recent advances in amyloidosis

Amyloidosis from basics to recent advances

Moderator- Dr Chaithra Presenter – Dr Dhanya A N

Contents • Introduction • Definition • Structure • Classification • Pathogenesis • Diagnosis• Morphology

– Gross – Microscopy

• Recent advances • Summary

Introduction

• The name amyloid -Rudolph Virchow in 19th century.

• Amyloid means “starch like” because it stained violet with iodine and sulphuric acid and thus attributed this to its cellulose or starch like nature.

Definition • It is a condition associated

with a number of inherited and inflammatory disorders in which extracellular deposits of fibrillar proteins are responsible for tissue damage and functional compromise.

• Produced by aggregation of misfolded proteins

Structure of amyloid protein

• Fibers are long,

• Straight or moderately curved

• Generally unbranched.

• Fibrils can appear as wavy filaments, rod shaped, with a diameter ranging from 5 to 25 nm.

• Amyloid fiber are formed by fibrillar subunits - “protofilaments”.

• In mature fiber, protofilaments can vary in number (2-6) and can twist one another forming an hollow fibril core.

• Mature fibers which forms amyloid are characterized by cross-beta sheet conformation

• Fibre showing four fibrils (there can be as many as six in each fibre) wound around one another with regularly spaced binding of the Congo red dye

Electron microscopic picture

Elongated, non branching, fibrillar filaments

Chemical nature of amyloid

• 95 % of amyloid consists of - fibril Proteins• 5 % of amyloid consists of - proteoglycans and

glycosaminoglycans – Heparin sulfate– Dermatan sulfate – Plasma protiens- serum amyloid P component (SAP)

Types of amyloidosis

3 major types 1. AL ( amyloid light chain )• Made up of immunoglobulin light chain • Most of AL protein composed of λ light chain

and occasionally қ chain • Secreted by a monoclonal population of

plasma cells, associated with plasma cell tumors

2. AA ( amyloid associated ) • Derived from non Ig protein, synthesized by the

liver • Are formed by proteolysis of larger precursor

serum amyloid associated (SAA), synthesized by liver

• SAA production increases in inflammatory conditions in response to stimuli from various proinflammatory cytokines, such as interleukin IL-1, IL-6, and tumor necrosis factor (TNF)

3. β- amyloid protein (Aβ)• Constitute core of the cerebral plaques found

in Alzheimer disease• Derived from proteolysis of larger trans

membrane glycoprotein, amyloid precursor protein (APP), due to genetic mutation of APP gene, on chromosome 21

Other types

1. Amyloid derived from Transthyretin (ATTR)

• Normal serum protein which transport thyroxine

and retinol.

• TTR gene on chromosome 18

• Mutated TTR (ATTR) deposited in familial amyloid

polyneuropathy, senile cardiac amyloidosis

2. Amyloid derived from β2 microglobulin (Aβ2 )

• Component of MHC class 1 molecule and a normal serum protein

• Seen in long term hemodialysis patients • It is not effectively filtered during

hemodialysis and hence its levels are more in these patients

3. Amyloid of prion protein(Aprp)• It is derived from precursor prion protein• PrP gene on chromosome 20• Mutated prion proteins are proteinacious infectious

particles lacking in RNA and DNA.• In prion diseases misfolded prion proteins aggregate

in the extracellular space and acquire the structural and staining characteristic of amyloid protein.

4. Amyloid from hormone precursor protein

• It includes amyloid derived from

– Calcitonin(Acal),

– Insulin(Ains),

– Prolactin(Apro),

– Lactoferrin(Alac)

