12th International Myeloma
Workshop, Washington DC,
February 28, 2009
Understanding pathogenesis to
to
improve care in amyloidosis
Giampaolo Merlini
gmerlini@unipv.it
Amyloid Research and Treatment Center
Fondazione IRCCS Policlinico San Matteo
University of Pavia, Italy
Faculty disclosure information
Consultant: Millenium, Novartis Vaccines and
Diagnostics
Grant/Research Support: No
Speakers Bureau: No
Major Stock Shareholder: No
Amyloidosis: protein misfolding disease
Limited
proteolysis
Atomic force microscopy
microscopy of ex-
vivo amyloid material
Glycosaminoglycans
SAP
Tissue targeting
Organ
Cytotoxicity
dysfunction
Specific tissue components
(collagen)
Metal ions
Dobson CM, Science 2004;304.1259-62 modif.
Most common systemic amyloidoses
1584 patients
Acquired amyloidoses: AL 71%, AA: 8%
Type
Precursor
Syndrome
(site of synthesis)
Immunoglobulin LC
Monoclonal LC
Primary amyloidosis,
amyloidosis (AL)
(BM plasma cells)
myeloma-associated: 10%
Reactive amyloidosis (AA)
Serum amyloid A (liver)
Chronic diseases
(rheumatoid arthritis,
infections, cancer)
Hereditary amyloidoses: 21%
Type
Precursor
Syndrome/involved tissue
(site of synthesis)
Familial transthyretin amyloidosis Variant transthyretin
FAP, heart
(ATTR)
(liver > 90%)
Familial apolipoprotein AI
Variant apolipoprotein A-I
Heart, liver,
amyli
lo d
idosis (AA
(AA A
po -1)
(liver, i t
n estiti )
ne
kidney, testis
LC = light chain; FAP = familial amyloidotic polyneuropathy.
Transthyretin amyloidosis
Approximately 100 mutations: incomplete
penetrance
p
and frequent
q
late-onset
· sensorimotor peripheral neuropathy
· autonomic nervous system: orthostatic
orthostatic
hypotension, altered GI motility
(diarrhea alternating with constipation),
it
impotence,
d
an
i
ur nary di
di t
s
b
ur ances
· cardiac (arrhythmias, CHF)
· some associated with renal, vitreous, or
leptomeningeal amyloid
Amyloid fibril composition is related to the phenotype of
hereditary transthyretin V30M amyloidosis
· fibrils composed of only full-length transthyretin were associated with early age of
onset (44.8 ± 12.9 years) and no clinical cardiac involvement
· presence of transthyretin fragments was associated with late age of onset (67.3
± 7.0 years) and signs of cardiac involvement
Ihse et al., J Pathol, 2008; 216: 253261
Apolipoprotein AI amyloidosis
N-terminal 83-93 residues in amyloid deposits
Mutation
Clinical Features
Geographic Kindreds
Gly26Arg
yg
PN, Nephro
,p
path
p
y
United States
Trp50Arg
Nephropathy
United Kingdom
Leu60Arg
Nephropathy
United Kingdom
Leu64Pro
Nephropathy
United States, Italy
del 60-71 ins Val/Thr
Hepatic
Spain
Del 70-
70 72
Nephropathy
South Africa
Africa
Primary hypogonadism, hepatic
Leu75Pro
Italy (70 families)
Tubulo-interstitial nephropathy
L 90P
eu
ro
Cd
Car i
di
t
omyopa h
thy, c t
u aneous, ll
laryngeal
France
Arg173Pro
Cardiomyopathy, cutaneous, laryngeal
United States
Leu174Ser
Cardiomyopathy
Italy
Ala175Pro
Laryngeal
United Kingdom
Leu178His
Cardiomyopathy, Laryngeal
France
Reactive (AA) amyloidosis
Novel approach: inhibition of amyloid fibrils formation
· Rheumatic Diseases
· Hd
Here it
ditary
t
au i
o f
n l
flammatory di
dis.
· Chronic infections
Castleman dis.
· Neoplasia
Waldenström M.
Sustained, marked
Schnitzler s.
Persistent
increase of SAA (SAA1)
Hairy cell leuk.
Inflammation Hodgkin lymph.
