TTR-FAP: Diagnosis and treatment Zürich June 19,2014. Ole B Suhr Umeå University and University Hospital Department of Medicine Umeå Sweden

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1 TTR-FAP: Diagnosis and treatment Zürich June 19,2014 Ole B Suhr Umeå University and University Hospital Department of Medicine Umeå Sweden

2 Diagnosis of ATTR amyloidosis Clinical symptoms of ATTR- amyloidosis Histopathological diagnosis Genetic testing

3 Clinical symptoms of hereditary ATTR Amyloidosis Extremely variable between and within different mutation Difficult clinically to distinguish between different types of amyloidosis (wild type ATTR, AL, etc.) Multiple organ system involvement Somatic and autonomic nervous system Heart (arrhythmia, hypertrophic restrictive cardiomyopathy) GI tract, kidney-urinary tract, eyes. Rarely CNS (Leptomeningeal). Often no family history

4 Case. Woman of African descend. No family history of amyloidosis Breast cancer operated Recurrence 2006, reoperated and treated. Heart enlargement noted. 2007, Coughing. No abnormalities detected. Heart enlargement is noted Severe diarrhoea/constipation, nausea and vomiting. Motility disturbances. Percutaneous endoscopic gastro-enterostomy (PEG). Develops faecal and urinary incontinence. Pseudoobstruction?

5 Case (Cont.-2) Onset of disabling pain in feet, later legs and hands-arms, rapidly progressing and not controlled by morphine. 2011, Oct. 5. Cardiac arrest. Resuscitated by husband. Evaluation shows hypertrophic heart. Arrhythmia probably caused by ventricular tachycardia. ICD-pacemaker treatment. Heart and fat biopsy: Transthyretin amyloid probably of hereditary type (P.W.). Transthyretin mutation Ala45Asp 5

6 Case (Cont - 3) Pronounced orthostatic hypotension (fainting) Morning plasma cortisol 210 nm (>400), adrenal insufficiency. Echocardiography: Pronounced hypertrophic heart; decreased heart func:on. DPD- scin:graphy: Intense uptake in myocardium. Not suitable for transplantation or Vyndaqel/diflunisal treatment because of a too advanced disease. Dies at the age of 42 after new heart arrest July

7 Muta7ons in Sweden! Note Wt- ATTR (Senile systemic amyloidosis most common form of ATTR amyloidosis. Predominantly cardiomyopathy TTR variant Coding region Phenotype Geographic focus (ethnic) origin Gly6Ser Exon 2 Polymorphism Universal Val30Met Exon 2 Polyneuropathy, gastrointestinal disturbances, eye deposits, cardiomyopathy, kidney complications. Val30Leu (GTG>TTG) Exon 2 Polyneuropathy, cardiomyopathy. Gastrintestinal disturbances Phe33Leu Exon 2 Polyneuropathy, cardiomyopathy, gastrointestinal disturbances, carpal tunnel syndrome Ala45Ser Ala45Asp Exon 2 Exon 2 Cardiomyopathy, carpal tunnel syndrome Gastrointestinal dysfunction, neuropathy, cardiomyopathy Universal Japan, Sweden (Finland) Sweden, USA (Poland, Lithuania) Sweden Ethiopia Gly53Glu Exon 3 CNS-involvement, cardiomyopathy France, Sweden Leu55Gln Exon 3 Polyneuropathy, cardiomyopathy, gastrointestinal disturbances Gly57Arg Tyr60Ala Exon 3 Exon 3 Cardiomyopathy Cardiomyopathy, Neuropathy Sweden, Serbia Sweden Sweden, Ireland, USA Tyr69His Exon 3 CNS involvement, eye deposits. USA, Sweden His88Arg Exon 3 Cardiomyopathy, carpal tunnel syndrome Ala97Ser Val122Ile Exon 4 Exon 4 Polyneuropathy, gastrointestinal disturbances, eye deposits, cardiomyopathy Cardiomyopathy Sweden, USA Malaysia/china Afro Americans

8 Diagnosis of ATTR amyloidosis Clinical symptoms of ATTR- amyloidosis Histopathological diagnosis Genetic testing

9 Preparation of surgical adipose tissue for diagnosis Westermark In: Amyloid proteins. Springer protocols

10 Per Westermark 10

11 Per Westermark 11

12 Typing of amyloid Immunohistochemistry: Sometimes good but there are pitfalls! Immune electron microscopy! Immunochemistry: Western blot, ELISA! Laser dissection microscopy followed by:! Mass spectrometry and/or N-terminal sequence analysis after protein extraction Per Westermark 12

13 Relationship between fibril composition and phenotype Commercial antibody Ihse%E%et%al.%J%Pahol,%2009% 13

14 Diagnosis of ATTR amyloidosis Clinical symptoms of ATTR- amyloidosis Histopathological diagnosis Genetic testing

15 Identification of TTR-mutation» Exclude wt-attr amyloidosis» To identify relatives at risk for disease» depend on penetrance of the trait and phenotype of the disease Note: Detection of a mutation and symptoms may prove unreliable for diagnosis without histopathological confirmation.

16 Case, Male 52 years old.» Father died from ATTR amyloidosis. Brother and cousin transplanted for the disease.» Since approximately 1 year, pain and sensory disturbances in lower legs.» Skin/fat punch (8mm) biopsy and GI biopsies negative for amyloid.» Evaluation:» Neurophysiology: Axonal neuropathy.» Echocardiography: slightly enlarged heart, but no hypertrophy. Preserved systolic and diastolic function, no arrhythmia.

