A Clinical Test for the Identification of Amyloid Proteins in Biopsy Specimens by a Novel Method Based on Laser Microdissection and Mass Spectrometry.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1480-1480 ◽  
Author(s):  
Julie A. Vrana ◽  
Jeffrey D. Gamez ◽  
Jason D. Theis ◽  
Timothy B. Plummer ◽  
Robert H. Bergen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bone Marrow ◽  
Light Chain ◽  
Laser Microdissection ◽  
Clinical Test ◽  
Immunoglobulin Light Chain ◽  
Accurate Identification ◽  
Biopsy Specimens ◽  
Specificity And Sensitivity ◽  
Novel Method

Abstract The management of systemic amyloidosis relies on the treatment of the underlying etiology and differs radically for different amyloid types. Therefore, given that at least 25 different proteins have been associated with amyloidosis, accurate identification of proteins deposited as amyloid fibrils is an important clinical problem. In this study, we describe a novel method that can characterize amyloid subtypes using laser microdissection (LMD) and mass spectrometry (MS) on routinely processed paraffin-embedded tissues. The study used 60 cases consisting of 16 transthyretin, 9 serum amyloid-associated protein, 20 immunoglobulin light chain lambda, 5 immunoglobulin light chain kappa, and 10 amyloid negative control samples. The biopsy specimens studied included heart, kidney, gastrointestinal tract, lung and decalcified bone marrow specimens. The amyloid type in all cases was previously characterized based on clinical findings, immunohistochemistry and, where indicated, by molecular testing for transthyretin mutations. Amyloid plaques were captured from an 10 micron paraffin section exhibiting positive Congo Red staining using LMD. Proteins were extracted, digested with trypsin and identified following MS/MS using the Mascot search algorithm analysis. MS correctly identified each of the 4 types of amyloidosis analyzed. Serum Amyloid P component and Apolipoprotein E were also identified as constituents of the amyloid deposition. The analysis was successful on all tissue types including decalcified bone marrow specimens and small biopsy specimens such as endomycardial biopsies and renal biopsies. The use of LMD from paraffin embedded biopsies and subsequent analysis by MS allows identification of the type of amyloid protein deposited with high specificity and sensitivity. This method promises to be a clinical test for accurate identification of amyloid proteins in routinely processed biopsy specimens and overcomes many of the specificity and sensitivity issues associated with current methods such as immunohistochemistry.

Classification of amyloidosis by laser microdissection and mass spectrometry–based proteomic analysis in clinical biopsy specimens

Blood ◽  
2009 ◽  
Vol 114 (24) ◽  
pp. 4957-4959 ◽  
Author(s):  
Julie A. Vrana ◽  
Jeffrey D. Gamez ◽  
Benjamin J. Madden ◽  
Jason D. Theis ◽  
H. Robert Bergen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Proteomic Analysis ◽  
Laser Microdissection ◽  
Training Set ◽  
Accurate Identification ◽  
Biopsy Specimens ◽  
Specificity And Sensitivity ◽  
Validation Set ◽  
Analytical Power

Abstract The clinical management of amyloidosis is based on the treatment of the underlying etiology, and accurate identification of the protein causing the amyloidosis is of paramount importance. Current methods used for typing of amyloidosis such as immunohistochemistry have low specificity and sensitivity. In this study, we report the development of a highly specific and sensitive novel test for the typing of amyloidosis in routine clinical biopsy specimens. Our approach combines specific sampling by laser microdissection (LMD) and analytical power of tandem mass spectrometry (MS)–based proteomic analysis. We studied 50 cases of amyloidosis that were well-characterized by gold standard clinicopathologic criteria (training set) and an independent validation set comprising 41 cases of cardiac amyloidosis. By use of LMD/MS, we identified the amyloid type with 100% specificity and sensitivity in the training set and with 98% in validation set. Use of the LMD/MS method will enhance our ability to type amyloidosis accurately in clinical biopsy specimens.

