Journal of

Clinical Medicine

Article
Screening for Occult Transthyretin Amyloidosis in Patients with
Severe Aortic Stenosis and Amyloid Red Flags
Aiste Monika Jakstaite 1,† , Julia Kirsten Vogel 1 , Peter Luedike 1 , Rolf Alexander Jánosi 1 , Alexander Carpinteiro 2,3 ,
Christoph Rischpler 4 , Ken Herrmann 5 , Tienush Rassaf 1 and Maria Papathanasiou 1, *

1 Department of Cardiology and Vascular Medicine, West German Heart and Vascular Center, University
Hospital Essen, Hufelandstrasse 55, 45147 Essen, Germany
2 Department of Hematology and Stem Cell Transplantation, West German Tumor Center, University Hospital
Essen, Hufelandstrasse 55, 45147 Essen, Germany
3 Institute of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany
4 Department of Nuclear Medicine, Klinikum Stuttgart, Kriegsbergstrasse 60, 70174 Stuttgart, Germany
5 Department of Nuclear Medicine, University Hospital Essen, Hufelandstrasse 55, 45147 Essen, Germany
* Correspondence: papathanasiou@[Link]; Tel.: +49-176-6343-4270; Fax: +49-69-6301-6374
† Current address: Department of Cardiology and Angiology, Hannover Medical School, Carl-Neuberg-Strasse
1, 30625 Hannover, Germany.

Abstract: Aims: The optimal strategy to identify transthyretin-type cardiac amyloidosis (ATTR-CA) in
patients with aortic stenosis (AS) is still unclear. This study aimed to investigate if targeted screening
for ATTR-CA in patients with severe AS and amyloid red flags is associated with higher detection
rates. Methods: The study prospectively enrolled patients ≥65 years with severe AS. Patients who
fulfilled ≥1 major (carpal tunnel syndrome (CTS), ruptured biceps tendon, spinal stenosis, N-terminal
pro B-type natriuretic peptide ≥1000 pg/mL, cardiac troponin >99th percentile) or ≥2 minor criteria
(diastolic dysfunction ≥2 grade/lateral e’ <10 cm/s, atrial fibrillation, atrioventricular conduction
disease/pacemaker) received bone scintigraphy and biochemical analysis for light chain amyloi-
dosis. Hypertensive patients (>140/90 mmHg) and those with interventricular septal thickness
Citation: Jakstaite, A.M.; Vogel, J.K.; (IVSd) ≤13 mm were excluded. Results: Overall, 264 patients were screened, of whom 85 were
Luedike, P.; Jánosi, R.A.; Carpinteiro, included in the analysis. Tracer uptake Perugini grade ≥1 was detected in nine patients (11%). An
A.; Rischpler, C.; Herrmann, K.; endomyocardial biopsy was additionally performed in four of nine patients, yielding a prevalence of
Rassaf, T.; Papathanasiou, M. 7% (n = 6). All patients with dual AS-ATTR were male. Syncope was more commonly reported in
Screening for Occult Transthyretin
AS-ATTR patients (50% vs. 6%, p = 0.010), who also tended to have more severe hypertrophy (IVSd
Amyloidosis in Patients with Severe
of 18 vs. 16 mm, p = 0.075). Pericardial effusion and CTS were more common in patients with dual
Aortic Stenosis and Amyloid Red
pathology (67% vs. 8%, p < 0.001, and 83% vs. 24%, p = 0.003, respectively). Conclusion: Targeted
Flags. J. Clin. Med. 2024, 13, 671.
screening for ATTR-CA in patients with AS and amyloid red flags does not yield higher detection
[Link]
jcm13030671
rates than those reported previously in all comers with AS.

Academic Editor: Gani Bajraktari Keywords: amyloidosis; ATTR amyloidosis; cardiomyopathy; heart failure; valvular heart disease
Received: 27 November 2023
Revised: 2 January 2024
Accepted: 22 January 2024
Published: 24 January 2024 1. Introduction
Degenerative aortic stenosis (AS) is a common disease in the elderly, affecting over
4% of individuals ≥70 years of age [1]. Transcatheter aortic valve replacement (TAVR) has
transformed the treatment and outcomes of AS in high-risk and inoperable patients with an
Copyright: © 2024 by the authors.
increasing interest to expand its application to all-risk patients [2]. According to previous
Licensee MDPI, Basel, Switzerland.
studies, transthyretin-type cardiac amyloidosis (ATTR-CA) has a prevalence of 8% to 16%
This article is an open access article
among all comers with severe AS undergoing TAVR and up to 4% in patients undergoing
distributed under the terms and
conditions of the Creative Commons
surgical aortic valve replacement (SAVR) [3–8]. Autopsy data report ATTR-CA in 25% of
Attribution (CC BY) license (https://
patients aged ≥85 years, suggesting that the prevalence may increase with age [9].
[Link]/licenses/by/ The diagnosis of CA in patients with severe AS remains challenging mainly due to the
4.0/). lack of disease-specific biomarkers and echocardiographic similarities between severe AS

J. Clin. Med. 2024, 13, 671. [Link] [Link]

J. Clin. Med. 2024, 13, 671 2 of 14

and CA. Routine screening for CA in all patients with AS using bone scintigraphy is not
feasible and cost-effective in everyday clinical practice. On the other hand, screening only
the subgroups of the AS population with a higher probability of ATTR-CA (e.g., low flow,
low gradient AS) may lead to underdiagnosis. Recently, a scoring system was proposed for
the identification of amyloidosis in AS patients. This included clinical (history of carpal tun-
nel syndrome (CTS), 3 points; age ≥ 85 years, 1 point), echocardiographic (left ventricular
septal wall thickness ≥18 mm, 1 point; E/A ratio > 1.4, 1 point), electrocardiographic (right
bundle branch block, 2 points; Sokolow–Lyon index < 1.9 mV, 1 point), and biomarker
(high-sensitivity troponin T > 20 ng/L, 1 point) indices. Sensitivity for scores ≥2 and ≥3
was 94% and 72%, and specificity 52% and 84%, respectively; thus, the optimal screening
strategy to precisely identify patients with AS-ATTR remains unclear [7]. Screening for
amyloid red flags in at-risk cohorts is recommended by international guidelines [10,11].
The predictive value of numerous amyloid red flags has been demonstrated in previous
studies across various patient cohorts. Conditions such as CTS and biceps tendon rupture
are common red flags that have been shown to occur 5–15 years before the manifestation
of CA [12,13]. Similarly, peripheral neuropathy is a common neurological disorder with
different etiologies and can also present as the initial symptom of amyloidosis [14,15]. The
current study aimed to test the hypothesis that a screening algorithm based on known
amyloid red flags leads to higher detection rates of ATTR-CA in patients with severe AS.

