JACC: CARDIOONCOLOGY VOL. 3, NO.

4, 2021

ª 2021 THE AUTHORS. PUBLISHED BY ELSEVIER ON BEHALF OF THE AMERICAN

COLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER

THE CC BY-NC-ND LICENSE (http://creativecommons.org/licenses/by-nc-nd/4.0/).

ORIGINAL RESEARCH

Concomitant Transthyretin Amyloidosis

and Severe Aortic Stenosis in Elderly
Indian Population
A Pilot Study

Aayush Kumar Singal, MD, DM,a Raghav Bansal, MD, DM,a Avinainder Singh, MD, MMSC,b
Sharmila Dorbala, MD, MPH,c Gautam Sharma, MD, DM,a Kartik Gupta, MD,d Anita Saxena, MD, DM,a
Balram Bhargava, MD, DM,a,e Ganesan Karthikeyan, MD, DM, MSC,a Sivasubramanian Ramakrishnan, MD, DM,a
Akshay Kumar Bisoi, MS, MCH,f Milind Padmakar Hote, MS, MCH,f Palleti Rajashekar, MS, MCH,f
Ujjwal Kumar Chowdhury, MS, MCH,f Velayoudam Devagourou, MS, MCH,f Chetan Patel, MD,g Ruma Ray, MD,h
Sudheer Kumar Arawa, MD,h Sundeep Mishra, MD, DMa

ABSTRACT

BACKGROUND Prevalence of both degenerative severe aortic stenosis (AS) and transthyretin cardiac amyloidosis
(ATTR-CA) increases with age. Dual disease (ASþmyocardial ATTR-CA) occurs in significant proportion of patients un-
dergoing surgical aortic valve replacement (SAVR).

OBJECTIVES This study aimed to determine the prevalence of ATTR-CA in severe AS in the Indian population, identify
noninvasive predictors of its diagnosis, and understand its impact on prognosis.

METHODS Symptomatic severe AS patients aged $65 years undergoing SAVR were enrolled. ATTR-CA diagnosis was
based on preoperative 99m-technetium pyrophosphate (PYP) scan and intraoperatively obtained basal interventricular
septum biopsy for myocardial ATTR-CA, and excised native aortic valve for isolated valvular ATTR-CA. Primary
amyloidosis was excluded by serum/urine protein electrophoresis with serum immunofixation.

RESULTS SAVR was performed in 46 AS patients (age 70  5 years, 70% men). PYP scan was performed for 32 patients,
with significant PYP uptake in 3 (n ¼ 3 of 32, 9.4%), suggestive of myocardial ATTR-CA. On histopathological exami-
nation, none of the interventricular septum biopsy specimens had amyloid deposits, whereas 33 (71.7%) native aortic
valves showed amyloid deposits, of which 19 (57.6%) had transthyretin deposition suggestive of isolated valvular
amyloidosis. Noninvasive markers of dual disease included low myocardial contraction fraction (median [interquartile
range], 28.8% [23.8% to 39.1%] vs 15.3% [9.3% to 16.1%]; P ¼ 0.006), deceleration time (215 [144 to 236] ms vs 88
[60 to 106] ms; P ¼ 0.009) and global longitudinal strain (-18.7% [-21.1% to -16.9%] vs -14.2% [-17.0% to -9.7%];
P ¼ 0.030). At 1-year follow-up, 2 patients died (4.3%); 1 each in myocardial ATTR-CA negative and positive groups
(3.4% vs 33.3%; P ¼ 0.477).

CONCLUSIONS Dual disease is not uncommon in India. Isolated valvular amyloidosis in severe AS is much
more common. (J Am Coll Cardiol CardioOnc 2021;3:565–576) © 2021 The Authors. Published by Elsevier on behalf
of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

From the aDepartment of Cardiology, AIIMS, New Delhi, India; bDepartment of Internal Medicine, Yale School of Medicine, New
Haven, Connecticut, USA; cDepartment of Radiology, Brigham and Women’s Hospital, Boston, Massachusetts, USA; dDepartment of
Medicine, Henry Ford Hospital, Detroit, Michigan, USA; eIndian Council of Medical Research, New Delhi, India, and Department of
Health Research (Ministry of Health and Family Welfare), Government of India, New Delhi, India; fDepartment of CTVS, AIIMS, New
Delhi, India; gDepartment of Nuclear Medicine, AIIMS, New Delhi, India; and the hDepartment of Pathology, AIIMS, New Delhi, India.

