Amyloidosis

Recent advances in AMYLOIDOSIS

1. AMYLOIDOSIS AND ITS RECENT ADVANCES Dr. SADAF KHAN

2. “Amyloid ”- starch like In 1854 Rudolph Virchow named it amyloid based of appearance of violet color after staining these proteins with iodine and sulfuric acid. AMYLOID

3. Disorder of protein folding Deposition of a pathologic acellular proteinaceous substance in extracellular space Organ tropism AMYLOIDOSIS

4. PROPERTIES Physical properties ( Electron microscopy) masses of extracellular, non-branched filaments random orientation. each fibril consists of two electron-dense filaments 2.5–3.5 nm in diameter, separated by a 2.5 nm space, giving a total diameter of 8–10 nm. X- ray crystallography- beta pleated sheet

5. Chemical properties Nomenclature : prefix “A” for amyloid, followed by an abbreviation derived from the name of the protein. PROTEIN GLYCOPROTEIN-P 85% 15%

6. MECHANISMS OF AMYLOIDOGENESIS

7. Recent evidence suggests “fibril deposition may not be necessary for cellular toxicity rather exposure to the precursor protein alone is sufficient for cytotoxicity” PATHOGENESIS

8. DIAGNOSIS

9. APPEARANCE STAIN APPEARANCE H and E Acellular, amorphous, eosinophilic, refractile , extracellular substance. CONGO RED On H&E – pink red On polarizing microscopy – apple green birefringence GOLD STANDARD THIOFLAVIN S Green

10. STAIN APPEARANCE ALCIAN BLUE Green amorphous CRYSTAL VIOLET Rose pink SIRIUS RED Pink red NOTE: Amyloid deposits do not stain with periodic acid Schiff (PAS) or silver stain, but spicules can be seen along the glomerular basement membranes on silver stain

11. MATERIALS frozen tissue is preferred for amyloid typing most sensitive -biopsy of a clinically affected organ Screening test- abdominal fat aspirate Other sites- *gastrointestinal tract: rectal or gastroduodenal- deep *labial salivary gland (LSG) Recently, a semiquantitative grading system ranging from 0 (negative) to 4+, for the amount of amyloid in fat tissue has been proposed by Hazenberg et al AMYLOID TYPING Establishing the type of amyloidosis has become a central issue as the current treatment of systemic amyloidosis directly depends on the molecular type of the amyloid protein

12. PRE- REQUISITES FOR CONGO RED strong light source rotating table darkened room for reading slides thicker sections (5–10 micro m) serial sections PITFALL : autofluorescence of collagen under polarization microscopy

13. IMMUNOHISTOCHEMISTRY Historically, potassium permanganate was used to distinguish AA from other forms of amyloid. Applying potassium permanganate to the tissue prevents Congo red from binding to AA fibrils but not AL. commercially available antisera directed against amyloid proteins PITFALLS- directed against constant region miss a number of hereditary amyloidosis s erum proteins in background ‘ overlay technique’’ Congo red stain and immunohistochemistry are performed on the same slide

14. LASER MICRODISSECTION laser microbe melts a thermoplastic ethyl vinyl acetate membrane that overlays the tissue sticks to the selected cells, which can then be lifted secured in a microfuge tube containing the appropriate extraction solution Gold standard PITFALL - Availability

15. FIBRIL TYPING BY PROTEOMICS Advances allow detection of a single amino acid change GENE SEQUENCING Hereditary amyloidosis For-transthyretin, fibrinogen alfa chain, Apo-A1, Apo-A2, Lysozyme, Gelsolin, serum amyloid A and Cystatin-C

16. CLASSIFICATION ORGAN DISTRIBUTION Systemic- > 1 organ involved Localised - only single organ ASSOCIATED CLINICAL FINDINGS Primary Secondary TUMOR ASSOCIATED MYELOMA ASSOCIATED HISTORICAL

