# Descriptive study of the clinical and myocardial status of a population with anatomopathological aortic valve amyloidosis

Jean-Baptiste Brette, Magali Colombat, Pauline Fournier, Maxime Moninhas, Bertrand Marcheix, Olivier Lairez, Eve Cariou, Laurent Alric, Christophe Bureau, Dominique Chauveau, et al.

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Jean-Baptiste Brette, Magali Colombat, Pauline Fournier, Maxime Moninhas, Bertrand Marcheix, et al. Descriptive study of the clinical and myocardial status of a population with anatomopathological aortic valve amyloidosis. Cardiovascular Pathology, 2024, 73, pp.107674. 10.1016/j.carpath.2024.107674. hal-04886336

HAL Id: hal-04886336

https://hal.science/hal-04886336v1

Submitted on 7 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Original Article

# Descriptive study of the clinical and myocardial status of a population with anatomopathological aortic valve amyloidosis☆

Jean-Baptiste Brette<sup>a,b</sup>, Magali Colombat<sup>d,e</sup>, Pauline Fournier<sup>a,b</sup>, Maxime Moninhas<sup>a,b</sup>, Bertrand Marcheix<sup>d,f</sup>, Olivier Lairez<sup>a,b,c,d,*</sup>, Eve Cariou<sup>a,b</sup>, On behalf of the Toulouse Amyloidosis Research Network collaborators<sup>1</sup>

<figure><img src="image_3.png" /></figure>

ARTICLE INFO

Article history:

Received 16 January 2024

Revised 6 July 2024

Accepted 7 July 2024

Keywords:

Aortic stenosis

Bone scintigraphy

Cardiac amyloidosis

Transthyretin amyloidosis

Valvular amyloid deposits

A B S T R A C T

**Background:** Aortic stenosis (AS) and transthyretin (ATTR) cardiac amyloidosis (CA) share the same clinical profiles and cardiac phenotype. Amyloid deposits have been frequently reported in aortic valves of patients with severe AS referred for surgical aortic valve replacement (SAVR). The aim of this study was to determine the clinical and myocardial status of patients with aortic valve amyloidosis after aortic valve surgery.

**Methods and results:** We performed a retrospective descriptive study of 46 patients who underwent SAVR for severe AS with amyloid deposits upon histological analysis. All patients were screened for cardiac involvement. Amyloid deposits typing was successful in 35 (76%) patients and 28 (80%) were ATTR. Two (4%) had positive bone scintigraphy and among the 5 myocardial biopsies performed during surgery, 80% were positive for ATTR deposits.

**Conclusion:** ATTR is the predominant type in the presence of amyloid deposits on the aortic valve after surgery for severe AS but is only rarely accompanied by cardiac uptake on bone scintigraphy. Early stages of myocardial involvement are frequent and myocardial biopsy is more sensitive for detection of mild amyloid deposits than bone scintigraphy

© 2024 The Author(s). Published by Elsevier Inc.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

1. Introduction

Amyloidosis are a rare group of diseases, which result from extracellular deposition of misfolded proteins and can affect all organs [1]. The type depends on the precursor protein, and they are two main types that can lead to heart involvement: light chain amyloidosis (AL), due to a systemic immunoglobulin light chain and transthyretin amyloidosis (ATTR), which can be hereditary

(ATTRv) or wild type (ATTRwt). Cardiac involvement is the leading cause of morbidity and mortality in amyloidosis. It occurs in about 50% of patients with AL and is the dominant feature in patients with ATTRv and ATTRwt [2]. Myocardial amyloid deposition results mainly in hypertrophic cardiomyopathy but all tissues such as the conduction system or valves are affected [3,4]. It is a progressive and fatal cardiomyopathy with the need for early diagnosis and treatment [5].

Abbreviations List: ⁹⁹mTc-HMPD, ⁹⁹mTc-hydroxymethylene-diphosphonate; AApo-A1, Apo-A1 amyloidosis; AL, Light chain amyloidosis; AS, Aortic stenosis; ATTR, Transthyretin amyloidosis; ATTRv, Variant transthyretin amyloidosis; ATTRwt, Wild-type transthyretin amyloidosis; CA, Cardiac amyloidosis; ECG, Electrocardiogram; IHC, Immunohistochemistry; NT-proBNP, N-terminal prohormone brain natriuretic peptide; NYHA, New York Heart Association; SAVR, Surgical aortic valve replacement; SD, Standard deviation; TTE, Transthoracic echocardiography; TTR, Transthyretin.