Immunoglobulin Light Chain Gene Constant Region Is An Invariable Part of Amyloid Deposits in AL Amyloidosis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3128-3128
Author(s):  
Jason D. Theis ◽  
Julie A. Vrana ◽  
Jeffrey D. Gamez ◽  
Angela Dispenzieri ◽  
Stephen R. Zeldenrust ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Light Chain ◽  
Proteomic Analysis ◽  
Laser Microdissection ◽  
Variable Region ◽  
Al Amyloidosis ◽  
Constant Region ◽  
Immunoglobulin Light Chain ◽  
Amyloid Deposits ◽  
Region I

Abstract Background: Amyloidosis caused by immunoglobulin light chain (IGLC) deposition, so-called AL-type or primary amyloidosis, is the most common type of amyloidosis. It has been long believed that IGLC variable regions form the core of the AL-type amyloid deposits and peptides derived from IGLC constant region peptides are only occasionally integrated into this core. For this reason, the scientific effort to identify thge risk factors for development of AL amyloidosis and the biochemical characteristics amyloid deposits has focused on IGLC variable region derived proteins. To understand the peptide constituents of AL amyloidosis better, we undertook a comprehensive study of AL amyloidosis using a novel mass spectrometry based proteomic analysis approach. Methods: Paraffin embedded tissue from 100 cases of AL amyloidosis was studied. In each case amyloid type was previously established by clinical and pathological examination. Congo red stained paraffin sections were prepared and amyloid deposits were microdissected by laser microdissection microscopy. The microdissected tissue fragments were processed and trypsin digested into peptides. The peptides were analyzed by nano-flow liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS). The resulting LC-MS/MS data were correlated to theoretical fragmentation patterns of tryptic peptide sequences from the Swissprot database using Scaffold (Mascot, Sequest, and X!Tandem search algorithms). Peptide identifications were accepted if they could be established at greater than 90.0% probability and protein identifications were accepted if they could be established at greater than 90.0% probability and contain at least 2 identified spectra. The identified proteins were subsequently examined for the presence or absence of amyloid related peptides. Results and Discussion: LC-MS/MS gave peptide profiles consistent with AL amyloidosis in each case. The analysis showed IGLC-lambda deposition in 66 cases and IGLC-kappa deposition in 34 of cases. In each case, LC MS/MS confirmed the previous clinicopathological diagnosis. Interestingly peptides representing IGLC constant region were present in each case. Using this LC-MS/MS methodology, theoretically it is possible to cover 78% of the IGLC-lambda and 87% IGLC-kappa constant regions. In our samples, the average coverage of the IGLC-lambda and IGLC-kappa constant regions were 40% (range 14–78%)and 55% (range 16–87%) respectively. Additionally, the distribution of the peptides suggested that in the majority of the cases whole of the IGLC constant region was deposited. LC MS/MS also identified IGLC-lambda variable region peptides in 37 of 66 cases and IGLC-kappa variable region peptides in 29 of 34 cases studied. The variable region coverage was more restricted and the peptides identified were frequently within the framework segments. It is likely that the peptides derived from CDR segments were present but not detected by the methodology as somatic hypermutation randomly alters the amino acid sequence in the CDR segments and such new sequences are not available in public databases used by algorithms for peptide identification. In the cases with the IGLC variable region hits, it was also possible to assign variable region family usage. IGLC-lambda cases frequently used IGLC-lambda variable region I, II and III families whereas, in IGLC-kappa cases, IGLC-kappa variable region I and III families dominated. Conclusions: AL amyloidosis can be accurately diagnosed using laser microdissection and LC-MS/MS based proteomic analysis in routine clinical specimens. AL amyloidosis invariably contains IGLC constant region peptides and, frequently, the whole of the constant region is deposited. This finding suggests that studies on molecular pathogenesis of amyloidosis should not only consider the IGLC-variable region but also the constant region. It is possible to identify IGLC variable region family usage in AL amyloidosis using LC MS/MS based proteomic analysis. In the clinical setting, this information may be helpful in predicting organ distribution and clinical outcome.