Classification of amyloidosis Clinicopathological category

Associated diseases Major fibril protein Precursor protein

Systemic amyloidosis

Immune dyscrasias with amyloidosis /Primary amyloidosis

Multiple myeloma and other plasma cell proliferation

AL Ig light chain , mostly λ type

Reactive systemic amyloidosis/ secondary amyloidosis

Chronic inflammatory conditions

AA SAA

Hemodialysis associated amyloidosis

Chronic renal failure Aβ2 m β2 microglobulin

Classification of amyloidosis Clinicopathological category

Associated diseases Major fibril protein Precursor protein

Hereditary amyloidosis

Familial Mediterranean fever

AA SAA

Familial amyloidotic neuropathies

ATTR transthyretin

Systemic senile amyloidosis

ATTR transthyretin

Classification of amyloidosis Clinicopathological category

Associated diseases Major fibril protein Precursor protein

Localized amyloidosis

Senile cerebral Alzheimers disease Aβ APP

Endocrine - Medullary ca of

thyroid- Islets of

langerhans

Type 2 DMAcal

AIAPP

Calcitonin

Islet amyloid poly peptide

Isolated atrial amyloidosis

AANF Atrial natriuretic factor

Pathogenesis

• Abnormal folding of proteins, which becomes insoluble, aggregate and deposit as fibrils in the extracellular tissue

• Normally, misfolded proteins are removed by proteasomes intracellularly and by macrophages extracellularly

• Any of these mechanism fail, amyloid fibril deposition occurs

• The proteins that form amyloid

– Normal proteins – tendency to form improper folding, and they do so when they are produced in large amount .

–Mutated proteins - prone to misfolding and aggregate to form fibrils

Primary amyloidosis (AL type)

• Associate with plasma cell disorder • Systemic distribution • Clonal proliferation of malignant plasma cell Ig proteins(unpaired free қ , λ light chain which has amyloidogenic potential) amyloidosis ( in the tissues)

Reactive systemic amyloidosis (AA type)

• Associated with – Tuberculosis, Bronchiectasis – Chronic osteomyelitis – Rheumatoid arthritis– Connective tissue disorders like ankylosisng

spondilitis – Inflammatory bowel diseases – Heroin abusers – Solid tumors like RCC and hodgkins lymphoma

Reactive systemic amyloidosis (AA type)

Chronic inflammatory conditions IL-1, IL-6, TNF SAA produced by liver normally Monocyte derived enzyme defect or Enzymes genetically abnormal SAA which resist degradationSoluble end product insoluble AA molecule

Pathogenesis for Aβ type of amyloidosis

Familial Mediterranean fever • Autosomal recessive, an systemic autoinflammatory

disorder• Activation of antigen-independent inflammatory

mechanisms, involving mediators and cells of the innate immune system due to genetic mutation of MEFV gene located on chromosome 16

• Mutated MEFV leads to the over secretion of inflammatory cytokines

• As a result SAA will be produced and AA fibrils are deposited in the tissues

Familial amyloidotic polyneuropathy

• Autosomal dominant • Amyloid fibrils are made up of mutated TTR• TTR gene is located on chromosome 18 • More than 100 mutations in the TTR protein

have been reported ,the mutations causes a single nucleotide substitution in the codon of TTR gene

Conti..

• Transthyretin (TTR) exists as a tetrameric plasma transport protein.

• The tetrameric structure has surface receptors for retinol-binding protein as well as binding sites for thyroxine

• The misfolding of the protein, cytotoxic and leads to inappropriate aggregation and accumulation in a variety of organ systems

Conti..

• These TTR produced in the– liver– retinal pigment epithelium of the eye– choroid plexus of the brain– neurons – peripheral nerve Schwann cells

Conti..• Nerve biopsies (sural nerve) reveal amyloid

deposits in the endoneurial and epineurial connective tissue, along with deposits in the endoneurial and epineurial blood vessel walls.

HAEMODIALYSIS ASSOCIATED AMYLOIDOSIS

• Seen in patients on long term hemodialysis for more than 10 years

• These patients have high concentration of β2 microglobulin in the serum because it cannot be filtered through dialysis membrane and gets accumulated in the synovium, joints or tendon sheath.

• Patient often presents with carpel tunnel syndrome because of amyloid deposition.

