Introduction
Systemic amyloidoses encompass a group of diseases characterized by deposition of abnormal, insoluble, misfolded, β-pleated protein fibrils in different organs. Amyloid protein positively stains with Congo red and demonstrates apple-green birefringence under polarized light. The deposition of the β-pleated amyloid fibrils causes disruption of tissue architecture and thereby results in organ dysfunction and eventually organ failure. The kidneys, heart, gastrointestinal tract, peripheral nerves, and liver are most commonly involved organs. Many different proteins can form amyloid fibrils and to date, 36 different proteins have been recognized to form amyloid fibrils in vivo. The different amyloidogenic proteins form the basis of classification of systemic amyloidosis. Systemic light chain (AL) amyloidosis (formerly known as primary amyloidosis ) is the most common type of systemic amyloidosis in the United States. AL amyloidosis may affect five to 12 people per million per year and the incidence may be higher based on autopsy results. Among 474 patients seen at the Mayo Clinic, 60% of patients were between 50 and 70 years of age and only 10% were under 50 years of age. Approximately 70% of patients with amyloidosis have kidney involvement at the time of presentation with 4% to 5% requiring dialysis. The precursor amyloidogenic protein in AL amyloidosis is a monoclonal immunoglobulin (Ig) light chain or its fragment produced by a clone of plasma cells in the bone marrow. The clonal plasma cell burden in the bone marrow is less than 10% in 50% of patients with AL amyloidosis and occurs in association with multiple myeloma in 10% to 15% of patients. The disease has an insidious onset with an extensive clinical heterogeneity. Therefore a high level of clinical suspicion is necessary to avoid a delay in diagnosis and initiation of therapy before irreversible organ damage develops.
Pathogenesis
Reduced folding stability is a unifying feature of amyloidogenic proteins. Polypeptide chains are synthesized in the endoplasmic reticulum, enter a funnel-like pathway, in which the conformational intermediates become progressively more organized as they merge, resulting in the most stable native state. Mutations result in destabilization of these polypeptides and in the extracellular environment, the mutant polypeptides change from a fully folded state to a partially folded state and then retrace the final part of the folding pathway, ultimately forming either a native or misfolded protein. Some of the extracellular influences that affect the fate of the mutated proteins and direct them toward the pathologic pathway include temperature, pH, metal ions, oxidation, and proteolysis. The partially folded proteins have high propensity to aggregate and form oligomers and eventually fibrils that are insoluble. The deposition of these insoluble fibrils in the extracellular space alters tissue architecture and causes amyloidosis.
The Ig light chain is the amyloidogenic precursor in AL amyloidosis. Solomon et al. used an in vivo animal model to investigate the nephrotoxic potential of Bence-Jones proteins from patients with AL amyloidosis or multiple myeloma. They could reproduce kidney lesions in mice similar to that observed in patients from whom the light chains were purified. The clonal plasma cells producing the amyloidogenic light chains undergo antigen driven selection resulting in a highly mutated Ig light chain variable (VL) domain. It is now believed that the alterations in the amino acid sequences in the Ig VL domain are responsible for the structure, stability, predisposition to aggregation, localization of deposits, and distinct ultrastructural organization of the fibrils. This explains the structural heterogeneity of the fibrils and the protean nature of the disease. Although definitive common structural motif has not yet been identified, both germline sequences of VL domain and acquired amino acid replacements during somatic mutations are thought to be involved in the reduction of the folding stability of the amyloidogenic light chains.