scholarly journals Apolipoprotein C-II Deposition Amyloidosis: A Potential Misdiagnosis as Light Chain Amyloidosis

2016 ◽  
Vol 2016 ◽  
pp. 1-5 ◽  
Author(s):  
Sadichhya Lohani ◽  
Emily Schuiteman ◽  
Lohit Garg ◽  
Dhiraj Yadav ◽  
Sami Zarouk
Keyword(s):  
Mass Spectrometry ◽  
Light Chain ◽  
Amyloid Fibrils ◽  
Plasma Cells ◽  
Laser Microdissection ◽  
Density Lipoprotein ◽  
Definitive Diagnosis ◽  
Diagnostic Challenge ◽  
Light Chain Amyloidosis ◽  
Apolipoprotein C

Hereditary amyloidoses are rare and pose a diagnostic challenge. We report a case of hereditary amyloidosis associated with apolipoprotein C-II deposition in a 61-year-old female presenting with renal failure and nephrotic syndrome misdiagnosed as light chain amyloidosis. Renal biopsy was consistent with amyloidosis on microscopy; however, immunofluorescence was inconclusive for the type of amyloid protein. Monoclonal gammopathy evaluation revealed kappa light chain. Bone marrow biopsy revealed minimal involvement with amyloidosis with kappa monotypic plasma cells on flow cytometry. She was started on chemotherapy for light chain amyloidosis. She was referred to the Mayo clinic where laser microdissection and liquid chromatography mass spectrometry detected high levels of apolipoprotein C-II, making a definitive diagnosis. Apolipoprotein C-II is a component of very low-density lipoprotein and aggregates in lipid-free conditions to form amyloid fibrils. The identification of apolipoprotein C-II as the cause of amyloidosis cannot be solely made with routine microscopy or immunofluorescence. Further evaluation of biopsy specimens with laser microdissection and mass spectrometry and DNA sequencing of exons should be done routinely in patients with amyloidoses for definitive diagnosis. Our case highlights the importance of determining the subtype of amyloidosis that is critical for avoiding unnecessary therapy such as chemotherapy.

scholarly journals Quantitative Immunoprecipitation Free Light Chain Mass Spectrometry (QIP-FLC-MS) Simplifies Monoclonal Protein Assessment and Provides Added Clinical Value in Systemic AL Amyloidosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4375-4375 ◽  
Author(s):  
Faye Amelia Sharpley ◽  
Hannah Victoria Giles ◽  
Richa Manwani ◽  
Shameem Mahmood ◽  
Sajitha Sachchithanantham ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Light Chain ◽  
Residual Disease ◽  
Renal Involvement ◽  
Light Chains ◽  
Al Amyloidosis ◽  
Free Light Chains ◽  
Accurate Identification ◽  
Monoclonal Protein ◽  
Serum And Urine

