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