ISSN 2666-0873 https://doi.org/10.1016/j.jaccao.2021.08.008

566 Singal et al JACC: CARDIOONCOLOGY, VOL. 3, NO. 4, 2021

Dual AS + ATTR-CA in Elderly Indian Patients OCTOBER 2021:565–576

T
ABBREVIATIONS he prevalence of both degenerative METHODS
AND ACRONYMS severe aortic stenosis (AS) and
transthyretin (TTR) cardiac amyloid- This is a single-center prospective observational pilot
99mTc-PYP = 99m-
technetium pyrophosphate
osis (ATTR-CA) increases with age (severe AS study. The study protocol was approved by the
in w3% of patients aged >75 years and ATTR- institutional ethics committee (reference number:
AS = aortic stenosis
CA in w25% of those >80 years) (1-3). Multi- IECPG-441/27.09.2018) and conforms to the ethical
AL-CA = light chain cardiac
amyloidosis ple studies suggest that ATTR-CA may act as guidelines of the 1975 Declaration of Helsinki. All
ATTR-CA = transthyretin
a disease and prognosis modifier in patients patients gave written informed consent. We recruited
cardiac amyloidosis with severe AS (4-7). The prevalence of all consecutive elderly patients ($65 years of age)
EMB = endomyocardial biopsy ATTR-CA is notably higher in patients with with severe AS being planned for SAVR at our center
GLS = global longitudinal severe AS, especially among those with low- between December 2018 and March 2020. Patients
strain flow, low-gradient (LfLg) AS. Up to 6% of who had suspicion of rheumatic etiology or those
IHC = immunohistochemistry those undergoing surgical aortic valve aged <65 years were excluded from the study.
LfLg AS = low-flow, low- replacement (SAVR) and 15% of those under- Because no research work in this field was previ-
gradient aortic stenosis going transcatheter aortic valve replacement ously reported from low and low-middle–income
SAVR = surgical aortic valve (TAVR) have been shown to have ATTR-CA countries such as ours, this was designed as a pilot
replacement
(5,6). Presence of ATTR-CA influences the study, targeting a sample size of 50 patients. By the
TAVR = transcatheter aortic management and clinical course of AS by dis- time the coronavirus disease-2019 pandemic began,
valve replacement
torting the severity assessment of AS and 46 patients had been recruited, and the study was
TTR = transthyretin
also by worsening the prognosis (5,8). stopped at that point. Figure 1 outlines the schematic
Whether this is by chance or there is a cause-and- flow of patients through the study. All patients un-
effect relationship is not clear as of now. However, derwent routine preoperative investigations,
it appears that this coexistence leads to worsening including blood tests, electrocardiogram (ECG), and
of symptoms and a less-than-expected response to basic echocardiographic study.
standard therapy. These patients tend to fare poorly ECHOCARDIOGRAPHY. A detailed 2-dimensional
as compared to those with lone AS, despite aortic (2D) transthoracic echocardiogram with tissue
valve replacement (TAVR or SAVR). Doppler imaging and speckle strain imaging was
With the advent of TAVR, the number of pa- performed (Phillips EPIQ 7C). All echo-based mea-
tients undergoing aortic valve replacement (AVR) surements were performed in accordance with the
is increasing, thus making it pertinent that this local lab protocols which are based on the 2017
group of patients with dual disease (AS þ ATTR- American Society of Echocardiography (ASE) and
CA) is identified preoperatively by noninvasive European Association of Cardiovascular Imaging
methods so that beyond AVR for severe AS, these guidelines (10). Various cut-offs for echo-based
patients are also considered for medications values were derived from the expert consensus rec-
directed against the accumulating myocardial TTR ommendations for multimodality imaging in
deposits. cardiac amyloidosis endorsed by the American Soci-
ATTR-CA is a disease with known racial and ethnic ety of Nuclear Cardiology (ASNC)/American Heart
variations in its distribution and prevalence (9). Association (AHA)/ASE (11). These included left ven-
Considering the lack of data regarding concomitant tricular ejection fraction (LVEF) #50%, myocardial
existence of ATTR-CA and severe AS in the Asian/ contraction fraction (MCF) (ie, the ratio of stroke
Indian context, we planned this study to determine volume to myocardial volume) #30%, relative wall
the prevalence rate of this dual disease in India, thickness (2 $ posterior wall thickness/left ventricular
identify noninvasive predictors of its diagnosis, and [LV] internal diameter) $0.42, E/A ratio $1.5, decel-
study its impact on prognosis. Taking into consider- eration time (dT) #150 ms, mitral annular tissue
ation the burden of senile severe AS in India which Doppler index (TDI) (mean of lateral þ medial
merits AVR, even a small relative percentage would annulus values of S’, e’, a’) all #5 cm/s, right ven-
amount to a large absolute number of patients with tricular S’ (lateral annulus) #10 cm/s, absolute mean
ATTR-CA. global longitudinal strain (GLS) #-15%. Relative

The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’
institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information,
visit the Author Center.

Manuscript received April 15, 2021; revised manuscript received August 15, 2021, accepted August 18, 2021.
JACC: CARDIOONCOLOGY, VOL. 3, NO. 4, 2021 Singal et al 567
OCTOBER 2021:565–576 Dual AS + ATTR-CA in Elderly Indian Patients

F I G U R E 1 Schematic Flow of Patients Through the Study

Symptomatic severe aortic stenosis (AS) patients aged $65 years undergoing surgical aortic valve replacement (SAVR) were enrolled and a
detailed 2-dimensional (2D) transthoracic echocardiogram including speckle tracking with longitudinal strain assessment was performed
preoperatively. Transthyretin cardiac amyloidosis (ATTR-CA) diagnosis was based on preoperative 99m-Technetium pyrophosphate radio-
nuclide scan (PYP) and intraoperatively obtained endomyocardial biopsy (EMB) for myocardial ATTR-CA, and excised native aortic valve for
isolated valvular ATTR-CA. Primary amyloidosis was ruled out by serum/urine protein electrophoresis with serum immunofixation. All
patients were followed up clinically post-SAVR. AVR ¼ aortic valve replacement; HPE ¼ histopathological examination.

