# Prevalence, clinical significance and prognosis value of liver stiffness measurement anomalies in transthyretin cardiac amyloidosis

Emmanuelle Lointier, Eve Cariou, Maxime Beneyto, Pauline Fournier, Yoan Lavie-Badie, Damien Eyharts, Christophe Bureau, Olivier Lairez

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## To cite this version:

Emmanuelle Lointier, Eve Cariou, Maxime Beneyto, Pauline Fournier, Yoan Lavie-Badie, et al. Prevalence, clinical significance and prognosis value of liver stiffness measurement anomalies in transthyretin cardiac amyloidosis. International Journal of Cardiology, 2024, 416, pp.132485. 10.1016/j.ijcard.2024.132485. hal-05093714

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# Prevalence, clinical significance and prognosis value of liver stiffness measurement anomalies in transthyretin cardiac amyloidosis

Emmanuelle Lointier<sup>a</sup>, Eve Cariou<sup>a,b</sup>, Maxime Beneyto<sup>a</sup>, Pauline Fournier<sup>a,b</sup>, Yoan Lavie-Badie<sup>a,b</sup>, Damien Eyharts<sup>a,b</sup>, Christophe Bureau<sup>c,d</sup>, Olivier Lairez<sup>a,b,d,*</sup>, On behalf of the Toulouse Amyloidosis Research Network collaborators

## ARTICLE INFO

### Keywords:

- Heart failure

- Liver stiffness measurement

- Prognosis

- Transthyretin cardiac amyloidosis

## ABSTRACT

**Background** – Laboratory liver anomalies are common in cardiac amyloidosis; however, their significance regarding liver stiffness is unknown. The aim of this study was to investigate the prevalence, clinical significance, and prognostic value of liver stiffness measurement (LSM) anomalies in transthyretin cardiac amyloidosis (ATTR-CA). Methods – Consecutive patients diagnosed with ATTR-CA who underwent liver stiffness assessment were included in the study. Demographic, clinical, laboratory, transthoracic echocardiography and liver stiffness data were retrospectively collected. LSM was obtained through either transient elastography or supersonic shear imaging. Patient cohort was divided in two groups according to a 10 kPa threshold. Follow up data were collected for the occurrence of hospitalization for heart failure and all-cause death. Results – Two hundred and eighty-four patients with ATTR-CA – 26 (9%) hereditary variant ATTR, 258 (91%) wild-type ATTR – were included. A LSM over 10 kPa was found in 4 (15%) and 98 (38%) patients with ATTRv and ATTRwt respectively (p = 0.02). Among patients with ATTRwt, high LSM was more frequent in advanced stages of ATTR-CA and was associated with increased risk of hospitalization for heart failure after multivariate analysis with a hazard ratio of 2.41 [1.05–5.55] (p = 0.04). Among patients with NYHA stage 1, 28% presented high LSM associated with high NT-proBNP levels. Integration of high LSM with NT-proBNP and estimated glomerular filtration rate provided a better estimate of patient survival. Conclusion – LSM over 10 kPa is found in up to 36% of patients with ATTR-CA and is associated with advanced stages of cardiomyopathy and increased risk of hospitalization for heart failure in ATTRwt patients.

## 1. Introduction

Amyloidosis is characterized by the accumulation of misfolded precursor protein, forming cross β sheet amyloid fibrils in the extracellular compartment of organs, leading to their dysfunction. Among the various organs involved, cardiac amyloidosis is a debilitating pathology that manifests in life-threatening heart failure. The two main forms of cardiac amyloidosis are immunoglobulin light chain and transthyretin

amyloidosis (ATTR), the latter being the most common, found in 25% of the population over the age of 85, and responsible for 15% of heart failure with preserved ejection fraction in autopsy series [1]. Transthyretin amyloidosis cardiomyopathy (ATTR-CA) is being increasingly diagnosed, especially since the development of bone scintigraphy as a non-invasive diagnostic tool [2]. The disease is classified depending on the sequence of the transthyretin gene, either ATTRwt when no mutation is found or ATTRv if a variant is discovered. ATTRv commonly

**Abbreviations:** 99mTc-DPD, 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid; ATTR-CA, Transthyretin cardiac amyloidosis; ATTRv, Hereditary variant transthyretin amyloidosis; ATTRwt, Wild type transthyretin amyloidosis; CAP, Controlled attenuation parameter; HLSM, High liver stiffness measurement; LLSM, Low liver stiffness measurement.

* Corresponding author at: Department of Cardiology, Rangueil University Hospital, 1, avenue Jean Poulhès, TSA 50032, 31059 Toulouse Cedex 9, France.

