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.

Figure 1.

Immunohistochemical staining and proteomic analysis showing 2 distinct patterns of amyloid within the cardiac biopsy. (A) Congo red staining of the myocardium. There was an area of dark Congo red staining that also stained with an antibody against λ light chain, but not transthyretin, and an area of lighter Congo red staining that stained with an antibody against transthyretin, but not λ light chain. (B) Transthyretin immunostaining confirming the presence of transthyretin. (C) Immunostaining with an antibody against λ light chain. (D) Proteomic analysis illustrating spectral counts and peptide coverage. Both samples showed the amyloid signature proteins apolipoprotein A-IV, serum amyloid P component, and apolipoprotein. (E) Sample X (left) shows abundant λ light chain (7 spectra, 57% protein coverage in yellow highlight [E]) without any transthyretin and sample Y (right) shows abundant transthyretin (10 spectra, 41% protein coverage in yellow highlight [F]) and some λ light chain (4 spectra, 14% protein coverage). The protein identification probability was >95% in each case.

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.

Authorship

Acknowledgments: The authors would like to gratefully acknowledge the patient and the staff at the National Amyloidosis Centre involved in his care.

Contribution: S.M., J.A.G., and J.D.G. performed the research and wrote the manuscript; N.R. performed the proteomic analysis; C.J.W., H.J.L., A.D.W., and P.N.H. treated the patient and contributed to writing the manuscript; and all authors approved the final version of the manuscript.

Conflict of interest disclosure: The authors declare no competing financial interests.

Correspondence: Julian D Gillmore, National Amyloidosis Centre, UCL Medical School (Royal Free Campus), Rowland Hill St, London, NW3 2PF United Kingdom; e-mail: j.gillmore@ucl.ac.uk.