17. Based on the chemical composition of the deposited amyloid protein and not neccesarily on the clinical phenotype . > 25 types RECENT CLASSIFICATION OFFICIAL ABBREVIATION AMYLOID TYPE/GENE DESCRIPTION AL Amyloid light chain ( λ ) AL amyloidosis / Multiple myeloma AA Serum amyloid A protein AA amyloidosis A β β amyloid Alzheimer’s disease A β 2M β 2 amyloid microglobulin Hemodialysis related amyloid ALect2 LECT2 protein LECT2 amyloidosis ATTR Transthyretin Familial amyloid polyneuropathies, Wild type transthyretin amyloidosis, leptomeningeal amyloidosis AIAPP amylin Type II diabetes APrP prion protein CJD, BSE ACys CST3 Cerebral Amyloid Angiopathy AGel GSN Finnish type amyloidosis AApoA1¹/ Afib²/ Alys³ APOA1¹/ FGA²/LYZ³ Familial visceral amyloidosis APro prolactin Prolactinoma AKer keratoepithilin Familial corneal amyloidosis AANF Atrial natriuretic factor Senile amyloid of the heart ACal Calcitonin Medullary carcinoma of the thyroid

18. Types of protein component AL Made up of immunoglobulin light chains ( λ chains) secreted by plasma cells. Found in plasma cell tumors AA Made from proteolysis of SAA synthesized in liver, c irculates in association with HDL. Deposited in inflammatory states as a part of acute phase response - acute phase protein β amyloid protein Derived from Amyloid Precursor protein. Core of cerebral plaques found in Alzheimer’s. Also deposited in walls of cerebral blood vessels TTR Bound to thyroxin and retinol, helps in transporting them Wild TTR -Senile systemic amyloidosis Mutant TTR - Familial amyloidotic polyneuropathy β 2 microglobulin It’s a component of MHC class I A β 2 m, deposited in patients on hemodialysis Prions Misfolded proteins accumulate in CNS in prion disease

19. DISTRIBUTION BY AMYLOID TYPE

20. most aggressive -13 mnths kidney and heart are most commonly affected H istologically , AL deposits can be found in all three compartments of the kidney - glomerulus:minimal to massive - interstitium -the wall of blood vessels . Treatment of AL - advanced considerably - first effective treatment : melphalan and prednisone (MP ). - high dose melphalan followed by autologous STC -renal transplantation AL

21. OTHER AMYLOIDOSIS MISTAKEN FOR AL TYPE ORGAN INVOLVEMENT DIAGNOSTIC KEY AA Kidney, liver, spleen, gut Anti protein AA fixation in IHC ATTR Heart, nerve, carpal tunnel syndrome Anti TTR fixation in IHC Genetic test AFib Kidney Pure glomerular pattern of the deposits Genetic test ALys Kidney, GIT, skin Anti- lysozyme fixation in IHC Genetic test AApoA1 Kidney, heart, liver, skin, larynx, nerve Anti apo A1 fixtaion in IHC Genetic test

22. The fibrils in AA are derived from serum amyloid A (SAA) protein Secondary to AA Infection TB, bronchiectasis , chronic osteomyelitis Inflammatory conditions Rheumatoid arthritis, Ankylosing spondylitis, inflammatory bowel disease, Crohn’s and ulcerative colitis skin popping in heroin abusers non immunocyte derived tumors renal cell carcinoma Hodgkin’s lymphoma HEREDITARY form- periodic fever syndromes Familial Mediterranean Fever ( FMF) - most common -MEFV gene mutation on chromosome 16p . TNF-receptor-associated periodic fever syndrome TNFRSF1A gene mutation :regulates the type 1 tumor necrosis factor (TNF) receptor Muckle–Wells syndr ome -only cryopyrinopathy -CIAS1 gene mutation Hyper IgD syndrome - mevalonate kinase (MVK) gene mutation

