Amyloidosis. Philip Hawkins National Amyloidosis Centre UCL & Royal Free Hospital, London

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1 Amyloidosis Philip Hawkins National Amyloidosis Centre UCL & Royal Free Hospital, London

2 Amyloid Abnormal extracellular fibrillar protein deposit in tissues Pathognomonic red-green birefringence after Congo red staining Different amyloid fibril proteins provide basis for classification and nomenclature

3 Amyloidosis Disease caused by amyloid deposits: local or systemic Systemic amyloidosis is usually fatal Causes about 1 per 1000 deaths Diagnosis & treatment are challenging Significant recent advances Still a major unmet medical need

4 Amyloid deposits Amyloid fibrils GAGs - heparan/dermatan sulphate Serum amyloid P component (SAP)

5 Amyloid fibril formation in vivo Sustained high concentration of normal protein: SAA AA amyloid (mainly renal) Prolonged presence of normal protein at normal concentration: TTR ATTR amyloid (cardiac) Acquired production of abnormal protein: cloncal Ig light chain AL amyloid (almost any organ) Hereditary production of variant protein: TTR, fibrinogen, apoai, lysozyme, gelsolin, etc Misfolding and highly ordered aggregation with characteristic amyloid cross-β core structure

6 Types of amyloidosis in UK Unclear 2% Hereditary 12% Localised 10% AA 15% Senile TTR 2% AL 59% >700 new patients per year currently seen at NAC

7 Monoclonal immunoglobulin / paraprotein Clonal plasma cell Bone marrow AL amyloidosis a tale of two diseases Light chains (antibody fragments) may form amyloid Kappa Heavy Chain Amyloid fibrils Light Chain Lambda

8 AL amyloidosis Protean clinical features

9 ATTR amyloidosis Transthyretin (TTR) Normal plasma protein, transports thyroxine and retinol binding protein Inherently amyloidogenic wild-type senile cardiac amyloidosis age yr. All by age 110 yr. >100 amyloidogenic TTR mutations cause familial amyloid polyneuropathy ~10,000 cases worldwide Val122Ile polymorphism in ~4% of black individuals; greatly increases susceptibility to senile cardiac amyloidosis

10 Diagnosing amyloid (NB: 10% false positive and false negative histology) Biopsy of affected organ Screening biopsy Abdominal fat needle aspirate variable sensitivity, 25-70% Rectal/gut biopsy more invasive, more sensitive. Provides better sample for immunohistochemical typing Van Gameren, Arth&Rheum 2006;54:2015 Ansari-Lari, Diagn Cytopathol 2004;30:178

11 123 I-labelled SAP scintigraphy AL AA AFib AGel

12 SAP scintigraphy using SPECT-CT

13 Complex contraction of LV - - Longitudinal Contraction Radial Contraction - Long Axis Deformation - Twisting Apex relatively fixed

14 Echocardiography in amyloidosis A thick walled heart, but how much is amyloid? Strain Tracks the longitudinal movement of the heart muscle

15 HCM HT Amyloid mucopolysaccharidosis Seward et al JACC 2010

16 Cardiac MRI: Diffuse Late Gadolinium Enhancement in a patient with senile cardiac amyloidosis

17 Cardiac MRI in amyloidosis CMR provides more accurate measurements of volume, mass and wall thickness than echocardiography Enables myocardial tissue characterisation Characteristic patterns of late gadolinium enhancement (LGE) T1 mapping through the measurement of the native (non-contrast) myocardial T1, post-contrast T1 and extracellular volume (ECV) T1 (longitudinal relaxation time) markedly elevated in the presence of cardiac amyloid

18 EELGE in Q-CMR Equilibrium contrast MRI to measure ECV Gd Gd Gd Gd Vd (b) Gd Gd Vd (m) Gd Gd in blood Eequal

19 ECV elevated in disease: fibrosis and amyloid Sado DM, Heart 2012

20 99m Tc-DPD scintigraphy in cardiac ATTR amyloid Negative Positive Fused SPECT/CT transaxial image

21 Serial DPD imaging ATTR progression y.o female TTR G47V mutation FH of cardiac amyloid Completely well in 2010 Normal echo (8mm) and CMR Normal echo (10mm) Normal Gd kinetics BUT EQ-CMR = cardiac amyloid Anterior S e p t a l L a t e r a l Inferior

22 Identification of amyloid fibril type - why is it necessary? Untreated systemic amyloidosis is usually progressive and fatal General treatment principals in amyloidosis Support or replace compromised organ function Reduce supply of the fibril precursor protein

23 Determining the amyloid fibril type Immunohistochemistry Standard method in clinical practice AA: sensitive and specific Hereditary: mostly diagnostic AL - false negatives in ~30% Mass Spectrometry Important development. Tiny amounts of tissue required

24 Clonal markers detectable in almost all patients with AL amyloidosis Urine BJP - 61% % patients IF - 17% 52% IF-21% 40% 79% Serum PP on SPEP/IF or Serum FLC - 69% Abnormal FLC ratio - 90% 0 Serum Urine FLC ~99% if all tests done Essential to request serum and urine immunofixation and serum free light chain assay