2. Methods
2.1. Study Design
The study prospectively enrolled patients with severe degenerative AS referred to our
center for TAVR. Patients were eligible for the study if aged ≥65 years old and fulfilled
at least one major or two minor prespecified risk criteria for ATTR-CA. Major criteria in-
cluded: (1) CTS, (2) a ruptured biceps tendon, (3) spinal stenosis, (4) N-terminal pro B-type
natriuretic peptide (NT-proBNP) ≥1000 pg/mL, and (5) high-sensitivity cardiac troponin I
(hs-cTnI) >99th percentile upper reference limit. As minor criteria, we defined the following:
(1) second or higher grade diastolic dysfunction, or e’ velocity in pulsed wave Doppler of
the lateral mitral valve annulus <10 cm/s, (2) atrial fibrillation, and (3) atrioventricular
conduction disease or history of conduction disorders warranting pacemaker implantation.
Exclusion criteria encompassed arterial hypertension at admission with resting arterial
blood pressure ≥140/90 mmHg, interventricular septal thickness ≤13 mm, inability to
provide informed consent, or inability to perform scintigraphic imaging for other reasons
(e.g., frailty, dementia). All study participants provided written informed consent. The in-
vestigation conforms with the principles outlined in the Declaration of Helsinki and received
approval from the local ethics committee. The study is registered at [Link]
(registry number NCT05693376).

2.2. Study Procedures

Patients who fulfilled the inclusion criteria received technetium-99m-labeled 3,3-
diphosphono-1,2-propanodicarboxylic acid (99m Tc-DPD) scintigraphy with single-photon
emission computed tomography/low-dose computed tomography (SPECT/CT) of the
chest and biochemical analysis of serum and urine for monoclonal gammopathy/light
chain amyloidosis (Figure 1). The latter included serum and urine immunofixation, serum
electrophoresis, and serum free light chain assay. Both scintigraphy and biochemical
analysis were performed during the index hospitalization for TAVR or shortly thereafter
but not later than 3 months post-TAVR. 99m Tc-DPD bone scintigraphy was performed using
a hybrid SPECT/CT gamma camera (Symbia T2 and Symbia Intevo, Siemens Medical
Solutions AG, Erlangen, Germany) with low-energy high-resolution collimators following
the weight-adjusted administration of 99m Tc-DPD (Dupharma A/S, Kastrup, Denmark).
The protocol consisted of early (1 h) and late (3 h) planar whole-body imaging with thoracic
SPECT/CT image acquisition after 3 h. The visual Perugini grading system was used for the
evaluation of myocardial tracer uptake on planar images with the following classification:
 not later than 3 months post-TAVR. Tc-DPD bone scintigraphy was performed using a
hybrid SPECT/CT gamma camera (Symbia T2 and Symbia Intevo, Siemens Medical Solu-
tions AG, Erlangen, Germany) with low-energy high-resolution collimators following the
weight-adjusted administration of 99mTc-DPD (Dupharma A/S, Kastrup, Denmark). The pro-
J. Clin. Med. 2024, 13, 671 tocol consisted of early (1 h) and late (3 h) planar whole-body imaging with thoracic 3 of 14
SPECT/CT image acquisition after 3 h. The visual Perugini grading system was used for the
evaluation of myocardial tracer uptake on planar images with the following classification: 0
=0no myocardial
= no myocardial uptake, 1 =1myocardial
uptake, = myocardial uptake lessless
uptake than thethe
than ribrib
uptake,
uptake,2 =2myocardial
= myocardial up-
take equal to rib uptake, 3 = myocardial uptake greater than the rib uptake
uptake equal to rib uptake, 3 = myocardial uptake greater than the rib uptake [16]. The [16]. The heart-
to-contralateral lung (H/CL)
heart-to-contralateral ratio (ratio
lung (H/CL) ratioof(ratio
heartofregions of interest
heart regions to contralateral
of interest lung re-
to contralateral
gion
lungofregion
interest mean counts)
of interest mean was used
counts) forused
was quantitative analysis analysis
for quantitative [17]. The[17].
diagnosis of ATTR-
The diagnosis
CA was established
of ATTR-CA accordingaccording
was established to currenttoguidelines in the caseinof
current guidelines Perugini
the case of grade
Perugini2 orgrade
3 up-
take
2 or with normal
3 uptake withserum
normalfree light free
serum chains kappa
light chainsand lambda
kappa andand free and
lambda lightfree
chain ratio
light and
chain
absence
ratio andofabsence
monoclonal band on serum
of monoclonal band and urine and
on serum immunofixation [10].
urine immunofixation [10].

Figure
Figure1.
[Link]
Screening algorithm
algorithm and
and study inclusion.
study inclusion.