A B S T R A C T

<footnote><sup>a</sup> Department of Cardiology, Toulouse University Hospital, France</footnote>

<footnote><sup>b</sup> Cardiac Imaging Center, Toulouse University Hospital, France</footnote>

<footnote><sup>c</sup> Department of Nuclear Medicine, Toulouse University Hospital, France</footnote>

<footnote><sup>d</sup> Medical School, Toulouse III Paul Sabatier University, Toulouse, France</footnote>

<footnote><sup>f</sup> Department of Cardiac Surgery, Toulouse University Hospital, France</footnote>

<footnote><sup>g</sup> Department of Pathology, IUCT Oncopôle, Toulouse France</footnote>

<footnote>ª This study is an ancillary study of the RACAMYL study funded by Pfizer France.</footnote>

<footnote>* Corresponding author: Olivier Lairez, Department of Cardiology, Rangueil University Hospital, 1, avenue Jean Poulhès, TSA 50032, 31059 Toulouse Cedex 9, France.</footnote>

<footnote>E-mail address: lairez.o@chu-toulouse.fr (O. Lairez).</footnote>

<footnote n="1">¹ For Details of Toulouse Amyloidosis Research Network collaborators click here.</footnote>
Degenerative aortic stenosis (AS), the most common valvular heart disease, is frequently associated with ATTRwt cardiac amyloidosis (CA) [6,7]. They are both age-related and responsible for similar cardiac remodeling [8]. Moreover, the combination of AS and CA worsens patient prognosis [9]. In surgical aortic valve replacement (SAVR) for the AS population, CA has a prevalence of 6 to 10% and of up to 32% in male groups undergoing transcatheter aortic valve replacement [10-12].

Furthermore, amyloid deposits have been reported in almost three quarters of the aortic valves of patients referred for surgery for aortic stenosis and recent progress in amyloid typing highlights frequent ATTR [13,14].

The aim of this study was to determine the clinical and myocardial status of patients with aortic valve amyloidosis after aortic valve surgery.

## 2. Methods

### 2.1. Study population

Between April 2017 and January 2023, all patients who underwent SAVR for severe AS at the University Hospital of Toulouse, (France) were retrospectively identified using the anatomopathology registry. Patients with aortic valvular amyloid deposits at histological analysis were considered for the study. The valves explored as part of the preliminary analyses used to design the RACAMYL study (ClinicalTrials.gov Identifier: NCT04636684) prior to the start of the study were associated with the valves explored as part of the RACAMYL study, which seeks to determine the prevalence of valvular deposits in severe degenerative AS, started in 2020 and is still ongoing. Patients who died before screening or without complete screening for cardiac amyloidosis were excluded. Finally, the study group included 46 patients. The study flow-chart is presented in Fig. 1. This work was approved by the local ethics committee in conformity with the Declaration of Helsinki and we made a retrospective register declaration at the start of the study (#RnIPH 2023-17).

### 2.2. Data collection

All data concerning preoperative cardiac assessment and surgery were retrospectively collected using electronic patient records. The data consisted of clinical findings, chemistry parameters, detailed analysis of preoperative electrocardiogram (ECG) and transthoracic echocardiography (TTE) results. The presence of a bicuspid aortic valve was collected at the time of surgery. For patients who underwent myocardial biopsy at the surgeon's discretion, the results of the histological analysis were correlated with the clinical findings.

### 2.3. Histological analysis

Immediately after surgical removal of the heart valve, the specimens were fixed in buffered formaldehyde solution (5%). Due to severe calcium deposits on the aortic valves, all specimens were treated with ethylenediaminetetraacetic acid (EDTA) solution for a few days until sufficient decalcification, embedded in paraffin and then stained with standard coloration. The search for amyloid deposits entailed Congo red staining and apple-green birefringence under cross-polarized light. The occurrence of amyloid deposits was graded semi-quantitatively by rough estimation using three levels: slight (+), moderate (++) and severe (+++). All valves with amyloid deposit explored as part of the preliminary analyses or as part of the RACAMYL study have undergone fibril typing using the proteomic method of mass spectrometry analysis of amyloidotic material, the new gold standard [1]. This method involves laser microdissection and capture of Congo red stained deposits from a fixed tissue section using a laser capture microscope followed by tryptic digestion, liquid chromatography and tandem mass spectrometry with mass spectrophot nanoLC-LTQ Orbitrap Velos (Thermo Fischer) in the Proteomic and Structural Biology Institute [15]. Computer algorithms (Mascot Deamon) then match the peptides to a protein reference database (Swiss Prot) to give amyloidosis typing [16]. For myocardial samples, Congo red staining was routinely performed, and the same process was used for amyloidosis typing.

<figure><img src="image_4.png" /><figcaption>Fig. 1. Flow chart.</figcaption></figure>
## 2.4. Screening for cardiac involvement

All patients with valvular amyloid deposits underwent screening for ATTR-CA with ⁹⁹mTc-hydroxymethylene-diphosphonate (⁹⁹mTc-HMPD) scintigraphy, serum and urine immuno-fixation and serum-free light chain assay according to the current guidelines [17]. Only patients with complete screening were selected for this study, and results of these investigations were correlated with clinical findings. In case of normal ⁹⁹mTc-HMPD scintigraphy and biology, no more exams were performed. In accordance with the current guidelines, patients with grades 2 or 3 ⁹⁹mTc-HMPD scintigraphy without monoclonal gammopathy were considered as having ATTR-CA [18].

