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Amyloidosis: Typing & Nomenclature
The Society for Cardiovascular PathologyCompanion Society MeetingUSCAP 2015 Annual Meeting
March 22, 2015
Joseph J. Maleszewski, MD, FCAP, FACCAssociate Professor of Pathology & Medicine
Divisions of Anatomic Pathology, Cardiovascular Diseases, & Molecular Genetics
Disclosures
•Relevant financial relationships
•None
•Off-label usage
•None
Outline
•Definition & Types
•Nomenclature
•Methods of Typing• Indirect methods•Direct methods
•Antibody-based methods
•Proteomic methods
•Conclusions
Outline
•Definition & Types
•Nomenclature
•Methods of Typing• Indirect methods•Direct methods
•Antibody-based methods
•Proteomic methods
•Conclusions
• “amylum” (Latin: starch)
• misfolded extracellular protein
• antiparallel ß-sheets
• fibrils
• 7.5 - 10 nm
• non-branching, rigid, insoluble
• binds Congo Red + birefringe
• characteristic cross ß-diffraction
on X-ray diffraction
• 31 recognized proteins (human)
AmyloidosisDefinition
Amyloid FibrilsAtomic-Force Microscopy
Merlini G, Bellotti V. N Engl J Med 2003;349:583-596.
Amyloid FibrilsElectron Microscopy
*
• intrinsic propensity to mis-fold
• aging
• high concentration
• mutation
• sporadic / de novo
• familial
• proteolytic remodeling of
precursor protein
• biophysical / functional amyloid
AmyloidosisDefinition
Transthyretin
Ig Light Chain
Apolipoprotein A-I
• Congo Red
• Thioflavin T/S
• Sulfated Alcian Blue
• Crystal violet or Methyl violet
AmyloidosisDefinition - Special Stains
Sulfated Alcian Blue
Crystal Violet
Hematoxylin & Eosin
Congo Red
AmyloidosisDefinition - Organs Involved
Heart Lung Kidney
Liver Spleen
• Texture
• Atria
• Ventricles
• firm
• waxy
• normal size or dilated
• subendocardial deposits
• thick walls
• VS: VFW
VS
Four-Chamber View
Short-Axis View
AmyloidosisCardiac - Gross Features
Histopathologic Findings (H&E)
AmyloidosisCardiac - Histologic Features
Nodular Pericellular Vascular
AmyloidosisCardiac - Histologic Features
AmyloidosisTypes - Overall
Protein Precursor Distribution Type Dx / Tissues
Aß Aß precursor localized acq / hered Alzheimer
APrP Prion localized acq / hered CJD
Aß2M ß2-microglobulin systemic acquired dialysis
AL Ig light chain sys / local acquired myeloma
AA Amyloid A systemic acquired infx / inflamm
ATTR Transthyretin systemic acq / hered FA / senile
AApoAI Apolipoprotein A-I systemic hereditary heart, liver
AGel Gelsolin systemic hereditary FHA
ALys Lysozyme systemic hereditary kidney, liver
AFib Fibrinogen systemic hereditary kidney
Adapted from Sipe JD et al. Amyloid fibril protein nomenclature: 2012 recommendations fromthe Nomenclature Committee of the International Society of Amyloidosis. Amyloid. 2012Dec;19(4):167-70.
Protein Precursor Distribution Type Dx / Tissues
AL Ig light chain sys / local acquired myeloma
ATTR Transthyretin systemic acq / hered HA / “senile”
AANF Atrial Natriuretic Factor localized acquired heart
AApoA4 Apolipoprotein A-4 systemic hereditary heart, liver
AA Amyloid A systemic acquired infx / inflamm
AGel Gelsolin systemic hereditary FHA
ALys Lysozyme systemic hereditary kidney, liver
AApoA1 Apolipoprotein A-1 systemic hereditary heart, liver
AmyloidosisTypes - Cardiac
Protein Precursor Distribution Type Dx / Tissues
AL Ig light chain sys / local acquired myeloma
ATTR Transthyretin systemic acq / hered HA / “senile”
AANF Atrial Natriuretic Factor localized acquired heart
AApoA4 Apolipoprotein A-4 systemic hereditary heart, liver
AA Amyloid A systemic acquired infx / inflamm
AGel Gelsolin systemic hereditary FHA
ALys Lysozyme systemic hereditary kidney, liver
AApoA1 Apolipoprotein A-1 systemic hereditary heart, liver
AmyloidosisTypes - Cardiac
80%18%
2%
AmyloidosisTypes - Endomyocardial Biopsy
ATTR62%
AL36%
Other2%
n = 1432
AmyloidTyping
• AL high-dose chemotx (+/- PBSCT)
• ATTR (hereditary) liver/heart transplant
• ATTR (wild-type) heart transplant / drugs
• SAA anti-inflammatory
Is Typing Important?
Outline
•Definition & Types
•Nomenclature
•Methods of Typing• Indirect methods•Direct methods
•Antibody-based methods
•Proteomic methods
•Conclusions
AmyloidosisNomenclature
• “A” followed by protein abbr.• systemic vs. localized
(AL, AH, ß2-Microglobulin)
Naming Scheme
Examples
Immunoglobin Light Chain = AL
AL, systemic
Atrial Natriuretic Factor = ANF
AANF, localized
Sipe JD et al. Nomenclature 2014: Amyloid fibril proteinsand clinical classification of the amyloidosis. Amyloid. 2014Dec;21(4):276-81.
