Citations

Abstract Background Immunoglobulin light chain-associated amyloidosis (AL) is caused by deposition of immunoglobulin light chain molecules with unique, clonotypic variable (V) regions. Detection and identification of the V region, however, has been challenging due heterogeneity inherent in V regions. We developed a new method for detecting IGKV and IGLV region fragments in AL deposits using protein mass spectrometry and novel bioinformatics approach. We report distinct IGKV and IGLV usage in localized versus systemic AL. Methods Shotgun protein mass spectrometry on amyloid plaques was performed as previously described (Blood. 2009; 114: 4957-9). Peptide tandem mass spectra (MS/MS) were searched against a composite sequence database containing SwissProt's human complete proteome augmented with 1764 IG V region sequences obtained from ImMunoGeneTics database and a Mayo Clinic internal database. Reversed sequences were appended to the database for estimating identification false discovery rates (FDRs). Peptide identifications were processed with Scaffold software. Confident protein identifications (probability > 0.9) with at least one unique peptide identifications and five MS/MS matches were considered. Detected variable gene family (if any) with most number of peptide and spectral matches is considered to be present in the deposit. The method was first validated in 8 cases of AL amyloidosis with known IGKV and IGLV region sequences and then applied on 1238 systemic and 393 localized AL cases. Differences between groups were measured by generating hypergeometric p-values. Results In all 8 AL cases with known IGKV and IGLV region usage, gene family identified in the AL deposit via proteomics matched the gene family inferred from bone marrow plasma cells by Sanger sequencing of IGLC genes. Armed with this method, we analyzed the amyloid proteomics data from 1238 patients with systemic AL amyloidosis and 393 patients with localized AL amyloidosis. The anatomical site distribution in systemic cohort was 296 GI tract, 364 heart, 225 kidney, 81 liver and 272 fat aspirate; Localized cohort has 78 bladder, 158 lung and 157 skin cases. Figure 1 shows the normalized frequency of the variable gene families detected between the systemic and localized AL amyloidosis. For AL-kappa, KV1 was more prevalent in systemic cases when compared to localized cases (p=7.2E-12) suggesting KV1 clones as a hallmark for systemic AL-kappa amyloidosis. KV3 was more frequently seen in localized AL-kappa cases when compared to systemic AL-kappa cases (p =2.8E-11). For AL-lambda, LV1 and LV2 gene families were more prevalent in localized cases when compared to systemic cases (p= 0.039 and 2.7E-05). LV6 was more prevalent in systemic AL-lambda cases (p=1.4E-07). We also detected a higher incidence of mixed AL/AH type in localized AL (18%) when compared to systemic AL (5%; Odds Ratio=4.2673, p<0.0001). We next turned to the organ specific IGKV and IGLV gene family usage patterns in patients with localized amyloidosis. For 147 localized AL-kappa cases, KV3 was most dominant in lung (64%) and KV1 was dominant in skin (38%). For 246 localized AL-lambda cases, LV2 was most prevalent in lung (34%) and LV3 was most prevalent in skin (36%). For bladder, both LV1 and LV2 had comparable prevalence (42% and 32%). For 361 systemic AL-kappa cases, KV1 consistently ranked at the top of gene families used in GI tract (55%), heart (71%), kidney (59%), liver (75%) and fat aspirate (50%). For 877 systemic AL-lambda cases, LV2 was predominant in GI tract (30%), LV3 in heart (35%) and LV6 in kidney (33%). The prevalence of LV1, LV2 and LV3 in liver is comparable (36%, 22% and 31%). LV6 and LV3 had comparable prevalence (22% and 24%) in AL-lambda fat aspirates. Conclusion The novel proteomics method detects IGLC V family usage in large cohorts of AL patients. It identifies unique profiles in systemic and localized cases, and in different organ sites. This information will be helpful in determining systemic versus localized nature of AL amyloidosis at diagnosis and to assess risk of specific end organ involvement. We found also a strong association between IGKV and IGLV gene usage and organ involvement. Disclosures: No relevant conflicts of interest to declare.

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 1Frequency of amyloid subtypes observed in the Mayo Amyloid Cohort (n=4139). Asterisk denotes non-canonical subtypesAmyloid SubtypeNumber (%) of casesAL2553 (61.68)ATTR1015 (24.52)AA151 (3.65)ALect2148 (3.58)AIns48 (1.09)Keratin*36 (0.87)AApoA130 (0.72)AH27 (0.65)AFib26 (0.63)TGFB1-IP*22 (0.53)AApoA420 (0.48)AANF14 (0.34)Ab2M12 (0.29)AGel12 (0.29)ASem112 (0.29)APro7 (0.17)ALys3 (0.07)ACal2 (0.05)Enfuvirtide*2 (0.05)AIAPP2 (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.

Aims Prognostication and treatment selection for cardiac amyloidosis requires accurate amyloid typing. The aim of this study was to investigate the utility of histomorphology for predicting type. Methods and results Autopsy cases with cardiac amyloidosis (1998–2010) were typed by the use of mass spectrometry‐based proteomics. Deposition patterns were correlated with amyloid type. Among 108 decedents (mean age 75 years; 69% men), 107 had a single type, including transthyretin (ATTR) (60 cases), amyloid light chain (AL) (32 λ; 12 κ), amyloid A (AA) (two), and apolipoprotein AIV (AApoAIV) (one). Interstitial deposition was more extensive in AL amyloidosis cases than in ATTR cases [odds ratio (OR) 6.8, P = 0.0004]. Histomorphological patterns of interstitial deposition were mixed in 61% of AL amyloidosis cases and in 61% of ATTR cases, but diffuse pericellular deposits favoured AL amyloidosis (OR 10.7, P = 0.0001), nodular deposits favoured ATTR (OR 3.1, P = 0.0229), and discrete pericellular deposits tended to partially favour ATTR (OR 1.7, P = 0.1970). Arterial and venous deposits each favoured AL amyloidosis (OR ranging from 9.3 to 192.0, P‐value ranging from 0.0022 to <0.0001), and were severe in AL amyloidosis. Endocardial deposits favoured AL amyloidosis (OR 46.3, P < 0.0001) and were also more severe in AL amyloidosis. Conclusions The extent and distribution of cardiac amyloidosis strongly correlate with amyloid type, suggesting fundamental differences in the pathobiology of deposition. The tendency for mixed patterns to occur limits the practicality and accuracy of using histopathology for amyloid typing.

Key Points Leukocyte cell-derived chemotaxin-2–associated amyloidosis (ALect2) is a common cause of systemic amyloidosis involving the liver. Recognition and accurate diagnosis of hepatic ALect2 amyloidosis is essential for accurate management of patients with hepatic amyloidosis.