apical strain was calculated as: [(mean apical strain)/ endorsed by the ASNC/AHA/ASE, Perugini grade II or
(mean basal strain þ mean mid strain)], and relative III, and H:CL ratio $1.50 were considered as signifi-
apical strain sparing was noted if this value was >1. cantly positive for diagnosis of myocardial ATTR-CA
(11). Perugini grade I was considered borderline posi-
RADIONUCLIDE SCINTIGRAPHY. Standard dose of
tive in this study.
99m-technetium pyrophosphate (PYP) for cardiac im-
aging (10 to 20 mCi) was injected intravenously. Image ENDOMYOCARDIAL BIOPSY. During SAVR, a tissue
acquisition was performed on a GE Infinia Hawkeye 4 sample was obtained under direct vision by per-
dual-head gamma camera mounted with low-energy forming a LV endomyocardial biopsy from the basal
all-purpose (LEAP) collimators with parallel holes. At segment of the interventricular septum (IVS). Also,
1-hour post-injection, both planar and single-photon the excised native aortic valve was sent for histo-
emission computed tomography (SPECT) (rest scans) pathological examination.
were acquired with heart in the center of field of view. HISTOPATHOLOGICAL EXAMINATION. Tissue sam-
If blood pool activity was suspected in the images at 1 ples thus obtained were formalin fixed. Congo red
hour, the scan was repeated at 3 hours post-injection. staining was performed to look for apple green bire-
Time for each projection was 15 to 20 seconds leading fringence suggesting amyloid deposits. Supplemental
to a total scan duration of approximately 20 to 25 mi- Table 2 summarizes the histopathological grading of
nutes. The same imaging procedure was repeated at 3 IVS tissue and aortic valve based on hematoxylin and
hours if required. The scans were visually assessed eosin staining and Congo red staining. For patients in
qualitatively (heart-to-bone ratio, Perugini score) whom the PYP scan was positive, the biopsy speci-
(Supplemental Table 1), and semiquantitatively (heart- mens were independently reviewed by a second se-
to-contralateral-lung [H:CL] uptake, ie, the H:CL ra- nior pathologist.
tio). Based on the expert consensus recommendations For subtyping the amyloid detected on congo red
for multimodality imaging in cardiac amyloidosis staining, immunohistochemistry (IHC) using specific
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Dual AS + ATTR-CA in Elderly Indian Patients OCTOBER 2021:565–576

antibodies against TTR protein was performed on 2D ECHOCARDIOGRAPHY. All the patients under-
samples showing apple green birefringence under went a detailed 2D transthoracic echocardiogram. The
polarized light microscope. TTR-specific antibodies mean LVEF was 55.6  13%. Twelve patients (26%)
(Invitrogen ) were used in a dilution of 1:50. Protocol had LV systolic dysfunction (LVEF #50%). The mean
for IHC analysis including tissue fixation, heat- indexed aortic valve area was 0.37  0.11 cm 2/m 2 and
induced antigen (epitope) retrieval, antibody bind- the mean gradient was 56.5  21 mm Hg. Eight pa-
ing, detection under microscope, and counterstaining tients (17.4%) had LfLg AS. Details are provided
is summarized in Supplemental Figure 1. in Table 1.

MONOCLONAL PROTEIN DETECTION. Primary light

HISTOPATHOLOGY. C o n g o r e d s t a i n i n g . IVS biopsy
chain cardiac amyloidosis and monoclonal gammop-
and excised native aortic valve were obtained from all
athy of unknown significance (MGUS) were ruled out
46 patients undergoing SAVR. Both the tissue sam-
in patients diagnosed to have amyloidosis by per-
ples were subjected to histopathological examination
forming serum protein electrophoresis, urine protein
per protocol. Amyloid deposition was suspected on
electrophoresis, and serum immunofixation. A bone
hematoxylin and eosin staining (Figure 2A). This was
marrow biopsy was planned to rule out monoclonal
confirmed on Congo red staining, under light micro-
cell expansion in cases where M-band was detected
scope (Figure 2B) and under polarized microscope
in either of these investigations.
(Figure 2C). In the IVS biopsy specimens, none of the
All these tests, including 2D echocardiography, PYP
samples showed positivity on Congo red staining
scan, and histopathological analysis were performed
(Figure 3A). In the aortic valve specimens, 33 (71.7%)
independent of each other and in a blinded fashion.
had positive uptake with Congo red stain and showed
For echocardiography, effective aortic orifice area,
a negative apple green birefringence on polarized
TDI, and strain imaging, findings were confirmed by a
microscopy. Amyloid deposition was primarily
second reviewer. PYP scan and histopathological ex-
observed adjacent to calcific deposits. These 33 valves
amination results were also independently verified by
were subjected to IHC to look specifically for
a second examiner.
TTR deposition.
STATISTICAL METHODS. Statistical analysis was
performed using SPSS V27 software. For continuous I H C . Staining with TTR-specific antibodies was per-
variables, median (25th and 75th percentiles [inter- formed on the tissue specimens with Congo red
quartile range]) was calculated and compared using positivity. Because none of the septum biopsy sam-
Wilcoxon rank sum test. For categorical variables, ples showed amyloid deposition, IHC was performed
frequency and percentage were calculated. Categori- only on the aortic valves that were Congo red–posi-
cal variables were compared using chi square or tive. Nineteen of 33 valves (58%) showed uptake of
Fisher exact test. A P value <0.05 was considered TTR antibodies. Of the total 46 patients, isolated
statistically significant. valvular amyloidosis was noted in 33 (72%), and iso-
lated valvular TTR amyloidosis was observed in 19
RESULTS
(41%).