Introduction Early diagnosis, effective therapy and precise monitoring are central for improving clinical outcomes in systemic light chain (AL) amyloidosis. Diagnosis and disease response assessment is primarily based on the presence of monoclonal immunoglobulins and free light chains (FLC). The ideal goal of therapy associated with best outcomes is a complete responses (CR), defined by the absence of serological clonal markers. In both instances, detection of the monoclonal component (M-component) is based on serum FLC assessment together with traditional serum and urine electrophoretic approaches, which present inherent limitations and lack sensitivity particularly in AL where the levels are typically low. Novel mass spectrometry methods provide sensitive, accurate identification of the M-component and may prove instrumental in the timely management of patients with low-level amyloidogenic light chain production. Here we assess the performance of quantitative immunoprecipitation FLC mass spectrometry (QIP-FLC-MS) at diagnosis and during monitoring of AL amyloidosis patients treated with bortezomib-based regimens. Methods We included 46 serial patients with systemic AL amyloidosis diagnosed and treated at the UK National Amyloidosis Centre (UK-NAC). All patients had detailed baseline assessments of organ function and serum FLC measurements. Baseline, +6- and +12-month serum samples were retrospectively analysed by QIP-FLC-MS. Briefly, magnetic microparticles were covalently coated with modified polyclonal sheep antibodies monospecific for free kappa light chains (anti-free κ) and free lambda light chains (anti-free λ). The microparticles were incubated with patient sera, washed and treated with acetic acid (5% v/v) containing TCEP (20 mM) in order to elute FLC in monomeric form. Mass spectra were acquired on a MALDI-TOF-MS system (Bruker, GmbH). Results were compared to serum FLC measurements (Freelite®, The Binding Site Group Ltd), as well as electrophoretic assessment of serum and urine proteins (SPE, sIFE, UPE and uIFE). Results Cardiac (37(80%) patients) and renal (31(67%) patients) involvement were most common; 25(54%) patients presented with both. Other organs involved included liver (n=12), soft tissue (n=4), gastrointestinal tract (n=3) and peripheral nervous system (n=2). Baseline Freelite, SPE, sIFE and uIFE measurements identified a monoclonal protein in 42(91%), 22(48%), 34(74%) and 21(46%) patients, respectively. A panel consisting of Freelite + sIFE identified the M-component in 100% of the samples. QIP-FLC-MS alone also identified an M-component in 100% of the samples and was 100% concordant with Freelite for typing the monoclonal FLC (8 kappa, 34 lambda). In 4 patients, QIP-FLC-MS identified an additional M-protein that was not detected by the other techniques. In addition, 4/8(50%) kappa and 4/38(11%) lambda patients showed a glycosylation pattern of monoclonal FLCs at baseline by mass spectrometry. Interestingly, the frequency of renal involvement was significantly lower for patients with non-glycosylated forms (25% vs 76%, p=0.01), while no similar relationship was found for any other organs. During the 1-year follow-up period, 17 patients achieved a CR; QIP-FLC-MS identified serum residual disease in 13(76%) of these patients. Conclusion In our series, QIP-FLC-MS was concordant with current serum methods for identifying the amyloidogenic light chain type and provided, against all other individual tests, improved sensitivity for the detection of the monoclonal protein at diagnosis and during monitoring. The ability to measure the unique molecular mass of each monoclonal protein offers clone-specific tracking over time. Glycosylation of free light chains is over-represented in AL patients which may allow earlier diagnosis and better risk-assessment of organ involvement. Persistence of QIP-FLC-MS positive M component in patients otherwise in CR may allow targeted therapy. Overall, QIP-FLC-MS demonstrates potential to be exploited as a single serum test for precise serial assessment of monoclonal proteins in patients with AL amyloidosis. Disclosures Wechalekar: GSK: Honoraria; Janssen-Cilag: Honoraria; Amgen: Research Funding; Takeda: Honoraria; Celgene: Honoraria.

Highly Restricted Usage of Immunoglobulin Light Chain IGKV3-20 with Stereotyped Sequence in Primary Cold-Agglutinin Disease (CAD)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4137-4137 ◽  
Author(s):  
Agnieszka Malecka ◽  
Gunhild Trøen ◽  
Anne Tierens ◽  
Ingunn Østlie ◽  
Ulla Randen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Heavy Chain ◽  
Light Chain ◽  
Cold Agglutinin ◽  
Antigen Binding ◽  
Gene Rearrangements ◽  
Cold Agglutinin Disease ◽  
Immunoglobulin Light Chain ◽  
Thermal Amplitude

