Journal of the American Society of Nephrology : JASN, Jan 13, 2016
Amyloidosis is characterized by extracellular deposition of misfolded proteins as insoluble fibri... more Amyloidosis is characterized by extracellular deposition of misfolded proteins as insoluble fibrils. Most renal amyloidosis cases are Ig light chain, AA, or leukocyte chemotactic factor 2 amyloidosis, but rare hereditary forms can also involve the kidneys. Here, we describe the case of a 61-year-old woman who presented with nephrotic syndrome and renal impairment. Examination of the renal biopsy specimen revealed amyloidosis with predominant involvement of glomeruli and medullary interstitium. Proteomic analysis of Congo red-positive deposits detected large amounts of the Apo-CII protein. DNA sequencing of the APOC2 gene in the patient and one of her children detected a heterozygous c.206A→T transition, causing an E69V missense mutation. We also detected the mutant peptide in the proband's renal amyloid deposits. Using proteomics, we identified seven additional elderly patients with Apo-CII-rich amyloid deposits, all of whom had kidney involvement and histologically exhibited no...
Apolipoprotein A-IV associated amyloidosis (AApoAIV amyloidosis) is a rare cause of amyloidosis w... more Apolipoprotein A-IV associated amyloidosis (AApoAIV amyloidosis) is a rare cause of amyloidosis with only a single reported case. Here we describe the clinical, biopsy, and mass spectrometry characteristics of 11 cases of renal AApoAIV amyloidosis encompassing 9 men and 2 women with a mean age at diagnosis of 63.5 years. Progressive chronic kidney disease (mean serum creatinine 2.9 mg/dl) was the most common cause for biopsy with proteinuria absent or minimal in all except one. Hematological and serological evaluation was negative in 9 patients, while 2 had a monoclonal gammopathy. The renal biopsy findings were striking and showed large amounts of eosinophilic Congo-red positive amyloid deposits restricted to the renal medulla with sparing of the renal cortex. In 6 cases, peritubular amyloid was noted in addition to the interstitial involvement. Immunofluorescence studies were negative for immunoglobulins. Electron microscopy showed nonbranching fibrils measuring 7 to 10 nm in diam...
Mutations in myofilament proteins, most commonly MYBPC3-encoded myosin-binding protein C and MYH7... more Mutations in myofilament proteins, most commonly MYBPC3-encoded myosin-binding protein C and MYH7-encoded beta-myosin heavy chain, can cause hypertrophic cardiomyopathy (HCM). Despite significant advances in structure-function relationships pertaining to the cardiac sarcomere, there is limited knowledge of how a mutation leads to clinical HCM. We, therefore, set out to study expression and localization of myofilament proteins in left ventricular tissue of patients with HCM. Frozen surgical myectomy specimens from 47 patients with HCM were examined and genotyped for mutations involving 8 myofilament-encoding genes. Myofilament protein levels were quantified by Western blotting with localization graded from immunohistochemical staining of tissue sections. Overall, 25 of 47 (53%) patients had myofilament-HCM, including 12 with MYBPC3-HCM and 9 with MYH7-HCM. As compared with healthy heart tissue, levels of myofilament proteins were increased in patients manifesting a mutation in either...
Shotgun proteomics of hereditary amyloid deposits generates all the information necessary to iden... more Shotgun proteomics of hereditary amyloid deposits generates all the information necessary to identify pathogenic mutant peptides and proteins. However, these mutant peptides are invisible to traditional database search strategies. We developed a two-pronged informatics workflow for detecting both known and novel amyloidogenic mutations from clinical proteomics data sets. We implemented the workflow in a CAP/CLIA certified clinical laboratory dedicated for proteomic subtyping of amyloid deposits extracted from formalin-fixed paraffin-embedded specimens. Performance of the workflow was characterized on a validation cohort of 49 hereditary amyloid samples, with confirmed mutations, and 85 controls. The sensitivity, specificity, positive predictive value, and negative predictive value of the known mutation detection workflow were determined to be 92%, 100%, 100%, and 96%, respectively. For novel mutation detection workflow, these performance parameters were 82%, 99%, 99%, and 90%, respectively. Validated workflow was applied to detect amyloidogenic mutations from a clinical cohort of 150 amyloid samples. The known mutation detection workflow detected rare frame shift mutations in apolipoprotein A1 and fibrinogen alpha amyloid deposits. The novel mutation detection workflow uncovered unanticipated mutations (W22G and C71Y) of the serum amyloid A4 protein present in patient amyloid deposits. In summary, clinical amyloid proteomics data sets contain mutant peptides of clinical significance that are recoverable with improved bioinformatics.