Intense research is currently being conducted on the physiochemical properties of amyloid fibrils to understand amyloidogenesis in the kidneys. Pathogenic light chains can affect the tubulointerstitium or the glomerulus. About 70% of the pathogenic light chains are deposited in the tubulointerstitial compartment and are called tubulopathic light chains, whereas the remainder of the pathogenic light chains are deposited in the mesangium and are called glomerulopathic light chains. The term AL amyloidosis was coined in 1970 by Glenner and colleagues, when he showed that light chains formed a type of amyloid. In 1981 Gise observed and documented glomerular amyloidosis begins in the mesangium. Mesangial cells of the kidneys play a key role in the pathogenesis of AL amyloidosis. They phenotypically transform as macrophages in the presence of pathogenic AL amyloidosis light chains and aid in the generation of more amyloid fibrils. In contrast, in light chain deposition disease, they have a phenotype similar to myofibroblasts. Ting et al. validated the pathogenesis of AL amyloidosis in vivo using an animal model, where mice were injected with amyloidogenic light chains purified from the urine of patients with biopsy-proven, light-chain associated glomerular amyloidosis. The sequential steps involved are internalization of the amyloidogenic light chains by mesangial cells using caveolae followed by trafficking to the mature lysosomal compartment where they aggregate because of their thermodynamic instability and form fibrils through proteolysis. The fibrils are then extruded into the extracellular space where they accumulate and disrupt normal mesangium. Increasing evidence is emerging that the precursor amyloidogenic proteins also have direct cytotoxicity and contribute to disease manifestations. Oligomers or protofibrils may mediate cellular toxicity through a mechanism that activates apoptosis in the cells of target tissues.
Although in the normal bone marrow there is a greater proportion of Kappa (κ) than Lambda (λ) expressing clonal plasma cells, λ isotype plasma cells are more commonly associated with amyloidosis (λ:κ is 3:1) and the λ VI light chains have shown to be those most frequently linked to amyloidosis in the kidneys. This association may be related to specific amino acid sequences in the Ig VL domain and hence λ VI light chains are associated with kidney involvement, κ with hepatic involvement, and λ VIII with soft tissue deposits.
Clinical presentation
AL amyloidosis initially presents with vague clinical features, such as generalized fatigue and weight loss. Suspicion arises only when it affects a specific organ system. The most frequently involved organ systems at the time of diagnosis include the kidneys and the heart presenting as nephrotic syndrome, with or without kidney dysfunction and congestive heart failure, respectively. However, AL amyloidosis can involve any organ system other than the central nervous system. The peripheral nervous system and gastrointestinal tract are also commonly involved.
Kidneys
Nearly 45% to 50% of patients with AL amyloidosis have dominant involvement of the kidneys. It is predominantly a glomerular lesion presenting as nephrotic syndrome except in a small proportion of patients (< 10%), where the amyloid deposition occurs in the kidney vasculature and tubulointerstitium, causing kidney dysfunction without nephrotic syndrome. Considerable albuminuria in the setting of multiple myeloma, as opposed to isolated Bence-Jones proteinuria, should alert the physician to investigate for AL amyloid. Clinical signs and symptoms are like that of any nephrotic syndrome and include peripheral edema, fatigue, pericardial effusions, pleural effusions, and edema/anasarca. Nearly 20% of patients with kidney involvement will require dialysis after a median interval of 13 to 14 months. The amount of proteinuria and the degree of kidney dysfunction at the time of diagnosis predicts the need for dialysis in the future.
Heart
Cardiac amyloidosis causes concentric ventricular wall thickening, impaired cardiac filling, and restrictive cardiomyopathy resulting in rapidly progressive heart failure associated with a poor prognosis. Low voltage QRS complexes in the standard leads of an electrocardiogram (EKG) are found in a high proportion of patients and may precede clinical manifestations. Echogenic hypertrophy on echocardiogram is characteristic. Cardiac silhouette appears normal in a chest x-ray. Clinical signs and symptoms are the result of right-sided heart failure and include elevated jugular venous pressure, peripheral edema, hepatomegaly, arrhythmias, and signs of low cardiac output state, such as orthostatic hypotension.
Peripheral and autonomic neuropathy
Neural involvement gives rise to a varied range of nonspecific clinical symptoms frequently resulting in a long delay from presentation to diagnosis. Peripheral symmetrical sensory neuropathy is common and presents as paresthesia and numbness that can progress to motor neuropathy. Compression lesions, such as carpal tunnel syndrome, is very common and may precede other symptoms by more than a year. Autonomic neuropathy is severely debilitating by causing severe postural hypotension, bladder and erectile dysfunction, or gastrointestinal motility disorders.
