AL Amyloidosis:
Diagnosis
d
an Prognosis
Giampaolo M li
er
i
n
Amyloidosis Center
Fondazione IRCCS Policlinico San Matteo
University of Pavia, Italy
QC
QC
Organ dysfunction
gy
Pathway Followed by a Newly Synthesized Polypeptide Chain in a Patient Who Is
Heterozygous for an Amyloidogenic Mutation.
N Engl J Med 2003;349:583-96
Mutations in light chain genes cause loss of variable domain
stability which
which is a major
major driving force
force in fibril formation
Open circles:
V fibril formers
L
Closed circles:
V that did not
L
form fibrils
(Protein Sci 1999; 8:509-17)
N Engl J Med 2003;349:583-96
Amyloid Proteins
SYSTEMIC
LOCALIZED
Ig light chain ( or )
Ig light chain ( or )
Ig heavy chain
APP
Serum Amyloid A (SAA)
PrP
2M
Procalcitonin
Transthyretin
Atrial Natriuretic Factor
Apo AI
AI
Amylin (IAPP)
Apo AII
Bri
Apo AIV
Keratoepithelin
Gelsolin
Semenogelin 1
Lysozyme
Prolactin
Fibrinogen A chain
Insulin
Cystatin C
Lactadherin
MOST COMMON SYSTEMIC AMYLOIDOSES
Type
Precursor
Sd
Syndrome
Acquired Amyloidoses
Immunoglobulin LC
Monoclonal LC
Primary
amyloidosis (AL)
(BP Plasma cells)
Myeloma associated
Reactive amyloidosis ()
(AA)
S(
SAA (liver)
Chronic diseases
(Rheum. Infect. Cancer)
Hereditary Amyloidoses
Familial transthyretin
Transthyretin
Familial
amyloidosis (
y(ATTR)
(Liver >90%
Senile systemic
Familial apolipoprotein AI
Apolipoprotein A-1
Familial
amyloidosis (AApoA-1)
(Liver, Intestine)
Familial fibrinogen A
Fibrinogen A chain
Familial nephropathy
chain amyloidosis (AFib)
(Liver)
Distribution of 1528 Amyloid Patients at the
Pavia Amyloid Center
AA
11%
Hereditary
20%
AL
69%
AL AMYLOIDOSIS
The age- and sex- adjusted annual incidence rate
rate of
of AL
AL in
in
Olmsted County, Minnesota, was determined to be
8.9/million person-year
The age adjusted rate for males was more than twice that for
females
Kyle et al, Blood 1992; 79:1817-1822
P
l
reva ence f
o AL
AL
l
amy id
o
i
os s in
ti
pa
t
en s
i
w th
ith MM: 12 15%
-
Systemic AL Amyloidosis
868 AL patients (494 males; median age 62, range 23-91)
kidney (72%)
IF anti
heart (63%)
()
Bl
Bone marrow plasma
l
ce lll clone
liver (26%)
synthesizing amyloidogenic light chain
No of organs involved:
GI (8%)
1 (25%)
PNS (19%)
2 (36%)
()
(36%)
3 or more (39%)
ANS (16%)
soft tissues (12%)
Clinical presentation in 868 patients with AL
%
Fatigue
68
Pi
Peri h
p
l
era
d
e ema
62
Weight loss (kg)
43
median 8 (2
(2 30)
-
Exertional dyspnea
40
Orthostatic hypotension
hypotension
27
Dysesthesias, Paresthesias
23
Dysgeusia
18
Macroglossia
14
Purpura
11
Diarrhea
9
AL amyloidosis presentation
Periorbital purpura 11%
Macroglossia 14%
Nail lesions 3%
Submandibular swelling (15%)
Alopecia (5%) and jugular vein
Shoulder pad 4%
distension (12%)
DIAGNOSING AMYLOIDOSIS
A clinician should suspect amyloidosis whenever a patient
presents with:
- proteinuria > 0.5/day with or without renal
if
insu fi
ff i
c ency
- congestive heart failure from restrictive cardiomyopathy,
- unexplained hepatomegaly, or functional hyposplenism
- progressive peripheral neuropathy
- orthostatic hypotension, autonomic neuropathy
- weight
g
loss
DIAGNOSING AMYLOIDOSIS
Key points:
· Diagnosis relies on
on demonstration on
on tissue biopsy
biopsy
of fibrillar deposits which are congophilic and
birefringent under polarized light, or typical, rigid,