## 2.5. Follow-up

Follow-up was performed in January 2023 by electronic chart review or by phone interview of the patient's general practitioner/cardiologist, patient, or family to assess all-cause mortality. This was determined using time to death for deceased subjects and time to last known follow-up for those last known to be alive.

## 2.6. Statistical analysis

Continuous variables were described as mean ± standard deviation (SD). Results of values not normally distributed were presented as medians with interquartile range. Nominal values were expressed as numbers and percentages. Statistical analyses were performed using SPSS V.20 software.

# 3. Results

## 3.1. Patients' characteristics

Over a period of 70 months, 2137 patients underwent surgery for severe AS, some of whose extracted valves were explored for amyloidosis. The search for amyloidosis was not performed consecutively but according to the availability of the surgeon and the sampling equipment, as well as the pathologist and the capacity for proteomic analysis. A retrospective analysis allowed to select 36 valves with amyloid deposits, used in preliminary analyses for the design of the RACAMYL study, which motivated inclusion in the study for a full cardiological work-up in search of cardiac amyloidosis. At the end of this period, 55 other valves were prospectively explored as part of the RACAMYL study. Among them, 21 (38%) showed amyloid deposits. Finally, 57 valves with amyloid deposit were screened, and 46 patients with valvular amyloid deposits and complete screening for cardiac amyloidosis were identified (Fig. 1). Mean age was 70 ± 9 years and 29 (85%) were men. Six (13%) patients were in New York Heart Association (NYHA) functional class III/IV and 31 (67%) were in functional class II. Three (7%) patients had carpal tunnel, 2 (4%) had lumbar spinal stenosis and none had bicipital rupture. Forty-four (96%) patients had severe AS and 2 (4%) underwent surgery for moderate AS associated with coronary bypass or aortic aneurysm surgery. Two (4%) patients had concomitant indication of mitral valve replacement for severe stenosis. Based on surgical findings, 18 (39%) had bicuspid aortic valves.

Regarding ECG, 6 (13%) patients had atrial fibrillation, 14 (30%) had left ventricular hypertrophy, 3 (7%) had first-degree atrioventricular block, 2 (4%) had right bundle branch block and 1 (2%) had low voltage.

Median level of N-terminal prohormone brain natriuretic peptide (NT-proBNP) was 486 [200–1476] pg/ml, median level of troponin was 20 [10–262] ng/L and 4 (9%) had monoclonal gammopathy.

<table><caption>**Table 1**
Echocardiographic characteristics</caption><thead><tr><th></th><th>n = 46</th></tr></thead><tbody><tr><td>Left ventricular ejection fraction, %</td><td>59 ± 11</td></tr><tr><td>Global Longitudinal Strain, - %</td><td>17 ± 5</td></tr><tr><td>Apical sparing, n (%)</td><td>8 (17)</td></tr><tr><td>Interventricular septum wall thickness, mm</td><td>13 ± 2</td></tr><tr><td>Posterior wall thickness, mm</td><td>12 ± 2</td></tr><tr><td>Left ventricular mass index, g/m²</td><td>141 ± 52</td></tr><tr><td>Right ventricular hypertrophy, n (%)</td><td>0 (0)</td></tr><tr><td>Aortic mean pressure gradient, mmHg</td><td>49 ± 12</td></tr><tr><td>Aortic peak transvalvular velocity, m/s</td><td>4.3 ± 0.6</td></tr><tr><td>Aortic valvular area, cm²</td><td>0.9 ± 0.4</td></tr><tr><td>Low flow, low gradient, n (%)</td><td>1 (2)</td></tr><tr><td>Pericardial effusion, n (%)</td><td>0 (0)</td></tr></tbody></table>

## 3.2. Echocardiography

All patients underwent a TTE to assess aortic stenosis before SAVR. TTE parameters are shown in Table 1.

## 3.3. Valvular histological analysis

All amyloid deposits viewed under optic microscopy were slight. Qualitatively, the amyloid deposits were found on remodeled valve tissue showing degenerative changes combining necrosis and calcifications.