Inflammation
Abnormal processing of SAA by mononuclear phagocytes
Matrix metalloproteases proteolytic processing
Interaction with tissue glycosaminoglycans
SAP
Kidney
GI tract
Amyloid
Spleen
Deposits
Liver
Systemic AL Amyloidosis
10/million person-year
1125 AL patients (620 males; median age 62, range 23-91)
No of organs involved:
Small clone
1 (25%)
(d
(me iB
dian MPC
BMPC 7%)
1 (25%)
7%)
2 (36%)
3 or more (39%)
75%
PNS (19%)
6a
k 25%
ANS (16%)
soft tissues (12%)
1c ?
kidney (72%)
Nephrotic s. 52%
US
U
S
Renal fail. 18%
heart (63%)
CHF 20%
GI (8%)
liver (26%)
Cardiotoxicity of amyloidogenic light chains
· Infusion of amyloidogenic light chains causes immediate diastolic
dysfunction in isolated mouse hearts Liao et al Circulation 2001;104;1594-1597
· Cellular response
response of
of cardiac fibroblasts to
to amyloidogenic
amyloidogenic light chains
Trinkaus-Randall et al, Am J Pathol 2005; 166:197-208
· Production of recombinant amyloidogenic complete light chains
· Purifi
ification f
o lili h
g t
ht chains from patients i
w th
ith severe cardiac involvement
Systolic Shorthening (m/sec)
Diastolic Lengthening (m/sec)
0
120
-20
100
*
-40
80
-60
60
-80
40
-100
20
*
-120
0
Control
Cardiac AL
Non Cardiac AL
Control
Cardiac AL
Non Cardiac AL
Isolated adult rat cardiomyocytes, n=14 per group, 24-hour exposure in collagen-coated wells
CARDIAC INVOLVEMENT IN AL AMYLOIDOSIS
Cardiac involvement is causing the death of ~ 80%
of AL patients
It is the most important prognostic factor
Cardiac biomarkers: N-terminal of natriuretic
peptide type
type B (NT-proBNP - BNP) and troponins
(cTnI or cTnT)
Palladini et al, Circulation 2003; 107:2440-45
Dispenzieri et al, Lancet 2003;361:1787-9
Staging system for AL amyloidosis
Dispenzieri et al, J Clin Oncol 2004; 22:3751-3757
Diagnosis of amyloidosis relies on tissue biopsy
· Tissue of choice: abdominal fat
· Av
A ailable from
from virtually
virtually all patients, innocuous,
fast, inexpensive: sensitivity 88%, specificity 97%
Congo red stain
Biopsy of the labial minor salivary glands
Biopsy of the organ involved
Typing of amyloid deposits is essential for the
choice of treatment
· Immunohistochemistry
pI
3
10
250
effective
ef
for
for AA, unreliable in AL ,A-
,A ApoAI, (ATTR)
(A
· Ultrastructural (EM) immunohistochemistry
sensitivity 93%, specificity 99%
6
MW
(kDa)
· Protein identification by MS
· DNA analysis
10
Amyloid deposits can regress
IF:
THE SYNTHESIS OF THE PRECURSOR PROTEIN IS
SUPPRESSED
AND/OR
CLEARANCE OF THE AMYLOID DEPOSITS IS
ENHANCED
Treatment for amyloidosis is increasingly
based on understanding the
pathophysiology of the disease
Therapy of ATTR amyloidosis
1.
Reduction of the supply of the amyloidogenic protein
·
liver transplantation (progression of cardiomyopathy after transplant)
·
gene repair
(single strand oligonucleotides, Nakamura et al, 2004)
·
inhibition of TTR synthesis
(antisense oligonucleotides, Benson et al, 2006 and small interfering RNA,
Kurosawa et al, 2005)
2.
Stabilization of the amyloidogenic protein precursor
·
diflunisal (Tojo et al, 2006)
·
Fx-1006A
3. Disruption/inhibition of amyloid fibrils
·
iododoxorubicin (Merlini 1995, Pahla et al, 2000)
·
doxycyclin (Cardoso et
et al
al, 2006)
·
immunization (Terazaki et al, 2006)
Reactive (AA) amyloidosis
Novel approach: inhibition of amyloid fibrils formation
· Rheumatic Diseases
Anti-TNF
· Hereditary autoinflammatory dis.
Anti-IL-1
· Chronic infections
Castleman dis.
· Neopl
eop as
asia
Wl
Wa d
ldensttröm M.
Sustained, marked
Schnitzler s.
Persistent
increase of SAA (SAA1)
Hairy cell leuk.
Ifl
ti
Hodgkin lymph
Inflammation
.