AmyloidosisNomenclature
• Variants named according to protein
• Use “hereditary” rather than “familial”
Mutations
Example
Transthyretin = TTR, V30M
ATTRV30MSipe JD et al. Nomenclature 2014: Amyloid fibril proteinsand clinical classification of the amyloidosis. Amyloid. 2014Dec;21(4):276-81.
Outline
•Definition & Types
•Nomenclature
•Methods of Typing• Indirect methods•Direct methods
•Antibody-based methods
•Proteomic methods
•Conclusions
AmyloidosisTyping - Indirect Methods
SPEP + IFEM-spike κ-LC
• Gammopathy does not 100% correlate with amyloid type
• Specificity ~75%• Only AL-type
• Inexpensive• Non-invasive
Strengths
Limitations
PEP + IFE
AmyloidosisTyping - Indirect Methods
• Does not 100% correlate with amyloid type
• Specificity ~90%• Only AL-type
• Inexpensive• Non-invasive
Strengths
Limitations
Serum FLC Assay
AmyloidosisTyping - Indirect Methods
Lachmann HJ1, Booth DR, Booth SE, etal. Misdiagnosis of hereditary amyloidosisas AL (primary) amyloidosis. N Engl JMed. 2002 Jun 6;346(23):1786-91.
Palladini G1, Russo P, Bosoni T, et al.Identification of amyloidogenic light chainsrequires the combination of serum-freelight chain assay with immunofixation ofserum and urine. Clin Chem. 2009Mar;55(3):499-504.
Maleszewski JJ, Murray DL, Dispenzieri A,et al. Relationship between monoclonalgammopathy and cardiac amyloid type.Cardiovasc Pathol. 2013 May-Jun;22(3):189-94.
Outline
•Definition & Types
•Nomenclature
•Methods of Typing• Indirect methods•Direct methods
•Antibody-based methods
•Proteomic methods
•Conclusions
AmyloidosisTyping - Direct Methods
• Large Ab panels (site)• High background staining• Conformational differences in fixed
vs. unfixed light chains• Antigen masking• Variable domains of LCs• Protein fragmentation during
amyloid formation
• Familiarity with technology• TAT
Strengths
Limitations
AL-λ Immunohistochemistry
ATTR Immunohistochemistry
Immunohistochemistry
AmyloidosisTyping - Direct Methods
• Reliable• Many labs have access to IF• Familiarity with technology• TAT
Strengths
Image courtesy of Dr. James R. StoneMassachusetts General Hospital
Amyloid IF Panel, ATTR+
• Frozen tissue• Large Ab panels needed
(depending on site)• Ab specificity for mutant
proteins not fully established
Limitations
Immunofluorescence
AmyloidosisTyping - Direct Methods
• Reliable, direct
Strengths
• Cost• Access to EM• TAT
Limitations
Immunoelectron Microscopy
Outline
•Definition & Types
•Nomenclature
•Methods of Typing• Indirect methods•Direct methods
•Antibody-based methods
•Proteomic methods
•Conclusions
AmyloidosisTyping - Direct Methods
• Reliable, direct• Single assay, no panels req
Strengths
• Cost• Availability of LC MS/MS• TAT• Low-level proteins often
missed
Limitations
2D-PAGE MS
AmyloidosisTyping - Direct Methods
• Reliable, direct• Single assay, no panels req• Reliable screen for mutations
Strengths
• Cost• Availability of LC MS/MS• TAT
Limitations
LC MS/MS
AmyloidosisTyping - Direct Methods
• Reliable, direct• Single assay, no panels req• Reliable screen for mutations
Strengths
• Cost• Availability of LC MS/MS• TAT
LimitationsLaser Microdissection
Proteomic Spectra
LC MS/MS
AmyloidosisTyping - Direct Methods
• Reliable, direct• Single assay, no panels req• Reliable screen for mutations
Strengths
• Cost• Availability of LC MS/MS• TAT
Limitations
SwissProt Databse Query
AmyloidTyping - Genetics
Electropherogram
AmyloidTyping - Genetics
Alamut Screenshot
Tandem Mass Spectrometry
• TTR, ApoA1, ApoA4, Gel• For known mutations
sensitivity 92%, specificity 100%
• mutations at tryptic site
• mutations not in tryptic peptides
• synonymous ∆ AA
• some isobaric mutations• For novel mutations
sensitivity 82%, specificity 99%
• not sequencing
AmyloidTyping - MS/MS Proteomics
Outline
•Definition & Types
•Nomenclature
•Methods of Typing• Indirect methods•Direct methods
•Antibody-based methods
•Proteomic methods
•Conclusions
AmyloidosisTake Home Points
• Increasingly recognized cause of heart disease
• Nomenclature standards set by ISA (2014)
• 31 recognized types, distinguishing is important
• AL & ATTR account for ~98% of cardiac amyloid• AL-type most common, but most likely to encounter
ATTR on biopsy material• Preferred typing method: direct (vs indirect)
• IF, MS, ImmunoEM
AmyloidosisAcknowledgements
• Paul J. Kurtin
• Ahmet Dogan
• Julie A. Vrana
• Jason D. Theis
• William D. Edwards
• Martha Grogan
• Angela Dispenzieri
• David L. Murray
• Mayo Clinic Amyloid Interest Group
Amyloidosis: Typing & Nomenclature
The Society for Cardiovascular PathologyCompanion Society MeetingUSCAP 2015 Annual Meeting
March 22, 2015
Joseph J. Maleszewski, MD, FCAP, FACCAssociate Professor of Pathology & Medicine
Divisions of Anatomic Pathology, Cardiovascular Diseases, & Molecular Genetics