Proteomic analysis by mass spectrometry on valvular specimens allowed a characterization of amyloidosis type in 35 (76%) patients. Two situations did not allow the type of amyloidosis to be identified by proteomic analysis: 1. the deposits were not abundant enough to perform the technique under good conditions (47% of cases); 2. the technique, although performed with enough material, did not allow a conclusion to be drawn (53% of cases). In this second case, the protein amyloid signature (serum amyloid P component, Apo-A4, and Apo-E) was present but no amyloid protein could be detected. Among specimens that could be typed by proteomic analysis, 28 (80%) were ATTR alone, 5 (14%) were ATTR associated with AL (4 kappa and 1 lambda), 1 (3%) was serum amyloid A amyloidosis (AA) and 1 (3%) was Apo-A1 amyloidosis (AApo-A1). Among the 5 patients with associated AL, one (with kappa deposits) had monoclonal gammopathy which was kappa light chain monoclonal gammopathy of undetermined significance (MGUS). The valve with AApo-A1 deposits had a macroscopic aspect of endocarditis. Among the 2 patients who underwent concomitant mitral surgery, 1 (50%) of them had the same amyloid ATTR and AL lambda deposits on the mitral as on the aortic valve, without associated monoclonal gammopathy.

Fig. 2 shows the typical proteomic results for the 2 main types of amyloidosis (i.e. ATTR and AL) found in the aortic valve.

## 3.4. ⁹⁹mTc-HMPD scintigraphy

Forty-five (98%) patients were screened with ⁹⁹mTc-HMPD scintigraphy after surgery and one before surgery. Two (4%) were positive and showed Perugini's visual score of 2. None of them had monoclonal gammopathy allowing to confirm ATTR-CA. All of them had ATTR on the valve and 1 had a myocardial biopsy, which confirmed ATTR on the myocardium. No patients had grade 1 ⁹⁹mTc-HMPD scintigraphy or grade 2 or 3 ⁹⁹mTc-HMPD scintigraphy with concomitant monoclonal gammopathy.

## 3.5. Myocardial biopsy

Five (11%) patients underwent concomitant myocardial biopsy at the surgeon's discretion, not as part of a surgical indication, but
<figure><img src="image_5.png" /><figcaption>Fig. 2. Typical proteomic results for the 2 main types of amyloidosis.</figcaption></figure>

<table><caption>**Table 2**
Main patient characteristics with confirmed CA</caption><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>Screening for cardiac amyloidosis</td><td colspan="5">99mTc-HMPD + myocardial biopsy</td></tr><tr><td>Main patient characteristics</td><td colspan="5"></td></tr><tr><td>Patient</td><td>A</td><td>B</td><td>C</td><td>D</td><td>E</td></tr><tr><td>Age, (y)</td><td>75</td><td>76</td><td>81</td><td>79</td><td>83</td></tr><tr><td>Sex</td><td>male</td><td>male</td><td>male</td><td>male</td><td>male</td></tr><tr><td>Carpal tunnel syndrome</td><td>yes</td><td>no</td><td>no</td><td>no</td><td>no</td></tr><tr><td>Mean LV wall thickness, (mm)</td><td>14</td><td>15</td><td>13</td><td>12</td><td>15</td></tr><tr><td>LVEF, (%)</td><td>61</td><td>67</td><td>55</td><td>60</td><td>66</td></tr><tr><td>GLS, (%)</td><td>-13</td><td>-7</td><td>-18</td><td>-21</td><td>-14</td></tr><tr><td>Myocardial biopsy</td><td>ATTR, severe</td><td>ATTR, slight</td><td>ATTR, slight</td><td>ATTR, slight</td><td>2</td></tr><tr><td>99mTc-HMPD grade</td><td>2</td><td>0</td><td>0</td><td>0</td><td>2</td></tr><tr><td>Valvular amyloidosis</td><td>ATTR, slight</td><td>ATTR, slight</td><td>NT, slight</td><td>ATTR, slight</td><td>ATTR, slight</td></tr></tbody></table>

Mean wall thickness: (septal wall thickness + posterior wall thickness / 2); LVEF: Left ventricular ejection fraction; GLS: Global longitudinal strain; 99mTc-HMPD: 99mTc-hydroxymethylene-diphosphonate scintigraphy; ATTR: Transthyretin amyloidosis; NT: not typed.

if amyloidosis was suspected prior to surgery or according to the appearance of the myocardium intraoperatively. The tissue sample was obtained under direct vision by performing a left ventricular endomyocardial biopsy from the basal segment of the interventricular septum. Four (80%) of them had myocardial amyloidosis: 1 (20%) was considered as severe at Congo red staining with apple-green birefringence under cross-polarized light and 3 (60%) as slight. Amyloid deposits were almost exclusively interstitial. All positive samples could be identified by proteomic and were typed as ATTR. Among the 4 patients with confirmed myocardial amyloidosis, 3 (75%) had ATTR on the aortic valve and 1 (25%) had undetermined type. The only patient with severe myocardial ATTR had positive 99mTc-HMPD scintigraphy allowing the noninvasive diagnosis of ATTR-CA.