nonbranching, 7.5-10 nm wide fibrils by electron
microscopy
· Unequivocal identification of amyloid protein is
essential for:
· appropriate treatment
· assess prog
pgnosis
· genetic counseling (when appropriate)
The diagnosis of amyloidosis relies on tissue biopsy
Fine-needle abdominal fat aspirate
a)
b)
Fine needle abdominal fat aspirate - Congo red staining
a) Light microscopy (200x); b) under polarized light
721 patients
Sensitivity
88%
Specificity
py
97%
biopsy of the
biopsy of the labial minor salivary glands
organ involved
(sensitivity 85-90%, specificity 90-98%)
Typin
yp g of amyloid deposits
p
· Immunohistochemistry
· Ultrastructural (EM) immunohistochemistry
· Chemical typing (MS)
· Proteomics
IMMUNOHISTOCHEMISTRY TYPING OF AMYLOID
DEPOSITS
Kidney - Immunohistochemical
Kidney - Immunohistochemical
labeling with anti-AA
labeling with anti-fibrinogen
Immunohistochemical labeling of tissue biopsies:
gp
effective for AA,
unreliable in AL (up to 62%), A-ApoAI, (ATTR)
Typing of amyloid deposits
Ultrastructural (EM) immunohistochemistry
sensitivity 93%, specificity 99%
anti- antibod
based y
anti- antibody
on
y 377 tests performed on
abdominal fat aspirates
anti-TTR antibody
anti-apolipoprotein A-I antibody
Typing of amyloid deposits
Chemical typing (Mass Spectrometry)
Microextraction and purification techniques applicable to chemical
characterization of amyloid proteins in minute amounts of tissue.
Kaplan et al. Methods Enzy
pymol. 1999;309:67-81.
Chemical typing of amyloid protein contained in formalin-fixed paraffin-
embedded biopsy specimens.
Murphy et al. Am J Clin Pathol 2001;116:135-142
Biochemical subtyping of amyloid in formalin-fixed tissue samples
confirms and supplements immunohistologic data.
Kaplan et al. Am J Clin
Clin Pathol
Pathol. 2004;121:794-800.
TYPING AMYLOIDOSIS: PROTEOMICS ON ABDOMINAL FAT
ASPIRATE
pI
3
10
pI
Patient
3
10
250
Control
250
)a
(kD
(kDa)
MW
MW
10
105-127
2503.3 (Ile122 TTR)
105-127
104-126
10
2489.3 (wt TTR)
104-126
2530.4 (Ile122 TTR)
2516.3 (wt TTR)
81-103
Intensity
+ 14
+ 14
2451.2
22-34
Val ­ Ile = + 14Da
1366.7
36-48
1394.6
2490
m/z
2540
35-48
Intensity
77-80
1522.7
104-127
583.25
2645.4 104-127
22-35
2659.4
16-21
1494.8
672.40
Transth
th
t
yre i
tin, V l122Il
a
e vari
t
an
49-76
42-48
47-62
3140.4
735.23
1674.78
(heterozygote)
500
m/z
3500
Lavatelli et al, 2007
TYPING AMYLOID DEPOSITS
· Characterization of amyloidosis as AL type
(demonstration of a clonal plasma cell disorder)
1) A MC is evident on serum electrophoretic pattern in only
56% of patients
2) Immunofixation electrophoresis of serum and urine,
sensitivity 96%, / = 29/71%
3) Bone marrow aspiration, median bone marrow plasma-
cell: 7% (1-
(1 30%)
-
, 6%
6% of
of patients having >20%
>20%, /
/ ratio
4) Immunonephelometric immunoglobulin serum free light
hi
chain (FLC)
(FLC) assay
TYPING AMYLOID DEPOSITS
· Characterization of amyloidosis as AL type
(demonstration of a clonal plasma cell disorder)
Finding amyloid deposits in an organ on biopsy in the
presence of monoclonal immunoglobulin-clone is strong but
not conclusive evidence of AL
The chance association
association of
of a monoclonal gammapathy in an
older patient with hereditary amyloidosis (2-10%) must be
kept in mind:
Val122Ile ATTR in 3.9% of African Amer.
