1. Amyloidosis Diagnosis and Classification in Native Kidney and Renal transplants Prof. Dr. B. Handan Özdemir Baskent University Ankara-Turkey

2. Definition Amyloidosis comprises a diverse group of systemic and local diseases Characterized by organ involvement by the extracellular deposition of fibrils composed of subunits of a variety of normal serum proteins

3. Amyloid fibril protein occurs in tissue deposits as rigid, non- branching fibrils 7-to 10 nm in dm When analysed by X-ray diffraction, the fibrils exhibit a characteristic cross Beta diffraction pattern Physical Nature

4. Chemical Nature  Pentagonal molecule  95% Protein Fibril  5% Glycoprotein P component

5. resorption Cofactors such as amyloid P component may have an important role in the tissue deposition and resorption Promoting fibrillogenesis Stabilization of the fibrils Binding to matrix proteins Affecting metabolism and proteolysis of formed fibrils Act by Husby G. Clin Immunol Immunopathol 1994:70:2 Genetic factors may be involved in predisposing to the development of fibrillogenesis and amyloidosis

6. What factors allow some proteins to aggregate into amyloid fibrils ? Patients with AA amyloidosis have levels of SAA protein no greater than those patients with inflammatory diseases who do not have amyloid Therefore some additional unknown stimulus is required for amyloid fibrils to form and precipate

7. Cast nephropathy versus Amyloidosis Certain light chains also may form high molecular weight aggregates in vitro In AL amyloidosis biochemical characteristics of the light chain is important in determining amyloid formation

8.   The established amyloid fibril nomenclature is based on the chemical nature of the fibril protein   Which is designated protein A and followed by a suffix that is an abbreviated form of the parent or precursor protein name   For example, when amyloid fibrils are derived from immunoglobulin light chains, the amyloid fibril is AL and the disease is AL amyloidosis Amyloid fibril protein nomenclature Amyloid, 2010; 17(3–4): 101–104

9.  At least 25 different precursor proteins are known  They are associated with variety of Inflammatory Immune Infectious Hereditary conditions Amyloid fibril protein nomenclature

11.  Typing of amyloid deposits is important because of the difference in their treatment strategies  Typing of the amyloid deposits can be performed with various techniques  The most definitive method used is IF or IHC staining of tissue using antibodies that are directed against known amyloid proteins TYPING OF RENAL AMYLOIDOSIS

12.  IHC typing of amyloidosis poses several problems and requires experience  Wide range of success in IHC amyloid typing, ranging from 38% to 87% was reported  IHC diagnosis of AA type is relatively reliable,  But there is a problem in the differentiation of AL and hereditary amyloidosis Kebbel A, Röcken C. Am J Surg Pathol. 2006;30:673 Amyloid Typing and Pitfalls

13. Commercial antibodies are raised against the constant regions of the Ig light chains A subset of AL, in which amyloid fibrils are derived from a truncated light chain “containing only variable regions” will be nonreactive with commercial antibodies Therefore, negative light chain staining does not rule out AL amyloidosis Therefore, negative light chain staining does not rule out AL amyloidosis Amyloid Typing and Pitfalls

14. The typical antibody panel should include The typical antibody panel should include Amyloid P component Amyloid P component Kappa & lambda Ig light chains Kappa & lambda Ig light chains Amyloid A protein Amyloid A protein Transthyretin Transthyretin Fibrinogen Fibrinogen Beta-2 microglobulin Beta-2 microglobulin This panel allowed definitive This panel allowed definitive typing of amyloid in 90% of kidney biopsies Immunopathology in Renal Amyloidosis

15. Systemic Amyloidosis Primary Amyloidosis Secondary Amyloidosis Classification of Amyloidosis Localized amyloidosis Senile cerebral Senile cardiac Type 2 diabetes

16. Primary Systemic Amyloidosis Disease name Type of amyloid Precursor M.Myeloma AL Light chains PrimaryAL Secondary Systemic Amyloidosis Disease name Type of amyloid Precursor Chronic inflammatory disease AAAAAAAASAA Hemodialysis associate Aβ2- micro globulin β2- micro globulin

17.  The most common type of amyloidosis depends on the population studied  In the USA and the Western World AL amyloidosis is the most prevelant, followed by AA amyloidosis  In Turkey AA amyloidosis with an underlying disease of FMF is the most common type  In developing countries AA amyloidosis is far more common than AL amyloidosis (Tbc !) Ozdemir AI. Am J Gastroenterol 1969; 51:311