Endomyocardial
Endomyocardial biopsybiopsy was
was additionally performed in in the
the case
caseofofPerugini
Peruginigrade
grade11
uptake
uptakeororinin patients
patients with
with abnormal immunofixation or free free light
light chain
chainassays.
[Link]-
Endomy-
ocardial biopsies were
ocardial biopsies wereperformed
performed in in
thethe supine
supine position
position viacommon
via the the common
femoral femoral vein
vein using
the standard
using Seldinger
the standard canulation
Seldinger technique
canulation and a long
technique and anon-flexible [Link].
long non-flexible A minimum
A min-
of 4 samples
imum were obtained
of 4 samples from the
were obtained fromright
theventricular (RV) septum
right ventricular under under
(RV) septum fluoroscopic
fluoro-
guidance using a flexible bioptome. Genetic testing was performed using
scopic guidance using a flexible bioptome. Genetic testing was performed using genomic genomic DNA
from peripheral
DNA blood blood
from peripheral in patients with ATTR-CA.
in patients The exons
with ATTR-CA. The of the transthyretin
exons (TTR)
of the transthyretin
gene were
(TTR) geneamplified and sequenced
were amplified by single-gene
and sequenced Sanger sequencing.
by single-gene Further laboratory
Sanger sequencing. Further la-
analyzes analyzes
boratory were performed as per institutional
were performed protocol prior
as per institutional to TAVR
protocol priorand included
to TAVR andNT-in-
proBNP, hs-cTnI, complete blood count, serum albumin, and renal and
cluded NT-proBNP, hs-cTnI, complete blood count, serum albumin, and renal and liverliver function testing.
Electrocardiograms
function were recorded before
testing. Electrocardiograms wereand within 24
recorded h after
before and TAVR.
withinThe24Sokolow–Lyon
h after TAVR.
index was calculated as the sum of the S-wave in lead V1
The Sokolow–Lyon index was calculated as the sum of the S-wave in lead and the tallest R-waveV1 in
and lead
the
V5 or V6 [18]. Low voltage was defined as a QRS amplitude of ≤0.5 mV in all peripheral
tallest R-wave in lead V5 or V6 [18]. Low voltage was defined as a QRS amplitude of ≤0.5
leads [19]. The voltage/mass ratio (VMR) was calculated by dividing the value of the
mV in all peripheral leads [19]. The voltage/mass ratio (VMR) was calculated by dividing
Sokolow–Lyon index on ECG by the LV mass index (LVMI) on echocardiography. Transtho-
the value of the Sokolow–Lyon index on ECG by the LV mass index (LVMI) on echocardi-
racic echocardiography for evaluation of AS severity, concomitant valvular pathologies,
ography. Transthoracic echocardiography for evaluation of AS severity, concomitant
measurements of cardiac diameters, and systolic and diastolic function were performed in
accordance with the latest recommendations [20–22]. Qlab 10 software (Philips Electronics,
Eindhoven, the Netherlands) was used for offline strain analysis. Apical two-, three-, and
four-chamber views were obtained and stored for the analysis of LV global longitudinal
strain (GLS).
 valvular pathologies, measurements of cardiac diameters, and systolic and diastolic func-
tion were performed in accordance with the latest recommendations [20–22]. Qlab 10 soft-
ware (Philips Electronics, Eindhoven, the Netherlands) was used for offline strain analy-
sis. Apical two-, three-, and four-chamber views were obtained and stored for the analysis
J. Clin. Med. 2024, 13, 671 of LV global longitudinal strain (GLS). 4 of 14

2.3. Statistical Analysis

2.3. Statistical
Continuous Analysis
variables were summarized as the mean ± standard deviation or median
Continuous
(interquartile variables
range (IQR))were summarized
and categorical as the mean
variables ± standard
as counts deviation orContinu-
and percentages. median
(interquartile
ous data wererange (IQR))for
evaluated andnormality
categorical
ofvariables as counts
distribution andShapiro–Wilk
with the percentages. Continuous
test and by
data were of
inspection evaluated
the [Link]
Thenormality
two-sidedoft-test
distribution
was usedwith the Shapiro–Wilk
for comparison test andnor-
of continuous, by
inspection of the plots. The two-sided t-test was used for comparison
mally distributed data, otherwise the non-parametric Mann–Whitney U test. The x test of continuous,
2

normally
was useddistributed data, otherwise
to test the association the non-parametric
between Mann–Whitney
categorical variables. x2 test
U test. Themethod
The Kaplan–Meier
was
was used
used to
fortest the association
survival between
analysis. The levelcategorical variables.
of significance was setThe Kaplan–Meier
at 0.05. All analyzesmethod
were
was used for survival analysis. The level of significance
performed using SPSS Version 28 (IBM Corp., Armonk, NY, USA). was set at 0.05. All analyzes were
performed using SPSS Version 28 (IBM Corp., Armonk, NY, USA).
3. Results
3. Results
3.1. Study
3.1. Study Population
Population
During aa 24-month
During 24-month period,
period, 101
101 of
of 264
264 consecutive
consecutive patients
patients met
met the
the study
study criteria
criteria and
and
providedwritten
provided writtenconsent.
[Link] Weexcluded
excluded1515 patients
patients whowho fulfilled
fulfilled thethe study
study inclusion
inclusion cri-
criteria
teria but did not undergo bone scintigraphy for logistical reasons (COVID-19
but did not undergo bone scintigraphy for logistical reasons (COVID-19 related restrictions, related re-
strictions, tracer availability issues) and one patient who died before the scheduled
tracer availability issues) and one patient who died before the scheduled scintigraphy. The scin-
tigraphy. The
analytical cohortanalytical
includedcohort included
85 patients 85 patients
(Figure (Figure 2).
2). The majority of The majority
patients of patients
fulfilled at least
one major criterion for study inclusion, and 42% had both two minor and atminor
fulfilled at least one major criterion for study inclusion, and 42% had both two and
least one
at leastcriteria.
major one major criteria.
Table Table 1 demonstrates
1 demonstrates the distribution
the distribution of thecriteria.
of the inclusion inclusion criteria.
The most
The most commonly
commonly documented documented criterion
criterion was was NT-proBNP
NT-proBNP >1000 pg/mL >1000(84%),
pg/mLfollowed
(84%), followed
by CTS
by CTSOnly
(28%). (28%). 3%Only 3% of patients
of patients were included
were included in the
in the study study
based based
only onlycombination
on the on the combi- of
nation of two
two minor [Link] criteria.

Figure 2.
Figure 2. Derivation of the analytic cohort of the study.
study.

Table 1. Distribution of major and minor criteria for inclusion in the study.

All Lone AS AS and ATTR-CA

p-Value
n = 85 n = 79 n=6
Major criteria
CTS, n (%) 24 (28) 19 (24) 5 (83) 0.003
Biceps tendon rupture, n (%) 16 (19) 15 (19) 1 (17) 0.887
Spinal stenosis, n (%) 16 (19) 14 (18) 2 (33) 0.379
J. Clin. Med. 2024, 13, 671 5 of 14

Table 1. Cont.