Gastrointestinal and hepatic involvement
Gastrointestinal symptoms depend on the site and extent of amyloid deposition. Hepatomegaly is present in about 25% of patients and could be caused by either direct amyloid infiltration or hepatic congestion from amyloid cardiomyopathy. Hepatomegaly from amyloid infiltration is usually massive, rock hard, and nontender. Substantial elevation of alkaline phosphatase, as compared with transaminases, is a typical trait of hepatic amyloid as the infiltration involves the sinusoids. Features of gastrointestinal amyloid include early satiety, diarrhea, chronic nausea, malabsorption, weight loss, gut perforation, and rectal bleeding. Some of the symptoms, such as early satiety and explosive postprandial diarrhea, may be related to gastrointestinal motility disorders from autonomic neuropathy. Macroglossia is a hallmark feature of AL amyloidosis, although found in only about 10% of patients and may lead to airway obstruction, sleep apnea, and difficulty eating.
Hemostatic abnormalities
Amyloidosis manifests with hemorrhage and an abnormal clotting screen at some point during the disease in about one-third of patients. Periorbital purpura presenting as raccoon eyes is characteristic. The mechanism is thought to be caused by vascular endothelial wall friability caused by amyloid deposits, increased fibrinolysis, reduced conversion of fibrinogen to fibrin, circulating anticoagulants, and loss of vitamin K dependent clotting factors via binding to amyloid deposits in the spleen causing a warfarin-like effect. Factor X deficiency is most common, affecting up to 9% of the patients. Although purpura is the most common presenting symptom of hemorrhage, serious life-threatening bleeding can also occur after diagnostic liver or kidney biopsy.
Other organ systems
Additional clinical findings include skin nodules, carpal tunnel syndrome, alopecia, nail dystrophy, splenomegaly, lymphadenopathy, and painful seronegative arthropathy. Endocrinopathies, such as hypothyroidism and hypoadrenalism, have been reported but are rare. Hoarseness of voice may occur because of infiltration of the vocal cords with amyloid deposits. Bone involvement can also occur resulting in lytic lesions and bone pain; pathologic fractures are rare in contrast to multiple myeloma. Localized AL amyloidosis happens infrequently in the upper respiratory and urogenital tracts.
Diagnosis
Because AL amyloidosis presents with a wide array of clinical features and has an insidious onset, physicians should have a high index of clinical suspicion for its timely diagnosis. Delay in diagnosis results in advanced organ dysfunction at the time of diagnosis, when the response to therapy and prognosis remain poor. AL amyloidosis should be suspected in any patient who presents with nondiabetic nephrotic syndrome, hepatomegaly with elevated alkaline phosphatase and normal liver on imaging, heart failure with preserved ejection fraction or nonischemic cardiomyopathy with normal cardiac silhouette on chest x-ray and hypertrophy on echocardiography, polyneuropathy with monoclonal protein, or monoclonal gammopathy of unknown significance (MGUS) with unexplained fatigue, weight loss. As a next step, electrophoresis and immunofixation of serum and urine, and serum-free light chain (FLC) assays should be performed. Amyloidosis is a histologic diagnosis. If a monoclonal protein is detected, then a bone marrow biopsy and abdominal fat pad aspiration are recommended next. Noninvasive fine needle aspiration of abdominal fat pad in combination with bone marrow biopsy may reveal amyloid deposits in more than 70% of patients. Other organs where noninvasive biopsies can be performed to aid diagnosis are salivary gland, rectum, gingiva, and skin. Bone marrow biopsy aids to determine the type and burden of plasma cells and rule in or out other plasma cell disorders, such as multiple myeloma and Waldenstrom macroglobulinemia. Negative bone marrow biopsy and fat pad aspiration do not rule out amyloidosis. Biopsy of the affected organ is often necessary to establish the diagnosis of amyloidosis in cases of high clinical suspicion. However, presence of amyloid deposits alone does not confirm the diagnosis of AL amyloidosis, because several other forms of amyloid deposition have been described that are caused by other proteins besides light chains. These non-AL amyloid proteins include transthyretin (senile amyloidosis) and serum amyloid A (AA amyloidosis). Therefore it is crucial for histologic diagnosis to be followed by identification of the type of amyloid fibril and the extent of organ involvement, because chemotherapy is contraindicated in non-AL amyloidosis. Identification of the amyloid protein may be accomplished by using immunohistochemistry, deoxyribonucleic acid (DNA) analysis, or protein sequencing and mass spectrometry. However, protein sequencing through mass spectrometry is considered gold standard. See Fig. 8.1 for the diagnostic algorithm of AL amyloidosis.