Shotgun proteomics technology has matured in the research laboratories and is poised to enter cli... more Shotgun proteomics technology has matured in the research laboratories and is poised to enter clinical laboratories. However, the road to this transition is sprinkled with major technical unknowns such as long-term stability of the platform, reproducibility of the technology and clinical utility over traditional antibody-based platforms. Further, regulatory bodies that oversee the clinical laboratory operations are unfamiliar with this new technology. As a result, diagnostic laboratories have avoided using shotgun proteomics for routine diagnostics. In this perspectives article, we describe the clinical implementation of a shotgun proteomics assay for amyloid subtyping, with a special emphasis on standardizing the platform for better quality control and earning clinical acceptance. This assay is the first shotgun proteomics assay to receive regulatory approval for patient diagnosis. The blueprint of this assay can be utilized to develop novel proteomics assays for detecting numerous other disease pathologies.
Examination of abdominal subcutaneous fat aspirates is a practical, sensitive and specific method... more 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 Journal of the American Society of Nephrology, 2010
Background and objectives: Amyloidosis is a group of disorders characterized by accumulation of e... more Background and objectives: Amyloidosis is a group of disorders characterized by accumulation of extracellular deposition of proteins as insoluble aggregates. The clinical management of amyloidosis is based on identifying the underlying etiology and accurate typing of the amyloid. Ig heavy chain amyloid involving the kidney is poorly recognized and often poses a diagnostic dilemma.
Nodular pulmonary amyloidosis, a rare localized form of amyloidosis, has been associated with imm... more Nodular pulmonary amyloidosis, a rare localized form of amyloidosis, has been associated with immunoglobulin light chains (AL type) and variably with low-grade lymphoma. The clinicopathologic features of 18 cases were investigated; 5 of 14 had autoimmune disease. In 14 cases monotypic plasma cells could be demonstrated by immunohistochemistry. By mass spectrometry analysis of the amyloid deposits, all 18 cases showed a peptide profile with an abundance of immunoglobulin light chains (12 κ, 4 λ, and 2 mixed κ and λ), with 13 also showing significant codeposition of heavy chains (10 γ, 2 α, 1 δ). Of 14 patients with follow-up, 3 developed recurrent pulmonary amyloidoma, 2 had pulmonary recurrence plus cutaneous extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) type with and without amyloid, and 1 had a history of parotid gland MALT lymphoma. This study highlights the unique features of this localized form of amyloidosis. AL κ type is more frequent than AL λ type, with a ratio of 3:1, in contrast to the AL λ predominance that characterizes systemic AL amyloidosis. In addition, the majority of nodular pulmonary amyloid is of mixed AL/AH type, a rare finding in systemic amyloidosis. The association of nodular pulmonary amyloidoma with autoimmune disease and lymphoma indicate the majority of these lesions relate to an underlying lymphoplasmacytic neoplasm in the spectrum of MALT lymphoma.
We report a 12-year-old boy with nephrotic syndrome due to renal AA amyloidosis. The AA amyloidos... more We report a 12-year-old boy with nephrotic syndrome due to renal AA amyloidosis. The AA amyloidosis was associated with a 3-year history of systemic-onset juvenile idiopathic arthritis. The presence of serum amyloid A protein was confirmed by laser microdissection of Congo Red-positive glomeruli and vessels followed by liquid chromatography and tandem mass spectrometry; this analysis excluded hereditary and familial amyloidosis. Aggressive management of the systemic-onset juvenile idiopathic arthritis resulted in improvement in clinical and laboratory parameters. The case represents an unusual cause of nephrotic syndrome in children. Early diagnosis of renal amyloidosis and management of systemic-onset juvenile idiopathic arthritis is paramount to preventing progression of kidney disease. Am J Kidney Dis. 62(4):834-838.