All Lone AS AS and ATTR-CA

p-Value
n = 85 n = 79 n=6
NT-proBNP > 1000 pg/mL 71 (84) 65 (82) 6 (100) 0.134
Hs-cTnI > 99th percentile 20 (24) 17 (22) 3 (50) 0.142
Minor Criteria
AF, n (%) 50 (58.8) 45 (57) 5 (83) 0.206
Diastolic dysfunction ≥2 grade or lateral e’ <10 cm/s 40 (52) 35 (49) 5 (83) 0.094
Atrioventricular block or previously implanted pacemaker 25 (29) 22 (28) 3 (50) 0.272
Inclusion reason (criteria)
1 major 21 (24.7) 21 (26.6) 0 (0) 0.059
2–3 major 24 (28.2) 23 (29.1) 1 (16.7) 0.493
4–5 major 1 (1.2) 1 (1.3) 0 (0) 0.701
2 minor only 3 (3.5) 3 (3.8) 0 (0) 0.504
2 minor + ≥1 major 36 (42.4) 31 (39.2) 5 (83.3) 0.032
AF = atrial fibrillation, AS = aortic stenosis, ATTR-CA = transthyretin cardiac amyloidosis, CTS = carpal tunnel
syndrome, hs-cTnI = high-sensitivity cardiac troponin I, NT-proBNP = N-terminal pro B-type natriuretic peptide.

3.2. Baseline Characteristics

Baseline clinical characteristics are demonstrated in Tables 2 and 3. The mean age was
82 years, and 60% of the patients were male. Study patients were highly symptomatic (88%
with NYHA class ≥3) and carried a high burden of comorbidities. The median EuroSCORE
II was 5.27 (IQR: 3.88–8.44). After evaluation by the heart team, 79 (93%) patients underwent
TAVR. Six patients (7%) who were initially referred for TAVR underwent surgical aortic
valve replacement (SAVR). The most common AS phenotype was classical high-gradient
AS (64%), followed by low-flow, low-gradient AS with reduced EF (20%), low-flow, low-
gradient AS with preserved EF (15%), and normal-flow, low-gradient AS with preserved
EF (1%).

Table 2. Baseline characteristics.

All Lone AS AS and ATTR-CA

p-Value
n = 85 n = 79 n=6
Age, years 81.7 ± 5.2 81.5 ± 5.2 84.8 ± 4.2 0.123
Male sex, n (%) 51 (60) 45 (57) 6 (100) 0.011
BMI, kg/m2 27.1 ± 4.9 27.3 ± 0.6 24.2 ± 1.5 0.138
Euroscore II 5.3 (3.9–8.4) 5.3 (3.8–8.4) 5.1 (4.4–15.6) 0.498
NYHA class, n (%)
I 1 (1) 1 (1) 0 (0)
II 9 (11) 9 (11) 0 (0) 0.593
III 73 (86) 67 (85) 6 (100)
IV 2 (2) 2 (3) 0 (0)
History of syncope, n (%) 8 (9) 5 (6) 3 (50) 0.010
AS phenotype
High gradient, n (%) 54 (64) 51 (65) 3 (50) 0.483
LFLG with reduced EF, n (%) 17 (20) 15 (19) 2 (33) 0.425
LFLG with preserved EF, n (%) 13 (15) 12 (15) 1 (17) 0.924
Normal-flow low-gradient with preserved EF, n (%) 1 (1) 1 (1) 0 (0) 0.701
J. Clin. Med. 2024, 13, 671 6 of 14

Table 2. Cont.

All Lone AS AS and ATTR-CA

p-Value
n = 85 n = 79 n=6
Comorbidities
Peripheral neuropathy, n (%) 9 (11) 8 (10) 1 (17) 0.638
Pre-interventional pacemaker, n (%) 11 (13) 9 (11) 2 (33) 0.177
Hypertension, n (%) 76 (89) 70 (89) 6 (100) 0.237
Diabetes mellitus, n (%) 19 (22) 18 (23) 1 (17) 0.720
Dyslipidaemia, n (%) 51 (60) 46 (58) 5 (83) 0.201
Atrial fibrillation, n (%) 50 (59) 45 (57) 5 (83) 0.206
Coronary artery disease, n (%) 62 (73) 57 (72) 5 (83) 0.534
Previous stroke, n (%) 6 (7) 6 (8) 0 (0) 0.340
Chronic kidney disease, n (%) 36 (42) 33 (42) 3 (50) 0.696
Anemia, n (%) 30 (35) 27 (34) 3 (50) 0.444
AS = aortic stenosis, ATTR-CA = transthyretin cardiac amyloidosis, BMI = body mass index, EF = ejection fraction,
LFLG = low-flow low-gradient, NYHA = New York Heart Association.

Table 3. Laboratory, echocardiographic, and electrocardiographic findings prior to TAVR.