### 3.6. Patients with confirmed myocardial amyloidosis

Finally, 5 (11%) patients were confirmed for ATTR-CA: 4 on the basis of myocardial biopsy and 1 on the basis of 99mTc-HMPD scintigraphy without monoclonal gammopathy. A genetic analysis of the TTR gene was carried out in all patients, with no mutations found. Table 2 summarizes the main characteristics of patients with confirmed CA and Fig. 3 shows typical findings of 2 patients with valvular and myocardial ATTR.

### 3.7. Follow-up

No patients died after a median follow up period of 13 [4–17] months and the patients with known MGUS have not evolved.
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Typical findings of 2 patients with valvular and myocardial ATTR deposits.

Patient A (Table 2) presents slight valvular amyloidosis and severe interstitial myocardial amyloidosis on Congo red-stained histological sections with concordant grade 2 positive bone scintigraphy and mild impaired GLS at -13%. Patient B (Table 2) presents slight valvular and interstitial myocardial amyloidosis on Congo red-stained histological sections with severe impaired GLS at -7% and normal bone scintigraphy.</figcaption></figure>

## 4. Discussion

Our study provides a clinical and myocardial description of 46 AS population patients with valvular amyloid deposits including 61% confirmed for ATTR alone using proteomic analyses and 24% undetermined. Among patients with valvular amyloid deposits, 11% were diagnosed with ATTR-CA: 2% based on positive bone scintigraphy and concordant positive myocardial biopsy, 2% with positive bone scintigraphy without monoclonal gammopathy and 7% with positive myocardial biopsy and negative bone scintigraphy. The central picture summarizes main study results.

The presence of amyloid deposits has been reported on cardiac valves most of the time in the aortic valve, particularly in the presence of severe stenosis [19–21]. The prevalence can reach 75 to 100% in SAVR for AS, generally identified by immunohistochemistry (IHC) and with contradictory results [14,22]. Singal et al. have recently reported a prevalence of 72% of amyloid deposits in a population of patients with severe AS, including 41% of ATTR [13]. In this study, amyloidosis typing was assessed by IHC with a success rate of 58%. In our study, the proteomic method allowed typing of amyloidosis in 76% of valve specimens. This yield is lower than the usual accuracy of the proteomic method which is reported to be almost 94% [23], which is explained by the technical difficulties inherent in calcification of the aortic valve.

One of the main mechanisms responsible for degenerative AS is an active inflammatory process with some similarities to atherosclerosis including lipid deposition [24]. Apo-A1 amyloidosis, derived from high density lipoprotein (HDL), seems to play a role in valvular degeneration [25]. Furthermore, Audet et al. report 100% of Apo-A1 amyloidosis identified by IHC, in a 70 calcific aortic valve cohort, the extent of which correlates with the valve pressure gradient and leads to mineralization [26]. However, in this study, only Apo-A1 amyloidosis was searched for, and it is likely that other types, such as ATTR, could have been found if they had been tested on aortic valves. Overdiagnosis of ApoA1 amyloidosis is a limitation of IHC and does not exist with proteomics, which explains why there was only one case in our study. It is conceivable that amyloid deposits are a part of the pathophysiology of degenerative AS.

Initially described as dystrophic amyloidosis, valvular amyloidosis did not appear to be related to systemic involvement [27]. The presence of vascular amyloid deposits, such as in the aorta, is common in the elderly and is not associated with organ damage [28,29]. However, recent findings identifying valvular amyloid deposits, predominantly TTR, and the increasingly well described relationship between AS and CA raises questions about the pathophysiology of these two infiltrative processes [10,11]. In our study, 11% of patients with valvular amyloid deposits had contemporary
myocardial involvement, mostly diagnosed by myocardial biopsy and probably underestimated due to the lack of sensitivity of bone scintigraphy. These results suggest a possible link between these two processes with the hypothesis of an initial infiltration at the valvular level, which may evolve into myocardial infiltration in a second phase. However, we have no formal proof of such a link, and it should be noted that in our population, few patients had signs of extra-cardiac involvement such as a history of lumbar spinal stenosis or carpal tunnel syndrome [30,31]. Conversely, Sin- gal *et al.* did not find positive myocardial biopsies in their series of ATTR valve amyloidosis, which they explain by the young age of the patients included (mean age 70 years) compared to other stud- ies [13]. Follow-up of carriers of ATTR valvular amyloid deposits should be considered to further investigate this relationship and to determine whether there is a pathophysiological continuum be- tween the two factors or whether valvular amyloidosis is an inde- pendent phenomenon.

In our study we reported a large proportion of bicuspid aor-
tic valves with a similar mean age as SAVR for AS cohorts with a
prevalence of 8%-24% [10,11,14]. Screening for valvular amyloid de-
posits was randomly performed regardless of AS etiology and does
not explain this difference. In this regard, Kristen et al. did not find
any statistical association between aortic valvular morphology and
the prevalence of amyloid deposits [14].