18.  Derived from the immunoglobulin light chain   Can occur alone or in association with   M. Myeloma  Malignant lymphomas  Macroglobulinemia AL Amyloidosis Kyle RA et al N Engl J Med. 2006;354:1362

19.   Light chain deposition disease has a similar pathogenesis with AL amyloidosis   Primary difference is that   Deposited light chain fragments do not form fibrils and do not engender deposition of amyloid cofactors AL Amyloidosis

20. Wide spectrum of organ system involvement Wide spectrum of organ system involvement The kidneys are commonly affected The kidneys are commonly affected Proteinuria Proteinuria Edema and hypoalbuminemia Edema and hypoalbuminemia Mild renal dysfunction is frequent Mild renal dysfunction is frequent Rapidly progressing renal failure is rare Rapidly progressing renal failure is rare AL Amyloidosis

21. Waxy appearance of intradermal amyloid deposition around the eye Macroglossia showing teeth indentations Peri-orbital haemorrhage (raccoon or panda eyes) Gross lymphadenopathy

22.  The AA amyloid proteins result from a proteolytic cleavage SAA protein  SAA is an acute-phase reactant produced by liver  Sustained high concentration of SAA prerequisite for AA amyloidosis AA Amyloidosis Benditt EP et al. Ann N Y Acad Sci. 1982;389:183

23. Acquired and hereditary diseases can lead to AA amyloidosis AA Amyloidosis Chronic inflammatory diseases Rheumatoid arthritis Inflammatory bowel disease FMF Bronchiectasis, Tuberculosis

24. Kidney is the most frequently affected target organ Kidney is the most frequently affected target organ The primary clinical manifestation is proteinuria The primary clinical manifestation is proteinuria The underlying disease usually is longstanding The underlying disease usually is longstanding Active inflammation typically is present when Active inflammation typically is present when amyloidosis becomes evident AA Amyloidosis

25.  Caused by deposition of genetically variant proteins  Associated with mutations in the genes for  Transthyretin  Apolipoprotein AI,  Apolipoprotein AII  Lysozyme  Fibrinogen A. Hereditary Amyloidosis

26.  Typically they associated with polyneuropathy and cardiac involvement but can affect kidneys  Renal deposits may be clinically silent  Isolated glomerular involvement with no amyloid in the tubules, interstitium, or vessels has been found to be characteristic of fibrinogen A  Renal failure develops rapidly Hereditary Amyloidosis Blood. 1997;90:799–805

27.  Clinically evident renal involvement occurs mainly in AA and AL amyloidosis  The deposition of Abeta2 M occurs in patients on prolonged dialysis  But diagnosis on the kidney biopsy is unexpected  Eight precursor fibrils are particularly important for kidney RENAL AMYLOIDOSIS

29.  The incidence of amyloid in patients with nephrotic syndrome or proteinuria was found in 2% to 12% of native renal biopsies RENAL AMYLOIDOSIS Arch Pathol Lab Med. 2007;131:917–922 Amyloidosis without therapy usually progresses to endstage kidney disease Deposits may also regress N Engl J Med. 2003;349:583–596. Ozdemir BH. Transplant Proc. 2006;38:432–434.

30. Diagnosis of Amyloidosis Can be very difficultCan be very difficult No blood test can diagnose or exclude amyloidosisNo blood test can diagnose or exclude amyloidosis Usually relies on clinical suspicionUsually relies on clinical suspicion Possibility supported byPossibility supported by Underlying chronic inflammatory state – AAUnderlying chronic inflammatory state – AA Underlying plasma cell dyscrasia – ALUnderlying plasma cell dyscrasia – AL Family history - hereditaryFamily history - hereditary Evidence of renal dysfunctionEvidence of renal dysfunction

31.  In H&E stained sections, amyloid is recognized as amorphous hyaline and eosinophilic material  Weakly PAS positive Detection of Renal Amyloid

32. Detection of Amyloid Amyloid do not stain by silver staining Amyloid do not stain by silver staining Occasionally may stain with silver stains and Occasionally may stain with silver stains and show spicules (Jones silver)

33. Detection of Amyloid is the gold standard Congo red is the gold standard Slides must be examined under polarized light Apple-green birefringent deposits is diagnostic