All Lone AS AS and ATTR-CA

p-Value
n = 85 n = 79 n=6
Laboratory testing
NT-proBNP (pg/mL) 2518 (1340.5–5319.5) 2449 (1256–5274) 4081.5 (1868.8–18771.5) 0.181
BNP (pg/mL) 265.7 (163.3–420) 251.6 (153–386) 589.9 (294.1–1007.4) 0.035
Hs-cTnI (ng/L) 18 (11–42.8) 18 (10.8–41.3) 47 (10.8–62.5) 0.305
eGFR (mL/min/1.73 m2 ) 56.5 ± 20.8 56.7 ± 20.8 53.7 ± 21.6 0.739
Creatinine (mg/dL) 1.3 ± 0.7 1.3 ± 0.7 1.4 ± 0.5 0.807
Haemoglobin (g/dL) 11.9 ± 1.9 11.9 ± 1.9 11.3 ± 2.1 0.463
Albumin (mg/L) 3.6 ± 0.4 3.6 ± 0.5 3.8 ± 0.2 0.374
Monoclonal immunoglobulin, n (%) 17 (20) 14 (18) 3 (50) 0.093
Echocardiography
LVEF (%) 52 ± 8.8 52 ± 9.1 52 ± 5.1 0.992
GLS (%) −12.9 ± 3.6 −13 ± 3.7 −12.1 ± 2.4 0.624
MCF (%) 28.3 ± 13.3 27.4 ± 12 24.1 ± 12.7 0.524
Pericardial effusion, n (%) 10 (12) 6 (8) 4 (67) <0.001
LVDd (cm) 4.8 ± 0.8 4.8 ± 0.9 4.7 ± 0.5 0.772
RVDd (mm) 4.10 ± 0.73 3.91 ± 0.71 4.5 ± 0.74 0.029
IVSd (mm) 16 ± 2.5 16 ± 2.4 18 ± 3.5 0.075
LVMI (g/m2 ) 150.6 ± 40.3 149.7 ± 40.5 162.1 ± 40.1 0.473
LAVI (ml/m2 ) 55.1 ± 23.4 55.5 ± 24.1 50.2 ± 11.7 0.590
RA dimension (mm) 4.4 ± 0.8 4.3 ± 0.7 5 ± 1.4 0.052
SV (mL) 69.9 ± 23.3 70.1 ± 23.4 67 ± 23.1 0.749
Aortic valve area, cm2 0.7 ± 0.2 0.7 ± 0.2 0.6 ± 0.2 0.301
AV MPG, mmHg 39.9 ± 16.7 40.5 ± 17.1 31.1 ± 7.2 0.184
AV Vmax, m/s 4 ± 0.8 4.1 ± 0.8 3.4 ± 1 0.062
E velocity (cm/s) 102.3 ± 40.7 102 ± 41.5 106.1 ± 33.3 0.813
E’ (cm/s) 8.6 ± 2.8 8.6 ± 2.9 8.2 ± 1.5 0.756
Lateral E/e’ 11.9 ± 4.7 11.8 ± 4.8 13.6 ± 1.5 0.471
Peak TR velocity, (m/s) 2.9 (2.5–3.4) 2.9 (2.6–3.4) 2.7 (2.3–3.6) 0.557
sPAP (mmHg) 42 ± 15.7 42.1 ± 14.9 41.7 ± 24.2 0.969
TAPSE (mm) 20.2 ± 5.4 20.2 ± 5.5 19.5 ± 4.7 0.749
Right ventricular s’ (cm/s) 10.2 ± 2.1 10.4 ± 2.3 9.4 ± 0.9 0.403
J. Clin. Med. 2024, 13, 671 7 of 14

Table 3. Cont.

All Lone AS AS and ATTR-CA

p-Value
n = 85 n = 79 n=6
ECG
Atrial fibrillation/flutter, n (%) 29 (34) 28 (35) 1 (17) 0.323
Heart rate (beats/min) 74 ± 12 74 ± 13 72 ± 10 0.693
AV block ≥2 grade, n (%) 17 (20) 15 (19) 2 (33) 0.425
PQ duration, ms * 191 ± 42 188 ± 39 235 ± 69 0.062
QRS duration, ms * 110 ± 36 108 ± 33 139 ± 56 0.038
RBBB, n (%) * 13 (15) 12 (15) 1 (17) 0.780
LBBB, n (%) * 10 (12) 10 (13) 0 (0) 0.252
LAFB, n (%) * 15 (18) 15 (19) 0 (0) 0.154
Low QRS voltage, n (%) * 9 (11) 9 (13) 0 (0) 0.376
Sokolow–Lyon index, mV * 2.1 ± 0.8 2.1 ± 0.8 2.6 ± 0.2 0.259
RWT/SaVR index * 0.07 (0.05–0.1) 0.07 (0.05–0.1) 0.08 (0.06–0.1) 0.398
VMR, mV/g/m2 × 10−2 * 1.5 ± 0.6 1.5 ± 0.7 1.7 ± 0.3 0.709
AS = aortic stenosis, ATTR-CA = transthyretin cardiac amyloidosis, AV = aortic valve, AV Vmax = aortic peak
velocity, BNP = B-type natriuretic peptide, ECG = electrocardiogram, eGFR = estimated glomerular filtration
rate, GLS = global longitudinal strain, hs-cTnI = high-sensitivity cardiac troponin I, IVSd = interventricular
septum thickness, LAFB = left anterior fascicular block, LAVI = left atrial volume index, LBBB = left bun-
dle branch block, LVDd = left ventricular end-diastolic diameter, LVEF = left ventricular ejection fraction,
LVMI = left ventricular mass index, MCF = myocardial contraction fraction, MPG = mean pressure gradient,
NT-proBNP = N-terminal pro B-type natriuretic peptide, RA = right atrium/right atrial, RBBB = right bundle
branch block, RVDd = right ventricular end-diastolic diameter, RWT = relative wall thickness, SaVR = ECG S-wave
from aVR, sPAP = systolic pulmonary artery pressure, SV = stroke volume, TAPSE = tricuspid annular plane sys-
tolic excursion, TAVR = transcatheter aortic valve replacement, TR = tricuspid regurgitation, TTE = transthoracic
echocardiography, VMR = voltage/mass ratio. * Analysis in patients with non-paced rhythm.

3.3. The Prevalence of CA

99m Tc-DPD scintigraphy detected myocardial uptake (Perugini grade ≥1) in nine
patients (11%). Grade 1 was detected in two, grade 2 in two, and grade 3 in five patients.
An endomyocardial biopsy was performed in four of nine patients to confirm or rule out
amyloidosis according to the current guidelines. In three of these patients no amyloid
was found by biopsy, yielding an ATTR-CA prevalence of 7%. Patients with ATTR-CA
had a significantly higher H/CL ratio (1.2 vs. 2.7, p < 0.001). There were no differences in
the amount of radioactivity used (Table 4). Monoclonal gammopathy or a pathologic free
light chain assay were detected in 17 (20%) patients who underwent further evaluation
by hematologists with expertise in amyloidosis and plasma cell diseases. Light chain
amyloidosis was excluded clinically or via biopsy/bone marrow studies in all cases. All
but one patient consented to TTR gene sequencing. In all five cases, wild-type ATTR-CA
was diagnosed.

Table 4. Scintigraphic imaging findings.