The ASCP designates this journal-based CME activity ("JMD 2014 CME Program in Molecular Diagnosti... more The ASCP designates this journal-based CME activity ("JMD 2014 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s)ä. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. 2 0 1 4 J M D C M E P r o g r a m
Amyloid is a characteristic histologic feature in medullary thyroid carcinomas (MTC). We utilized... more Amyloid is a characteristic histologic feature in medullary thyroid carcinomas (MTC). We utilized a novel mass spectrometry-based proteomic analysis to determine if we could identify specific proteins associated with amyloid in MTC. We studied 9 MTC (1 multiple endocrine neoplasia type 2A, 1 familial MTC, and 7 sporadic). Laser microdissection was utilized to sample the amyloid which was then trypsin digested and evaluated by liquid chromatography electrospray tandem MS (LC-MS/MS) which identified the presence of amyloidogenic proteins in all cases of MTC. High levels of calcitonin were identified in all 9 cases of MTC. Secretogranin-1 was identified in 6 of 9 MTC. Calcitonin gene-related peptide was identified in 4 of 9 cases of MTC. LC-MS/MS proteomic analysis provides a rapid, highly specific, and sensitive method for identification of the specific type of amyloid in these endocrine tumors. This approach may allow classification of different forms of endocrine amyloid present in ...
Prognostication and treatment selection for cardiac amyloidosis requires accurate amyloid typing.... more Prognostication and treatment selection for cardiac amyloidosis requires accurate amyloid typing. Study aimed to investigate the utility of histomorphology to predict type. Autopsy cases with cardiac amyloidosis (1998-2010) were typed by 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 ATTR (60 cases), AL (32 λ, 12 κ), AA (2), and AApoAIV (1). Interstitial deposition was more extensive with AL versus ATTR (odds ratio [OR]=6.8, p=0.0004). Histomorphologic patterns of interstitial deposition were mixed in 61% of AL and 61% of ATTR cases, but diffuse pericellular deposits favored AL (OR=10.7, p=0.0001), nodular deposits favored ATTR (OR=3.1, p= 0.0229), and discrete pericellular deposits tended to partially favor ATTR (OR=1.7, p=0.1970). Arterial and venous deposits each favored AL (OR ranging from 9.3 to 192.0, p=0.0022 to <0.0001), and were severe in AL. End...
To determine the impact of amyloid on the prognosis of patients with hypertrophic cardiomyopathy ... more To determine the impact of amyloid on the prognosis of patients with hypertrophic cardiomyopathy (HC), we reviewed outcomes of patients who underwent septal myectomy for HC from March 7, 1996, to October 9, 2012, with amyloid deposits identified in operative specimens. Amyloid subtypes were differentiated by mass spectrometry-based proteomics. The survival rate was compared with that of an age-matched population (2:1) without amyloid who underwent septal myectomy for HC. Sixteen patients (mean age ± SD 71 ± 8 years; 12 men) met study criteria. All 16 had intraventricular peak systolic gradients reduced intraoperatively from 105 ± 53 mm Hg to 3 ± 7 mm Hg (p <0.001). Amyloid deposits in specimens ranged from minimal to mild. Nine patients had senile (transthyretin-type) amyloidosis, 4 had immunoglobulin-associated amyloidosis, 2 had apolipoprotein A4 amyloidosis type, and 1 had serum amyloid A type. There were no deaths before 30 days. Twelve patients had New York Heart Association...
Amyloidosis is caused by deposition in tissues of abnormal protein in a characteristic fibrillar ... more 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 diagn...