Abstract Primary cold agglutinin disease (CAD) is a type of hemolytic anemia mediated by anti-I autoantibodies. Patients suffer from anemia as well as circulatory problems. However, the severity of disease differs greatly between patients. We recently demonstrated that primary CAD is caused by an underlying low grade B cell lymphoproliferative disease of the bone marrow with a typical histology that is different from lymphoplasmacytic lymphoma and, accordingly, does not display the MYD88 L265P mutation (Randen et al., Haematologica, 2013). The majority of patients display circulating monoclonal antibodies encoded by the immunoglobulin heavy chain gene IGHV4-34. The disease severity does not correlate with antibody titers, but seems to be determined by the thermal amplitude, i.e., the highest temperature at which the cold agglutinin binds to the antigen. The framework region 1 of IGHV4-34 encodes for a sequence that binds to I antigen. However, this does not explain the molecular basis of disease heterogeneity. We studied 27 patients with well-characterized primary CAD and sequenced immunoglobulin heavy as well as immunoglobulin light chains to find additional consensus regions that may determine anti-I reactivity. Bone marrow aspirates, or frozen bone marrow trephine biopsies and blood from 27 patients with well-documented primary CAD were collected. Monoclonal B cells were isolated by flow sorting (FACS Aria Ilu High speed sorter, Becton Dickinson). Viable cells were detected using the forward scatter versus side scatter dot plot. Subsequently, CD45 bright events with low side scatter features representing lymphocytes, were selected. Then, CD5 positive and CD19 negative events, i.e. T cells, were gated out using a CD5 versus CD19 dot plot leaving only B cells. Finally, monoclonal B cells were selected using the immunoglobulin light chain gate, either k or l. Clonally rearranged IGH genes were detected using the Somatic Hypermutation Assay v2.0 (Invivoscribe) and were then sequenced. Immunoglobulin light chain genes (IGL) were amplified by an in-house diagnostic protocol based on Biomed-2 primers (van Dongen et al., Leukemia, 2003). All sequences were analyzed using the IMGT database (www.imgt.org). Productive IGHV4-34 gene rearrangements were identified in 22/27 patients. In 4 patients, no productive rearrangement was identified, while in one patient a productive IGHV3-23 was seen. No significant homology of complementarity determining region 3 (CDR3) regions was found between IGHV sequences. The N-glycosylation sequence within the CDR2 region, affecting antigen-binding, was mutated in 8 patients whereas no mutations were present in 7 patients and mutations in flanking residues were seen in 6 patients. The latter mutations may modulate glycosylation efficacy. Clonal rearrangement of the IGKV3-20 was detected in 16/27 patients, clonal IGKV3-15 gene rearrangements were identified in 4/27 patients whereas other IGL genes were rearranged in 4/27 patients. No clonal IGL gene rearrangement was found in 3/27 patients. Of interest, 7 of the patients with IGKV3-20 rearrangement displayed highly homologous CDR3 regions. The latter was highly associated with an un-mutated N-glycosylation sequence of the respective IGHV4-34 sequence. In conclusion, our data show that in addition to IGHV, also IGLV usage is highly restricted in CAD. Furthermore, stereotyped IGLV sequences are seen that are mutually exclusive with mutated N-glycosylation sequences in the IGHV CDR2 sequence. These data indicate that multiple regions within the immunoglobulin heavy chain as well as immunoglobulin light chain contribute to I-antigen binding. The data suggest that subtle differences in these multiple binding sequences may contribute to the differences in thermal amplitude of I antigen binding of the antibody. The highly restricted usage of IGKV3-20 provides a rationale for vaccination with IGKV3-20 proteins, known to be immunogenic and being considered for treatment in other lymphoproliferative diseases (Martorelli et al., Clin Cancer Res, 2012). Disclosures No relevant conflicts of interest to declare.

A comparison of immunohistochemistry and mass spectrometry for determining the amyloid fibril protein from formalin-fixed biopsy tissue

2015 ◽  
Vol 68 (4) ◽  
pp. 314-317 ◽  
Author(s):  
Janet A Gilbertson ◽  
Jason D Theis ◽  
Julie A Vrana ◽  
Helen Lachmann ◽  
Ashutosh Wechalekar ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amyloid Fibrils ◽  
Accurate Identification ◽  
Protein Precursor ◽  
Biopsy Specimens ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Amyloid Fibril Protein ◽  
The Uk ◽  
Formalin Fixed