Finally, our study shows the limits of the performance of ⁹⁹mTc-
HMPD scintigraphy in the detection of ATTR-CA by showing no car-
diac uptake in 3 patients despite the presence of slight deposits on
the biopsy. Although it is reported as an effective test in the di-
agnosis of ATTR-CA, especially when there is an echocardiographic
phenotype [18]. It seems that the bone scintigraphy lacks sensitiv-
ity in early forms of amyloidosis. The expert consensus that vali-
dated scintigraphy for the noninvasive diagnosis of amyloidosis in-
cluded symptomatic patients referred to cardiologists for suspected
amyloidosis suggesting advanced forms of the disease [18]. In car-
riers of the TTR mutations with neurological tropism, it has been
shown that the bone scintigraphy could be negative at an early
stage of cardiac involvement despite cardiac sympathetic denerva-
tion evidenced by decreased metaionobenzylguanidin (MIBG) scan
cardiac uptake [32]. Furthermore, false-negative bone scintigraphy
has been reported in some specific TTR mutations [17,33]. In our
study, none of the patients diagnosed with CA had a TTR mu-
tation, confirming the possibility of false-negative scintigraphy in
early stage ATTRwt-CA.

## 4.1. *Limitations*

There are several limitations in our study. First, the study has
all the limitations associated with retrospective, single-center and
limited sample studies. Although all surgical specimens benefit
routinely from a standard histological analysis, the screening
for amyloid deposits is not systematic and is only performed
randomly, which explains the low number of patients over this
period. Systematic screening for ATTR-CA was performed by
bone scintigraphy and blood and urine tests, but patients with
valvular deposits of AL or who could not be typed did not have
a peripheral biopsy to rule out systemic AL. However, the pres-
ence of undiagnosed AL is unlikely since no patient died during
the follow-up period. Finally, since myocardial biopsies were not
routinely performed, and since most bone scintigraphies were neg-
ative, we cannot exclude other forms of early cardiac involvement
that would not have been diagnosed by bone scintigraphy. In this
context, it is not possible reach a conclusion on the performance
of the bone scintigraphy in the early stages of the disease, which
would require a prospective study with systematic myocardial
biopsies.

5. Conclusion

ATTR is the predominant type in the presence of amyloid de-
posits on the aortic valve after surgery for severe AS but is only
rarely accompanied by cardiac uptake on bone scintigraphy. On the
other hand, in 60% of the cases explored by biopsy, there are slight
ATTR myocardial deposits, which suggests that myocardial biopsy
is more sensitive for detection of mild amyloid deposits than bone
scintigraphy.

Toulouse Amyloidosis Research Network collaborators

Laurent Alric, MD, PhDª; Christophe Bureau, MD, PhDᵇ; Diminique Chauveau, MD, PhDᶜ; Pascal Cintas, MD, PhDᵈ; Audrey Delas, MDᵉ; Delphine Dupin-Deguine, MDᵉ; Stanislas Faguer, MD, PhDᶜ; Antoine Huart, MDᶜ; Bénédicte Puissant, MDg; Grégory Pugnetª; Grégoire Prévot, MDʰ; David Ribes, MDᶜ; Laurent Sailler, MD, PhDᵢ

a–Department of Internal Medicine and Digestive Diseases, Pur-
pan University Hospital, Toulouse, France

b–Department of Hepatology-Gastroenterology, Rangueil Uni-
versity Hospital, Toulouse, France

c–Department of Nephrology and Referral Center for Rare Dis-
eases, Rangueil University Hospital, Toulouse, France

d–Department of Neurology, Purpan University Hospital,
Toulouse, France

e–Department of Pathology, IUCT Oncopôle, Toulouse, France

f–Department of Genetic, Toulouse University Hospital,
Toulouse, France

g–Immunology Laboratory, Toulouse University Hospital,
Toulouse, France

h–Department of Pneumology, Toulouse University Hospital,
Toulouse, France

i–Department of Internal Medicine, Toulouse University Hospi-
tal, Toulouse, France

Declaration of competing interest

Jean-Baptiste Brette: none, Magali Colombat: grant from Pfizer France, Pauline Fournier: consultancies form Pfizer France, Maxime Moninhas: none, Bertrand Marcheix: none, Olivier Lairez: consultancies form Pfizer France and Alnylam, Eve Cariou: consultancies form Pfizer France.

CRediT authorship contribution statement

**Jean-Baptiste Brette:** Writing – original draft, Investigation, Data curation. **Magali Colombat:** Resources, Formal analysis. **Pauline Fournier:** Resources, Formal analysis, Data curation. **Maxime Moninhas:** Investigation, Data curation. **Bertrand Marcheix:** Writing – review & editing, Validation. **Olivier Lairez:** Writing – review & editing, Validation, Resources, Methodology, Conceptualization. **Eve Cariou:** Writing – review & editing, Supervision, Formal analysis, Conceptualization.

