CHAPTER 18: AMYLOIDOSIS

Amyloidosis is the term for diseases caused by the extracellular deposition of insoluble polymeric protein fibrils in tissues and organs. These diseases are a subset of a growing group of disorders attributed to misfolding of proteins. Among these are Alzheimer's disease and other neurodegenerative diseases; transmissible prion diseases; and genetic diseases caused by mutations that lead to misfolding, aggregation, and protein loss of function, such as certain of the cystic fibrosis mutations. Amyloid fibrils share a common β-pleated sheet structural conformation that confers unique staining properties. The term amyloid was coined by the pathologist Rudolf Virchow around 1854, who thought such deposits were cellulose-like under the microscope.

Amyloid diseases are defined by the biochemical nature of the protein in the fibril deposits and are classified according to whether they are systemic or localized, acquired or inherited, and by their clinical patterns (Table 18-1). The accepted nomenclature is AX, where A indicates amyloidosis and X represents the protein in the fibril. AL is amyloid composed of immunoglobulin light chains (LCs) and has been called primary systemic amyloidosis; it arises from a clonal B cell disorder and may be associated with myeloma or lymphoma. AF groups the familial amyloidoses, most commonly due to mutations in transthyretin, the transport protein for thyroid hormone and retinol-binding protein. AA amyloid is composed of the acute-phase reactant serum amyloid A protein, occurs in the setting of chronic inflammatory or infectious diseases, and has been termed secondary amyloidosis. Aβ₂M is amyloid composed of β₂-microglobulin and occurs in individuals with end-stage renal disease (ESRD) of long duration. Aβ is the most common form of localized amyloidosis. Aβ is deposited in the brain in Alzheimer's disease and is derived from abnormal proteolytic processing of the amyloid precursor protein (APP).

Diagnosis and treatment of the amyloidoses rest upon the pathologic diagnosis of amyloid deposits and immunohistochemical or biochemical identification of amyloid type (Fig. 18-1). In the systemic amyloidoses, the 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 examined, but the most easily accessible tissue, positive in more than 80% of patients with systemic amyloidosis, is fat. After local anesthesia, needle aspiration of fat from the abdominal wall can be expelled onto a slide and stained, avoiding even a minor surgical procedure. If the results for this material is negative, biopsy of kidney, heart, liver, or gastrointestinal tract can be considered. The regular β-sheet structure of amyloid deposits exhibits a unique green birefringence by polarized light microscopy when stained with Congo red dye; the 10-nm-diameter fibrils can be seen directly by electron microscopy of paraformaldehyde-fixed tissue. Once amyloid is found, the protein type must be determined, usually by immunohistochemistry, immunoelectron microscopy, or extraction and biochemical analysis by mass spectrometry or other technique. Careful evaluation of the patient's history, physical findings, and clinical presentation, including age and ethnic origin, organ system involvement, underlying diseases, and family history, can provide helpful clues to the type of amyloid.

The mechanisms of fibril formation and tissue toxicity remain controversial. Factors that contribute to fibrillogenesis include variant or unstable protein structure; extensive β-sheet conformation of the precursor protein; proteolytic processing of the precursor protein; association with components of the serum or extracellular matrix (e.g., amyloid P-component, apolipoprotein E, or glycosaminoglycans); and local physical properties, including pH of the tissue. Monomeric proteins appear to go through an oligomeric aggregation step and then form higher order polymers. Once the polymers reach a critical size, they become insoluble and deposit in extracellular tissue sites as fibrils. These large macromolecular deposits interfere with organ function and, due to cellular uptake of oligomeric amyloid precursors, may be toxic to target cells.

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 renal involvement will usually have proteinuria, which can be as much as 30 g/d, producing hypoalbuminemia that can be profound. Patients with cardiac involvement will often have elevation of brain natriuretic peptide (BNP), pro-BNP, and troponin. These can be useful for monitoring disease activity and have been proposed as prognostic factors; they can be falsely elevated in the presence of renal insufficiency. Patients with liver involvement, even when it is advanced, usually develop cholestasis with an elevated alkaline phosphatase but minimal elevation of the transaminases and preservation of synthetic function. In AL amyloidosis, endocrinopathies can occur, with laboratory testing demonstrating hypothyroidism, hypoadrenalism, or even hypopituitarism. None of these findings are specific for amyloidosis. Thus, a diagnosis of amyloidosis rests upon a tissue biopsy that, after Congo red staining, shows "apple-green" birefringence on polarization microscopy.

