Amyloidosis: From Fundamentals to Therapeutic Decisions

History

In the mid-19th century, Rudolf Virchow, known as the father of modern pathological anatomy, was the first to apply the term amyloid, introduced by the German botanist Matthias Jakob Schleiden, to describe these tissue deposits. Although the term, translated from Latin, means starch, the deposits observed by Virchow stained with iodine in the same way as cellulose (1). From that point onward, a new staining method for the microscopic diagnosis of amyloidosis was discovered approximately every 30 years. In 1883, Paul Böttinger introduced Congo red staining, and in the 1920s, Bennhold improved this method when it was observed that, under polarized light, the protein deposits displayed an apple-green color. Some time later, in the 1940s, thioflavin T also became widely used in diagnostics; when interacting with amyloid fibrils, it stains the sample a greenish-yellow color (Figure 1).

In 1959, electron microscopy was used for the first time to examine amyloid deposits. These deposits were found to consist of straight, non-branching fibers 8–10 nm in diameter, in which protofilaments can be visualized at higher resolution (2, 3). However, these methods are insufficient to determine the type of amyloid fibril, and immunohistochemical methods are therefore used (4, 5).

Pathogenesis

Genetic factors are among the most important factors in many types of amyloidosis. Specific point mutations (missense and nonsense) cause structural changes in the encoded proteins, resulting in a tendency toward fiber formation (fibrillogenesis), which may eventually progress to localized or systemic amyloidosis. It is important to note that different pathogenetic mechanisms may operate in different types of amyloidosis, such as pathological changes in protein structure (antiparallel β-sheets) or inhibition of their degradation pathways (6–9).

Factors or disorders unrelated to fibril formation are no less important, particularly in systemic amyloidosis. In chronic inflammation or plasma cell dyscrasia, increased synthesis of amyloidogenic proteins may substantially exceed the capacity of degradation mechanisms. If these processes persist, amyloid deposits eventually form in multiple areas of the body.

Fibril Formation

The mechanisms of fibrillogenesis are associated with the formation or failure to form the complete structure of the precursor protein. Both processes are thought to be facilitated by an acidic environment in the body and the resulting protein denaturation (10). In addition to fibrils, other non-fibrillar structures are frequently detected in areas of deposition, including glycosaminoglycans (GAGs, particularly heparan sulfate), serum amyloid P component (SAP), and specific apolipoproteins (E and J) (11, 12). These cofactors may directly influence any stage of fibrillogenesis, including not only changes in precursor conformation and deposition but also reabsorption (13).

Genetics

Many types of amyloidosis have a clear genetic origin. Numerous familial forms of this disorder are associated with missense mutations in the nucleotide sequences of precursor proteins. Although more than 30 human and 10 animal precursor proteins from which amyloid fibrils can form have now been identified (14), each of these types can be assigned to 1 of 4 major clinical groups:

• mutations in genes encoding proteins that are more likely to participate in fibrillogenesis than their unaltered counterparts;
• polymorphisms of cofactors, such as apolipoprotein E, or protein subunits;
• inherited disorders that promote the production of amyloidogenic proteins and the formation of deposits, such as presenilin mutations in familial Alzheimer’s disease;
• inherited disorders characterized by chronic inflammation and protein deposition in predisposed patient groups, such as tumor necrosis factor (TNF) receptor mutations.

Nevertheless, these classifications must be interpreted critically because some disorders in these groups are also characterized by other types of mutations, including nonsense point mutations and deletions (15). An example is essentially all heterozygous familial forms of amyloidosis presenting with renal, nervous system, and cardiovascular disorders. These diseases are characterized by an interesting phenomenon: both unaltered and mutated proteins are found within amyloid deposits.

Types of Amyloidosis

AL (primary) and AA (secondary) amyloidosis are the types most commonly encountered in tertiary and outpatient healthcare centers. The former results from plasma cell dyscrasia, whereas the latter is most commonly associated with prolonged inflammation in chronic diseases. Other forms of the disease, including dialysis-related, senile, hereditary, and organ-specific amyloidosis, are encountered much less frequently in clinical practice.