Immunoglobulin light chain (LC) amyloidosis (AL) is caused by deposition of clonal LCs produced b... more Immunoglobulin light chain (LC) amyloidosis (AL) is caused by deposition of clonal LCs produced by an underlying plasma cell neoplasm. The clonotypic LC sequences are unique to each patient and they cannot be reliably detected by either immunoassays or standard proteomic workflows that target the constant regions of LCs. We addressed this issue by developing a novel sequence template-based workflow to detect LC variable (LCV) region peptides directly from AL amyloid deposits. The workflow was implemented in a CAP/CLIA compliant clinical laboratory dedicated to proteomic subtyping of amyloid deposits extracted from either formalin-fixed paraffin-embedded tissues or subcutaneous fat aspirates. We evaluated the performance of the workflow on a validation cohort of 30 AL patients, whose amyloidogenic clone was identified using a novel proteogenomics method, and 30 controls. The recall and negative predictive value of the workflow, when identifying the gene family of the AL clone, was 93...
Some patients with immunoglobulin paraproteinemic neuropathy have intra-nerve deposits that morph... more Some patients with immunoglobulin paraproteinemic neuropathy have intra-nerve deposits that morphologically mimick amyloid, but do no stain with Congo red. Patients with amyloid-like deposits were identified. The nerve amyloid-like aggregates were studied by laser microdissection and dual mass spectrometry. Three male patients, all with IgM gammopathy, and neuropathy were identified. Follow-up, disease duration was 5, 19, and 7 years, respectively. All had progressive asymmetric sensory-onset distal axonal polyneuropathy with late motor involvement. Autonomic symptoms occurred in only one after 13 years of symptoms. None had clinical cardio-renal involvement. One had skin papules with dermal amyloid-like deposits. Endoneurial amyloid-like deposits had granulo-fibrillar ultrastructure. Mass spectrometry of laser-dissected deposits identified IgM pentameric macroglobulin (heavy, light, and joining chains) without amyloid-associated proteins including absent apolipoprotein E and serum amyloid P-component. Amyloid-like neuropathy has distinct clinical, pathologic, and proteomic features which expand the spectrum of IgM neuropathies. Patients have favorable survival, relative absence of autonomic features, and distinct proteomic profiles of the infiltrative protein in nerve.
Journal of the American Society of Nephrology : JASN, Jan 13, 2016
Amyloidosis is characterized by extracellular deposition of misfolded proteins as insoluble fibri... more Amyloidosis is characterized by extracellular deposition of misfolded proteins as insoluble fibrils. Most renal amyloidosis cases are Ig light chain, AA, or leukocyte chemotactic factor 2 amyloidosis, but rare hereditary forms can also involve the kidneys. Here, we describe the case of a 61-year-old woman who presented with nephrotic syndrome and renal impairment. Examination of the renal biopsy specimen revealed amyloidosis with predominant involvement of glomeruli and medullary interstitium. Proteomic analysis of Congo red-positive deposits detected large amounts of the Apo-CII protein. DNA sequencing of the APOC2 gene in the patient and one of her children detected a heterozygous c.206A→T transition, causing an E69V missense mutation. We also detected the mutant peptide in the proband's renal amyloid deposits. Using proteomics, we identified seven additional elderly patients with Apo-CII-rich amyloid deposits, all of whom had kidney involvement and histologically exhibited no...
Apolipoprotein A-IV associated amyloidosis (AApoAIV amyloidosis) is a rare cause of amyloidosis w... more Apolipoprotein A-IV associated amyloidosis (AApoAIV amyloidosis) is a rare cause of amyloidosis with only a single reported case. Here we describe the clinical, biopsy, and mass spectrometry characteristics of 11 cases of renal AApoAIV amyloidosis encompassing 9 men and 2 women with a mean age at diagnosis of 63.5 years. Progressive chronic kidney disease (mean serum creatinine 2.9 mg/dl) was the most common cause for biopsy with proteinuria absent or minimal in all except one. Hematological and serological evaluation was negative in 9 patients, while 2 had a monoclonal gammopathy. The renal biopsy findings were striking and showed large amounts of eosinophilic Congo-red positive amyloid deposits restricted to the renal medulla with sparing of the renal cortex. In 6 cases, peritubular amyloid was noted in addition to the interstitial involvement. Immunofluorescence studies were negative for immunoglobulins. Electron microscopy showed nonbranching fibrils measuring 7 to 10 nm in diam...