Seventy-two patients aged $65 years who were RADIONUCLIDE SCINTIGRAPHY. Of the total 46 pa-
symptomatic with severe AS were screened, of which tients, PYP imaging was performed in 32 patients
46 underwent SAVR and are included in the present (70%). Qualitatively, the most common Perugini
analysis. The remaining 26 patients were excluded grade was 0, noted in 23 of 32 patients (71.9%), fol-
due to various reasons, including refusal to undergo lowed by grade I (n ¼ 6 of 32, 18.8%). Perugini grades
AVR (n ¼ 15), loss to follow-up preoperatively (n ¼ 5), II or III were observed in 3 patients (9.4%, 95% con-
death before SAVR (n ¼ 4), and suspected rheumatic fidence interval: 2.0% to 25.0%). Different Perugini
heart disease (n ¼ 2) (Central Illustration). These 46 grades 0, I, II, and III are shown in Figure 4. Quanti-
patients underwent the protocol-mandated preoper- tatively, the mean H:CL ratio was 1.28  0.14. Char-
ative investigations and a biopsy from the IVS was acteristics of the study cohort as distributed between
obtained intraoperatively. Their baseline character- PYP grades 0 or I versus grades II or III are presented
istics are presented in Table 1. The mean age of the in Table 1. Patients with grade II or III uptake were
group was 70  5 years, 70% were men, and majority considered to have myocardial TTR amyloidosis; this
of the patients were in New York Heart Association group had significantly low MCF (36  17% vs 14 
functional class II (76%). None of the patients had 4%; P ¼ 0.020), deceleration time (207  80 ms vs 85
carpal tunnel syndrome, ruptured biceps tendon, or  23 ms; P ¼ 0.003) and GLS (-19.3  4.3% vs -13.6 
atrial fibrillation. 3.7%; P ¼ 0.027).
JACC: CARDIOONCOLOGY, VOL. 3, NO. 4, 2021 Singal et al 569
OCTOBER 2021:565–576 Dual AS + ATTR-CA in Elderly Indian Patients

C ENTR AL I LL U STRA T I O N Study Recruitment and Results

Singal, A.K. et al. J Am Coll Cardiol CardioOnc. 2021;3(4):565–576.

Seventy-two patients with severe aortic stenosis were screened, of which 46 and 32 underwent surgical aortic valve replacement (SAVR) and 99m technetium py-
rophosphate (PYP) scan, respectively. Significant PYP uptake was observed in 3 patients (n ¼ 3 of 32; 9%), suggestive of myocardial transthyretin (TTR) cardiac
amyloidosis (ATTR-CA). On histopathological examination, none of the interventricular septum biopsy specimens had amyloid deposits, whereas 33 (72%) native aortic
valves showed amyloid deposits, of which 19 (58%) had TTR deposition suggestive of isolated valvular amyloidosis. At the 1-year follow-up examination, 2 had
patients died (4.3%); 1 each in myocardial ATTR-CA-negative and –positive groups (3% vs 33%, P ¼ 0.477). CR ¼ Congo red stain.
570 Singal et al JACC: CARDIOONCOLOGY, VOL. 3, NO. 4, 2021

Dual AS + ATTR-CA in Elderly Indian Patients OCTOBER 2021:565–576

T A B L E 1 Procedural Details, Baseline Parameters, Histopathological, and Radionuclide Scintigraphic Study Assessment of the
Study Patients

Overall Population (N ¼ 46)a Isolated AS (n ¼ 29)a Dual AS þ ATTR-CA (n ¼ 3)a P Value