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

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

<footnote><sup>b</sup> Cardiac Imaging Centre, Rangueil University Hospital, France</footnote>

<footnote><sup>c</sup> Hepatology department, Rangueil University Hospital, Toulouse, France</footnote>

<footnote><sup>d</sup> Medical School, Toulouse III Paul Sabatier University, Toulouse, France</footnote>
results in concomitant cardiomyopathy and polyneuropathy, the clinical presentation being conditioned by the variant [3]. Predominant polyneuropathy presentation typically arises from Val30Met variant, while Val122Ile is the most common variant associated with ATTR-CA [4]. ATTR remains nonetheless a systemic disease and can impact other organs [5]. In patients with ATTR-CA, liver function test disturbances have been observed, despite absence of history of liver disease. In such cases, liver elastography is recommended to explore liver stiffness and rule out cirrhosis. However, in patients with ATTR-CA, the current state of knowledge does not allow us to conclude whether these results could be the consequence of congestion secondary to heart failure or linked to amyloid protein infiltration of the liver. Furthermore, the prognostic impact of liver stiffness anomalies is unknown in the presence of ATTR-CA.

The aim of this study was to investigate the prevalence, clinical significance and prognostic value of liver stiffness anomalies explored by elastography in patients with ATTR-CA.

## 2. Methods

### 2.1. Study population

All patients diagnosed with ATTR-CA who underwent liver stiffness assessment between March 2019 and April 2023 at Rangueil Hospital, Toulouse, France, were retrospectively included.

Medical records were reviewed to confirm the diagnosis of ATTR-CA, either variant or wild-type, established by a positive 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid (99mTc-DPD) scintigraphy with a Perugini grade 2 or 3 in absence of monoclonal gammapathy or either a positive non-cardiac or endomyocardial biopsy for ATTR in presence of monoclonal gammopathy or a positive ⁹⁹mTc-HMPD scintigraphy with a Perugini grade 1 [6]. All patients underwent transthyretin genetic testing. Patients with ATTR without cardiac involvement, non-transthyretin-related amyloidosis or previously known liver disease were excluded. We collected demographic, clinical, laboratory, electrocardiogram, transthoracic echocardiography, and liver stiffness data.

This study conforms with the principles outlined in the Declaration of Helsinki. Following French law on ethics, all patients were informed that their codified data would be used for this study. After validation by the data protection officer and according to the General Data Protection Regulation, the study is covered by the MR-004 and have been registered at the Toulouse University Hospital (CNIL number: 2206723 v 0).

### 2.2. Transthoracic echocardiography

Transthoracic echocardiography was performed with either a Vivid E95 or Vivid S70 ultrasound system (GE Healthcare) and images analyzed offline using the EchoPAC V.204 software (GE Medical Systems). Two-dimensional and Doppler echocardiography measurements were performed to collect left ventricular systolic and diastolic parameters and functions including global longitudinal strain, tricuspid regurgitation, elevated right atrial pressure and estimated systolic pulmonary arterial pressure according to the American Society of Echocardiography guidelines (i.e. a diameter of the inferior vena cava ≤21 mm associated to a sniff test inducing more than 50 % collapsibility suggesting normal right atrial pressure, while in other cases right atrial pressure was considered elevated) [7].

### 2.3. Liver stiffness measurement

LSM was obtained through either transient elastography or supersonic shear imaging, providing a result in kilopascals (kPa).

Transient elastography was performed by Fibroscan® [8]. The technique consists of an ultrasound transducer coupled to a vibrator axis, allowing for the creation of vibration waves (i.e. shear waves) transmitted to the liver tissues, while the pulse-echo ultrasound device

measures the velocity of the wave propagation. In the presence of fibrous tissue, which is stiffer than normal liver, there is a variation in shear wave velocity, which makes it possible to quantify liver fibrosis. Controlled attenuation parameter (CAP) in dB/m was developed, combined to Fibroscan technology and assessed simultaneously with LSM, as a complementary parameter allowing for diagnosis of liver steatosis by estimating ultrasonic attenuation upon propagation through liver tissue. CAP cut off values of 238 dB/m, 259 dB/m and 293 dB/m are retained for S1, S2 and S3 steatosis, respectively [9,10].

Supersonic shear imaging uses acoustic radiation force to generate shear waves moving through the tissue at supersonic speed. Tissue stiffness is computed from speed of shear waves travelling across the tissue. A local velocity map is then obtained through speckle tracking technique. This technique allows for the description of liver stiffness as well as viscosity, a mechanical property of tissue [11].