Mutations in myofilament proteins, most commonly MYBPC3-encoded myosin-binding protein C and MYH7... more Mutations in myofilament proteins, most commonly MYBPC3-encoded myosin-binding protein C and MYH7-encoded beta-myosin heavy chain, can cause hypertrophic cardiomyopathy (HCM). Despite significant advances in structure-function relationships pertaining to the cardiac sarcomere, there is limited knowledge of how a mutation leads to clinical HCM. We, therefore, set out to study expression and localization of myofilament proteins in left ventricular tissue of patients with HCM. Frozen surgical myectomy specimens from 47 patients with HCM were examined and genotyped for mutations involving 8 myofilament-encoding genes. Myofilament protein levels were quantified by Western blotting with localization graded from immunohistochemical staining of tissue sections. Overall, 25 of 47 (53%) patients had myofilament-HCM, including 12 with MYBPC3-HCM and 9 with MYH7-HCM. As compared with healthy heart tissue, levels of myofilament proteins were increased in patients manifesting a mutation in either...
Shotgun proteomics of hereditary amyloid deposits generates all the information necessary to iden... more Shotgun proteomics of hereditary amyloid deposits generates all the information necessary to identify pathogenic mutant peptides and proteins. However, these mutant peptides are invisible to traditional database search strategies. We developed a two-pronged informatics workflow for detecting both known and novel amyloidogenic mutations from clinical proteomics data sets. We implemented the workflow in a CAP/CLIA certified clinical laboratory dedicated for proteomic subtyping of amyloid deposits extracted from formalin-fixed paraffin-embedded specimens. Performance of the workflow was characterized on a validation cohort of 49 hereditary amyloid samples, with confirmed mutations, and 85 controls. The sensitivity, specificity, positive predictive value, and negative predictive value of the known mutation detection workflow were determined to be 92%, 100%, 100%, and 96%, respectively. For novel mutation detection workflow, these performance parameters were 82%, 99%, 99%, and 90%, respectively. Validated workflow was applied to detect amyloidogenic mutations from a clinical cohort of 150 amyloid samples. The known mutation detection workflow detected rare frame shift mutations in apolipoprotein A1 and fibrinogen alpha amyloid deposits. The novel mutation detection workflow uncovered unanticipated mutations (W22G and C71Y) of the serum amyloid A4 protein present in patient amyloid deposits. In summary, clinical amyloid proteomics data sets contain mutant peptides of clinical significance that are recoverable with improved bioinformatics.
Shotgun proteomics technology has matured in the research laboratories and is poised to enter cli... more Shotgun proteomics technology has matured in the research laboratories and is poised to enter clinical laboratories. However, the road to this transition is sprinkled with major technical unknowns such as long-term stability of the platform, reproducibility of the technology and clinical utility over traditional antibody-based platforms. Further, regulatory bodies that oversee the clinical laboratory operations are unfamiliar with this new technology. As a result, diagnostic laboratories have avoided using shotgun proteomics for routine diagnostics. In this perspectives article, we describe the clinical implementation of a shotgun proteomics assay for amyloid subtyping, with a special emphasis on standardizing the platform for better quality control and earning clinical acceptance. This assay is the first shotgun proteomics assay to receive regulatory approval for patient diagnosis. The blueprint of this assay can be utilized to develop novel proteomics assays for detecting numerous other disease pathologies.
Examination of abdominal subcutaneous fat aspirates is a practical, sensitive and specific method... more 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 Journal of the American Society of Nephrology, 2010
Background and objectives: Amyloidosis is a group of disorders characterized by accumulation of e... more Background and objectives: Amyloidosis is a group of disorders characterized by accumulation of extracellular deposition of proteins as insoluble aggregates. The clinical management of amyloidosis is based on identifying the underlying etiology and accurate typing of the amyloid. Ig heavy chain amyloid involving the kidney is poorly recognized and often poses a diagnostic dilemma.