34.   Caution is advised regarding ‘‘overinterpreting’’ collagen as amyloid   Because deposits of amyloid are frequently very focal and irregularly distributed in tissue sections   Multiple and thicker (5–10 m) sections may need to be examined Picken MM. Curr Opinion Nephrol Hypertens. 2007;16:196 Detection of Amyloid

35.   Other stains, or techniques   Fluorescence   Thioflavin S and T   Methyl violet   Sulphonated Alcian blue   They are less specific Curr Opinion Nephrol Hypertens. 2007;16:196 Detection of Amyloid Sen S. Arch Pathol Lab Med 2010: 134: 532

36. Characterized by randomly disposed, rigid, nonbranching, variably long, 7-to 10nm-dm fibrils Characterized by randomly disposed, rigid, nonbranching, variably long, 7-to 10nm-dm fibrils Ultrastructural immunogold labelling can depict the precursor protein in amyloif fibrils Ultrastructural immunogold labelling can depict the precursor protein in amyloif fibrils Electron Microscopy

37. Rare cases exhibit massive aggregates of amyloid fibrils in subendothelium Rare cases exhibit massive aggregates of amyloid fibrils in subendothelium They arranged in thightly packed, electron-dense structures and can be easily confused with They arranged in thightly packed, electron-dense structures and can be easily confused with MPGN MPGN Cryoglobulinemic glomerulopathy Cryoglobulinemic glomerulopathy These cases are usually associated with monoclonal kappa light cahins These cases are usually associated with monoclonal kappa light cahins Electron Microscopy

38. Enlarged kidneys Enlarged kidneys Pale, waxy appearing cut surfaces Pale, waxy appearing cut surfaces Increase in the weight of kidney Increase in the weight of kidney Gross Pathology of Renal Amyloidosis Weight of the kidney did not correlate with Renal function The site of renal amyloid deposition Inversely proportional to the amount of amyloid

39. Amyloid can be found in any of the renal compartment Amyloid can be found in any of the renal compartment The most common and the earliest site of amyloid deposition in the kidney is the glomeruli The most common and the earliest site of amyloid deposition in the kidney is the glomeruli Glomerular amyloid formations begins in the mesangium Glomerular amyloid formations begins in the mesangium Extends into capillary walls Extends into capillary walls Microscopy of Renal Amyloidosis

40. Amyloid deposition in glomeruli may occur Amyloid deposition in glomeruli may occur Segmental Segmental Diffuse mesangial Diffuse mesangial Nodular Nodular Pure basement membrane patterns Pure basement membrane patterns Microscopy of Renal Amyloidosis

41. Renal amyloidosis was divided into 6 classes Similar to the classification of SLE A proposed histopathologic classification Sen S. Arch Pathol Lab Med 2010: 134: 532

42. Early segmental deposits are small and confined to mesangium without creating nodularity Early segmental deposits are small and confined to mesangium without creating nodularity It is very easy to miss this early form It is very easy to miss this early form Microscopy of Renal Amyloidosis

43. In the diffuse form In the diffuse form The mesangium is uniformly expanded by deposit The mesangium is uniformly expanded by deposit Microscopy of Renal Amyloidosis

44. In the nodular form In the nodular form Mesangium is asymmetrically expanded by amyloid Mesangium is asymmetrically expanded by amyloid Distinguish from diabetic nephropathy Distinguish from diabetic nephropathy Other forms of nodular glomerulosclerosis Other forms of nodular glomerulosclerosis Microscopy of Renal Amyloidosis

45. Subepithelial amyloid deposition Subepithelial amyloid deposition Associated with spikes Can be seen at the periphery of mesangial areas Microscopy of Renal Amyloidosis

47. Rarely cresents can be seen Rarely cresents can be seen Highlighting the fact that capillary wall rupture has occured Microscopy of Renal Amyloidosis

48. Interstitial amyloid are seen in 50% cases Interstitial amyloid are seen in 50% cases Generally begins in areas adjacent to blood vessels Generally begins in areas adjacent to blood vessels Medullary amyloid deposits are more frequent Medullary amyloid deposits are more frequent Microscopy of Renal Amyloidosis

49. AA amyloidosis AA amyloidosis Certain mutants of transthyretin Certain mutants of transthyretin May show amyloid deposition limited to the interstitium and medulla May show amyloid deposition limited to the interstitium and medulla Such patients present with renal failure not associated with proteinuria Such patients present with renal failure not associated with proteinuria Microscopy of Renal Amyloidosis

