• Download figure

 

• Open in new tab

 

• Download powerpoint

Figure 1

Diagnosing and typing amyloidosis. (A) A Congo red stained renal biopsy (left) shows the apple-green birefringence in polarized light (right) indicative of amyloidosis, 200× (images were acquired using an Olympus BX51 microscope equipped with a 20×/0.5 NA objective and mounted DP12 digital camera, and were further processed [cut out and white balanced] with Adobe Photoshop CS2). (B) Characteristic amyloid fibrils extracted from the postmortem spleen of a patient with rapidly progressive κ AL are shown in a transmission electron micrograph, 100 000× (image was obtained at the Sloan-Kettering Institute Core Electron Microscopy Facility). Fibrils are 7 to 10 nM in diameter and variable in length. (C) Exon 4 of TTR shows heterozygosity (left, arrow) in an African American man with amyloidosis due to the Val122Ile variant (codons for aa 121, 122, and 123 are shown). Homozygotes (right, arrow) are rare but can present with severely symptomatic disease in their early 50s. (D) An example of immunogold electron microscopy (IEM) is shown, 50 000× (courtesy of Dr Carl O'Hara, July 2008; the image was obtained at the Boston University School of Medicine Experimental Pathology Laboratory Service Core). Tissue sections on grids were treated overnight with primary polyclonal rabbit anti–human antibodies (for κ and λ light chains and for TTR) and then with goat anti–rabbit gold secondary antibody conjugates. In this instance the patient had a κ light chain paraprotein and mutant TTR. IEM demonstrated ATTR. Note how the gold beads sit along the fibrils. (E) The peptides of the FR1 portion of the κ light chain from panel B are identified by liquid chromatography and mass spectrometry of the extracted amyloid protein (liquid chromatography/tandem mass spectrometry were performed at the Proteomics Resource Center at Rockefeller University).

This patient was treated before the FLC assay became available. The serum FLC assay, combined with serum and urine immunofixation, has become essential to the management of patients with AL, identifies the pathologic FLC, and provides the target for therapy.31^,32 It reports absolute levels of κ and λ-FLCs and the κ-to-λ ratio. FLCs are metabolized by renal tubular cells, and their measurement is influenced by renal function.31^,33 Persons with renal insufficiency without monoclonal gammopathies have κ-to-λ ratios that remain within a broad normal range.34

The clonal plasma cell disease in AL is usually λ (ratio of κ-to-λ clones is 1:4) probably because the 3r and 6a (IGLV6–57) λ variable region germline donors account for 40% of cases.35^,36 Pathologic FLCs are widely distributed in body tissues and misfold, forming intermediates possibly injurious to normal cells.37^,38 In this view, fibrils may be less toxic than FLC intermediates until the fibrillar burden overwhelms organs and patient.6 These processes remain black boxes, but computational biology and empiric experimental approaches may reveal their mechanisms.39^,40

Case 2: An Elderly Man With Heart Failure, Orthostasis, And Proteinuria

Six months after stenting of 2 coronary arteries, a 71-year-old black man reported new-onset dyspnea, lightheadedness, and abdominal cramping. Echocardiogram showed diastolic dysfunction, concentric left ventricular hypertrophy, septal thickness 1.4 cm, and left ventricular ejection fraction 60%. On examination, he was orthostatic by blood pressure but not pulse, had jugular venous distension, reduced aeration at the right base, hepatomegaly, epigastric discomfort, and 2+ edema to the knees. Complete blood count and PT/PTT were normal. Electrocardiogram showed loss of voltage compared with 6 months previous. Endomyocardial biopsy showed amyloidosis. BNP was 3520 pg/mL (normal, 0-100 pg/mL) and troponin I 0.69 ng/mL (normal, < 0.06 ng/mL). Proteinuria was 2550 mg/day and creatinine clearance 74 mL/min. Alkaline phosphatase was elevated at 315 U/L. Marrow showed 23% λ-restricted plasma cells and λ-FLCs were 1260 mg/L with an abnormal ratio. Transthyretin gene sequence was wild-type.