Amyloidosis is caused by deposition in tissues of abnormal protein in a characteristic fibrillar form. There are many types of amyloidosis, classified according to the soluble protein precursor from which the amyloid fibrils are derived. Accurate identification of amyloid type is critical in every case since therapy for systemic amyloidosis is type specific. In ∼20–25% cases, however, immunohistochemistry (IHC) fails to prove the amyloid type and further tests are required. Laser microdissection and mass spectrometry (LDMS) is a powerful tool for identifying proteins from formalin-fixed paraffin-embedded tissues. We undertook a blinded comparison of IHC, performed at the UK National Amyloidosis Centre, and LDMS, performed at the Mayo Clinic, in 142 consecutive biopsy specimens from 38 different tissue types. There was 100% concordance between positive IHC and LDMS, and the latter increased diagnostic accuracy from 76% to 94%. LDMS in expert hands is a valuable tool for amyloid diagnosis.

How I treat amyloidosis: the importance of accurate diagnosis and amyloid typing

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3206-3213 ◽  
Author(s):  
Nelson Leung ◽  
Samih H. Nasr ◽  
Sanjeev Sethi
Keyword(s):  
Mass Spectrometry ◽  
Cardiac Mri ◽  
Amyloid Fibrils ◽  
Soft Tissues ◽  
Laser Microdissection ◽  
Mutational Analysis ◽  
Advanced Technique ◽  
Specificity And Sensitivity ◽  
Commercial Antibodies ◽  
Rare Group

Abstract Amyloidosis is a rare group of diseases characterized by deposition of amyloid fibrils in soft tissues. More than 28 types of amyloid have been identified. They all share common ultrastructural and chemical characteristics. Treatments are available for many types but are type specific. Therefore, confirmation and typing of amyloid are essential before initiating treatment. Monoclonal protein studies should be performed on suspected cases, but the diagnosis requires a tissue biopsy. Congo red stain and electron microscopy are helpful to discriminate between amyloid and other pathologic fibrils. Once amyloid is confirmed, typing should be performed. Immunofluorescence and immunohistochemistry are frequently used and are helpful, but this approach has limitations, such as availability, specificity and sensitivity of commercial antibodies. Genetic mutational analysis is vital for ruling in and out hereditary amyloidoses but is unhelpful in nonmutated forms. The most advanced technique of amyloid typing is laser microdissection followed by mass spectrometry. Using proteomics, laser microdissection followed by mass spectrometry can directly identify proteins with or without mutations. Finally, imaging studies, such as cardiac MRI with gadolinium and 123I-labeled SAP scintigraphy not only assist in evaluation of patients with known amyloidosis but cardiac MRI has detected amyloid in patients previously unsuspected of the disease.

scholarly journals Clinical Characteristics and Outcome of Patients with Wild-Type Transthyretin and AL Cardiac Amyloidosis Confirmed By Mass Spectrometry

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3126-3126
Author(s):  
Stephen Parkin ◽  
Michael A. Seidman ◽  
Margot Davis ◽  
Kevin Song
Keyword(s):  
Mass Spectrometry ◽  
Bone Marrow ◽  
Light Chain ◽  
Nyha Class ◽  
Complete Response ◽  
Al Amyloidosis ◽  
Cell Percentage ◽  
Bone Marrow Plasma ◽  
Kappa Light Chains ◽  
Al Amyloid