Procedural details, demographic profile,

blood investigations, and ECG parameters
Surgical aortic valve replacement 46 (100) 29 (100) 3 (100) 1.000
Coronary artery bypass graft 13 (28.3) 9 (31.0) 1 (33.3) 0.935
Permanent pacemaker implantation 4 (8.7) 3 (10.3) 0 (0) 0.558
Median age (IQR), y 69.5 (66.0-73.0) 69.0 (66.0-73.0) 70.0 (70.0-75.0) 0.450
Men 32 (69.6) 22 (75.9) 1 (33.3) 0.682
NYHA functional class II 35 (76.1) 21 (72.4) 2 (66.7) 0.642
Diabetes mellitus 8 (17.4) 6 (20.7) 2 (66.7) 0.089
Hypertension 17 (37.0) 12 (41.4) 1 (33.3) 0.783
Coronary artery disease 14 (30.4) 9 (31.0) 2 (66.7) 0.938
Cerebrovascular disease 1 (2.2) 1 (3.4) 0 (0) 0.525
Body surface area, m2 1.6 (1.5-1.7) 1.7 (1.6-1.7) 1.7 (1.3-1.8) 0.770
Hemoglobin, gm/dL 12.6 (11.6-13.6) 12.5 (11.7-13.6) 13.7 (11.2-15.0) 0.460
Creatinine, mg/dL 0.9 (0.8-1.1) 1.0 (0.9-1.1) 0.8 (0.6-1.0) 0.130
Troponin I, pg/mL 14.7 (8.1-29.3) 14.4 (9.5-33.4) 26.8 (16.3-53.1) 0.280
NT-proBNP, pg/mL 261.2 (99.7-519.8) 354.9 (94.4-916.5) 517.7 (261.2-519.8) 0.670
Low voltage complexes 4 (9.5) 2 (7.4) 1 (50.0) 0.724
Right bundle branch block 3 (7.1) 2 (7.4) 0 (0) 0.397
QRS duration, ms 95.0 (90.0-106.0) 100.0 (90.0-110.0) 93.5 (75.0-112.0) 0.600
2D echocardiogram parameters
Ejection fraction, % 55.6 (48.3-63.5) 54.4 (50.5-62.1) 33.5 (28.5-57.9) 0.110
LV systolic dysfunction (<50%) 12 (26.1) 6 (20.7) 2 (66.7) 0.112
MCF, % 29.4 (23.0-40.0) 28.8 (23.8-39.1) 15.3 (9.3-16.1) 0.006
Abnormal MCF (#30%) 31 (67.4) 22 (75.9) 3 (100) 1.000
dT, ms 185.0 (109.0-229.0) 215.0 (144.0-236.0) 88.0 (60.0-106.0) 0.009
E/e’ 20.1 (16.3-27.1) 21.9 (18.5-27.6) 22.9 (22.8-38.1) 0.420
Indexed LA volume, mL/m2 32.1 (27.1-41.4) 32.4 (27.1-42.8) 41.4 (25.4-41.7) 0.790
Septum thickness, mm 14.1 (12.0-16.0) 14.2 (12.4-16.9) 14.5 (14.1-15.1) 0.900
Low mitral annular S’ (#5 cm/s) 15 (32.6) 20 (69.0) 3 (100) 0.682
LV TDI 5-5-5 rule (all #5 cm/s) 3 (6.5) 2 (6.9) 1 (33.3) 0.119
RV S’, cm/s 10.3 (8.9-12.7) 10.4 (9.3-12.7) 9.3 (8.2-11.1) 0.350
Abnormal RV S’ (#10 cm/s) 10 (22.7) 12 (42.9) 2 (66.7) 0.457
GLS, % -18.4 (-20.9 to -15.0) -18.7 (-21.1 to -16.9) -14.2 (-17.0 to -9.7) 0.030
Abnormal GLS ($-15%) 6 (13.0) 4 (13.8) 2 (66.7) 0.020
Relative apical strain 0.7 (0.7-0.8) 0.7 (0.7-0.8) 0.7 (0.6-0.8) 0.460
Relative apical strain sparing 0 0 0 NA
Mean pressure gradient, mm Hg 56.5 (45.0-70.0) 60.0 (53.0-72.0) 52.0 (36.0-70.0) 0.330
Indexed aortic valve area, cm2/m2 0.4 (0.3-0.4) 0.4 (0.3-0.4) 0.2 (0.2-0.4) 0.099
High gradient severe AS 38 (82.6) 26 (89.7) 2 (66.7) 0.882
Low-flow, low-gradient severe AS 8 (17.4) 3 (10.3) 1 (33.3) 0.882
Classical low-flow, low-gradient AS 4 (8.7) 0 (0) 1 (33.3) 0.104
Paradoxical low-flow, low-gradient AS 4 (8.7) 3 (10.3) 0 (0) 0.255
Histopathological and radionuclide scintigraphic
study assessment
Congo red positive — aortic valve 33 (71.7) 22 (75.9) 3 (100) 0.976
IHC-positive — aortic valve 19 (41.3) 15 (51.7) 2 (66.7) 0.863
Congo red–positive — IVS 0 (0) 0 (0) 0 (0) NA
IHC-positive — IVS 0 (0) 0 (0) 0 (0) NA
PYP — Perugini grade 0a 23 (71.9) 23 (79.3) 0 (0) —
PYP — Perugini grade Ia 6 (18.8) 6 (20.7) 0 (0) —
PYP — Perugini grade II or IIIa 3 (9.4) 0 (0) 3 (100) —
PYP — H:CL ratioa 1.3 (1.2-1.4) 1.2 (1.2-1.3) 1.6 (1.6-1.6) —
1-y mortality rate 2 (4.3) 1 (3.4) 1 (33.3) 0.477

Values are n (%) or median (interquartile range). aOf the total 46 patients who underwent SAVR, only 32 patients underwent a PYP scan (due to logistical constraints).
Significant radiotracer uptake was seen in 3 patients (dual AS þ ATTR-CA), and radiotracer uptake was not significant in the remaining 29 patients (isolated AS).
2D ¼ 2-dimensional; AS ¼ aortic stenosis; ATTR-CA ¼ transthyretin cardiac amyloidosis; dT ¼ deceleration time; ECG ¼ electrocardiogram; GLS ¼ global longitudinal strain;
IHC ¼ immunohistochemistry; IVS ¼ interventricular septum; LA ¼ left atrial; LV ¼ left ventricular; MCF ¼ myocardial contraction fraction; NA ¼ not available; NT-proBNP ¼ N-
terminal pro-B-type natriuretic peptide; NYHA ¼ New York Heart Association; PYP ¼ 99m-technetium pyrophosphate radionuclide scan; TDI ¼ tissue Doppler index.
JACC: CARDIOONCOLOGY, VOL. 3, NO. 4, 2021 Singal et al 571
OCTOBER 2021:565–576 Dual AS + ATTR-CA in Elderly Indian Patients

F I G U R E 2 Native Aortic Valve Subjected to Histopathological Examination

(A) Hematoxylin and eosin stain showed grade 2 amyloid deposition that was multifocal, with both nodular (thick arrow) and jagged edges
(thin arrow). (B) Ordinary light microscopy with Congo red special stain highlights amyloid deposition. (C) Polarized microscopy shows apple
green birefringence in Congo red–positive amyloid deposition. (D) Immunohistochemistry with transthyretin monospecific antibody shows
positive staining (brown) in grade 2 valve amyloid.