25 SAP scintigraphy D

26 Diagnostic pitfalls Presence of plasma cell dyscrasia and amyloid does not prove AL type 10-15% of older population have a plasma cell dyscrasia using sensitive techniques required in AL amyloidosis Immunohistochemistry has limitations 30% AL cases cannot be confirmed definitively Variable penetrance in hereditary amyloidosis there is often no family history

27 Amyloid proteomics at NAC Cut out tiny fragments of amyloid from biopsies with Laser dissection microscope Liquid chromatography and mass spectrometry to determine precise molecular size of protein, which reveals the identity of amyloid protein

28 Fibril protein and amyloid signature Mascot data

29 Organ involvement and staging in amyloidosis History autonomic symptoms, bowel disturbance Clinical examination macroglossia, neuropathy etc ECG Echocardiogram Cardiac MRI scan Cardiac biomarkers NTpro-BNP and troponin 6 minute walk test Urine protein, GFR Liver function tests Postural BP (at 5 minutes)

30 Cardiac biomarkers for staging AL amyloidosis Both Normal NT-proBNP <39 pmol/l Troponin-T <0.03 ng/l Either NT-proBNP or Trop-T abnormal Both abnormal Dispenzieri A et al, J Clin Oncol 2004;22:3751-7

31 Heart failure with echocardiogram and/or cardiac magnetic resonance imaging (CMR) suggesting/indicating cardiac amyloid Bone scintigraphy with DPD/HMDP/PYP Grade 0 Grade 1 Grade 2 to 3 Serum immunofixation + Urine Immunfixation + serum free light chain assay No Yes Yes No Yes No Cardiac Amyloidosis unlikely Need specialized assessment for Diagnosis : Histological confirmation and typing of amyloid Cardiac ATTR amyloidosis TTR genotyping Cardiac AL amyloidosis Variant ATTR Wild-Type TTR

32 Treatment strategies in amyloidosis

33 Regression of amyloid AA AL Hawkins et al. Arthritis Rheum 1993;36: Hawkins et al. N Engl J Med 1990;323:

34 Survival in AA amyloidosis in relation to response of SAA Complete remission (n=42) Probability of survival (%) Persistent inflammation (n=38) 20 P < Months

35 FLC response as a predictor of cardiac outcome in AL amyloidosis and change in cardiac function Cardiac Outcomes Progression Unchanged Response dflc response to 3 cycles NR 9 (69%) 2 (15%) 2 (15%) Fisher s Exact test P=0.16 PR 5 (25%) 7 (35%) 8 (40%) CR/VGPR 5 (20%) 7 (28%) 13 (52%) Fisher s Exact test P=0.04 Gillmore et al, NAC 2012,

36 Increasing choice of chemotherapy regimen for AL amyloidosis Options Oral Melphalan-Dex CycloThalDex Velcade based regimens CycloVelDex Lenalidomide containing regimens High dose melphalan with autologous stem cell rescue (Anti-CD38 and new IMiDs & proteasome inhibitors in trial) Factors to consider Speed of clonal response Depth of clonal response Duration of clonal response Anticipated side effects Anticipated treatment related mortality Response to previous treatments

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38 Regression of hereditary ApoAI amyloid after liver transplantation Before transplantation 1 year post transplantation 4 years post transplantation

39 Prospects for treatment of ATTR amyloidosis Liver transplantation for patients with early symptoms associated with TTR Met30. Amyloidogenic property of wildtype TTR limits this approach in patients with cardiac involvement (most cases!) TTR stabilizing drugs shows some promise, though limited clinical efficacy demonstrated to date Reducing product of TTR RNAi and ASO therapies in Phase III. Reduction of TTR synthesis by >80%.

40 CPHPC O O OH N N HO O O

41 Depletion of SAP with CPHPC CPHPC developed to remove SAP from amyloid but, despite almost total plasma depletion, does not clear all SAP from amyloid deposits Pepys et al. (2002). Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis. Nature 417:

42 Clearance of mouse AA amyloid deposits by anti- SAP antibody following depletion of plasma SAP Control antibody Anti-SAP antibody Bodin K et al, Nature 2010;468:93-7

43 Original Article Therapeutic Clearance of Amyloid by Antibodies to Serum Amyloid P Component Duncan B. Richards, D.M., Louise M. Cookson, B.Sc., Alienor C. Berges, Pharm.D., Sharon V. Barton, M.Sc., Thirusha Lane, R.N., M.Sc., James M. Ritter, D.Phil., F.Med.Sci., Marianna Fontana, M.D., James C. Moon, M.D., Massimo Pinzani, M.D., Ph.D., Julian D. Gillmore, M.D., Ph.D., Philip N. Hawkins, Ph.D., F.Med.Sci., and Mark B. Pepys, Ph.D., F.R.S. N Engl J Med Volume 373(12): September 17, 2015

44 Anti-SAP antibody following depletion of circulating SAP with CPHPC SAP scintigraphy AL amyloidosis: Pt with large liver amyloid load. Complement depletion & acute phase response day 0 day 42 Liver ECV EQ-MRI (med normal 29%) 36% 29% Liver Stiffness (median normal 5.3 kpa) 5.7 kpa 2.8 kpa

45 Acknowledgments Patients and NHS Colleagues and collaborators Medical Research Council The Wolfson Foundation The Wellcome Trust UCL Amyloidosis Research Fund