All Lone AS AS and ATTR-CA

p-Value
n = 85 n = 79 n=6
DPD scintigraphy parameters
Perugini grade, n (%)
0 76 (89.4) 76 (96.2) 0 (0)
1 2 (2.4) 2 (2.5) 0 (0) <0.001
2 2 (2.4) 1 (1.3) 1 (16.7)
3 5 (5.9) 0 (0) 5 (83.3)
H/CL ratio 1.4 ± 0.6 1.2 ± 0.4 2.7 ± 0.9 <0.001
Radioactivity (MBq) 567.5 ± 83.7 565.8 ± 85.8 589.7 ± 48.9 0.505
AS = aortic stenosis, ATTR-CA = transthyretin cardiac amyloidosis, DPD = 99m technetium-3,3-diphosphono-1,2-
propanodicarboxylic acid, H/CL ratio = heart-to-contralateral lung ratio.
J. Clin. Med. 2024, 13, 671 8 of 14

3.4. Clinical Features of Lone AS vs. AS-ATTR

Patients with dual AS-ATTR pathology were male (100% in AS-ATTR vs. 57% in
lone AS, p = 0.011). No differences were observed between ATTR amyloidosis and non-
amyloidosis patients regarding the NYHA functional class (Table 2). Syncope was reported
more frequently in AS-ATTR patients (50% vs. 6%, p = 0.010). No differences were observed
between ATTR and AS-ATTR patients regarding the above-mentioned major and minor
criteria/amyloid red flags, except for CTS, which was more common among patients
with amyloidosis (83% vs. 24%, p = 0.003). Levels of BNP were significantly higher
in AS-ATTR patients (251.6 pg/mL (IQR: 153–386) vs. 589.9 pg/mL (IQR: 294–1007),
p = 0.035) and a trend toward higher levels of NT-proBNP (2449 pg/mL (IQR: 1256–5274) vs.
4081.5 pg/mL (IQR: 1868.8–18771.5), p = 0.181) and troponin I (18 pg/mL (IQR: 10.8–41.3)
vs. 47 pg/mL (IQR: 10.8–62.5)) was shown in patients with ATTR-CA compared to those
without amyloidosis (Table 3).
Regarding the electrocardiographic features, no differences were observed between
the two groups. None of the AS-ATTR patients met electrocardiographic criteria for low
voltage. VMR and Sokolow–Lyon, among other indices, did not differ between the groups.
There were no differences in hemodynamic measures of AS (aortic valve area 0.7 cm2
vs. 0.63 cm2 , p = 0.301, mean pressure gradient 40.54 mmHg vs. 31.08 mmHg, p = 0.184,
peak aortic valve velocity 4.08 m/s vs. 3.44 m/s, p = 0.062). The prevalence of either
classical low-flow, low-gradient AS with reduced EF (19% vs. 33.3%, p = 0.425) or low-flow,
low-gradient AS with preserved EF (15.2% vs. 16.7%, p = 0.924) was similar among the
two groups. Pericardial effusion was more common in the AS-ATTR group (8% vs. 67%,
p < 0.001). There were no differences in the measures of LV systolic function (LVEF 52% vs.
52%, p = 0.992, MCF 27.38% vs. 24.12%, p = 0.524, GLS −12.98% vs. −12.05%, p = 0.624) and
right ventricular (RV) function (TAPSE 20.24 mm vs. 19.5 mm, p = 0.749, RV s’ 10.43 cm/s
vs. 9.4 cm/s, p = 0.403). Patients with dual AS-ATTR had higher septal thickness, but
this did not reach clinical significance (IVST 1.56 cm vs. 1.75 cm, p = 0.075, PWT 1.28 cm
vs. 1.37 cm, p = 0.503, LVMI 149.72 g/m2 vs. 162.08 g/m2 , p = 0.473, RWT 0.55 vs. 0.6,
p = 0.562). The measures of diastolic function did not differ significantly (Table 3).

3.5. Periprocedural Complications and Mortality

Complications between lone AS and dual AS-ATTR occurred with similar rates, includ-
ing new-onset conduction disorders requiring pacemaker implantation that were observed
in eight subjects without amyloidosis (11%). One-third of AS-ATTR patients had a pace-
maker implanted before the valve procedure (Table 5). Thirty-day survival after TAVR was
100%. Three patients died within the first 30 days post-SAVR. During a median follow-up
of 18 (10–23) months, nine (11%) patients (all with lone AS) died. The all-cause mortality of
lone AS vs. AS-ATTR was 12% and 0%, respectively, p = 0.228 (Figure 3).

Table 5. Procedural complications.

All Lone AS AS and ATTR-CA

p-Value
n = 85 n = 79 n=6
New-onset conduction disorders
8/74 (10.8) 8/70 (11.3) 0/4 (0) 0.332
warranting pacemaker implantation, n (%) *
Access-site bleeding, n (%) 6 (7.1) 6 (7.7) 0 (0) 0.336
Acute kidney injury, n (%) 11 (12.9) 11 (14.1) 0 (0) 0.186
Stroke/TIA, n (%) 1 (1.2) 1 (1.3) 0 (0) 0.699
ATTR-CA = transthyretin-type cardiac amyloidosis, AS = aortic stenosis, TIA = transient ischemic attack,
TAVR = transcatheter aortic valve replacement. * No pacemaker prior to TAVR: 74 in total study population, 70
with lone AS and 4 with AS and ATTR-CA.
J.J. Clin.
Clin. Med.
Med. 2024,
2024, 13,
13, 671
x FOR PEER REVIEW 9 of 14
9 of 14

Figure 3. Kaplan–Meier
Kaplan–Meiersurvival
survivalanalysis
analysisfor
forlone ASAS
lone [Link].
dual AS-ATTR
dual AS-ATTRpathology. AS =AS
pathology. aortic
= aortic
stenosis, AS-ATTR
stenosis, AS-ATTR =
= dual
dual pathology
pathology of
of aortic stenosis and
aortic stenosis and transthyretin
transthyretin cardiac
cardiac amyloidosis.
amyloidosis.