Abstract INTRODUCTION: Cardiac involvement is common in both wild-type transthyretin (wATTR) and AL amyloidosis and these entities can have overlapping clinical features (Banypersad et al, JAHA 2012). Accurate diagnosis is vital given differences in spectrum of disease, management, and prognosis. We examined the presenting clinical features and survival outcome of patients diagnosed with cardiac wATTR and AL amyloidosis confirmed by mass spectrometry. MATERIALS & METHODS: Patients diagnosed between January 2014 and September 2017 with biopsy proven wATTR or AL amyloidosis and cardiac involvement by consensus criteria (Gertz et al, Amyloid 2010) were included. Mass spectrometry testing was performed at the Mayo Clinic Laboratories to confirm amyloid subtype in all cases. Patient records were retrospectively reviewed to identify clinical characteristics as well as details regarding treatment and survival. RESULTS: Forty-three patients were identified (wATTR n=27, AL n=16) with site of biopsy: 41 cardiac (95%); 1 gastric (2%); 1 skin (2%). Two (13%) patients with AL amyloid had coexisting multiple myeloma. Thirty-five (81%) patients were male, with a strong male predominance in wATTR patients (100% vs 50% for AL patients, p=<0.01). Median age at diagnosis was 73 years (range 45-86), with a trend towards older age in wATTR patients (median 75 vs 62 years for AL patients, p=0.09). No difference was seen in presenting NYHA class or levels of troponin and ntProBNP between amyloid subtypes. BNP trended towards higher values in AL patients (median 514 vs 249 ng/L for wATTR, p=0.09). wATTR amyloid patients had lower median presenting left ventricular ejection fraction (43 vs 53%, p=0.05). Two (7%) patients with wATTR amyloid had an M-protein on SPEP compared to 7 (44%) with AL amyloid. Only 2 patients with AL amyloid had M-protein greater than 5 g/L while both wATTR patients had less than 5 g/L. In those with serum free light chain testing available (wATTR n=24, AL n=16), median lambda light chain level was higher in AL patients (176.0 vs 16.9 mg/L for wtATTR, p=<0.01). No difference was seen in kappa light chains (16.5 vs 23.8 mg/L for wATTR, p=0.60), though the only patient with kappa AL amyloid had kappa light chains significantly elevated at 1640 mg/L. The kappa/lambda ratio was abnormal in 15% of wATTR patients (all kappa predominant) compared to 100% in AL amyloid patients (94% lambda predominant) (p=<0.01). The median difference between involved and uninvolved light chain (dFLC) was 22 mg/L for wtATTR patients compared to 249 mg/L for AL patients. In patients with bone marrow biopsy results available (AL n=15, wATTR n=7), bone marrow plasma cell percentage was higher in patients with AL amyloid (median 7% vs 2%, p=0.01). No monoclonal plasma cells were seen on bone marrow in wATTR patients by immunophenotype. Treatment for patients with AL amyloid was: bortezomib, cyclophosphamide, and dexamethasone (VCD) in 11 (69%); VCD followed by melphalan 200 mg/m2 autologous transplantation in 1 (6%); melphalan/dexamethasone in 1 (6%); no treatment in 2 (13%); unknown in 1 (6%). In evaluable patients, hematologic response rate was 54% (complete response n=4, very good partial response n=2, partial response n=1, no response n=6). With a median follow-up of 1.8 years for surviving patients, 1 year overall survival (OS) was 76% for the entire cohort. Patients with AL amyloidosis had significantly poorer 1 year OS (41% vs 92% for wATTR patients, p=<0.01). Patients with NYHA class 3-4 had significantly worse 1 year OS (54% vs 96% for those 1-2, p=<0.01), and this held true for both AL and wATTR patients. In AL patients, revised Mayo stage (Kumar et al, JCO 2012) of III-IV predicted for poor 1 year OS (19% vs 75% for stages I-II, p=0.03), as did not achieving complete response to primary treatment (1 year OS 33% vs 67% for CR patients, p=0.06). DISCUSSION & CONCLUSIONS: Male sex, older age, and lower LVEF were more common in patients with wATTR compared to AL amyloidosis. Higher levels of BNP, larger dFLC with lambda predilection, and higher bone marrow plasma cell percentage were more common in AL amyloid patients. Overall survival was significantly worse for patients with AL amyloidosis, particularly those with high NHYA class, advanced revised Mayo stage, and suboptimal response to primary therapy. These results highlight the importance of accurate amyloid sub-typing in patients with suspected cardiac amyloidosis. Disclosures No relevant conflicts of interest to declare.

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