Abstract

Amyloidosis is a group of hereditary and acquired disorders of diverse pathogenesis united by end organ damage due to tissue deposition of misfolded proteins. The diagnosis is typically based on demonstrating Congo red positive amyloid deposits in tissue biopsies of clinically involved sites. Because treatment options for amyloidosis patients are highly varied and intricately tied to the identity of the protein that constitutes the amyloid deposits, adequate diagnostic evaluation now requires identification of the amyloidogenic protein. Traditionally, immunohistochemistry with antibodies to amyloid-associated proteins was the clinical technique of choice for subtyping amyloid deposits in tissue sections. This technique suffers from poor analytical sensitivity and specificity and is a suboptimal technique on which to base treatment decisions for amyloidosis patients.

We developed a highly accurate and reproducible liquid chromatography electrospray tandem mass spectrometery (LC-MS/MS)-based shotgun proteomics assay for subtyping amyloid deposits (Blood. 2009; 114: 4957-9) from microdissected paraffin embedded tissue samples. Implementation of this assay in a CAP/CLIA clinical laboratory has subsequently transformed the standard of amyloidosis care at Mayo Clinic. Since 2008, we utilized the assay in routine practice to identify 20 different amyloid types in tissue samples from diverse anatomic sites from 4139 patients (Table 1). Although AL and ATTR were most common, the cases include a large variety of unusual, and in many cases clinically unsuspected amyloid types. In addition, the amyloid protein subtypes had different frequencies in different anatomic sites (A partial list of tissue sites and their amyloid types analyzed in this study includes: bone marrow: AL 82%, ATTR 15%, AA 2%, other 1%; kidney: AL 51%, ALect2 18%, AA 17%, AH 5%, AFib 4%, other 5%; liver: AL 64%, ALect2 23%, AApoA1 6%, AA 3%, ATTR 2%, other 2%; heart: ATTR 60%, AL 37%, AANF1%, other 2%; GI tract: AL 78%, ATTR 13%, AA 6% AApoA4 1%, other 2%; eye [primarily adnexae] AL 63%; eye [primarily cornea] TGFB induced protein ig-h3 34%, other 3%). One caveat is that these distributions of amyloid types may not accurately reflect population prevalence. More likely, the findings reflected a composite prevalence that included challenging amyloidosis cases.

Table 1

Frequency of amyloid subtypes observed in the Mayo Amyloid Cohort (n=4139). Asterisk denotes non-canonical subtypes

Amyloid Subtype Number (%) of cases 
AL 2553 (61.68) 
ATTR 1015 (24.52) 
AA 151 (3.65) 
ALect2 148 (3.58) 
AIns 48 (1.09) 
Keratin* 36 (0.87) 
AApoA1 30 (0.72) 
AH 27 (0.65) 
AFib 26 (0.63) 
TGFB1-IP* 22 (0.53) 
AApoA4 20 (0.48) 
AANF 14 (0.34) 
Ab2M 12 (0.29) 
AGel 12 (0.29) 
ASem1 12 (0.29) 
APro 7 (0.17) 
ALys 3 (0.07) 
ACal 2 (0.05) 
Enfuvirtide* 2 (0.05) 
AIAPP 2 (0.05) 
Amyloid Subtype Number (%) of cases 
AL 2553 (61.68) 
ATTR 1015 (24.52) 
AA 151 (3.65) 
ALect2 148 (3.58) 
AIns 48 (1.09) 
Keratin* 36 (0.87) 
AApoA1 30 (0.72) 
AH 27 (0.65) 
AFib 26 (0.63) 
TGFB1-IP* 22 (0.53) 
AApoA4 20 (0.48) 
AANF 14 (0.34) 
Ab2M 12 (0.29) 
AGel 12 (0.29) 
ASem1 12 (0.29) 
APro 7 (0.17) 
ALys 3 (0.07) 
ACal 2 (0.05) 
Enfuvirtide* 2 (0.05) 
AIAPP 2 (0.05) 

The prevalence of diverse and unexpected amyloid types in many tissues suggest that shotgun proteomic-based analysis may be the only way to accurately assign amyloid subtype in tissue samples. The technique avoids the analytical problems of immunohistochemistry and the bias introduced by assigning probable amyloid type based on clinical presentation and disease distribution. It provides the clinician with an amyloid subtype that allows for confident individualized patient management. Accurate amyloid subtyping by LC-MS/MS is not only possible, but has proven to be a robust routine clinical tool for the evaluation of numerous amyloidosis patients in our practice.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.