Histology
Amyloid is usually diagnosed by biopsy of an affected organ and staining with Congo red dye. Amyloid deposits stain positive with Congo red dye ( Fig. 8.2 A ) and give a pathognomonic apple-green birefringence under polarized light microscopy ( Fig. 8.2 B ). Observation of the amyloid deposits through electron microscopy reveals the presence of haphazardly arranged nonbranching fibrils, with a diameter in the range of 8 to 10 nm and 30 to 1000 nm long ( Fig. 8.3 A ). They may also be present as organized parallel bundles of nonbranching fibrils ( Fig. 8.3 B ). Each fibril is composed of two twisted 3 nm filaments, with an antiparallel β-pleated sheet configuration stabilized by covalent bonding. Glycosaminoglycans and serum amyloid-P component (SAP) provide additional stabilization to the fibrils.
Immunohistochemistry and DNA analysis
Immunohistochemical staining for Ig light chain has less than 60% sensitivity and hence diagnosis is made by exclusion of hereditary types of amyloidosis through DNA sequencing and of AA and transthyretin types through immunohistochemical staining. The low sensitivity to detect Ig light chain may be caused by light chain epitopes that interact with antisera to κ, and λ light chains are lost during processing of the specimen or during fibril formation. It is imperative that immunohistochemical staining be performed in experienced laboratories, as it is very common to have false negative or positive results. Immunohistochemical staining is rarely possible in an abdominal fat specimen, so kidney or gastrointestinal tract specimens are preferred for this purpose.
Certain types of hereditary amyloidosis have clinical features that mimic AL amyloidosis and seem to occur much more frequently, making diagnosis challenging. Hereditary fibrinogen A α-chain amyloidosis and hereditary transthyretin amyloidosis need mention in this context. A family history of amyloidosis is often absent because of variation in penetrance. Hereditary fibrinogen A α-chain amyloidosis presents with exclusive kidney involvement and has a characteristic kidney biopsy pattern with extensive glomerular involvement in the absence of significant extraglomerular amyloid.
Protein sequencing and mass spectrometry
Amino acid sequencing and mass spectrometry confirms the amyloid protein composition and is the definitive method of identifying the protein subunits of the amyloid deposits. It can also identify genes involved in hereditary amyloidosis. The technique can be applied to any tissue type and can detect both light and heavy chains. Mass spectrometry is considered superior to immunohistochemistry in typing the protein subunits.
Imaging and other tests
Imaging of amyloid deposits is challenging, and progress has been very slow. A comprehensive assessment of the extent of organ involvement can be done by performing the SAP scanning when feasible. SAP is involved in the stabilization of all amyloid fibrils and this is the basis of a novel disease assessment tool called the SAP scintigraphy . Radiolabeled 123I-SAP scintigraphy localizes amyloid deposits rapidly and specifically in proportion to the amount of deposits. SAP scintigraphy is useful for diagnosis and quantification of amyloid deposits, although cardiac and nerve amyloid are poorly visualized. Treatment effects and the extent of organ involvement can be monitored using the SAP scintigraphy. However, the technical complexity of the procedure has limited its wider usability outside expert centers.
Extent of cardiac involvement is a major determinant of amyloidosis outcome. Although histologic diagnosis is the gold standard, it is not always feasible in cardiac amyloid, as endocardial biopsy is a technically challenging and high-risk procedure. Cardiac imaging is the preferred modality of diagnosis in such situations. Echocardiogram, EKG, chest x-ray, cardiac magnetic resonance imaging (MRI) (an emerging gold standard), 99m Tc-labelled 3,3-diphosphono-1,2-propanodicarboxylic acid (99m Tc-DPD) scintigraphy, and 99m Tc-labelled pyrophosphate (99m Tc-PYP) scintigraphy are used for cardiac amyloid imaging. Cardiac MRI is especially useful in differentiating amyloidosis induced left ventricular hypertrophy/thickening from other causes, such as hypertension. Late gadolinium enhancement is highly characteristic of cardiac amyloid. Radionuclide imaging with 99m Tc-DPD and 99m Tc-PYP helps differentiate between AL cardiac amyloidosis (no uptake) and Transthyretin (TTR) or senile cardiac amyloidosis (2+ or greater uptake). More recently, positron emission tomography tracers (18F-florbetapir) used in the diagnosis of Alzheimer disease are used to study cardiac and extracardiac involvement of amyloidosis. Nerve conduction studies, and autonomic function tests, should be performed if neuropathy is suspected. Box 8.1 provides a list of the diagnostic tests used in the evaluation of AL amyloidosis.