Localized Amyloidosis

a)Senile Cardiac(ATTR), isolated artial

amyloidosis (AANF)

b)Senile Cerebral(Aβ,APrP)

c)Endocrine Amyloidosis

d)Localised Tumour forming amyloid(AL)

Senile cardiac amyloidosis

• Individual > 70 yrs • Mutated TTR (ATTR)• In isolated cardiac amyloidosis – atrial

natriuretic peptides are deposited • Causes restrictive cardiomyopathy

Senile Cerebral(Aβ,APrP)

• Spongiform Encephalopathies (APrP Amyloid )

-Kuru -Creutzfeldt-Jakob disease (CJD), -Gerstmann-Straussler-Sheinker disease (GSS), • Down’s syndrome(Aβ)• Alzheimer’s Disease(Aβ)

Endocrine Amyloid(hormoneprecursors)

• Microscopic deposits of localised amyloids incertain endocrine lesions –

- Medullary carcinoma of thyroid -calcitonin - Islet tumors of pancreas -Islet amyloid polypeptide or Amylin) - Type 2 diabetes mellitus -Proinsulin(Ains) - Pitutary amyloid -Prolactin(Apro)

Localized Tumor forming amyloid(AL)

• Isolated tumor like formation of amyloid(AL) deposits. Seen as nodular masses in-– lungs,– larynx,– skin,– urinary bladder,– tongue, eye

Clinical features

Renal involvement • Proteinuria • Renal failure and uremia Associated with multiple myeloma• Serum and urine bence jones protein • Bony lytic lesions• Hypercalcemia

Conti..

Cardiac involvement • Features of congestive heart failure • Conduction defects like arrhythmias • Restrictive cardiomyopathy Gastrointestinal tract • Tongue- macroglossia • Stomach, duodenum – malabsorption • Diarrhea • Bleeding

conti..

Familial mediterranean fever • Repeated attacks of fever lasting for 1-3 days

and resolve spontaneously Familial amyloidotic polyneuropathy • Numbness, Paresthesia, and pain • Compression of peripheral nerves, especially

the median nerve within the carpal tunnel,• Localized sensory changes

Conti..

Skin manifestations • Periorbital Purpura Bleeding diathesis • Mechanisms include factor X deficiency due to

binding to amyloid fibrils primarily in the liver and spleen

• Decreased synthesis of coagulation factors in advanced liver disease;

• Amyloid infiltration into blood vessels.

Diagnostic approach • Clinical features laboratory findings • Lab findings – Monoclonal protein study in serum by electrophoresis – shows

M band – Bence jones proteins estimation – Creatinine clearance – reduced – Bony lytic lesion – x-ray – ECG shows arrhythmias, low voltage wave seen – Serum troponin T – increased – Serum brain natriuretic peptide – increased – Liver enzymes – AST, ALT, ALK elevated

Serum electrophoresis

Arrow indicates M band in gamma region , immunoglobulin light chain lambda type

Tissue biopsy

• Any of the following tissue, – Abdominal fat pad aspirate – Rectal biopsy – Bone marrow biopsy – Gingival biopsy - not routinely performed ,

uncomfortable to patient • Specific organs biopsy

Abdominal fat aspiration

Abdominal fat pad aspiration ,congo red staining, apple green birefringence

Staining characteristics Specific stains 1)Congo red Red-pink stain that shows typical green birefringence in polarized light.

2)Methyl violet/ crystal violet Metachromatic -rose pink

3)Thioflavin Uv light - Fluorescence

Non specific stains a) Toluidine blue- Orthochromatic pale blueb) Alcian Blue Blue Greenc) PAS Pink

• H and E stain showing dense amorphous eosinophilic deposition in the glomeruli

• Congo Red positivity in diagnosing amyloid deposition

• When stained with Congo Red, the sections show a typical apple-green birefringence under polarized light

• Crystal violet stains amyloid deposits pink color, that are present in both glomeruli and vessels

• Glomeruli and vessels appear diffusely positive when stained with fluorescent Thioflavin T

H and E stain showing pink amorphousdeposit (arrow) around the distortedmyocytes

Toluidine blue stain shows pale purple-blue staining around the distortedmyocytes

H and E of cardiac muscle showing amorphous pink deposit

Alcian blue staining of same section showing greenish blue color of the deposit

• Amyloid appears as an amorphic, eosinophilic, PAS negative or scantly positive, extracellular substance.

