although none of these findings is specific for amyloidosis, the presence of abnormalities in multiple organ systems should raise suspicion regarding this diagnosis.

Etiology and Incidence

AL amyloidosis is most frequently caused by a clonal expansion of bone-marrow plasma cells that secrete a monoclonal immunoglobulin LC depositing as amyloid fibrils in tissues. Whether the clonal plasma cells produce a LC that misfolds and leads to AL amyloidosis or an LC that folds properly, allowing the cells to inexorably expand over time and develop into multiple myeloma (Chap. 107), may depend upon primary sequence of the clonal LC or other genetic or epigenetic factors. AL amyloidosis can occur with multiple myeloma or other B lymphoproliferative diseases, including non-Hodgkin’s lymphoma (Chap. 104) and Waldenström’s macroglobulinemia (Chap. 107). AL amyloidosis is the most common type of systemic amyloidosis diagnosed in North America. Its incidence has been estimated at 4.5 cases/100,000 population; however, ascertainment continues to be inadequate, and the true incidence may be much higher. AL amyloidosis, like other plasma cell diseases, usually occurs after age 40 and is often rapidly progressive and fatal if untreated.

Pathology and Clinical Features

Amyloid deposits are usually widespread in AL amyloidosis and can be present in the interstitium of any organ outside the central nervous system. The amyloid fibril deposits are composed of full-length 23-kDa monoclonal immunoglobulin LCs as well as fragments. Accessory molecules co-deposited with LC fibrils (as well as with other amyloid fibrils) include serum amyloid P component, apolipoproteins e and AIV, glycosaminoglycans, and metal ions. Although all kappa and lambda LC subtypes have been identified in AL amyloid fibrils, lambda subtypes predominate. The lambda 6 subtype appears to have unique structural properties that predispose it to fibril formation, often in the kidney.

AL amyloidosis is often a rapidly progressive disease that presents as a pleiotropic set of clinical syndromes, recognition of which is key for initiation of the appropriate workup. Nonspecific symptoms of fatigue and weight loss are common; however, the diagnosis is rarely considered until symptoms referable to a specific organ develop. The kidneys are the most frequently involved organ and are affected in 70–80% of patients. Renal amyloidosis usually manifests as proteinuria, often in the nephrotic range and associated with hypoalbuminemia, secondary hypercholesterolemia and hypertriglyceridemia, and edema or anasarca. In some patients, interstitial rather than glomerular amyloid deposition can produce azotemia without proteinuria. The heart is the second most commonly affected organ (50–60% of patients), and cardiac involvement is the leading cause of death from AL amyloidosis. Early on, the electrocardiogram may show low voltage in the limb leads with a pseudo-infarct pattern. Echocardiographic features of disease include concentrically thickened ventricles and diastolic dysfunction with an abnormal global longitudinal strain pattern; a “sparkly” appearance has been described but is often not seen with modern high-resolution echocardiographic techniques. Poor atrial contractility occurs even in sinus rhythm, and patients with cardiac amyloidosis are at risk for development of atrial thrombi and stroke. Cardiac MRI can show increased wall thickness, and characteristic delayed enhancement of the subendocardium has been described following injection of gadolinium contrast. Nervous system symptoms include peripheral sensorimotor neuropathy and/or autonomic dysfunction manifesting as gastrointestinal motility disturbances (early satiety, diarrhea, constipation), dry eyes and mouth, impotence, orthostatic hypotension, and/or neurogenic bladder. Macroglossia (Fig. 108-2A), a pathognomonic sign of AL amyloidosis, is seen in only ~10% of patients. Liver involvement causes cholestasis and hepatomegaly. The spleen is frequently involved, and there may be functional hyposplenism in the absence of significant splenomegaly. Many patients experience “easy bruising” due to amyloid deposits in capillaries or deficiency of clotting factor X due to binding to amyloid fibrils; cutaneous ecchymoses appear, particularly around the eyes, producing another uncommon but pathognomonic finding, the “raccoon-eye” sign (Fig. 108-2B). Other findings include nail dystrophy (Fig. 108-2C), alopecia, and amyloid arthropathy with thickening of synovial membranes in the wrists and shoulders. The presence of a multisystemic illness or general fatigue along with any of these clinical syndromes should prompt a workup for amyloidosis.