Screening tests
Serum and urine electrophoresis and immunofixation
Serum-free light chains and their ratio
Imaging tests
Cardiac – CXR, EKG, ECHO, cardiac MRI
Radionuclide – SAP scintigraphy, PYP scan, DPD scan, florbetapir PET
Histology
Bone marrow biopsy and fat pad aspirate
Organ biopsy
Mass spectrometry and protein sequencing
Other tests
24-hour urine for albumin
Complete blood count, basic chemistry profile, alkaline phosphatase
cTnT and NT-proBNP levels
Nerve conduction studies
Autonomic function tests
Treatment and prognosis
Markers of prognosis and treatment response
Deeper understanding of the pathogenesis and organ involvement in AL amyloidosis has led to major advances in risk stratification, disease monitoring, and expanding therapeutic strategies. It is promising that the treatment outcomes and long-term survival rates are improving because of this advancement. However, the 1-year survival rate continues to be low, likely because of delayed diagnosis. As previously mentioned, the extent of cardiac involvement is the major determinant of prognosis of AL amyloidosis. N-terminal pro-brain natriuretic peptide (NT-proBNP) alone or in conjunction with serum troponin T are strong predictors of survival. Assessment of serum FLC burden is also a key prognostic indicator. High sensitivity cardiac troponin T assays at presentation, and reduction in serum FLC burden and NT-proBNP post chemotherapy are strong predictors of long-term outcome. Recent studies reveal that low differences between amyloidogenic (involved) and nonamyloidogenic (uninvolved) FLC levels (< 50 mg/L) have lower rates of cardiac involvement and increased rates of kidney involvement and thereby have a more favorable prognosis and outcome. The staging system for AL amyloidosis based on cardiac biomarkers is shown in Table 8.1 . This system was proposed over a decade ago and was later revised to include the difference between involved and uninvolved FLCs (dFLC). Scores are calculated by assigning one point for each of the following: cardiac troponin T greater than 0.025 ng/mL, NT proBNP greater than 1800 pg/mL, and dFLC levels greater than 180 mg/L. This provides a staging system of I, II, III, IV based on the number of points assigned (0,1,2,3). In patients with kidney dysfunction, BNP is preferred over NT-proBNP because of less effects on serum levels by reduced glomerular filtration rate (GFR). Concomitant presence of multiple myeloma confers an adverse prognosis. The degree of nephrotic range proteinuria and kidney dysfunction at presentation, along with response to chemotherapy, determine the long-term need for dialysis in patients with kidney involvement. Overall, the presence of kidney involvement is associated with longer survival mainly because of lower proportion of patients with cardiac involvement. Based on the estimated GFR (< 50 mL/min/1.73 m 2 ) and 24-hour proteinuria (> 5 g/day) at presentation, a staging system has been validated to distinguish three groups for risk stratification of dialysis requirement. Both values below cut-off is stage I, one value above cut-off is stage II, and both values above cut-off is stage III with 1%, 12%, and 48% risk of dialysis at 2 years, respectively. Kastritis et al. also validated this staging system and identified the ratio of 24-h proteinuria to estimated GFR, as a sensitive marker of kidney risk and progression to dialysis. λ Light chains are known to be more nephrotoxic than the κ type and have worse outcomes. Response to therapy criteria have been established for uniform reporting, and to monitor disease activity and therapeutic outcomes ( Table 8.2 ).
| Criteria a | Staging |
|---|---|
| Both markers below threshold | I |
| One marker above threshold | II |
| Both markers above threshold | III |
a Threshold values of NT-proBNP and cTnT are < 332 ng/L, and < 0.035 µg/L, respectively.
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