4. Discussion
Table 5. Procedural complications.
4.1. The Prevalence of ATTR-CA in Patients with Severe AS
All Lone AS AS and ATTR-CA
The diagnosis of concomitant amyloidosis in patients with severe AS is of paramount p-Value
n = 85 n = 79 n=6
importance and enables the implementation of disease-modifying therapies in addition to
New-onset conduction disorders warranting
valve replacement to achieve 8/74optimal
(10.8) long-term outcomes. Previous
8/70 (11.3) studies reported
0/4 (0) 0.332the
pacemaker implantation, n (%) of
coexistence * amyloidosis in up to 16% of patients with severe AS. Since then, multiple
Access-site bleeding, n (%)have been made to
attempts 6 (7.1)
characterize this 6 (7.7)
population and derive 0 (0) scoring systems 0.336
Acute kidney injury, n (%)
for the identification of dual 11 (12.9) 11 (14.1)
pathology [3–5,7,23,24]. A previously 0 (0)proposed score 0.186for
Stroke/TIA, discrimination
n (%) of lone AS from 1 (1.2) 1 (1.3) CA was shown0to
AS with concomitant (0)have high specificity
0.699
but low sensitivity,
ATTR-CA thus limiting
= transthyretin-type its broader
cardiac clinical
amyloidosis, AS =implementation [7]. =The
aortic stenosis, TIA current
transient study
ischemic
attack, TAVR
shows = transcatheter
that despite prospectiveaortic valve replacement.
systematic screening*for Noamyloid
pacemaker redprior
flags,toan
TAVR: 74 in total
approach that
study
is population, 70
recommended bywith lone AS and
international 4 with AS and
guidelines andATTR-CA.
consensus documents for patients older
than 65 years old [10,11], the ATTR-CA detection rate in severe AS remains lower than that
4. Discussion
previously reported in all comers with severe AS. With this approach, fourteen patients with
severe
4.1. The Prevalence ofAS
degenerative should undergo
ATTR-CA in Patients diagnostic
with Severe work-up
AS for one patient to be diagnosed
with The
ATTR-CA.
diagnosis Theoflow observedamyloidosis
concomitant ATTR-CA prevalence
in patientsinwithoursevere
studyAS may beparamount
is of attributed
to the slightly younger patient cohort of the current study. The
importance and enables the implementation of disease-modifying therapies in addition mean age was 82 years,
to
lower than that reported in previous studies (83.6–85 years) [4,6,7].
valve replacement to achieve optimal long-term outcomes. Previous studies reported This hypothesis is also
the
supported by CA screening studies in SAVR patients [3,24] that demonstrated a much lower
coexistence of amyloidosis in up to 16% of patients with severe AS. Since then, multiple
prevalence, e.g., 6% in a patient cohort with a median age of 75 years. Post-mortem studies
attempts have been made to characterize this population and derive scoring systems for
confirm a higher prevalence of amyloidosis in octogenarians and further support the age-
the identification of dual pathology [3–5,7,23,24]. A previously proposed score for dis-
dependent prevalence of ATTR-CA [9]. Further, it is possible that previous studies on all
crimination of lone AS from AS with concomitant CA was shown to have high specificity
comers with severe AS overestimated the prevalence of ATTR-CA due to selection bias or
but low sensitivity, thus limiting its broader clinical implementation [7]. The current study
false positive scintigraphic findings. The latter was also observed in our study in a patient
shows that despite prospective systematic screening for amyloid red flags, an approach
with grade 2 uptake who was later proven negative for amyloid by an endomyocardial
that is recommended by international guidelines and consensus documents for patients
biopsy. It is not known if AS and the accompanying myocardial fibrosis could lead to
older than 65 years old [10,11], the ATTR-CA detection rate in severe AS remains lower
false-positive DPD scintigraphy, but false-positive studies were previously reported in
than that previously reported in all comers with severe AS. With this approach, fourteen
patients after acute myocardial infarction [25].
patients with severe degenerative AS should undergo diagnostic work-up for one patient
to beThe
4.2. diagnosed
Predictivewith ATTR-CA.
Value of Amyloid TheRedlow observed ATTR-CA prevalence in our study may
Flags
be attributed to the slightly younger
The current findings provide insight into patient cohort of the current
the potential study.
predictive The of
value mean age
clinical
was 82 years, lower
manifestations known thanto that
servereported
as amyloid in previous
red flagsstudies (83.6–85
in patients who years) [4,6,7].toThis
are thought carryhy-
a
pothesis is also supported by CA screening studies in SAVR patients
higher risk, such as those with severe AS. In this cohort, CTS and NT-proBNP >1000 pg/mL [3,24] that demon-
strated
were thea two
much lower
most prevalence,
prevalent e.g.,
criteria 6% in a patient
associated cohort with
with ATTR-CA in athis
median
cohort; age of 75 years.
however, the
Post-mortem studies confirm a higher prevalence of amyloidosis in octogenarians and
J. Clin. Med. 2024, 13, 671 10 of 14

low number of diagnoses does not allow for the derivation of a predictive model. CTS is a
common condition with a prevalence of 5% in the general population aged 65–74 years [26].
It has been shown that CTS is associated with a future diagnosis of CA and is diagnosed
10–15 years before cardiac impairment [27]. In an extensive analysis of the Danish national
registries, CTS was associated with a 12-fold higher future risk of amyloidosis [28]. Our
results corroborate these findings. However, 80% of patients with AS who had CTS had a
negative DPD scan, suggesting that other etiologies of CTS may limit its predictive value in
older patients. Similar to CTS, a biceps tendon rupture may represent ATTR deposition. It
was observed in 33–44% of patients with diagnosed ATTR-CA and occurred 5 years before
the diagnosis of cardiac involvement [13,29]. The value of age-related conditions such as
spinal stenosis and biceps tendon rupture in discriminating ATTR-CA from lone AS is still
unclear, and prospective data regarding their exact prevalence in systemic amyloidosis are
lacking. A possible hypothesis is that an isolated soft-tissue type of ATTR-CA may be more
prevalent than ATTR with organ involvement, thus limiting the overall predictability of
soft-tissue manifestations.