(Comenzo et
et al
al, Blood
Blood 2006;107:3489-91)
Senile systemic cardiac amyloidosis (wt TTR): 8-25% older
than 80
Cli i
n
ll
ca y, it is diffi
diffi
lt
cu to di
di t
s i
ti
i
ngu h
s b t
e ween
the various types of amyloidosis
Clinical manifestations of the most common forms
of systemic amyloidoses
AL
AA
ATTR
AApoAI
AFib
Kidney
++
++
(+)
++
++
Heart
++
(+)
++
++
-
Peripheral NS
+
-
++
+
-
Autonomic NS
+
+
++
-
-
Liver
+
+
-
++
(+)
GI
+
+
-
-
-
Central NS
-
-
+
-
-
Tes
Te t
s i
t s
-+-
++
-
Immunoelectron microscopy DNA studies
AL amyloidosis: assessment of organ involvement
Organ
Pf
Performed
t
rou i
ti
l
ne y
As l
c ii
linicalllly id
in i
dicated
Kidney
Proteinuria, s. Creat.,
Renal vein Doppler US
creat. clear., Cystatin C
Heart
NT-proBNP, cTn, ECG,
Echo, chest x-ray
Liver
ALP, ALT
AL , Bili bi
ru n,
CT
Echo
Gastrointestinal t. Blood in stool
Endoscopy, biopsy,
Functional tests
Nerve
EMG
Lung
Chest x-ray
CT, Biopsy, Functional
tests
Hemostasis
PT, Factor X
Endocrine glands TSH
s. Cortisol
Gertz et al, Am J Hematol 2005; 79:319-28
Monitoring amyloid load - SAP scan
Radiolabelled SAP scintigraphy: posterior images of a 52
52-year-old woman
woman with systemic
AL kappa amyloidosis, before (left) and 1 year after (right) HDM chemotherapy.The serum
concentration of free kappa light chains had fallen from 551 mg/l to 52 mg/l.
Lachmann et al, Br. J. Haematol, 2003, 122:78­84
Cardiovascular Magnetic Resonance in Cardiac Amyloidosis
Maceira et al, Circulation 2005;111:186-93
CMR in a patient with systemic AL amyloidosis. Top row shows diastolic frames from cines
showing a thickened LV and pleural effusions (Pl eff) and pericardial effusions (Pc eff)
associated with heart failure
failure. Bottom row
row shows late
late gadolinium subendocardial
subendocardial enhancement
images in the same planes.
The sensitivity of this technique for detecting early cardiac involvement is not known, and the
specificity is low in an unselected population of patients (Falk RH, Circulation 2005;112:2047-60)
CARDIAC AMYLOID IMAGING
ACCURACY OF 99mTc-APROTININ SCINTIGRAPHY FOR
THE DETECTION OF MYOCARDIAL AMYLOIDOSIS:
LONG-
LONG TERM FOLLOW-UP OF 108 PATIENTS
· SENSITIVITY
0.95
· SPECIFICITY
09
0. 7
97
· ACCURACY
0.96
Aprile et al,
p, Eur J Nucl Med. 1995;22:139
;
3-401
Focal accumulations of 99mTc aprotinin
were seen in different organs of 22 patients.
Sci ti
n
h
grap y revealed "silent" amyloid
id
deposits in at least 5 patients who
developed clinical symptoms 5 to 8
months later.