References

[1] Wechalekar AD, Gillmore JD, Hawkins PN. Systemic amyloidosis. Lancet (London, England) 2016;387:2641-54. doi:10.1016/S0140-6736(15)01274-X.

[2] Falk RH. Diagnosis and management of the cardiac amyloidoses. Circulation 2005;112:2047-60. doi:10.1161/CIRCULATIONAHA.104.489187.

[3] Damy T, Maurer MS, Rapezzi C, Plante-Bordeneuve V, Karayal ON, Mundayat R, et al. Clinical, ECG and echocardiographic clues to the diagnosis of TTR-related cardiomyopathy. Open Hear 2016;3:e000289. doi:10.1136/openhrt-2015-000289.

[4] Sabzwari SRA, Tzou WS. Systemic diseases and heart block. Card Electrophysiol Clin 2021;13:721-40. doi:10.1016/j.ccep.2021.06.011.

[5] Gillmore JD, Damy T, Fontana M, Hutchinson M, Lachmann HJ, Martinez-Naharro A, et al. A new staging system for cardiac transthyreтин amyloidosis. Eur Heart J 2018;39:2799-806. doi:10.1093/eurheartj/ehx589.
[6] Longhi S, Lorenzini M, Gagliardi C, Milandri A, Marzocchi A, Marrozzini C, et al. Coexistence of degenerative aortic stenosis and wild-type transthyretil-related cardiac amyloidosis. JACC Cardiovasc Imaging 2016;9:325-7. doi:10.1016/j.jcmg.2015.04.012.

[7] Nietlispach F, Webb JG, Ye J, Cheung A, Lichtenstein SV, Carere RG, et al. Pathology of transcatheter valve therapy. JACC Cardiovasc Interv 2012;5:582-90. doi:10.1016/j.jcin.2012.03.012.

[8] Castano A, Narotsky DL, Hamid N, Khalique OK, Morgenstern R, DeLuca A, et al. Unveiling transthyretil cardiac amyloidosis and its predictors among elderly patients with severe aortic stenosis undergoing transcatheter aortic valve replacement. Eur Heart J 2017;38:2879-87. doi:10.1093/eurheartj/ehx350.

[9] Ternacle J, Krapf L, Mohty D, Magne J, Nguyen A, Galat A, et al. Aortic stenosis and cardiac amyloidosis: JACC review topic of the week. J Am Coll Cardiol 2019;74:2638-51. doi:10.1016/j.jacc.2019.09.056.

[10] Treibel TA, Fontana M, Gilbertson JA, Castelletti S, White SK, Scully PR, et al. Occult transthyretil cardiac amyloid in severe calcific aortic stenosis: prevalence and prognosis in patients undergoing surgical aortic valve replacement. Circ Cardiovasc Imaging 2016;9:e005066. doi:10.1161/CIRCIMAGING.116.005066.

[11] Allen RD, Edwards WD, Tazelaar HD, Danielson GK. Surgical pathology of subaortic septal myectomy not associated with hypertrophic cardiomyopathy: a study of 98 cases (1996-2000). Cardiovasc Pathol Off J Soc Cardiovasc Pathol 2003;12:207-15. doi:10.1016/s1054-8807(03)00057-7.

[12] Cavalcante JL, Rijal S, Abdelkarim I, Althouse AD, Sharbaugh MS, Fridman Y, et al. Cardiac amyloidosis is prevalent in older patients with aortic stenosis and carries worse prognosis. J Cardiovasc Magn Reson 2017;19:98. doi:10.1186/s12968-017-0415-x.

[13] Singal AK, Bansal R, Singh A, Dorbala S, Sharma G, Gupta K, et al. Concomitant transthyretil amyloidosis and severe aortic stenosis in elderly Indian population: a pilot study. JACC CardioOncology 2021;3:565-76. doi:10.1016/j.jaccao.2021.08.008.

[14] Kristen A V, Schnabel PA, Winter B, Helmke BM, Longerich T, Hardt S, et al. High prevalence of amyloid in 150 surgically removed heart valves-a comparison of histological and clinical data reveals a correlation to atheroinflammatory conditions. Cardiovasc Pathol 2010;19:228-35. doi:10.1016/j.carpath.2009.04.005.

[15] Dogan A. Amyloidosis: insights from proteomics. Annu Rev Pathol 2017;12:277-304. doi:10.1146/annurev-pathol-052016-100200.

[16] Colombat M, Holifanjaniaina S, Onifarasoaniaina S, Valleix S, Maisonneuve H, Kahn JE. [Proteomics, a new tool for an accurate typing of amyloidosis]. La Rev Med interne 2015;36:346-51. doi:10.1016/j.revmed.2014.11.006.