ETIOLOGY AND INCIDENCE

AL amyloidosis is most frequently caused by a clonal expansion of plasma cells in the bone marrow that secrete a monoclonal immunoglobulin LC that deposits as amyloid fibrils in tissues. It may be purely serendipitous whether the clonal plasma cells produce a LC that misfolds and leads to AL amyloidosis or folds properly, allowing the cells to inexorably expand over time and develop into multiple myeloma (Chap. 17). It is also possible that the two processes have diverse molecular etiologies. AL amyloidosis can occur with multiple myeloma or other B lymphoproliferative diseases, including non-Hodgkin's lymphoma (Chap. 15) and Waldenström's macroglobulinemia (Chap. 17). AL amyloidosis is the most common type of systemic amyloidosis in North America. Its incidence has been estimated at 4.5 per 100,000; 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 years and is often rapidly progressive and fatal if untreated.

PATHOLOGY AND CLINICAL FEATURES OF AL AMYLOIDOSIS

Amyloid deposits are usually widespread in AL amyloidosis and can be present in the interstitium of any organ outside of the central nervous system. The amyloid fibril deposits are composed of intact 23-kDa monoclonal Ig LCs or smaller fragments, 11–18 kDa in size, representing the variable (V) region alone, or the V region and a portion of the constant (C) region. 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 usually a rapidly progressive disease that presents with a pleiotropic set of clinical syndromes, recognition of which is key to initiating 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 affected organ, in 70–70% of patients. Renal amyloidosis is usually manifested as proteinuria, often in the nephrotic range and associated with significant hypoalbuminemia, secondary hypercholesterolemia, and edema or anasarca. In some patients, tubular rather than glomerular deposition of amyloid can produce azotemia without significant proteinuria. The heart is the second most commonly affected organ, in 50–50% of patients, and the leading cause of death. Early on, the electrocardiogram may show low voltage in the limb leads, with a pseudo-infarct pattern. Eventually, the echocardiogram will display concentrically thickened ventricles and diastolic dysfunction, leading to a restrictive cardiomyopathy; systolic function is preserved until late in the disease. A "sparkly" appearance is usually not seen using modern high-resolution echocardiography equipment. Cardiac magnetic resonance imaging can show an increased wall thickness and a characteristic subendocardial enhancement with gadolinium. Nervous system symptoms include a peripheral sensory neuropathy and/or autonomic dysfunction with gastrointestinal motility disturbances (early satiety, diarrhea, constipation) and orthostatic hypotension. Macroglossia, with an enlarged, indented, or immobile tongue, is pathognomonic of AL amyloidosis but is seen only in ~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 have "easy bruising" due to amyloid deposits in capillaries or to deficiency of clotting factor X, which can bind to amyloid fibrils; cutaneous ecchymoses appear, particularly around the eyes, giving the "raccoon-eye" sign. Other findings include nail dystrophy, alopecia, and amyloid arthropathy with thickening of synovial membranes in the wrists and shoulders (Fig. 18-2). The presence of a multisystem illness or general fatigue along with any of these clinical syndromes should prompt a workup for amyloidosis.

DIAGNOSIS

Identification of the underlying B lymphoproliferative process and clonal LC is key to the diagnosis of AL amyloidosis. The serum protein electrophoresis (SPEP) and urine protein electrophoresis (UPEP) are NOT useful screening tests if AL amyloidosis is suspected because the clonal LC or whole immunoglobulin, unlike in multiple myeloma, is often not present in sufficient quantity in the serum to produce a monoclonal "M-spike" or in the urine to cause LC (Bence Jones) proteinuria. However, more than 90% of patients have a serum or urine monoclonal LC or whole immunoglobulin that can be detected by immunofixation electrophoresis of serum (SIFE) or urine (UIFE) (Fig. 18-3A). Assaying for free immunoglobulin LCs circulating in the serum unbound to heavy chains using commercially available nephelometric (FreeLite^©) assay demonstrates an elevation and abnormal free kappa:lambda ratio in more than 75% of patients. Examining the ratio as well as the absolute amount is essential, because in renal insufficiency LC clearance is reduced, and both types of LCs will be elevated. In addition, an increased percentage of plasma cells in the bone marrow, typically 5–30% of nucleated cells, is noted in about 90% of patients. Kappa or lambda clonality can be demonstrated by flow cytometry, immunohistochemical staining, or in situ hybridization for LC mRNA (Fig. 18-3B).

A monoclonal serum protein by itself is not diagnostic of amyloidosis, since monoclonal gammopathy of uncertain significance (MGUS) is common in older patients (Chap. 17). However, when MGUS is present in patients with biopsy-proven amyloidosis, the AL type should be strongly suspected. Similarly, patients thought to have "smoldering myeloma" because of modest elevation of bone marrow plasma cells should be screened for AL amyloidosis if they have evidence of organ dysfunction. Accurate typing is essential for appropriate treatment. Immunohistochemical staining of the amyloid deposits is useful if they bind one light chain antibody in preference to the other; some AL deposits bind many antisera nonspecifically. Immunoelectron microscopy is more reliable, and mass-spectrometry-based microsequencing of small amounts of protein extracted from fibril deposits can also be done. In ambiguous cases, other forms of amyloidosis should be thoroughly excluded with appropriate genetic and other testing.