In the 3 patients with significant PYP uptake, his- patients had amyloid deposits in the aortic valve on
topathological examination of the aortic valve histopathological examination but only 1 had
revealed all were Congo red–positive (amyloid posi- TTR deposits.
tive), but only 2 of these stained positively with TTR
DISCUSSION
antibodies on IHC (ie, TTR positive).
MONOCLONAL PROTEIN DETECTION. None of the In a cohort of 46 elderly patients older than 65 years
patients showed presence of M band or other features who were symptomatic with severe AS and under-
suggestive of clonal cell expansion, thus ruling out went SAVR, PYP imaging was performed in 32 pa-
primary light chain cardiac amyloidosis and mono- tients (70%) that revealed significant radiotracer
clonal gammopathy of unknown significance. uptake (Perugini grade II or III) in 3 (9.4%) patients,
FOLLOW-UP. One-year follow-up was complete for suggesting myocardial TTR amyloidosis. However,
all patients (n ¼ 46, 100%). At the time of the 12- none of the patients showed amyloid deposition in
month follow-up, 2 patients had died (4.3%); 1 in the IVS biopsy specimens. Thirty-three patients (72%)
the myocardial TTR-negative and 1 in the -positive showed Congo red stain positivity in the aortic valves
group (3% vs 33%; P ¼ 0.477). Both the patients had suggestive of isolated valvular amyloid deposition. Of
elevated N-terminal pro–B-type natriuretic peptide these, 19 patients (58%) had positive staining with
(NT-proBNP) and low MCF preoperatively, and both TTR-specific antibodies on IHC, suggestive of isolated
died within 3 months of surgery. Both of these valvular TTR amyloidosis (Central Illustration).
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Dual AS + ATTR-CA in Elderly Indian Patients OCTOBER 2021:565–576

F I G U R E 3 Basal Interventricular Septal Biopsy Subjected to Histopathological Examination

(A) Hematoxylin and eosin stain shows normal myocardium without any amyloid deposition. (B) Immunohistochemistry with transthyretin
monospecific antibody shows negative transthyretin immunostain in the myocardium.

Although mass spectrometry–based shotgun prote- during IHC and found Apo-A1 in all their severe AS
omics would have helped to identify the different patients. This consistency of Congo red detection and
types of amyloid in the excised aortic valves, this was variance of amyloid subtyping across studies is likely
performed due to logistical and resource constraints due to the different methodologies used, as well as
(12). the varying racial/demographic profiles. The Apo-A1
Projecting these findings to the overall Indian deposition in the calcific aortic valves was previ-
population, the results suggest a huge unmet need for ously explained by the atherogenic milieu, and is
this dual disease’s awareness, detection, and treat- thought to be promoted by dyslipidemia. This is
ment. Extrapolating conservative estimates from our mainly due to local inflammation within the athero-
study (n ¼ 3 [9.3%] patients with significant PYP up- sclerotic plaques in the aortic valve which oxidizes
take of 32 patients for whom PYP scan was performed) the methionine residues of Apo-A1 and transforms
to the 2019 United Nations estimate for Indian pop- them into amyloidogenic proteins (16). These amy-
ulation suggests 90 million elderly patients >65 years loid depositions are believed to cause mineralization
old (6.6% of 1.36 billion), of whom 4.5 million would of calcified aortic valves by promoting apoptosis of
have severe AS (13). Nine percent of this amounts to valvular interstitial cells (15). However, there is no
w400,000 patients expected to have dual AS-ATTR- benefit of lipid-lowering therapy (statins) on severity
CA (range: 90,000 to 1,125,000 considering a 95% or progression of aortic stenosis (17). TTR deposition
confidence interval of 2.0% to 25%). may be explained by the shear stress caused by the
We found isolated valvular amyloidosis in the accelerated flow of blood across the stenosed
aortic valves of 33 (72%) patients. This is similar to valve predisposing it to amyloidogenesis. Also, TTR
the amyloid deposition in aortic valves of patients deposition was qualitatively observed more promi-
with calcific AS which has been noted previously, nently adjacent to high calcific burden sites. In
ranging from 75% to 100% (14,15). However, the ma- contrast to our study, none of these previous 2
jority of these amyloid deposits had been stained studies studying aortic valves looked at amyloid
with apolipoprotein A1 (Apo-A1) antibody; this is in deposition in the IVS (14,15). On the contrary,
contrast to our Congo red–positive patients, where 19 myocardial amyloidosis, without looking at valvular
patients (58%) stained positively with TTR antibody. amyloidosis, has been diagnosed on IVS biopsy by
Whereas Kristen et al (14) noted non-amyloidotic Treibel et al (5).
Apo-A1 deposition in 74% of severe AS patients, No previous study has looked at severe AS patients
Audet et al (15) did not use anti-TTR antibodies for concomitant ATTR-CA by performing
JACC: CARDIOONCOLOGY, VOL. 3, NO. 4, 2021 Singal et al 573
OCTOBER 2021:565–576 Dual AS + ATTR-CA in Elderly Indian Patients