23. Histologically , o ne feature that is particular but not unique to AA is the formation of crescents - most commonly in AA secondary to rheumatoid arthritis . O ften associated with rapidly progressive glomerulonephritis and rapid loss in renal function. Corticosteroids- effective at stabilizing and reversing the rapid loss in renal function Treatment - eliminating or controlling the underlying disease Reversibl e

24. AD inheritance most commonly- transthyretin gene Familial amyloidotic polyneuropathy Familial amyloidotic cardiomyopathy >130 mutations-Val30Met Microscopy amyloid deposition in endoneurium of nerve trunks , plexuses and sensory and autonomic ganglia destruction of the myelin sheath Treatment- liver transplantation FAMILIAL AMYLOIDOSIS

25. HEREDITARY RENAL AMYLOIDOSIS FEATURES AFib ALys Aapo AI and II Incidence Most common Heritance AD AD Mutations 6 4 13 and 4 Organs Kidney, liver, sleen , perpheral nerves and heart GIT, kidney Kidney, liver, skin, gonads Features Glomerular Sparing of extraglomerular compartments Liver rupture Bleeding Visual impairment Extraglomerular - medullary deposits Treatment OLT Renal transplant - Recurrence + + +

26. LECT2 Amyloidosis (ALECT2) N ative protein is leukocyte chemotactic factor 2 (LECT2) M ean age of presentation - 67 years Tandem mass spectrometry Kidney,liver , spleen, colon, and adrenal gland In the kidney, ALECT2 is highly congophilic and is deposited extensively in all compartments No treatment is currently available LATEST AMYLOIDOSIS

27. ORGAN WISE AMYLOID ANGIOPATHY CNS Localised form Familial hereditary amyloidosis Beta amyloid Features- leukoencephalopathy - microinfarcts -hemosiderin deposits

28. THYROID Medullary carcinoma Amyloid goitre Deposition in stroma, glandular and periglandular blood vessels CUTANEOUS Localised - macular -reticular -nodular Primary systemic first basement membranes of the blood vessels and adnexas followed by the replacement of the collagen of the dermal papilla .

29. CARDIAC restrictive cardiomyopathy congestive heart failure remains refractory to maximal medical therapy low-voltage changes on electrocardiogram associated with marked LV hypertrophy myocardial dysfunction in the setting of known plasma cell dyscrasia or connective tissue disorder. MICROSCOPY - interstitial and perimyocyte deposition -myocyte attenuation -nodular aggregates -walls of blood vessels: subendocardial

30. GIT Primary (AL)- blood vessel walls and muscularis propria Secondary(AA)- blood vessel wall and lamina propria lacks the surface epithelial damage and epithelial lymphocytosis

31. SPLEEN Two patterns Deposition in follicles Deposition in walls of sinuses (SAGO SPLEEN ) In red pulp - LARDACEOUS SPLEEN Later fusion of both areas gives the appearance of map like areas in the spleen

32. RENAL AMYLOIDOSIS Can involve- the glomeruli(earliest, commonest), - the blood vessels, -the interstitium , and -the tubular basement membranes

33. RENAL PATHOLOGY

34. Despite the similarity in the physical characteristics of the fibrils , amyloidosis is a tremendously heterogeneous group of diseases with differences ranging from amyloidogenesis to organ tropism. Amyloid typing is important as a number of novel approaches directly targeting the amyloid fibrils or the processes of amyloidogenesis have shown very promising early clinical or pre-clinical results. Given the complexity inherent in the diagnosis of amyloidosis, as well as the current need for accurate and early diagnosis, “loss of chance” doctrine might be employed in determining liability in some amyloidosis-related medical malpractice lawsuits CONCLUSION

35. Sternberg’s D iagnostic Surgical Pathology 16 th edition Bancroft’s Theory and Practice of Histological Techniques Robbins and Cotran Pathologic Bais of Disease 8 th edition Recent Advances in Transthyretin E volution, Structure and Biological functions-Samantha J. Richardson Protein Aggregation and F ibrillogenesis in Cerebral and Systemic A myloid Diseases-J. Robin Harris Amyloid journal REFERENCES