4.3. Prognostic Implications of Dual Pathology: AS and ATTR-CA

Long-term prognosis of patients with severe AS and ATTR-CA are not available so
far. The coexistence of ATTR-CA has been associated with notably higher rates of HF
hospitalization compared to that with lone AS [7,30]. Regarding mortality, inconsistent
data have been reported. Several studies have reported similar survival of patients with
dual pathology and lone AS [6,7,31]. In a retrospective study on patients with CA with
moderate and severe AS, ATTR-CA was associated with higher mortality at one year
(56% vs. 20%, p < 0.0001). After adjustment for potential confounders, CA remained
an independent predictor of all-cause mortality [30]. Nitsche et al. reported a trend
toward higher 1-year mortality (25.0% vs. 13.9%, log-rank p = 0.05) [7]. TAVR significantly
improves outcomes in both lone AS and AS-ATTR patients [6,7,32] but AS-ATTR patients
remain more symptomatic at 12 months after TAVR [33], further justifying the need for
disease-modifying pharmacotherapies in AS-ATTR. From a pathophysiological point of
view, it is possible that TAVR could lead to partial stabilization of amyloidogenesis after
the reduction of AS-induced myocardial strain that could favor amyloid tissue infiltration.
On the other hand, reducing amyloid load could potentially play a role in the management
of AS. Amyloid may contribute to AS progression, as demonstrated in a study where
surgically resected heart valve specimens were analyzed, revealing amyloid deposits in
55% of the specimens, with the highest prevalence observed in AS [34]. In our study, we
found no difference in all-cause mortality at the 18-month follow up, although the small
study sample limits the interpretation of this finding. The ATTR stabilizer tafamidis was
prescribed in the ATTR-CA group and may have catalyzed this outcome.
All patients in our study with a dual pathology of AS-ATTR were highly symptomatic
(NYHA stage III in all). At the current stage, it is challenging to determine whether
screening for ATTR-CA in such highly symptomatic patients is prognostically useful, as
therapeutic options are limited. On the other hand, therapies currently in development to
induce amyloid removal in ATTR-CA could potentially be used to treat patients in more
advanced disease stages.
Recently, machine learning approaches based on clinical, laboratory, and imaging
data have increasingly been employed to suspect CA. Machine learning has enabled the
early identification of ATTRv and helped identify patients with neuropathy who should
undergo genetic testing for ATTRv [35,36]. Furthermore, an ECG-based tool has effectively
suspected CA with an area under the curve of 0.97 [37]. Similarly, a deep learning approach
discriminated between amyloidosis and hypertrophic cardiomyopathy [38]. Given the
increasing number of patients, automated machine learning approaches could potentially
be employed for the early identification of patients with cardiac amyloidosis in the future.
It remains challenging to identify patients with AS who have the highest probability
of dual pathology of AS and ATTR-CA. We have demonstrated that the classical amyloi-
J. Clin. Med. 2024, 13, 671 11 of 14

dosis features were not of high predictive value in a population of patients with AS. Our
findings highlight the unmet need for novel and specific amyloidosis markers in patients
with AS. The true prevalence of ATTR-CA in AS should be prospectively investigated in
larger studies based on a method that ensures the highest diagnostic accuracy, such as
endomyocardial biopsy.

4.4. Limitations
This study was conducted at a referral tertiary center, thus inducing a potential
selection bias and leading to the underrepresentation of the general population of AS
patients. The study had a relatively high dropout rate. The small patient numbers in the
AS-ATTR group limit statistical power to allow for a multivariate predictive model. A
control group of patients without red flags and a larger study sample would be necessary
for this study to be conclusive regarding the true prevalence of ATTR-CA and the value
of this red flag-based screening approach. Given the false-negative scintigraphy findings
in patients with certain mutations, this could potentially have led to the underestimation
of ATTRv, as genetic testing was only performed in patients with positive (Perugini > 1)
bone scans, despite its low prevalence in the elderly. We excluded patients with high
blood pressure values in whom hypertension-induced hypertrophy may exist. Although
patients with ATTR-CA, especially in more advanced disease stages, typically experience
low blood pressure, some might still be hypertensive. This could potentially have led to
an underdiagnosis of ATTR-CA in this patient cohort. We used a septal thickness cutoff
of >13 mm based on the recommendations available at the time when study inclusion
was ongoing. However, increasing evidence suggests that in some cases, ventricular
hypertrophy might only be mild. Our results should be considered hypothesis generating
and emphasize that more precise screening algorithms are needed beyond clinical red flags
to effectively identify ATTR-CA in patients with AS.

5. Conclusions
Targeted screening for ATTR-CA in patients with AS and amyloid red flags does not
yield higher detection rates than those reported previously in unselected AS populations.
Unmasking CA in patients with AS using red flags proves to be challenging. A comprehen-
sive approach is needed to enhance diagnostic accuracy. Beyond traditional manifestations
linked to amyloidosis, precise profiling of this dual entity is needed to identify specific
disease markers. The true prevalence of ATTR-CA in AS remains unclear and has to be
investigated in prospective studies and compared with age-matched controls.

Author Contributions: A.M.J., M.P., P.L. and T.R. were responsible for the study design, data
preparation and interpretation, and drafted the manuscript. J.K.V., R.A.J., A.C., C.R. and K.H. drafted
a part of the manuscript and revised it for important intellectual content. All authors have read and
agreed to the published version of the manuscript.
Funding: This work was supported by the Universitaetsmedizin Essen Clinician Scientist Academy
(UMEA) and the German Research Foundation (DFG, Deutsche Forschungs-Gemeinschaft) (grant nr:
FU356/12-1 to M.P. and A.M.J.).
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki and approved by the Ethics Committee of University of Duisburg-Essen
(protocol code 20-9268-BO, date of approval 21 July 2020).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data availability is constrained by the absence of patient approvals,
thereby precluding the sharing of research data in compliance with ethical considerations.
Conflicts of Interest: The authors declare no conflicts of interest.
J. Clin. Med. 2024, 13, 671 12 of 14

Abbreviations

AS-ATTR (dual pathology of) aortic stenosis and transthyretin-type cardiac amyloidosis
ATTR-CA transthyretin-type cardiac amyloidosis
DPD 99m technetium-3,3-diphosphono-1,2-propanodicarboxylic acid

hs-cTnI high-sensitivity cardiac troponin I

NT-proBNP N-terminal pro B-type natriuretic peptide
SAVR surgical aortic valve replacement
TAVR transcatheter aortic valve replacement
TTR transthyretin

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