P-444: A comparison of immunohistochemistry and laser microdissection tandem mass spectrometry to identify the amyloid fibril protein from formalin-fixed paraffin embedded biopsy samples.

Introduction: Correct diagnosis of amyloidosis subtype is a critical step to direct patient management, inform prognosis and guide targeted genetic testing. Laser capture microdissection and tandem mass spectrometry (LMD-MS) analysis of formalin-fixed paraffin-embedded (FFPE) biopsy samples is emerging as the new gold-standard diagnostic technique in amyloid subtyping, with likely superiority to conventional immunohistochemistry (IHC) based approaches.

Methods: To assess this, a novel LMD-MS assay was developed. In brief, 10-micron sections were cut from FFPE biopsies, deparaffinised and stained with Congo red. Laser microdissection was performed of Congo red positive deposits. Proteins from dissected tissue were digested to peptides and analysed with high-performance liquid chromatography (HPLC) coupled with a ThermoFisher scientific Q Exactive plus mass spectrometer and peptide matches assessed against the Swiss-Prot/Uniprot human protein database with the addition of the Kabat library. 121 patient samples were assessed using both LMD-MS and an IHC panel consisting of 4 commercial antibodies: kappa, lambda, transthyretin and serum amyloid A. Each IHC was assessed independently by two experienced pathologists and graded quantitatively. LMD-MS was reported using institutional bioinformatic reporting algorithms.

Results: Biopsy sites were most frequently renal (25.6%), cardiac (17%) and gastrointestinal tract (25.6%). IHC assessment was considered non-diagnostic in 44% of samples. 121 samples were assessed for LMD-MS and 110 were samples were suitable for analysis with the assay . An amyloid subtype was confidently identified in 96% of samples analysed, with only 4 samples not identifying an amyloid forming protein. Concordance was assessed between LMD-MS and IHC. 3 cases of IHC typed light chain amyloidosis were reclassified as non-AL amyloid types and 3 cases of IHC typed AA amyloidosis were reclassified as AL-amyloidosis, both with potentially significant therapeutic implications.

Conclusions: Proteomic assessment of amyloid biopsy with laser capture microdissection and mass spectrometry is superior to immunohistochemical analysis using commercial antibodies for amyloidosis subtyping.