Diagnosis

Identification of an underlying clonal plasma cell or B lymphoproliferative process and a clonal LC are key to the diagnosis of AL amyloidosis. Serum protein electrophoresis and urine protein electrophoresis, although of value in multiple myeloma, are not useful screening tests if AL amyloidosis is suspected because the clonal LC or whole immunoglobulin often is not present in sufficient amounts to produce a monoclonal “M-spike” in the serum or LC (Bence Jones) protein in the urine. However, more than 90% of patients with AL amyloidosis have serum or urine monoclonal LC or whole immunoglobulin detectable by immunofixation electrophoresis of serum (SIFE) or urine (UIFE) (Fig. 108-3A) or by nephelometric measurement of serum “free” LCs (i.e., LCs circulating in monomeric form rather than in an immunoglobulin tetramer with heavy chain). Examining the ratio as well as the absolute amount of serum-free LCs is essential, as renal insufficiency reduces LC clearance, nonspecifically elevating both isotypes. In addition, an increased percentage of plasma cells in the bone marrow—typically 5–30% of nucleated cells—is found in ~90% of patients. Kappa or lambda clonality should be demonstrated by flow cytometry, immunohistochemistry, or in situ hybridization for LC mRNA (Fig. 108-3B).

A monoclonal serum protein by itself is not diagnostic of amyloidosis, since monoclonal gammopathy of uncertain significance is common in older patients (Chap. 107). However, when monoclonal gammopathy of uncertain significance is found in patients with biopsy-proven amyloidosis, the AL type should be ruled out. Similarly, patients thought to have “smoldering myeloma” because of a modest elevation of bone-marrow plasma cells should be screened for AL amyloidosis if they have signs or symptoms of renal, cardiac, or neurologic disease. Accurate tissue amyloid typing is essential for appropriate treatment. Immunohistochemical staining of the amyloid deposits is useful if they selectively bind one LC antibody in preference to the other; some AL deposits bind antibodies nonspecifically. Immunoelectron microscopy is more reliable, while mass spectrometry–based microsequencing of small amounts of protein extracted from fibril deposits has become the diagnostic standard. In ambiguous cases, other forms of amyloidosis should be thoroughly excluded with appropriate genetic and other testing.

Treatment

Etiology and Incidence

AA amyloidosis can occur in association with almost any chronic inflammatory state (e.g., rheumatoid arthritis, inflammatory bowel disease, familial Mediterranean fever [Chap. 362], or other periodic fever syndromes) or chronic infections such as tuberculosis or subacute bacterial endocarditis. In the United States and Europe, AA amyloidosis has become less common, occurring in fewer than 2% of patients with these diseases, presumably because of advances in anti-inflammatory and antimicrobial therapies. It has also been described in association with Castleman’s disease, lymphomas, renal cell carcinoma, emphasizing the diagnostic importance of CT scanning to look for such tumors as well as serologic and microbiologic studies. In up to 30% of patients, AA amyloidosis can also be seen without any identifiable underlying disease. AA is the most frequent systemic amyloidosis that occurs in children.

Pathology and Clinical Features

Organ involvement in AA amyloidosis usually begins in the kidneys. Hepatomegaly, splenomegaly, and autonomic neuropathy can also occur as the disease progresses; cardiomyopathy occurs in later disease. The symptoms and signs of AA disease cannot be reliably distinguished from those of AL amyloidosis. AA amyloid fibrils are usually composed of an 8-kDa, 76-amino-acid N-terminal portion of the 12-kDa precursor protein SAA. This acute-phase apoprotein is synthesized in the liver and transported by high-density lipoprotein (HDL3) in the plasma. Several years of an underlying inflammatory disease causing chronic elevation of SAA levels usually precede fibril formation, although infections can lead to AA deposition more rapidly.

Treatment

The familial amyloidoses are autosomal dominant diseases in which, beginning in midlife, a variant (FINE) plasma protein forms amyloid deposits. These diseases are rare, with an estimated incidence of <1 case/100,000 population in the United States, although founder effects in isolated areas of Portugal, Sweden, and Japan have led to a much higher incidence. The most common form of AF amyloidosis is ATTRm in the updated nomenclature, caused by mutation of the abundant plasma protein transthyretin (TTR, also known as prealbumin). More than 120 TTR mutations are known, and most are associated with ATTR amyloidosis. One variant, V122I, has a carrier frequency of nearly 4% in the African-American population and is associated with late-onset cardiac amyloidosis. The actual incidence and penetrance of disease in the African-American population is the subject of ongoing research, but ATTR amyloidosis warrants consideration in the differential diagnosis of African-American patients who present with concentric cardiac hypertrophy and evidence of diastolic heart failure, particularly in the absence of a history of hypertension. Other familial amyloidoses, caused by variant apolipoproteins AI or AII, gelsolin, fibrinogen Aα, or lysozyme, are reported in only a few families worldwide. New amyloidogenic serum proteins continue to be identified periodically, including recently the leukocyte chemotactic factor LECT2, a cause of renal amyloidosis in Hispanic and Pakistani populations. To date, no mutation in the coding sequence for the LECT2 gene has been identified, so the heritability of ALECT2 remains uncertain.