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 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 <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, and patients with AA amyloidosis should have computed tomography to look for such tumors, as well as serologic and microbiologic studies. AA amyloidosis can also be seen without any identifiable underlying disease. AA is the only type of systemic amyloidosis that occurs in children.

PATHOLOGY AND CLINICAL FEATURES

Deposits are more limited in AA amyloidosis than in AL amyloidosis; they usually begin in the kidneys. Hepatomegaly, splenomegaly, and autonomic neuropathy can also occur as the disease progresses; cardiomyopathy occurs albeit rarely. However, the symptoms and signs 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 a 12-kDa precursor protein, serum amyloid A (SAA). SAA is an acute-phase apoprotein 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 usually precedes fibril formation, although infections can lead to AA deposition more rapidly.

The familial amyloidoses are autosomal dominant diseases in which a variant plasma protein forms amyloid deposits beginning in midlife. These diseases are rare, with an estimated incidence of <1 per 100,000 in the United States, although there are isolated areas of Portugal, Sweden, and Japan, where founder effects have led to a much higher incidence of the disease. The most common form of AF is caused by mutation of the abundant plasma protein transthyretin (TTR, also known as prealbumin). More than 100 TTR mutations are known, and most are associated with ATTR amyloidosis. One variant, V122I, has a carrier frequency that may be as high as 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 it would be wise to consider this in the differential diagnosis of African-American patients who present with concentric cardiac hypertrophy and evidence of diastolic dysfunction, particularly in the absence of a history of hypertension. Even wild-type TTR can form fibrils, leading to so-called senile systemic amyloidosis (SSA) in older patients. It can be found in up to 25% of autopsies in patients older than age 80 years, and it can produce a clinical syndrome of amyloid cardiomyopathy that is similar to that occurring in younger patients carrying a mutant TTR. 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.

In ATTR and in other forms of familial amyloidosis, the variant structure of the precursor protein is the key factor in fibril formation. The role of aging is intriguing, since patients born with the variant proteins do not have clinically apparent disease until middle age despite the lifelong presence of the abnormal protein. Further evidence of an age-related "trigger" is the occurrence of SSA in the elderly caused by the deposition of fibrils derived from normal TTR.

CLINICAL FEATURES AND DIAGNOSIS

AF amyloidosis has a variable presentation but is usually consistent within affected kindreds with the same mutant protein. A family history makes AF more likely, but many patients present sporadically with new mutations. ATTR usually presents as a syndrome of familial amyloidotic polyneuropathy or familial amyloidotic cardiomyopathy. Peripheral neuropathy usually begins as a lower-extremity sensory and motor neuropathy and progresses to the upper extremities. Autonomic neuropathy is manifest by gastrointestinal symptoms of diarrhea with weight loss and orthostatic hypotension. Patients with TTR V30M, the most common mutation, have normal echocardiographic findings but may have conduction defects and require a pacemaker. Patients with TTR T60A and several other mutations have myocardial thickening similar to that caused by AL amyloidosis, although heart failure is less common, and the prognosis is better. Vitreous opacities caused by amyloid deposits are pathognomonic of ATTR amyloidosis.

Typical syndromes associated with other forms of AF 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, and AF carriers can develop AL, or conversely, AF patients can develop MGUS. Thus, it is important to screen for both plasma cell disorders and for mutations in some patients with amyloidosis. Variant TTR proteins can usually be detected by isoelectric focusing, but DNA sequencing is now standard for diagnosis of ATTR and the other AF mutations.

Aβ₂M amyloidosis is composed of β₂-microglobulin, the invariant chain of class I human leukocyte antigens, and produces rheumatologic manifestations in patients on long-term hemodialysis. β₂-Microglobulin is excreted by the kidneys, and levels become elevated in ESRD. 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 newer high-flow dialysis techniques.

Aβ₂M amyloidosis usually presents with carpal tunnel syndrome, persistent joint effusions, spondyloarthropathy, or cystic bone lesions. Carpal tunnel syndrome is often the first symptom of disease. In the past, persistent joint effusions accompanied by mild discomfort were seen in up to 50% of patients on dialysis for more than 12 years. Involvement is bilateral, and large joints (shoulders, knees, wrists, and hips) are more 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.

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 using Congo red staining of aspirated abdominal fat or of an involved organ biopsy specimen. Accurate typing using a combination of immunologic, biochemical, and genetic testing is essential to choosing the appropriate therapy (see algorithm for workup, Fig. 18-1). Tertiary referral centers can provide specialized diagnostic techniques and access to clinical trials for patients with these rare diseases.