Abnormal processing of SAA by mononuclear phagocytes
Matrix metalloproteases proteolytic processing
Interaction with tissue glycosaminoglycans
SAP
Goal: maintain the
Kidney
serum concentration
of SAA below the
Amyloid
GI tract
Spleen
threshold of 10 mg/L
Deposits
Liver
GAG mimetics for the treatment of AA amyloidosis
1,3 pro
,p panedilsulfonate
p
Eprodisate
Amyloid + GAGs:
GAG mimetics should prevent
Amyloid + GAGs:
p
Amyloid fibril formation leads
amyloid fibril formation,
to progression of disease
deposition and associated
toxicity in the organs
Therapy of amyloidosis:
from bench to bedside
Conclusions:
Eprodisate slows the
the decline of
of renal
renal function in AA amyloidosis
Therapy of AL amyloidosis
Chemotherapy
Clonal,
hematologic
New drugs
response
Anti-sense and
siRNA strategies
Immunotherapy
Organ
response
Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.Merlini & Stone Blood 2006;108:2520-2530
FLC and NT-proBNP response in 115
AL patients with cardiac amyloidosis
py
Myocardial shortening fraction (%)
NT
BNP
1. in
100
patients with reduction
-proBNP
reduction
Haematological
decreased
of NTproBNP > 30% unchanged IVS
0.9
response
> 30%
42
rviving 0.8
Cl
Complete response
18/21 pt
u
ts
40
s 0.7
p < 0.05
(elimination of the
(86%)
p = 0.001
amyloidogenic light chain)
38
0.6
ortionp 05
0.5
Partial response
29/50 pts
36
> 30% reduction
0.4
pro
(reduction to < 50% of the
(58%)
of NT-proBNP
(20 patients)
amyloidogenic light chain)
34
0.3
ative 0.2
NT-proBNP stable
32
or increased
No response
1/44 pts
0.1
(31 patients)
(2%)
Cumul 300.00 Baseline
5
10 15
A
20 fter
25 treatment
30 35 40
Survival time (months)
Palladini et al, 2008.
Palladini et al, Blood. 2006;107:3854-8.
Survival according to haematological
and cardiac response
p
Overall survival stratified by rise or fall in NT-ProBNP (n = 164)
(UK National
National Amyloid
Amyloid Centre and Italian
Italian Amyloid
Amyloid Centre, Pavia)
1.0
CR (n = 34)
0.8
PR with fall in NT-ProBNP (n = 24)
0.6
esurvival
Non-responders (n = 85)
0.4
PR with rise in NT-ProBNP (n = 21)
mulativu 02
0.2
p < 0 0001
.
C
0
010
20
30
40
50
Months
Wechalekar & Palladini, ASH 2008.
A new paradigm for treatment strategies
Aim of therapy
· obtain durable improvement of AL amyloidosis-
related organ function extend survival
Monitoring response to therapy
· Chemotherapy guided by
by frequent assessment of FLC and
cardiac biomarkers (every 2 cycles)
· Organ response:
- NT-proBNP, troponins, rapid
- Kidney markers (proteinuria
(proteinuria, s. creatinine) may be
be
delayed by > 3 mos up to 1 yr
Clinical suspicion
Document amyloid deposit in tissues by Congo
red staining or EM
Type the amyloid deposits:
EMimmuno
Mass Spectrometry
DNA
Tra
Tr nst
s h
t y
h re
r t
e i
t n, Apo
AI, or
o
AA
other hereditary amyloidosis
Age
Identif
Iden y underlying
TTR: variant, ,age
g , ,PF, TTR fragm.
underlying
g
No of organs involved
disease
ApoAI: variant, age
Renal funct. BNP, Tn
Chemothera
Chemother py
p
Tt
Treat
t
men f
o undl
derl i
y ng
Liver transplantation
ASCT
disease, colchicine,
(+ heart, kidney)
New drugs
eprodisate
experimental drugs
TARGETING
Therapy of
AMYLOID DEPOSITS
amyloidosis: drugs in the
pip
ppeline
Targeted pharmacological depletion of serum amyloid P
component for treatment of human amyloidosis
Open-label studies in
patients with AA, AL and
hd
here it
ditary
l
amy i
o d
idosis
are ongoing at NAC-UK
The structure of the complex of CPHPC with SAP
Pepys et al, Nature 2002;417:254
TARGETING AMYLOID DEPOSITS
Therapy of amyloidosis - drugs in the
pip
ppeline: doxycy
yycline analogs
g
doxicycline
+ 100M doxicycline
Mario Salmona Negri Institute Milan
Amyloidosis: from molecular mechanisms
to therapy
Difl
i
un
l
Protease
sal,
Liver
Fx-1006A,
(secretase)
transplant,
inhibitors,
ASCT,
MPACs
chemotherapy
(proteasome
inhibitors,
lenalidomide),
gene therapy
gpy
Immuno-
Immuno
therapy
Eprodisate
CPHPC
doxycycline,
immunotherapy
Organ dysfunction
MPAC = metalprotein attenuating compound. Adapted from Merlini G, Bellotti V. N Engl J Med. 2003;349:583-96.
University of Pavia and University Hospital San Matteo
Amyloid
y
Research and Treatment Center
Vittorio Bellotti
Vittorio Perfetti
Laura Obici
Giovanni Palladini
Francesca Lavatelli
Paola Russo
Mario Nuvolone
Laura Verga
Monica Stoppini
Irene Zorzoli
Palma Mangione
Sofia Giorgetti
Simona Casarini
Simona Donadei
Valentina Navazza
Gabriele Sarais
Sara Marini
Alberto Bovera
Fondazione
IRCCS Policlinico
San Matteo
Pavia
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