AL Amyloidosis

This plasma cell dyscrasia, in which a monoclonal protein is detected by immunofixation in urine and/or serum in more than 95% of cases, may occur either as an independent disorder or as a consequence of other diseases, including multiple myeloma and, less commonly, lymphoplasmacytic or non-Hodgkin lymphomas. In extremely rare cases, amyloid deposits are formed from fragments of the heavy chains of these immunoglobulins; this is known as AH amyloidosis (16).

AA Amyloidosis

Although no specific cause of AA amyloidosis can be identified in a small proportion of patients, this complication is relatively common in clinical practice in association with autoimmune diseases, such as rheumatoid arthritis, spondyloarthropathies, and inflammatory bowel diseases, as well as chronic infectious and oncological diseases. It results from the deposition of serum amyloid A in tissues due to its excessive production by the liver during prolonged inflammation (17).

AA amyloidosis most commonly affects the kidneys, in approximately 80% of cases. Microscopically, deposits are observed in the renal glomeruli and clinically manifest as nephrotic syndrome or other syndromes characteristic of this organ system.

Dialysis-Related Amyloidosis

In chronic renal failure, long-term dialysis leads to an increase in the concentration of β2-microglobulin in the blood, with deposits most commonly observed in tissues of the musculoskeletal system (18, 19). Patients with dialysis-related amyloidosis complain of shoulder pain due to inflammation of the tissues surrounding the joint and infiltration of the rotator cuff muscles, as well as symptoms characteristic of carpal tunnel syndrome.

Age-Related (Senile) Amyloidosis

In this proteinopathy, deposits composed of proteins whose structure theoretically does not differ from that of transthyretin (TTR) normally found in human blood most commonly form around the striated muscle fibers of the heart (20). Experts use this term only when asymptomatic amyloid deposits are identified microscopically during autopsy in patients who died from heart failure due to cardiomegaly or infiltrative cardiomyopathy.

Compared with AL amyloidosis associated with chronic diseases, the life expectancy prognosis in senile amyloidosis is considerably better. Although protein deposits cause thickening of all myocardial walls in both conditions, the mean survival of the latter study group was almost 7 times longer. Specialists believe that this is due to the infrequent occurrence of renal damage in senile amyloidosis (21).

Organ-Specific Amyloidosis

The main difference from systemic amyloidosis lies in the origin of the components forming the deposits. In this case, locally synthesized proteins, rather than circulating precursor proteins, accumulate, for example, immunoglobulin light chains. Some of the most common examples include:

• Alzheimer’s disease – deposits of β protein (Aβ), formed through enzymatic cleavage of amyloid precursor protein (APP) by β- and γ-secretases, in cerebral blood vessels;
• isolated bladder amyloidosis – a localized form of immunoglobulin light-chain amyloidosis (AL) (22);
• leukocyte cell-derived chemotaxin 2 (LECT2)-associated amyloidosis: in the United States alone, this disease accounts for approximately one-quarter of amyloid deposits in the liver (23).

Clinical Manifestations

The predominant form of amyloidosis has been observed to be closely associated with geographic location: AL amyloidosis is the most common form in developed countries, whereas AA amyloidosis predominates in developing countries. Specialists believe that this is associated with the high prevalence of chronic infectious diseases such as tuberculosis, leprosy, and osteomyelitis (24).

