Clinical diagnosis and typing of systemic amyloidosis in subcutaneous fat aspirates by mass spectrometry-based proteomics

Abstract

Examination of abdominal subcutaneous fat aspirates is a practical, sensitive and specific method for the diagnosis of systemic amyloidosis. Here we describe the development and implementation of a clinical assay using mass spectrometry-based proteomics to type amyloidosis in subcutaneous fat aspirates. First, we validated the assay comparing amyloid-positive (n=43) and -negative (n=26) subcutaneous fat aspirates. The assay classified amyloidosis with 88% sensitivity and 96% specificity. We then implemented the assay as a clinical test, and analyzed 366 amyloid-positive subcutaneous fat aspirates in a 4-year period as part of routine clinical care. The assay had a sensitivity of 90%, and diverse amyloid types, including immunoglobulin light chain (74%), transthyretin (13%), serum amyloid A (%1), gelsolin (1%), and lysozyme (1%), were identified. Using bioinformatics, we identified a universal amyloid proteome signature, which has high sensitivity and specificity for amyloidosis similar to that of Congo red staining. We curated proteome databases which included variant proteins associated with systemic amyloidosis, and identified clonotypic immunoglobulin variable gene usage in immunoglobulin light chain amyloidosis, and the variant peptides in hereditary transthyretin amyloidosis. In conclusion, mass spectrometry-based proteomic analysis of subcutaneous fat aspirates offers a powerful tool for the diagnosis and typing of systemic amyloidosis. The assay reveals the underlying pathogenesis by identifying variable gene usage in immunoglobulin light chains and the variant peptides in hereditary amyloidosis.Clinical diagnosis and typing of systemic amyloidosis in subcutaneous fat aspirates by mass spectrometry-based proteomics ABSTRACT Subcutaneous fat aspiration for diagnosis of systemic amyloidosisSpecimens were obtained, stained with Congo red and interpreted according to previously established protocols described elsewhere.5 For the validation cohort one half of each specimen was used to make a smear and stained with Congo red. The other half, irrespective of whether the smear was positive for amyloid by Congo red, was processed for MS-based proteomic analysis. For the clinical cohort, one half of each specimen was used to make a smear and stained with Congo red. The other half was processed for MS-based proteomic analysis only if the first half stained positive for amyloidosis, as required by the established clinical practice and test validation criteria. Mass spectrometry-based proteomic analysis of subcutaneous fat aspiratesThe SFA specimens were processed for MS analysis using a modification of a previously established protocol.26 Briefly, the aspirate was solubilized and digested in trypsin and used for protein identification by nano-flow liquid chromatography electrospray tandem MS.9 Peptide spectra present in the raw data files were matched against a composite protein sequence database using three different search engines (Sequest Measurement of serum immunoglobulin free light chainsSerum immunoglobulin ...

Clarifying immunoglobulin gene usage in systemic and localized immunoglobulin light-chain amyloidosis by mass spectrometry

“…The dissected tissue was digested into tryptic peptides and analyzed by LC-MS. For each fat aspirate specimen, 3 to 4 spicules were individually solubilized and digested in trypsin without undergoing LMD. 12 Tandem MS spectra present in each sample's raw file were identified with 3 different database search engines: Sequest, 15 X!Tandem, 16 and Mascot, 17 all of which had been augmented with known protein sequences from human IGVL genes and families. The method for identifying IGVL gene was validated in an independent population (n 5 30) by comparing it with mRNA sequencing performed on bone marrow samples, 11 and it was found to be 100% specific.…”

“…[10][11][12] Briefly, for patients with solid tissue specimens, laser microdissection (LMD) was performed on 3 to 4 Congo red-positive areas of formalin-fixed paraffin-embedded tissue blocks. The dissected tissue was digested into tryptic peptides and analyzed by LC-MS. For each fat aspirate specimen, 3 to 4 spicules were individually solubilized and digested in trypsin without undergoing LMD.…”

“…[10][11][12] This method has been in routine use in a CAP/CLIA-compliant clinical laboratory in our institution since 2008. This method was expanded to identify clonotypic peptides from AL deposits and identify the IGVL gene and gene family with 100% specificity.…”

Hepatic adenomas with synchronous or metachronous fibrolamellar carcinomas: both are characterized by LFABP loss

“…5 We have successfully used this method to discover biomarkers for detecting amyloid deposits present in fat aspirate specimens 6 and use this method for subtyping amyloid deposits present in clinical specimens. 7 We utilized the same method for detecting differentially expressed proteins between fibrolamellar carcinoma and paired normal liver.…”

Advances in clinical applications of tissue proteomics: opportunities and challenges

“…The second test the author's group developed was a screening assay for diagnosis and classification of amyloidosis using blindly obtained fresh abdominal fat aspirate specimens from patients suspected to have amyloidosis. The assay classified amyloidosis with 88% sensitivity and 96% specificity, well-beyond the analytical requirements for a screening clinical test [12]. Since the implementation of the tests as clinical platforms in 2008-2009, both assays have been extensively used for clinical management of thousands of patients with amyloidosis and performed within the analytical parameters established by the validation studies.…”