Determining the type of amyloid

Immunohistochemical –• A panel of antibodies against four major

amyloid fibril proteins• Includes serum AA protein, kappa light chain,

lambda light chain and transthyretin (TTR), antibody to amyloid P (AP) component also added to the panel of antibodies

Direct Immunofluorescence microscopy

• show deposition of a monoclonal (lambda or kappa) light chain in AL amyloidosis.

• Frozen sections were cut at 3- m 𝛍• Direct IF staining was performed using

fluorescein isothiocyanate conjugated primary polyclonal rabbit anti-human antibodies to lambda and kappa light chain

• Fluorescent microscopy is used, the antigen – antibody complex absorbs UV light and emits visible light.

• The intensity of staining was graded on a scale from 0 to 3 (1+, mild staining; 2+ , moderate staining; 3+, to strong staining; +/- indicated trace staining)

IF for kappa light chain IF for lambda light chain(1 + ) ( 3 + )

Indirect Immunoperoxidase

• Staining for AA type• Sections were deparaffinized and rehydrated• Sections were stained with primary polyclonal

rabbit antihuman amyloid A antibody• Followed by goat anti-rabbit secondary

antibody• counterstained with hematoxylin

Indirect Immunoperoxidase positive congo red showing apple green birefringencefor AA protein in glomeruli of kidney on polarized microscope of same section

Morphology

Kidney• Can have both primary (AL) and secondary (AA)

types of deposits • Gross – normal or enlarged in the early stage

shrunken in late stage- due to ischemia and narrowing of blood vessels

• microscopy – amyloid deposits seen– Glomeruli – Interstitial peritubular region– Arteries and arterioles

H and E

Congo red

Heart

Gross • Firm, rubbery • Chambers are normal or dilated • Wall thickened • Nodule resembling drips of wax in endocardium Microscopy • On H and E amorphous pink hyaline material

seen around the myocytes

H and E Congo red

Spleen (sago spleen, lardaceous spleen)

Always an expression of systemic amyloidosis Gross • Mild to moderate splenomegaly • Cut section, pale wax like deposition, resemble

sago grains Microscopically • Deposits involve walls of splenic sinuses,

small arteries, connective tissue.

H and E Congo red

Liver

Seen in systemic amyloidosis Gross • Marked hepatomegaly • Cut section –pale waxy appearance Microscopy - • Deposition first at space of disse• Encroaches adjacent liver parenchyma• Around the blood vessels and sinusoids

Congo red

H and E

Brian

Commonly associate with alzheimers diseaseGross • Variable degree of cortical atrophy • Widening of cerebral sulci • Enlarged ventricles secondary to atrophy Microscopically• Plaque are focal, arranged around the central

amyloid core

Gastrointestinal tract

• Common sites are the second part of the duodenum, the stomach and colorectum and the esophagus

• Deposition seen in muscularis mucosae, submucosa, and muscularis propria leads to polypoid protrusions and thickening of the valvulae conniventes l/t obstruction

H and E of duodenum showing pink amorphous deposition in lamina propria

Congo red stain showing amyloid in lamina propria and around blood vessels

Medullary carcinoma thyroid

H and E stain, pink amorphous depositbetween the tumor cells of medullary carcinoma thyroid

Apple green birefringence for congo red stain seen under polarized microscope

Islets of pancreas

H and e stain, pink amorphous deposit around the islets of pancreas

Congo res stain, red deposit around the islets of pancreas

Bone marrow biopsy in multiple myeloma

Bone marrow biopsy stained with PAS (left) shows pink deposition around the sinusoidsAnd also shows malignant plasma cells > 30 % and few multinucleated giant cells Same section stained with congo red (right) shows red color around the sinusoids

Conti..