17 Case 2, cont.» Deteriorates. Want to be transplanted as soon as possible?» Repeated punch skin biopsy: negative.

18 Case 2, cont.» Repeated punch skin biopsy: negative.» Deteriorates. Want to bo be transplanted as soon as possible?» Phosfatidyletanol (PEth) = 2.1» Marker of alcohol consumption ( normal consumption )» Alkohol related neuropathy and cardiomyopathy, ATTR amyloidosis?

19 Treatment of ATTR amyloidosis Liver (heart) transplantation TTR- Stabilisers Gene therapy Other

20 Number of Patients Transplanted/Year

21 Dec$2010$

22 Survival(a)er(transplanta/on(for(most(common( non4attr(val30met(muta/ons((n>10)( 100 Patient survival, % Ser77Tyr Thr60Ala Tyr114Cys Val71Ala Leu111Met Ser50Arg Leu58His Glu89Gln Years after transplantation Ser77Phe

23 Treatment of ATTR amyloidosis Liver (heart) transplantation TTR- Stabilisers Gene therapy Other 23

24 TTR-stabilisers» Tafamidis /Vyndaqel : Approved by EMA for neuropathic ATTR amyloidosis (Stage I (walking without aid)).» Diflunisal. Not approved by EMA, but proven efficacy in controlled clinical trial (Berk et al. JAMA 2013)» The 2 trials difficult to compare: Tafamidis ATTRV30M patients, stage I versus diflunisal patients: different ATTRmutations and clinical stages (I-III). Both Disease modifiers, no regression of symptoms.

25 Treatment of ATTR amyloidosis Liver (heart) transplantation TTR- Stabilisers Gene therapy Other 25

26 Gene silencing, prevailing methods under testing: Silencing RNA Antisence 26

27 Updated#Pa+siran#Phase#2#Study#Results# Silencing#RNA# Robust knockdown of both wild-type and mutant TTR in ATTR patients Open label, multi-center, multi-dose, dose escalation study Results (n=29) show up to 96% TTR knockdown; 84% and 87% mean TTR knockdown after 1 st and 2 nd doses in 0.30 mg/kg q3w cohort (p<0.001 vs mg/kg) 1:1 Knockdown of wild-type and mutant TTR; Similar efficacy in patients with TTR stabilizers A20# Dose Response and Duration of TTR Knockdown 200# Wild Type vs. Mutant % Mean Serum TTR Knockdown Relative to Baseline (SEM) 0# 20# 40# 60# 80# 100# 0# 30# 60# 90# 120# Cohorts#0.01A0.30#mg/kg#q4w# Days#Since#First#Visit# Cohort#0.30#mg/kg#q3w# Pa=siran## Treatment#Groups# 0.01#mg/kg#q4w#(n=4)# 0.05#mg/kg#q4w#(n=3)# 0.15#mg/kg#q4w#(n=3)# 0.30#mg/kg#q4w#(n=6*)# 0.30#mg/kg#q3w#(n=12)# *Excludes post-day 28 data from one patient that experienced drug extravasation during second infusion TTR#V30M#(ug/mL)# 150# 100# 50# 0# 0# 50# 100# 150# 200# 250# TTR#WT#(ug/mL)# R 2 #=##0.89## p#<#0.0001# 27 39#

28 %%Mean%TTR%Knockdown%% %Rela8ve&to&Baseline&(SEM)&& Pa8siran&Phase&2&OLE&Preliminary&Study&Results*& 0& 20& 40& 60& 80& Sustained&TTR&Knockdown&of&~80%&at&Pre%Dose&Measurement& Study%% Day% N%available%% to%graph% 1& 18& 3& 21& 7& 20& 17& 19& 42& 3& 84& 3& 168& 1& 100& 0& 25& 50& 75& 100& 125& 150& 175& Dose%1% (pre%&and&post%dose)& Dose%3% (pre%dose)& Dose%5% (pre%dose)& Dose%9% (pre%dose)& *Preliminary results as of April 3, 2014 Days%Since%First%Visit% 28 40&

29 Gene-therapy.» Phase III trials ongoing for SiRNA and antisence, efficacy not evaluated yet. 29

30 Treatment of ATTR amyloidosis Liver (heart) transplantation TTR- Stabilisers Gene therapy Other 30

31 Other therapies» Doxycycline/Tauro-ursodeoxycholic acid» Efficacy in open label trails, not evaluated in controlled trials» Antibody therapy» anti-sap 31

32 Conclusions ATTR amyloidosis» Multiple different presentations of the disease. NB, red flags: Combination of different symptoms: Cardiomyopathy, neuropathy (autonomic and somatic), carpal tunnel syndrome, GI-tract disturbances.» Genetic testing and histopathology important to secure the diagnosis of hereditary ATTR amyloidosis. 32

33 Conclusions, cont.» Proven treatments:» Liver transplantation, especially for early onset V30M ATTR amyloidosis» Tafamidis: ATTR with neuropathy in stage I» Diflunisal, proven in controlled trial on neuropathy» Not proven: Doxycycline/Taurodeoxycholic acid.» In pipeline: Si-RNA, Antisence 33