Nodular pulmonary amyloidosis, a rare localized form of amyloidosis, has been associated with imm... more Nodular pulmonary amyloidosis, a rare localized form of amyloidosis, has been associated with immunoglobulin light chains (AL type) and variably with low-grade lymphoma. The clinicopathologic features of 18 cases were investigated; 5 of 14 had autoimmune disease. In 14 cases monotypic plasma cells could be demonstrated by immunohistochemistry. By mass spectrometry analysis of the amyloid deposits, all 18 cases showed a peptide profile with an abundance of immunoglobulin light chains (12 κ, 4 λ, and 2 mixed κ and λ), with 13 also showing significant codeposition of heavy chains (10 γ, 2 α, 1 δ). Of 14 patients with follow-up, 3 developed recurrent pulmonary amyloidoma, 2 had pulmonary recurrence plus cutaneous extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) type with and without amyloid, and 1 had a history of parotid gland MALT lymphoma. This study highlights the unique features of this localized form of amyloidosis. AL κ type is more frequent than AL λ type, with a ratio of 3:1, in contrast to the AL λ predominance that characterizes systemic AL amyloidosis. In addition, the majority of nodular pulmonary amyloid is of mixed AL/AH type, a rare finding in systemic amyloidosis. The association of nodular pulmonary amyloidoma with autoimmune disease and lymphoma indicate the majority of these lesions relate to an underlying lymphoplasmacytic neoplasm in the spectrum of MALT lymphoma.
We report a 12-year-old boy with nephrotic syndrome due to renal AA amyloidosis. The AA amyloidos... more We report a 12-year-old boy with nephrotic syndrome due to renal AA amyloidosis. The AA amyloidosis was associated with a 3-year history of systemic-onset juvenile idiopathic arthritis. The presence of serum amyloid A protein was confirmed by laser microdissection of Congo Red-positive glomeruli and vessels followed by liquid chromatography and tandem mass spectrometry; this analysis excluded hereditary and familial amyloidosis. Aggressive management of the systemic-onset juvenile idiopathic arthritis resulted in improvement in clinical and laboratory parameters. The case represents an unusual cause of nephrotic syndrome in children. Early diagnosis of renal amyloidosis and management of systemic-onset juvenile idiopathic arthritis is paramount to preventing progression of kidney disease. Am J Kidney Dis. 62(4):834-838.
The ASCP designates this journal-based CME activity ("JMD 2014 CME Program in Molecular Diagnosti... more The ASCP designates this journal-based CME activity ("JMD 2014 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s)ä. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. 2 0 1 4 J M D C M E P r o g r a m
Amyloid is a characteristic histologic feature in medullary thyroid carcinomas (MTC). We utilized... more Amyloid is a characteristic histologic feature in medullary thyroid carcinomas (MTC). We utilized a novel mass spectrometry-based proteomic analysis to determine if we could identify specific proteins associated with amyloid in MTC. We studied 9 MTC (1 multiple endocrine neoplasia type 2A, 1 familial MTC, and 7 sporadic). Laser microdissection was utilized to sample the amyloid which was then trypsin digested and evaluated by liquid chromatography electrospray tandem MS (LC-MS/MS) which identified the presence of amyloidogenic proteins in all cases of MTC. High levels of calcitonin were identified in all 9 cases of MTC. Secretogranin-1 was identified in 6 of 9 MTC. Calcitonin gene-related peptide was identified in 4 of 9 cases of MTC. LC-MS/MS proteomic analysis provides a rapid, highly specific, and sensitive method for identification of the specific type of amyloid in these endocrine tumors. This approach may allow classification of different forms of endocrine amyloid present in ...