Strong positive staining with λ light chain immunoglobulin antibody (immunoperoxidase with hematoxylin counterstain

Recent advances in amyloidosis

Laser microdissection with mass spectrometry

• Tissue is fixed in formalin and processed in routine method and paraffin blocks are prepared

• Sections of 10 thickness are prepared and stained

• With the help of software, area is selected for microdissection

• Microdissected fragments were digested into tryptic peptides and analyzed by liquid chromatography Mass Spectrometry

Laser microdissection

Immuno-gold electron microscopy

• This is particularly useful for AL amyloidosis• Its one of the staining technique used in electron

microscopy • The high electron density of the gold particles are

used to identify the target molecule • These will increase the electron scatter to give

high contrast dark spots • Different sized gold probes are available ranging

from 1-40 nm

SAP scintigraphy (SAP scan)

• In 1987 Sir Mark Pepys invented• Professor Philip Hawkins developed it for routine

clinical use.• This technique shows the distribution and amount

of amyloid in the organs throughout the body without the need for biopsies

• The basis for the test is injection of a small amount of radio-labelled SAP which binds on amyloid deposits throughout the body.

• The deposited radio labeled SAP transmits a radioactive signal.

• This signal is picked up by a detector, gamma camera. All body parts where radioactive signal is detected will contain amyloid deposits

SAP scan showing amyloid deposit strongly in liver and sleep (left) and Same person SAP scan (right) showing reduced amyloidosis after treatment

SAP scintigraphy (SAP scan)

Genetic testing

• Mutations have been identified in all of the hereditary amyloidosis, such as – TTR gene on chromosome 18– APP gene on chromosome 21– PrP gene on chromosome 20– Apo A-1 gene on chromosome 11– Apo A II on chromosome 1– Fibrinigen A alpha on chromosome 4– Gelsolin gene on chromosome 9 – Cystatin C gene on chromosome 20

Conti..

• Patient’s peripheral blood collected

• Genes that are routinely sequenced in the laboratory by PCR

• Determine the mutated genes

Diagnostic criteria for AL type

• Mayo Clinic and the International Myeloma Working Group Presence of all of the following four criteria

1. Presence of an amyloid-related systemic syndrome.

2. Positive amyloid staining by Congo red in any tissue or the presence of amyloid fibrils on electron microscopy

Conti..

3. Evidence that the amyloid is light chain-

related established by direct examination of the

amyloid using spectrometry-based proteomic

analysis or immunoelectron microscopy.

Conti..

4. Evidence of a monoclonal plasma cell

proliferative disorder (eg, presence of a serum or

urine M protein, abnormal serum free light

chain, or clonal plasma cells in the bone

marrow)

Diagnostic algorithm

clinical features

lab findings

biopsy ( abdominal fat)

congo red stain

Conti..

congo red

negative positive

specific organ biopsy

positive typing of amyloidosis

Conti..

typing of amyloidosis

immnohistochemistry immunoelectron microscopy immunofloroscenc laser microdissection and mass spectrometry SAP scan genetic tests

Prognosis

• Serious disease with high mortality • Overall median survival rate after diagnosis is < 2

years in most of the cases • Patient with coexistent multiple myeloma has

poor prognosis • Survival time depends on the type of

predominantly involved organ • Cardiac involvement is the major determinant of

survival prognosis – major cause of death

Summary

• Rare, uncommon disease with poor long term survival.

• Associate with hereditary and inflammatory conditions.

• Symptoms are vague and its necessary to diagnose and to type the amyloidosis.

• Diagnosis is evolved with many recent advance techniques.

References

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7. Sachchithanantham S, Wechalekar AD. Imaging in systemic amyloidosis. Br Med Bull. 2013;107:41-56. 8. Kebbel A, Röcken C. Immunohistochemical classification of amyloid deposits in surgical pathology. Am J Surg Pathol. 2006 Jun;30(6):673-83

9. Satoskar AA, Burdge K, Cowden DJ, Nadasdy GM, Hebert LA, Nadasdy T. Typing of amyloidosis in renal biopsies: diagnostic pitfalls. Arch Pathol Lab Med. 2007 Jun;131(6):917-22.10. Hinton DR, Polk RK, Linse KD, Weiss MH, Kovacs K, Garner JA. Characterization of spherical amyloid protein from a prolactin producing pituitary adenoma. Acta Neuropathol. 1997 Jan;93(1):43-9

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