The diagnosis of AL amyloidosis involving heart, autonomic nervous system, kidneys, and liver was inferred based on the extent and tempo of disease. He began 4 consecutive days per month of oral melphalan (0.18 mg/kg per day) and dexamethasone (20 mg/day), and continued diuretics with daily weight and vital sign monitoring, compression stockings, and a bowel regimen to prevent constipation. After 2 months of therapy, λ-FLCs were 115 mg/L, BNP 2650 pg/mL, and troponin I 0.39 ng/mL. He completed 1 year of therapy to a total oral melphalan dose of 544 mg. Orthostasis and constipation resolved, and he became New York Heart Association class IIA. Diuretics (torsemide and eplerenone) were adjusted depending on weight. λ-FLC nadired at 42 mg/L, BNP at 1640 pg/mL, and proteinuria at 570 mg/day. After 2 years, he reported worsening dyspnea. λ-FLCs were 220 mg/L, and BNP and diuretic requirement increased. He was treated with lenalidomide and low-dose dexamethasone but developed fatigue and pneumonia so therapy was changed to low-dose thalidomide and cyclophosphamide without response. Echocardiogram showed worsening left ventricular function. He was then treated with 2 cycles of bortezomib, melphalan, and dexamethasone. λ-FLC decreased to 30 mg/L, but recurrent abdominal bloating and ileus led to discontinuation of therapy. Left ventricular function continued to decline despite the reduction in λ-FLC. He remains New York Heart Association class III surviving nearly 5 years from diagnosis.

Comments

Typing of cardiac amyloidosis in elderly men can be challenging because of the frequency of senile systemic amyloidosis (SSA) resulting from wild-type transthyretin (TTR). In addition, mutant TTR is the most common cause of hereditary amyloidosis, and 4% of blacks carry the gene for an amyloid-forming mutant TTR in which isoleucine replaces valine at position 122 (Val122Ile, Figure 1C).41^,42 TTR is produced in the liver and minimally in the choroid plexus, and more than 100 amyloid-forming variants have been identified. Sequencing exons 2, 3, and 4 of TTR from genomic DNA allows rapid identification of wild-type and mutants. Mutations cause substitutions that enable the tetrameric TTR protein to dissociate, increasing the concentration of aberrant amyloidogenic monomers.43 Why wild-type TTR forms amyloid fibrils and why SSA occurs with male gender exclusivity remain unknown. Demonstration of wild-type TTR by gene sequencing is helpful in ruling out hereditary ATTR and in diagnosing SSA.44 The drugs diflunisal and tafamidis stabilize the tetramer in vitro and in vivo and are in clinical testing for ATTR.45^,46

In Portugal and Japan, Val30Met ATTR can present as polyneuropathy in patients 20 to 40 years old, leading to disability and early death unless treated with orthotopic liver transplantation.47 In blacks, Val122Ile ATTR often presents in the sixth or seventh decade with progressive heart failure and/or neuropathy but not with proteinuria, although glomerulosclerosis can mimic AL renal involvement. ATTR variants are inherited as autosomal dominant traits with high penetrance. Because blacks have higher incidences of both Val122Ile and monoclonal gammopathies, a tissue diagnosis of amyloidosis requires testing for 2 potential precursor proteins.48 What happens if the patient has both Val122Ile and a monoclonal gammopathy? In our experience, only one of the precursors causes amyloid disease per patient.8 Tissue typing of amyloid fibrils is then required to determine which is causing disease, using techniques such as immunogold electron microscopy or protein separation and identification by mass spectrometry (Figure 1D-E).49^,50 Accurate diagnosis is important for the patient because therapy for AL is marrow toxic, and for the patient's family because Val122Ile ATTR is hereditary. In this case, TTR was wild-type and the extent and tempo of disease allowed the diagnosis of AL (not SSA) to be inferred with confidence, permitting prompt initiation of therapy. The short-term electrocardiographic changes in this case are not unusual (Figure 2).