Prognostication and treatment selection for cardiac amyloidosis requires accurate amyloid typing.... more Prognostication and treatment selection for cardiac amyloidosis requires accurate amyloid typing. Study aimed to investigate the utility of histomorphology to predict type. Autopsy cases with cardiac amyloidosis (1998-2010) were typed by 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 ATTR (60 cases), AL (32 λ, 12 κ), AA (2), and AApoAIV (1). Interstitial deposition was more extensive with AL versus ATTR (odds ratio [OR]=6.8, p=0.0004). Histomorphologic patterns of interstitial deposition were mixed in 61% of AL and 61% of ATTR cases, but diffuse pericellular deposits favored AL (OR=10.7, p=0.0001), nodular deposits favored ATTR (OR=3.1, p= 0.0229), and discrete pericellular deposits tended to partially favor ATTR (OR=1.7, p=0.1970). Arterial and venous deposits each favored AL (OR ranging from 9.3 to 192.0, p=0.0022 to <0.0001), and were severe in AL. End...
To determine the impact of amyloid on the prognosis of patients with hypertrophic cardiomyopathy ... more To determine the impact of amyloid on the prognosis of patients with hypertrophic cardiomyopathy (HC), we reviewed outcomes of patients who underwent septal myectomy for HC from March 7, 1996, to October 9, 2012, with amyloid deposits identified in operative specimens. Amyloid subtypes were differentiated by mass spectrometry-based proteomics. The survival rate was compared with that of an age-matched population (2:1) without amyloid who underwent septal myectomy for HC. Sixteen patients (mean age ± SD 71 ± 8 years; 12 men) met study criteria. All 16 had intraventricular peak systolic gradients reduced intraoperatively from 105 ± 53 mm Hg to 3 ± 7 mm Hg (p <0.001). Amyloid deposits in specimens ranged from minimal to mild. Nine patients had senile (transthyretin-type) amyloidosis, 4 had immunoglobulin-associated amyloidosis, 2 had apolipoprotein A4 amyloidosis type, and 1 had serum amyloid A type. There were no deaths before 30 days. Twelve patients had New York Heart Association...
Amyloidosis is caused by deposition in tissues of abnormal protein in a characteristic fibrillar ... more 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 diagn...
Immunoglobulin light chain (LC) amyloidosis (AL) is caused by deposition of clonal LCs produced b... more Immunoglobulin light chain (LC) amyloidosis (AL) is caused by deposition of clonal LCs produced by an underlying plasma cell neoplasm. The clonotypic LC sequences are unique to each patient and they cannot be reliably detected by either immunoassays or standard proteomic workflows that target the constant regions of LCs. We addressed this issue by developing a novel sequence template-based workflow to detect LC variable (LCV) region peptides directly from AL amyloid deposits. The workflow was implemented in a CAP/CLIA compliant clinical laboratory dedicated to proteomic subtyping of amyloid deposits extracted from either formalin-fixed paraffin-embedded tissues or subcutaneous fat aspirates. We evaluated the performance of the workflow on a validation cohort of 30 AL patients, whose amyloidogenic clone was identified using a novel proteogenomics method, and 30 controls. The recall and negative predictive value of the workflow, when identifying the gene family of the AL clone, was 93...
Some patients with immunoglobulin paraproteinemic neuropathy have intra-nerve deposits that morph... more Some patients with immunoglobulin paraproteinemic neuropathy have intra-nerve deposits that morphologically mimick amyloid, but do no stain with Congo red. Patients with amyloid-like deposits were identified. The nerve amyloid-like aggregates were studied by laser microdissection and dual mass spectrometry. Three male patients, all with IgM gammopathy, and neuropathy were identified. Follow-up, disease duration was 5, 19, and 7 years, respectively. All had progressive asymmetric sensory-onset distal axonal polyneuropathy with late motor involvement. Autonomic symptoms occurred in only one after 13 years of symptoms. None had clinical cardio-renal involvement. One had skin papules with dermal amyloid-like deposits. Endoneurial amyloid-like deposits had granulo-fibrillar ultrastructure. Mass spectrometry of laser-dissected deposits identified IgM pentameric macroglobulin (heavy, light, and joining chains) without amyloid-associated proteins including absent apolipoprotein E and serum amyloid P-component. Amyloid-like neuropathy has distinct clinical, pathologic, and proteomic features which expand the spectrum of IgM neuropathies. Patients have favorable survival, relative absence of autonomic features, and distinct proteomic profiles of the infiltrative protein in nerve.
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