Schaadt et al, J Nucl Med. 2003;44:177-83
Survival of 868 patients with AL amyloidosis
1.0
0.9
median survival 3.8 years
g 0.8
invirv 0.7
u
Cumulative proportion surviving:
S
· 1 year:
73%
0.6
on
·5 years:
46%
ti
y
· 10 years:
31%
0.5
ropor
P 0.4
e 0
eivat 0.3
ul
um 02
C 0.2
C
0.1
0.0
01
234
567
89
10
11
12
Survival time (years)
Survival of 467 patients with AL amyloidosis according to serum NT-proBNP
concentration
10
1.0
median survival 16 months vs. not reached
0.9
p<0.0001
g 08
0.8
iniv 0.7
rvu
Sn 06
0.6
tio
0.5
roporPe 0.4
ivatul 0.3
umC 0.2
NT-proBNP>=1287 ng/L (277 pts)
0.1
NT-proBNP <1287 ng/L (190 pts)
0.0
0
20
40
60
80
100
120
140
160
Survival time (months)
Factors associated with survival in 868 patients with AL amyloidosis
(Cox univariate analysis)
Variable
p
Serum alkaline phosphatase
0.03
Serum creatinine
0.02
Proteinuria
00
0. 2
02
Macroglossia
0.01
Hepatomegaly
0.008
Age
0.002
Weight loss
0.001
Syncope
0.0002
Postural hypotension
0.0002
Plasma BNP
0 0
. 001
0001
Fatigue
<0.0001
Number of organs involved
<0.0001
Ejection
j
fraction
<0.0001
Interventricular septum thickness
<0.0001
Serum cTnI
<0.0001
Serum NT-proBNP
<0.0001
Heart involvement (echo + NT-proBNP)
<0.0001
Congestive heart failure
<0.0001
Cox multivariate analysis of survival
on the whole AL amyloid population
treated with low-intensity therapy
· heart involvement:
p < 0.0001
· hematologic response:
p < 0.0001 (protective)
Prognostic factors in patients treated with ASCT
Cardiac involvement
involvement (Echo, cTnT,
cTnT NT-
NT proBNP)
Number of organs affected
FLC baseline concentration
Weight gain > 2% during stem cell mobilization
Absolute lymphocyte count 15 d after ASCT
Autonomic neuropathy
Liver involvement
Renal response
Degree of response (NR, PR, CR)
FLC absolute level achieved after therapy
Moreau et al, Br J Haematol 1998;101:766-9 ­ Comenzo & Gertz, Blood 2002; 99:4276-82 -Gertz et al,
Bone Marrow Transpl 2004;34:1025-31 ­ Skinner et al, Ann Intern Med 2004;140:85-93 ­ Sanchorawala et
al, Bone Marrow Transpl
T
2004;33:381-
2004;33:381 8
- - Porrata et
et al
al, Clin
Clin Cancer Res. 2005;11
2005;1 :1210
1
-8
- ­ Leung et al,
Blood 2005; 106:3353-7 - Goodman et al, Br J Haematol 2006; 134:417-25 ­ Perfetti et al, Haematologica
2006; 91:1635-43 ­ Dispenzieri et al Blood 2006; 107:3378-83 ­ Gertz et al, Curr Opin Oncol 2007; 19:136-
41
Survival of 570 patients with AL amyloidosis according to hematologic
response to chemotherapy
10
1.0
0.9
median survival 21 vs. 108 months
g 08
0.8
in
p<0.0001
iv 0.7
rvu
Sn 06
0.6
tio
0.5
roporPe 0.4
ivatul 0.3
umC 0.2
0.1
Responders (293 pts)
Non responders (277 pts)
0.0
0
20
40
60
80
100
120
140
160
180
Survival time (months)
Outcome in systemic AL amyloidosis in relation to changes
in concentration of circulating free immunoglobulin light
hi
chains fl
fo lllowing h
c
t
emo h
therapy
Lachmann et al, Br J Haematol. 2003;122:78-84.
Survival of 337 patients with cardiac AL amyloidosis according to
hematologic response to chemotherapy
10
1,0
0,9
median survival 12 vs. 70 months
08
0,8
ginv
p<0.0001
0,7
rvi
Sun 06
median NT-
NT proBNP 3221 ng/L
0,6
tio
(386-76927 ng/L)
0,5
roporPe 0,4
iv
median NT-proBNP 3109 ng/L
ulat
(374-56764 ng/L)
0,3
umC 0,2
Responders (176 pts)
Non responders (161 pts)
0,1
0,0
0
20
40
60
80
100
120
140
160
Survival time (months)
NT-proBNP m odification in patients who reached com plete rem is s ion, partial
rem is s ion, or did not res pond to chem otherapy
300
M e dian
25 %-7 5 %
Mi n -Ma x
)
250
%(
rapy
200
e
oth
em
p=0.001
h
150
c
ftera 100
tion
median +47%
a
(range -75% - +1862%)
fic
50
p=0.04
odimPN 0
median -31%
oB
(range -80% - +94%)
-prT -50
N
medi
m
an
edi
5
- 3%
53
(range -95% - -30%)
-100
Amyloidogenic FLC
FLC reduction >50%,
FLC stable or increased
disappearance at immunofixation amyloidogenic FLC still present
29 pts
9 patients
at immunofixation
13 patients
Palladini et al, Blood. 2006;107:3854-8.