[17] Garcia-Pavia P, Rapezzi C, Adler Y, Arad M, Basso C, Brucato A, et al. Diagnosis and treatment of cardiac amyloidosis: a position statement of the ESC working group on myocardial and pericardial diseases. Eur Heart J 2021;42:1554-68. doi:10.1093/eurheartj/ehab072.

[18] Gillmore JD, Maurer MS, Falk RH, Merlini G, Damy T, Dispenzieri A, et al. Nonbiopsy diagnosis of cardiac transthyretil amyloidosis. Circulation 2016;133:2404-12. doi:10.1161/CIRCULATIONAHA.116.021612.

[19] Goffin Y. Microscopic amyloid deposits in the heart valves: a common local complication of chronic damage and scarring. J Clin Pathol 1980;33:262-8. doi:10.1136/jcp.33.3.262.

[20] Iwata T, Nakamura H, Nagasawa T, Kamel T, Fujihara S, Yokota T, et al. Amyloid deposits in heart valves. Acta Pathol Jpn 1982;32:23-9. doi:10.1111/j.1440-1827.1982.tb02024.x.

[21] Ladefoged C, Rohr N. Amyloid deposits in aortic and mitral valves. A clinicopathological investigation of material from 100 consecutive heart valve operations. Virchows Arch A Pathol Anat Histopathol 1984;404:301-12. doi:10.1007/BF00694895.

[22] Falk E, Ladefoged C, Christensen HE. Amyloid deposits in calcified aortic valves. Acta Pathol Microbiol Scand A 1981;89:23-6. doi:10.1111/j.1699-0463.1981.tb00182.x.

[23] Gilbertson JA, Theis JD, Vrana JA, Lachmann H, Wechalekar A, Whelan C, et al. A comparison of immunohistochemistry and mass spectrometry for determining the amyloid fibril protein from formalin-fixed biopsy tissue. J Clin Pathol 2015;68:314-17. doi:10.1136/jclinpath-2014-202722.

[24] Otto CM, Kuusisto J, Reichenbach DD, Gown AM, O'Brien KD. Characterization of the early lesion of "degenerative" valvular aortic stenosis. Histological and immunohistochemical studies. Circulation 1994;90:844-53. doi:10.1161/01.cir.90.2.844.

[25] Mathieu P, Boulanger M-C. Basic mechanisms of calcific aortic valve disease. Can J Cardiol 2014;30:982-93. doi:10.1016/j.cjca.2014.03.029.

[26] Audet A, Côté N, Couture C, Bossé Y, Després J-P, Pibarot P, et al. Amyloid substance within stenotic aortic valves promotes mineralization. Histopathology 2012;61:610-19. doi:10.1111/j.1365-2559.2012.04265.x.

[27] Cooper JH. Localized dystrophic amyloidosis of heart valves. Hum Pathol 1983;14:649-53. doi:10.1016/s0046-8177(83)80208-1.

[28] Cornwell GG 3rd, Murdoch WL, Kyle RA, Westermark P, Pitkanen P. Frequency and distribution of senile cardiovascular amyloid. A clinicopathologic correlation. Am J Med 1983;75:618-23.

[29] Tanskanen M, Peuralinna T, Polvikoski T, Notkola I-L, Sulkava R, Hardy J, et al. Senile systemic amyloidosis affects 25% of the very aged and associates with genetic variation in alpha2-macroglobulin and tau: a population-based autopsy study. Ann Med 2008;40:232-9. doi:10.1080/07853890701842988.

[30] Bay K, Gustafsson F, Maiborg M, Bagger-Bahnsen A, Strand AM, Pilgaard T, et al. Suspicion, screening, and diagnosis of wild-type transthyretil amyloid cardiomyopathy: a systematic literature review. ESC Hear Fail 2022;9:1524-41. doi:10.1002/ehf2.13884.

[31] Inomata T, Tahara N, Nakamura K, Endo J, Ueda M, Ishii T, et al. Diagnosis of wild-type transthyretil amyloid cardiomyopathy in Japan: red-flag symptom clusters and diagnostic algorithm. ESC Hear Fail 2021;8:2647-59. doi:10.1002/ehf2.13473.

[32] Piekarski E, Chequer R, Algalarrondo V, Eliahou L, Mahida B, Vigne J, et al. Cardiac denervation evidenced by MIBG occurs earlier than amyloid deposits detection by diphosphonate scintigraphy in TTR mutation carriers. Eur J Nucl Med Mol Imaging 2018;45:1108-18. doi:10.1007/s00259-018-3963-x.

[33] Tahara N, Lairez O, Endo J, Okada A, Ueda M, Ishii T, et al. (99m) Technetium-pyrophosphate scintigraphy: a practical guide for early diagnosis of transthyretil amyloid cardiomyopathy. ESC Hear Fail 2022;9:251-62. doi:10.1002/ehf2.13693.