• Renal involvement: asymptomatic proteinuria or clinically apparent nephrotic syndrome. However, if the primary protein deposits accumulate in larger renal blood vessels or tubules, patients who develop renal failure may have only minimal amounts of protein in the urine (25).
• Cardiomyopathy: systolic or diastolic dysfunction and signs characteristic of heart failure. When amyloid accumulates in the coronary arteries, patients may experience syncope due to arrhythmias or varying degrees of heart block, as well as symptoms characteristic of angina or myocardial infarction (26). It is important to note that isolated atrial amyloidosis, consisting of deposits formed from atrial natriuretic peptide, is an age-related change that is usually clinically asymptomatic (27).
• Pulmonary involvement: hoarseness, stridor, airway obstruction, and dysphagia resulting from infiltration of the larynx and bronchi; persistent accumulation of fluid in the pleural cavity; parenchymal nodules (amyloidomas); and, in rare cases, pulmonary hypertension (28–43).
• Gastrointestinal involvement: hepatomegaly with or without splenomegaly is the predominant manifestation of amyloidosis involving this system. Less commonly, patients may experience bleeding due to vascular fragility and an impaired vascular response to injury, gastroparesis, constipation, malabsorption, and intestinal pseudo-obstruction caused by reduced gastrointestinal motility (44).
• Neurological changes:
  • peripheral and autonomic neuropathy is most characteristic of specific hereditary forms and AL amyloidosis. Sensory disturbances, paresthesias, and pain may occur in both non-specific and specific locations, for example, compression of the median nerve by amyloid deposits in the carpal tunnel. Bowel or bladder dysfunction and orthostatic hypotension suggest involvement of the autonomic nervous system (45);
  • central nervous system involvement is generally divided into 2 groups: sporadic or familial Alzheimer’s disease, characterized by pathological changes in cortical structures causing progressive dementia, and cerebral amyloid angiopathy, which causes spontaneous bleeding in cortical and subcortical structures in older patients (46).
• Musculoskeletal involvement: muscle pseudohypertrophy caused by tissue infiltration with protein and arthropathies resulting from amyloid deposits in the joints and surrounding tissues. The most characteristic examples are enlargement of the tongue (macroglossia) with prominent dental impressions along its margins, a sign of AL amyloidosis, and enlargement of the anterior surface of the shoulder caused by excess fluid in the glenohumeral joint due to deposits in the synovial membrane or other periarticular tissues (47).
• Hematological changes: increased risk of bleeding due to reduced factor X activity, vascular infiltration by amyloid, and impaired liver function (48–53). Other hematological changes depend on the extent of tissue damage, for example, secondary anemia associated with renal failure or multiple myeloma and thrombocytopenia caused by splenomegaly.
• Skin changes: keratoses, bruising (ecchymoses), and subcutaneous nodules. A relatively specific sign of AL amyloidosis is periorbital purpura developing after minor trauma or performance of the Valsalva maneuver (Figure 2) (54). Amyloid deposits in adipose tissue generally do not cause symptoms, making it one of the principal sites for biopsy.
• Other manifestations: some hereditary forms of amyloidosis are associated with visual or hearing impairment, hematuria, and symptoms of urinary tract irritation (22). AL amyloidosis involving blood vessels has been observed to manifest with signs otherwise relatively specific to other disorders, including jaw claudication associated with giant cell arteritis or ischemic heart disease. Therefore, comprehensive differential diagnosis is essential to avoid inappropriate therapeutic decisions (55, 56).

Diagnosis

Biopsy of affected tissues followed by microscopic analysis is the only test capable of objectively confirming a diagnosis of amyloidosis. Tissue samples may be obtained both from potentially affected sites without clinical symptoms, such as subcutaneous adipose tissue, minor salivary glands, or rectal mucosa, and from organs with impaired function, such as the kidneys or a nerve.

According to specialists, subcutaneous adipose tissue should be the first-choice sampling site in cases of amyloidosis involving more than one organ system. Examination of these samples using polarized light and standard microscopy with Congo red staining has a sensitivity of 57–85% and a specificity of 92–100% for primary (AL) or secondary (AA) amyloidosis (57–60). However, in cases of amyloidosis involving only a single organ, adipose tissue is not an appropriate material for examination because the sensitivity of the test is markedly reduced (61).

Although intestinal mucosal biopsy is now used relatively rarely, it is considerably more reliable, with a retrospective study involving more than 200 patients with objectively confirmed primary or secondary amyloidosis reporting a sensitivity of 84% (62). The sensitivity of kidney, liver, and carpal tunnel biopsies exceeded 90% in the same study population; however, these procedures should be avoided because of the high risk of bleeding or mechanical nerve injury.

Microscopic examination of a minor labial salivary gland biopsy, which is used in the diagnosis of primary Sjögren syndrome, is also suitable for confirming both AA and AL amyloidosis, particularly when changes occur in nearby soft-tissue structures, such as enlargement of the tongue and submandibular salivary glands or symptoms of joint involvement (63).

When a tissue sample is examined by light microscopy, amyloid deposits appear as an amorphous translucent mass. Protein fibrils bind specifically to Congo red, displaying an apple-green color under polarized light, and to thioflavin T, producing a greenish-yellow color. In some clinical cases, immunohistochemical studies are used to determine the type of protein subunit (64, 65). This method is most useful for diagnosing AA and TTR amyloid deposits. Because AL deposits lose the constant immunoglobulin-chain epitopes, or antigenic determinants, during proteolytic remodeling, they do not always react with the κ and λ antibodies used in these tests, substantially increasing the risk of a false-negative result. Antibodies that specifically bind to the variable regions of the immunoglobulins forming the amyloid provide an effective solution to this problem (66). The most accurate diagnostic methods for determining the type of amyloid—amino acid sequencing or mass spectrometry—are performed only in specialized centers (67).