Amyloid deposits composed of unmutated precursor protein (wild type TTR) occur with aging. Due to a rapidly aging world population, wild-type ATTR (ATTRwt) is being diagnosed with increasing frequency in Caucasian men >65 years of age with amyloid cardiomyopathy. Formerly termed senile systemic amyloidosis, ATTRwt has been found at autopsy in 25% of hearts from men older than age 80 years. Why a wild type protein becomes amyloidogenic, and why patients bearing mutant TTR genes do not express disease until adulthood, remains a mystery.

Clinical Features and Diagnosis

AF amyloidosis has a presentation that is variable but is usually consistent within kindreds affected by the same mutant protein. A family history makes AF disease more likely, but many patients present with previously unrecognized familial disease or sporadically with new mutations. ATTRm amyloidosis typically presents as a syndrome of familial amyloidotic polyneuropathy or familial amyloidotic cardiomyopathy. Peripheral neuropathy begins as a small-fiber length-dependent lower-extremity sensorimotor neuropathy and progresses to the upper extremities. Autonomic neuropathy manifests as diarrhea with weight loss and orthostatic hypotension. Patients with ATTR V30M, the most common mutation, often develop conduction defects late in the disease. Patients with ATTR T60A and several other mutations have myocardial thickening similar to that caused by AL amyloidosis, although heart failure is less common and long-term untreated survival rates are usually better. Vitreous opacities caused by amyloid deposits are pathognomonic for ATTR amyloidosis.

Typical syndromes associated with other forms of AF disease include renal amyloidosis with mutant fibrinogen, lysozyme, or apolipoproteins; hepatic amyloidosis with apolipoprotein AI; and amyloidosis of cranial nerves and cornea with gelsolin. Patients with AF amyloidosis can present with clinical syndromes that mimic those of patients with AL disease. Rarely, AF carriers can develop AL disease or AF patients may have monoclonal gammopathy without AL. Thus, it is important to screen both for plasma cell disorders and for mutations in patients with amyloidosis. Variant TTRs can usually be detected by isoelectric focusing, but DNA sequencing is now standard for diagnosis of ATTRm and other AF mutations.

Treatment

Aβ₂M amyloid is composed of β₂-microglobulin, the invariant chain of class I human leukocyte antigens, and produces rheumatologic manifestations in patients undergoing long-term hemodialysis. β₂-Microglobulin is excreted by the kidney, and levels become elevated in end-stage renal disease. The molecular mass of β₂M is 11.8 kDa—above the cutoff of some dialysis membranes. The incidence of this disease appears to be declining with the use of newer membranes in high-flow dialysis techniques. Aβ₂M amyloidosis usually presents as carpal tunnel syndrome, persistent joint effusions, spondyloarthropathy, or cystic bone lesions. Carpal tunnel syndrome is often the first symptom. In the past, persistent joint effusions accompanied by mild discomfort were found in up to 50% of patients who had undergone dialysis for >12 years. Involvement is bilateral, and large joints (shoulders, knees, wrists, and hips) are most frequently affected. The synovial fluid is noninflammatory, and β₂M amyloid can be found if the sediment is stained with Congo red. Although less common, visceral β₂M amyloid deposits do occasionally occur in the gastrointestinal tract, heart, tendons, and subcutaneous tissues of the buttocks. There is no specific therapy for Aβ₂M amyloidosis, but cessation of dialysis after renal allografting may lead to symptomatic improvement.

To date, treatment strategies have focused on limiting formation of amyloidogenic proteins. Disruption of existing amyloid by targeting ubiquitous components of the tissue deposits may offer means to improving major end organ function. Two antibody approaches—one involving an epitope exposed during immunoglobulin light chain misfolding (AL disease) and the other directed against serum amyloid P component—are undergoing evaluation in early phase III studies.

A diagnosis of amyloidosis should be considered in patients with unexplained nephropathy, cardiomyopathy (particularly with diastolic dysfunction), neuropathy (either peripheral or autonomic), enteropathy, or the pathognomonic soft tissue findings of macroglossia or periorbital ecchymoses. Pathologic identification of amyloid fibrils can be made with Congo red staining of aspirated abdominal fat or of an involved-organ biopsy specimen. Accurate typing by a combination of immunologic, biochemical, and genetic testing is essential in selecting appropriate therapy (Fig. 108-1). Systemic amyloidosis should not be considered an untreatable condition, as anti–plasma cell chemotherapy is highly effective in AL disease and targeted therapies are being developed for AA and ATTR disease. The combination of precursor and end organ amyloid therapeutics may permit not only disease control but also functional improvement to patients with amyloidosis. Tertiary referral centers can provide specialized diagnostic techniques and access to clinical trials for patients with these rare diseases.

ACKNOWLEDGMENT

This chapter represents a revised version of chapters co-authored by Drs. Martha Skinner, David C. Seldin, and John L. Berk in previous editions of Harrison’s Principles of Internal Medicine.