The FIB-4 score, a non-invasive test to assess liver fibrosis was calculated according to current guidelines [12,13].

The cohort was divided in two groups according to a 10 kPa liver stiffness threshold, usually consistent with advanced liver fibrosis (METAVIR score F3 or F4) [8,14].

### 2.4. Liver biopsies and histology

Liver biopsies were performed through transjugular access. After catheterization of the middle or right hepatic vein under fluoroscopy guidance, contrast-enhanced venography was performed to determine the anatomy and optimal positioning for biopsy. A core of tissue was obtained with a needle pass and specimens were then fixed in formalin.

The METAVIR scoring system was used in order to describe liver biopsies [14]. A refers to inflammation, ranging from A0 (no activity) to A3 (severe activity), and F describes fibrosis of liver tissues, from F0 (no fibrosis) to F4 (cirrhosis).

Amyloid deposition was searched for using Congo red dye and congestion was defined on visually dilated sinusoid as reported after examination by an expert pathologist.

### 2.5. Follow-up and prognosis

The patient cohort was divided into prognosis subgroups using a 45 ml/min glomerular filtration rate and 3000 pg/ml NT-proBNP thresholds, applicable to hereditary as well as wild-type group, following Gillmore's staging system [15]. In order to integrate the prognosis value of troponin, the Grogan staging system – combination of 3000 pg/ml NT-proBNP and 0.05 ng/ml T troponin thresholds - was also applied to the wild-type cohort [16].

Follow up data were collected up to July 2023 for the occurrence of hospitalization for heart failure and all-cause death.

### 2.6. Statistical analysis

Continuous variables were presented as mean ± standard deviation when normally distributed or as median [interquartile range] otherwise. Categorical variables were as counts (percentage). Continuous variable differences were tested with t-tests when normally distributed or Wilcoxon signed rank tests in other cases. Categorical variables were compared across groups with Fisher's exact tests or Kruskal Wallis tests in case of comparison across more than two independent groups.

Survival curves were plotted using the Aalen-Johansen estimator and survival analysis consisted of competitive risks models considering two absorbing states: hospitalization for heart failure and all-cause death. A LSM threshold of 10 kPa was used as the explanatory variable for death and heart failure. A log rank test was used to assess the impact of discrete variables on the occurrence of the event of interest. Extended Cox proportional hazard models were performed for continuous variables; applicability was verified with Schoenfeld tests.

All tests were two-sided and p-values <0.05 were considered
statistically significant. All statistical analyses were performed with R version 4.3.0 (R Foundation for Statistical Computing, Vienna, Austria). Survival analysis relied on the *survival* package, Area Under Curves (AUC) estimation relied on the *pROC* package.

# 3. Results

## 3.1. Whole population

Among the 534 patients referred for initial workup of ATTR-CA during the inclusion period, 284 (53 %) received systematic follow-up without selection criteria at our cardiac amyloidosis reference centre and underwent LSM. The remaining patients underwent outpatient follow-up without LSM. Finally, the study population consisted of 284 patients with ATTR-CA in whom LSM was performed. Among them, 258 (91 %) were ATTRwt and 26 (9 %) were ATTRv. The mean age was 83 ± 6 years old and 235 (83 %) patients were men. One hundred and sixty-eight (59 %) patients had arterial hypertension, 38 (13 %) diabetes mellitus, 91 (32 %) had history of coronary artery disease and 157 (55 %) had a glomerular filtration rate below 60 ml/min. One hundred and ninety-five (69 %) patients had supraventricular arrhythmia, mainly atrial fibrillation (91 %), and 90 (31 %) had a pacemaker implantation due to conduction disease. One hundred and sixteen (41 %) patients had polyneuropathy occurring from the disease, either clinical or diagnosed through electromyogram; most of them were subclinical. Among ATTRv, Val122Ile was the most common variant, found in 13 (50 %) patients, followed by Val30Met for 7 (27 %) patients. Other variants were Ala120Ser, Glu62Lys, His56Arg, Ile107Val, Ser77Phe and The69Ile. Globally, patients with ATTRv were 77 ± 7 years old, had a median eGFR of 80 ml/min [63–87] and NT-proBNP of 1002 ng/l [277–1657] and a mean global longitudinal strain of −13.6 ± 4.7 %. Gillmore staging system showed stage 1, 2 and 3 for 20 (78 %), 5 (19 %) and 1 (3 %) patient, respectively.