Circulating amyloidogenic free light chains and serum N-
terminal natriuretic peptide type B decrease simultaneously in
association with improvement of survival in AL
Palladini et al, Blood. 2006;107:3854-8.
STRATEGIES FOR AL TREATMENT
Monitoring organ response
· Cardiac markers (BNP, Trop
(ponins) rapid
· Kidney markers (median time to response 1 year)
- Proteinuria - Serum creatinine ­ Cystatin C
· Liver marker
marker (ALP)
(ALP)
Survival of 108 patients with cardiac AL amyloidosis according to response to therapy
1,0
0,9
g 0,8
invirv 0,7
u
S 0,6
tion
0,5
ropor
Pe 0,4
viatul 0,3
umC
Non res ponders (46 pts ) m edian NT-proBNP 1802 ng/L (range 337-16054)
C
p(
p )
p
g
(
g
02
0,2
Partial res pons e no organ res pons e (20 pts ) m edian NT-proBNP 3590 ng/L (range 359-26014)
Partial res pons e plus organ res pons e (22 pts ) m edian NT-proBNP 2222 ng/L (range 506-34049)
0,1
Com plete rem is s ion (20 pts ) m edian NT-proBNP 3875 ng/L (range 629-35563)
0,0
0
102030405060
Survival time (months)
See PO-910
STRATEGIES FOR AL TREATMENT
Aim f
o th
therapy
· obtain durable improvement of AL amyloidosis-
related organ function
extend survival
Goal
· CR
· fine balance between clonal and organ response
/treatment toxicity
Conclusions
· Early diagnosis
· Accurate classification
· Use of biomarkers for optimizing therapy
combined with new therapies should lead to
better outcome
Italian Amyloidosis Study Group
· 68 medical centers participate
in the network
Societą Italiana per l'Amiloidosi
· Web site: www.amiloidosi.it
· A common
common diagnostic
diagnostic and
therapeutic protocol is
periodically discussed and
updated
last update May 26 2007
University of Pavia and University Hospital San Matteo
Amyloid Research and Treatment Center
Vittorio Bellotti
Vittorio Perfetti
Laura Obici
Giovanni Pallladi
dini
Francesca Lavatelli
Paola Russo
Mario Nuvolone
Laura Verga
Monica Stoppini
Irene Zorzoli
Palma Mangione
S f
o i
fia Gi
tti
orge
Simona Casarini
Simona Donadei
Valentina Navazza
Silvia Gabb
bba
Sara Marini
Alberto Bovera
Chiara Crosignani
Survival of 868 patients with AL amyloidosis
1.0
medi
m
an s r
u i
v a
v l
al 3.8 years
0.9
median follow-up of living patients 27 months (range 1-182 months)
g 0.8
invirv 0.7
u
Cumulative proportion surviving:
S
· 1 year:
73%
0.6
on
·5 years:
46%
ti
y
· 10 years:
31%
0.5
ropor
P 0.4
e 0
eivat 0.3
ul
um 02
C 0.2
C
0.1
Patients at risk
792
473
336
248
179
134
91
57
41
29
19
12
0.0
01
234
567
89
10
11
12
Survival time (years)
Survival of 868 patients with AL amyloidosis
1.0
medi
m
an s r
u i
v a
v l
al 3.8 years
0.9
median follow-up of living patients 27 months (range 1-182 months)
g 0.8
invirv 0.7
u
Cumulative proportion surviving:
S
· 1 year:
73%
0.6
on
·5 years:
46%
ti
y
· 10 years:
31%
0.5
ropor
P 0.4
e 0
eivat 0.3
ul
um 02
C 0.2
C
0.1
Patients at risk
792 473 336 248 179 134 91
57
41
29
19
12
7
5
3
0.0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Survival time (years)