50. Renal vessels are often involved Renal vessels are often involved Arteriolar deposits being most frequent Arteriolar deposits being most frequent Followed by deposits in arteries, PTCs and veins Followed by deposits in arteries, PTCs and veins Microscopy of Renal Amyloidosis

51. Therapies are not successful in patients diagnosed at advanced stages of amyloidosis Supportive therapy is essential There are two choices for the therapy Hemodialysis Transplantation RENAL AMYLOIDOSIS

52. The survival of patients begining dialysis is poor Mean survival is only 33 months Worse than patients with other renal diseases Hemodialysis in Renal Amyloidosis

53. The European experience showed a 76% survival at 2 years for all patients compared with 53% among amyloidosis patients Similarly in our center the 2-year survival was 50% among patients with amyloidosis The mean survival of our patients on hemodialysis was 33 months Hemodialysis in Renal Amyloidosis

54. Common complications of amyloidosis are Extrarenal progression of amyloidosis Infections Major causes of death Hemodialysis in Renal Amyloidosis

55. It has been reported that 21 of the 31 deaths in the group that underwent dialysis were due to extrarenal progression of amyloidosis, namely to cardiac amyloidosis Similarly, we have observed that infection and extrarenal progressive amyloid deposition was the cause of death in 15 of our 30 patients who died approximately 9 months after starting hemodialysis Hemodialysis in Renal Amyloidosis Ozdemir BH et al. Transplant Int 2004: 17: 241–246

56. Other Choice of Therapy is Transplantation

57. The outcomes of renal tx in patients with amyloidosis is still controversial Pasternack 3-year survival Patients with amyloidosis 51% Patients with glomerulonephritis 79% Transplantation in Renal Amyloidosis Pasternack A et al. Transplantation 1986; 42:598

58. Pras noted similar graft and patient survival rates among patients with amyloidosis and GN Similarly our 5-year survival rates for amyloidosis patients (78%) were equal to the survival rates of patients with GN Transplantation in Renal Amyloidosis Pras M et al. Adv Nephrol Necker Hosp 1984; 13:261 Ozdemir BH et al. Transplant Proc 2006: 38, 432 Ozdemir BH et al. Transplant Int 2004: 17: 241–246

59.  The rates of recurrent amyloidosis vary from center to center (13 to 50%)  This is mainly because only a small number of patients have been studied in single-center series Recurrence of Amyloidosis The rates of recurrence is dependent on the indication of graft biopsy Since early amyloidosis could exist without urinary abnormalities or impairment of renal function

60.  In our center we observed higher rates of amyloid recurrence than have been reported previously  Recurrent amyloidosis was diagnosed by renal allograft biopsy in 20 of our 30 cases  Of 20 grafts, 18 were from living and 2 were from cadaveric donors (P<0.01)  Four of the five patients (80%) with HLA-identical LRDs showed amyloid recurrence Recurrence of Amyloidosis Ozdemir BH et al. Transplant Int (2004) 17: 241–246

61.  Previous reports document amyloid recurrence in renal grafts at 6 months to 4 years after tx Recurrence of Amyloidosis Transplantation 1981; 32:6  In our patients, amyloidosis developed in the grafts 18 months to 10 years after tx  This is a very wide range, emphasising that amyloid recurrence can develop at any time after tx Arch Intern Med 1979; 139:1135 Ozdemir BH et al. Transplant Int (2004) 17: 241–246

62.  Recipients with LRD showed shorter times to recurrence (33.6±5.2 months) than for those with cadaveric grafts (78±24 months; P<0.01)  The overall 3-, 5- and 10-year graft survival rates for the recipients with amyloidosis recurrence were 77%, 70%, and 36%, respectively  The corresponding patient survival rates were 93%, 78%, and 35% Recurrence of Amyloidosis Ozdemir BH et al. Transplant Int (2004) 17: 241–246

63. Why so many patients showed such a high incidence of amyloidosis recurrence, even though they were taking colchicine ? Part of the reason for this may be poor Drug compliance Doubting the need for medication Preference for self-care measures other than medication Convenience, side effects and lack of demonstrated benefit

64. Amyloidosis patients maintained on chronic dialysis have high mortality rates Better survival was noted among renal transplant patients even with a recurrence These results encourage transplantation for renal end-stage disease due to amyloidosis CONCLUSION