Two types of amyloid in a single heart

To the editor:

Amyloidosis is a heterogeneous group of diseases, in which amyloidogenic precursor proteins misfold and adopt a β-pleated sheet conformation.¹  Several proteins can form amyloid fibrils in vivo including transthyretin,²  apolipoprotein A-I and A-II, lysozyme, fibrinogen, serum amyloid A protein and immunoglobulin light chains, but “cross-fibril” seeding appears to be rare such that 2 types of amyloid are rarely identified in the same individual.³  Congo red (CR) staining with apple green birefringence under polarized light is used to confirm the presence of amyloid,⁴  whereas immunohistochemistry, staining the biopsy tissue with a panel of monospecific antibodies against known amyloidogenic proteins, is the technique most widely used to characterize the amyloid fibril protein, but it is flawed.⁵  Proteomic analysis involves proteolytic cleavage of proteins within microdissected amyloidotic tissue and identification by mass spectometry.⁶  This additional tool is being increasingly used in conjunction with immunohistochemistry to identify the amyloid fibril protein.

We describe a case in which 2 amyloid fibril proteins were isolated in an individual patient both by immunohistochemistry and by proteomic analysis. An 81-year-old man was referred to our center with a 6- to 7-month history of exertional dyspnea and New York Heart Association class II symptoms. Baseline investigations included an echocardiogram showing characteristic features of cardiac amyloidosis with a thickened interventricular wall of 18 mm, moderate diastolic dysfunction, and preserved left ventricular ejection fraction of 58% accompanied by elevated serum cardiac biomarkers (N-terminal fragment brain natriuretic peptide 1966 ng/L and troponin T 0.09 µg/mL). ¹²³I-labeled serum amyloid P component scintigraphy did not show visceral amyloid deposits,⁷  but ^99mTc-dicarboxypropane diphosphonate (^99mTc-DPD) scintigraphy showed abnormal (Perugini grade 2) cardiac uptake, typical of cardiac transthyretin amyloidosis⁸  and unusual in light chain (AL) amyloidosis.⁹  The κ-serum free light chain concentration was 13.1 mg/L, λ was 644 mg/L, and λ BJP was present. A bone marrow biopsy showed 6% plasma cells and immunophenotyping isolated a CD19⁻, CD56⁺, CD27⁺ plasma cell clone. Sequencing of the transthyretin gene was wild-type. The differential diagnosis was between wild-type cardiac transthyretin (ATTR) amyloidosis (senile systemic amyloidosis) and cardiac AL amyloidosis, the management and prognosis of which differ substantially. A cardiac biopsy was undertaken to differentiate between these diagnoses. Figure 1 shows CR and immunohistochemical staining of the specimen using antibodies to λ light chains and transthyretin, and the results of proteomic analysis. Interestingly, there were 2 distinct patterns of amyloid within the same specimen: 1 showed honeycomb morphology, lighter CR staining, and stained with antibody against transthyretin, but not λ light chains, and the other showed more diffuse, but darker CR staining, and stained with antibody against λ light chains, but not transthyretin. The 2 distinct areas were separately captured by laser microdissection and analyzed by tandem mass spectrometry. Amyloid was identified by its “signature proteins” in both samples, but in 1 there was abundant transthyretin with low level λ light chain and in the other there was abundant λ light chain without transthyretin (Figure 1D), thus confirming the immunohistochemistry results of 2 types of amyloid in the same heart. Our patient is due to receive chemotherapy for AL amyloidosis shortly.

In summary, the exceptionally rare occurrence of 2 different amyloid fibril proteins was suggested by immunohistochemistry in this patient. The coexistence of 2 amyloid types in the same heart was confirmed by laser capture microdissection and proteomic analysis of 2 distinct areas.