In patients without a previously diagnosed plasma cell dyscrasia in whom other possible causes of amyloidosis have already been excluded, plasma cells are evaluated first. Serum and urine protein electrophoresis (Figures 3 and 4) and immunofixation (Figures 5 and 6) are used to exclude the presence of a monoclonal population (68, 69). In addition to microscopic, immunohistochemical, and flow cytometric methods, this possibility may also be confirmed or excluded by bone marrow biopsy (Figure 7).

Amyloidosis is also associated with non-specific changes visible on radiological examinations. Examples include a mottled appearance of the myocardium on echocardiography, marked Tc99 accumulation in cardiac tissues, delayed subepithelial gadolinium enhancement on cardiac magnetic resonance imaging (MRI), lytic bone lesions characteristic of multiple myeloma, and cystic bone changes associated with long-term dialysis. However, because of the low specificity of these findings, they are insufficient to confirm the diagnosis.

Treatment

In clinical practice, the therapeutic strategy depends directly on the cause of excessive fibril formation. In primary (AL) amyloidosis, the main objective is to treat the plasma cell dyscrasia; in secondary amyloidosis, the underlying infectious or inflammatory disease; and in dialysis-related proteinopathy, to modify hemodialysis procedures or perform kidney transplantation. At present, considerable attention is being focused on promoting the clearance of amyloid deposits and inhibiting fibrillogenesis.

A beneficial change in tissue amyloid deposits has been observed following organ transplantation. In hereditary amyloidoses in which the genetically altered amyloid precursor protein is produced in the liver, such as transthyretin or fibrinogen Aα, liver transplantation may not only stop further deposit formation but also reduce the amount of fibrils in existing deposits (70–72). The best outcomes are achieved when organ transplantation is performed within 1 year of the onset of the first symptoms characteristic of amyloidosis. In addition, in patients who develop end-stage organ failure due to sporadic or hereditary amyloidosis, transplantation of the decompensated organ together with the liver has produced highly favorable outcomes and subsequent disease prognosis (71, 73, 74).

Agents that inhibit fibrillogenesis and promote the breakdown of protein deposits are currently being actively developed. Some are being investigated in vitro, whereas others have already reached phase II clinical trials. Their mechanisms of action are diverse, ranging from inhibition of the synthesis of amyloidogenic precursors, such as transthyretin, to activation of amyloid deposit degradation, for example through immunotherapy, or disruption of interactions between amyloidogenic proteins and connective tissue molecules such as proteoglycans.

TTR synthesis can be inhibited using inotersen, a medication approved in the European Union. This oligonucleotide—a very short segment of synthetic genetic material—is designed to bind to and inhibit genetic material in liver cells responsible for transthyretin production. As a result, less transthyretin is produced in the human body, leading to reduced amyloid formation and alleviation of symptoms associated with hereditary amyloidosis (75, 76). It is important to note that monitoring of platelet counts is recommended in treated patients, as more than half of the patients observed in studies developed some degree of thrombocytopenia.

Stabilization of TTR tetramers is another approach to inhibiting amyloid formation. One of 2 agents is most commonly used for this purpose: the specific agent tafamidis meglumine or diflunisal, a generic nonsteroidal anti-inflammatory drug. In randomized trials, both effectively alleviated symptoms characteristic of familial amyloid polyneuropathy (77–79).

Biological therapies used for rheumatoid arthritis or other autoimmune inflammatory disorders, such as TNF inhibitors, anakinra, an interleukin-1 (IL-1) inhibitor, and tocilizumab, an IL-6 receptor antagonist, may also alleviate symptoms caused by AA amyloidosis associated with rheumatic diseases (80–82). Specific immunotherapy targeting AA and AL amyloid fibrils and oligomers is still being investigated in bioengineering studies…

Ričardas Kundelis

Lithuanian University of Health Sciences

Read more in Internistas, No. 10, 2018.