F I G U R E 4 SPECT Images From PYP Scan Showing Different Perugini Grades

Images show grades (A) 0, (B) I, (C) II, and (D) III uptake. A standard dose of 99m technetium-pyrophosphate (10 to 20 mCi) was injected
intravenously, and planar and single-photon emission computerized tomography (SPECT) images were acquired at 1 hour (and 3 hours, if
suspicion of blood pool activity). The scans were visually assessed qualitatively (heart-to-bone ratio, Perugini score) (Supplemental Table 1).
Perugini grades II or III were considered as significantly positive for diagnosis of myocardial ATTR-CA. SPECT ¼ single-photon emission
computed tomography; other abbreviations as in Figure 1.

histopathological examination of cardiac tissue and (5-8,18-21). In the absence of tissue histopatholog-
PYP imaging prospectively and independently. There ical examination, red flags from clinical features,
is only 1 previous study that looked at the presence of ECG, echo, biomarkers (NT-proBNP and troponin),
ATTR-CA in patients undergoing SAVR; it showed a PYP/DPD scan, and cardiac magnetic resonance im-
prevalence of 5.6% (5). Here, detection of amyloid on aging should guide amyloid screening in these pa-
histopathology was the gold standard, and the same tients (22).
was later correlated with postoperative 3,3- Radionuclide scintigraphy has become the stan-
diphosphono-1,2-propanodicarboxylic acid (DPD) dard for diagnosis of ATTR-CA as per the guidelines
scans. endorsed by ASNC/AHA/ASE (11). Specifically, grade II
With the advent of TAVR, an increasing number of or III uptake, in the absence of clonal cell expansion,
patients are undergoing AVR without open heart is considered to be highly specific and sensitive
surgery. This is reflected in the recent studies, all of (>95%) for ATTR-CA (23). In our cohort, this was seen
which have attempted to diagnose ATTR-CA by in 3 patients (9.4% of the 32 patients where PYP im-
radionuclide scintigraphic imaging tests instead of aging was performed), suggesting significant
using IVS biopsy specimens (6,7,18,19). These studies myocardial ATTR-CA. There was no difference in our
are briefly summarized in Supplemental Table 3 patients with ATTR-CA (grade II or III uptake) versus
574 Singal et al JACC: CARDIOONCOLOGY, VOL. 3, NO. 4, 2021

Dual AS + ATTR-CA in Elderly Indian Patients OCTOBER 2021:565–576

those without ATTR-CA in terms of age, sex, and had Congo red positivity in aortic valves, only 1 of
grade of symptoms. Variables which differed signifi- them had significant Perugini grade II or III uptake.
cantly across this included MCF, deceleration time Lone AS patients fared better than dual disease (3% vs
(dT), GLS, and abnormal GLS. Similar results showing 33%, respectively). Previous studies have also shown
significant diastolic dysfunction (decreased dT) and an overall poorer long-term survival despite AVR in
significantly poor LV function (low MCF) have been severe AS patients who also had myocardial ATTR-CA
shown in previous studies as well (6,18,19,24,25). (5,8). It might be prudent to consider TTR amyloid
Histological alterations in the cardiac valves, targeting molecules such as tafamidis in patients with
including amyloid deposition, are beyond the reso- dual AS-ATTR-CA if AVR alone is shown to be less
lution of scintigraphic scans. PYP scan detects the beneficial in this group of patients as compared to
myocardial-based amyloid deposits, irrespective of those with lone AS. However, benefit from
the valvular amyloid. PYP grade I uptake might treatment of isolated valvular amyloidosis has not
represent a very early phase of myocardial ATTR-CA been shown until now. Bearing in mind that >40%
whose clinical relevance has not yet been proven. of total severe AS patients in this study had isolated
However, what has been shown previously is that the valvular TTR amyloidosis and the possibility of
prevalence of this subclinical grade I uptake is much valvular amyloidosis progressing to myocardial
higher in patients with severe AS than in age-matched amyloidosis, it may be worthwhile to consider the
controls without severe AS (21). The same is reflected impact of agents targeting TTR on the rate of
in our severe AS patients in whom grade I uptake was progression of AS severity. Also, early removal of
observed in 6 patients (19%). the hemodynamic stress imposed by the stenosed
The significant PYP uptake noted in 3 patients with valve (early AVR) may halt/delay the progression of
negative IVS biopsy specimens suggests substantial TTR amyloid deposition in severe AS patients with
myocardial involvement. The lack of amyloid detection in isolated valvular amyloidosis. These therapeutic
the IVS biopsy samples is likely due to chance factor and/or possibilities are thought-provoking and merit
sampling error. Also, the fact that our study population is at further studies to delineate the therapeutic ap-
least a decade younger than the other studies (mean age 70 proaches for both dual AS-ATTR-CA patients and
years vs >80 years) may account for a relatively lower severe AS patients with isolated valvular amyloidosis.
burden of amyloid deposition in the myocardium. Impor- Previous studies, primarily recruiting White pop-
tantly, the absence of amyloid deposition in the IVS of our ulations, have shown the prevalence of concomitant
patients must be explored further by inclusion of more ATTR-CA in severe AS patients to be approximately
patients who test positive on PYP imaging and by subject- 15% (Supplemental Table 3). Black Americans are
ing these patients to IVS biopsy intraoperatively during known to have even higher TTR penetrance (9).
SAVR. Before the present study, the distribution of ATTR-CA
Based on our findings of younger age of the study in an Asian population was unknown. As emphasized
participants with positive PYP scans but negative IVS above, conservatively estimating a prevalence of dual
biopsy specimens, a plausible hypothesis which AS-ATTR-CA as 9.4% (n ¼ 3 of 32) of those with severe
merits consideration is that the aortic valve is the site AS implies a tremendous unidentified burden of TTR
of initial accumulation of TTR deposits, which later amyloidosis (w400,000 patients with dual AS-ATTR-
progress to involve the myocardium; with progres- CA). Although hypothetical as of now, it is intuitive
sion of age and disease process, more florid deposi- that targeted management of myocardial TTR
tion of TTR amyloid would occur across the amyloid will have positive prognostic implications
myocardium, which might account for the improved over and above AVR in patients with dual AS-ATTR-
likelihood of amyloid detection on IVS biopsy, as CA. Considering this and despite the current cost
noted by Treibel et al (5) (amyloid detected in 5.6% of (>$100,000 USD annually) and difficult availability
patients with mean age of 75 years), or the higher rate of TTR-targeting molecules, it is quintessential to
of scintigraphic scan–based identification of TTR consider their use for these specific dual-disease
deposition, as noted by Castano et al (6) (amyloid patients.
detected in 16% of patients with mean age of 84 STUDY LIMITATIONS. ATTR-CA prevalence increases
years). However, it is not possible to establish the with age. Ours is a younger cohort (mean age: 70
same from the present study and hence, this remains years), compared to other studies (mean age: 80
a hypothetical possibility requiring validation from years). This may have been a potential reason behind
further research. the negative IVS biopsy specimens. Studying an older
At the 1-year follow-up examination, 4.3% patients population might have increased the prevalence of
had died post-SAVR. Although both these patients myocardial TTR deposits. Amyloid subtyping was
JACC: CARDIOONCOLOGY, VOL. 3, NO. 4, 2021 Singal et al 575
OCTOBER 2021:565–576 Dual AS + ATTR-CA in Elderly Indian Patients

restricted to TTR only. This was performed with IHC FUNDING SUPPORT AND AUTHOR DISCLOSURES
using TTR-specific antibodies alone, without use of
monospecific antibodies for other amyloid subtypes. Dr Dorbala has received consulting fees from Pfizer, GE Healthcare,
and Ionetix; and has received grant support from Pfizer, GE Health-
Also, laser microdissection and mass spectroscopic
care, and Attralus. All other authors have reported that they have no
analysis were not performed, which would have hel- relationships relevant to the contents of this paper to disclose.
ped to identify the other different types of amyloid in
the excised aortic valves. Being both the first study to ADDRESS FOR CORRESPONDENCE: Dr Sundeep
look at ATTR-CA in resource-constrained settings and Mishra, Department of Cardiology, Cardio Thoracic Sciences
a pilot study, the sample size and follow-up duration Center, All India Institute of Medical Sciences (AIIMS), Sri
is limited. The current follow-up, along with the Aurobindo Marg, Ansari Nagar East, New Delhi 110029,
limited sample size, is too small to justifiably draw India. E-mail: drsundeepmishranic@gmail.com. Twitter:
inferences regarding the predictors of mortality post- @Singal_Aayush, @AvinainderSingh, @Dorbala-
SAVR in this group of patients. Also, no adjustment Sharmila, @bansalrags, @ProfBhargava, @drkar-
for multiple testing was performed because of the thik2010, @aiims_newdelhi.
limited sample size, and results should be inter-
preted accordingly.

CONCLUSIONS PERSPECTIVES

The present study shows that concomitant existence

COMPETENCY IN MEDICAL KNOWLEDGE: Patients with
of myocardial ATTR-CA in severe AS is not uncom-
severe AS should be screened for TTR amyloidosis, especially in
mon, as detected by 99m-technetium pyrophosphate
the presence of red flags. Patients with dual disease should be
imaging (9%). Noninvasive markers of this dual dis-
monitored closely even after AVR, considering the trend towards
ease include significantly low myocardial contraction
worse post AVR survival seen in many studies.
fraction, deceleration time, and GLS. Amyloid depo-
sition in the aortic valves of patients with severe AS is TRANSLATIONAL OUTLOOK: The unique finding of isolated
much more common, as detected by histopathological valvular TTR amyloid (>40%) in addition to concomitant
examination (72%), significance of which remains myocardial amyloid merits future studies to examine the impact
unknown. The majority of the patients with amyloid of TTR-directed therapies in slowing the progression of valvular
in aortic valves showed deposition of TTR as detected disease, or to study the role of early AVR to halt/delay the pro-
by immunohistochemistry (58%). Future larger gression of myocardial TTR amyloid deposition in severe AS pa-
studies are warranted to actively investigate this tients with isolated valvular amyloidosis. Further research is
concept and its clinical significance. required to study the prognostic relevance of the dual AS-ATTR-
CA compared to AS alone on medium- to long-term follow-up
ACKNOWLEDGMENTS The authors thank the senior
post-AVR.
residents of the Departments of Cardiology and CTVS,
AIIMS, New Delhi, India, for their support.

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