LSM was obtained through transient elastography for 90 (32 %) patients and supersonic shear imaging for 194 (68 %) patients. Median liver stiffness was 7.7 kPa [5.6–13.4], without significant discrepancy between LSM medians obtained through either technique in ATTRwt (7.5 vs 8.4 kPa, *p* = 0.07) and ATTRv (5.5 vs 6.7 kPa, *p* = 0.17) patients. Median viscosity was 2.2 Pa/s [1.8–2.6] for the patients assessed by supersonic shear imaging and CAP 238 dB/m [202–283] for those assessed through transient elastography. Median FIB-4 score was 2.29 [1.77–2.99].

Using a 10 kPa threshold, prevalence of a high liver stiffness measurement (HLSM) was 36 %. HLSM was found in 4 (15 %) patients with ATTRv and 99 (38 %) patients in the ATTRwt (*p* = 0.03). Liver stiffness was significantly lower among ATTRv patients in comparison to ATTRwt (6.3 and 8.0 kPa respectively, *p* = 0.009), which was also the case for FIB-4 (1.80 and 2.29, *p* = 0.002). A similar pattern was observed for viscosity without quite reaching statistical significance (2.0 and 2.3 Pa/s, *p* = 0.08). Finally, CAP was non significantly higher in ATTRv patients compared to ATTRwt ones (248 and 238 dB/m respectively, *p* = 0.07).

## 3.2. Wild-type subgroups according to LSM

HLSM was found in 98 (38 %) of ATTRwt patients. Patients' characteristics are presented in Table 1. HLSM patients were more often men, with lower body mass index and showed more frequent supraventricular arrhythmia. NT-proBNP and troponin levels were higher in patients with HLSM as compared to patients with LSM. There was no difference for renal function. Liver data showed discrepancies between groups: HLSM group presented higher transaminases levels, alkaline phosphatase, γ-glutamyl transferase and bilirubin compared to LSM group. Regarding transthoracic echocardiography, patients from the HLSM group presented lower left ventricular ejection fraction, higher global longitudinal strain, a higher grade of tricuspid regurgitation, and

<table><caption>**Table 1**
- Baseline characteristics of ATTRwt patients.</caption><thead><tr><th></th><th>ATTRwt</th><th>LLSM</th><th>HLSM</th><th>p value</th></tr></thead><tbody><tr><td>Population</td><td>n = 258</td><td>n = 160</td><td>n = 98</td><td></td></tr><tr><td>Age, y</td><td>84 ± 6</td><td>84 ± 7</td><td>85 ± 6</td><td>0.14</td></tr><tr><td>Male, n (%)</td><td>214 (83)</td><td>126 (79)</td><td>88 (90)</td><td>0.03</td></tr><tr><td>Body mass index, kg/m2</td><td>25.3 ± 3.6</td><td>25.7 ± 3.8</td><td>24.6 ± 3.2</td><td>0.046</td></tr><tr><td>Liver assessment</td><td></td><td></td><td></td><td></td></tr><tr><td>Liver stiffness, kPa</td><td>8.0<br>[5.7–13.9]</td><td>6.1<br>[4.9–7.6]</td><td>16.4<br>[13.2–21.5]</td><td></td></tr><tr><td>Liver viscosity, Pa/s</td><td>2.3 [1.8–2.6]</td><td>1.9<br>[1.7–2.2]</td><td>2.6 [2.4–2.9]</td><td>&lt; 0.0001</td></tr><tr><td>CAP, dB/m</td><td>238<br>[201–283]</td><td>250<br>[204–286]</td><td>217<br>[189–247]</td><td>0.07</td></tr><tr><td>FIB-4</td><td>2.29<br>[1.77–2.99]</td><td>2.13<br>[1.73–2.77]</td><td>2.58<br>[1.92–3.45]</td><td>0.005</td></tr><tr><td>Functional evaluation</td><td></td><td></td><td></td><td></td></tr><tr><td>NYHA status, n (%)</td><td></td><td></td><td></td><td>0.16</td></tr><tr><td>I, n (%)</td><td>47 (18)</td><td>34 (21)</td><td>13 (13)</td><td></td></tr><tr><td>II, n (%)</td><td>162 (63)</td><td>100 (63)</td><td>62 (64)</td><td></td></tr><tr><td>III, n (%)</td><td>49 (19)</td><td>26 (16)</td><td>23 (23)</td><td></td></tr><tr><td>Past medical history</td><td></td><td></td><td></td><td></td></tr><tr><td>Arterial hypertension, n (%)</td><td>157 (61)</td><td>102 (64)</td><td>55 (56)</td><td>0.24</td></tr><tr><td>Diabetes mellitus, n (%)</td><td>38 (15)</td><td>27 (17)</td><td>11 (11)</td><td>0.39</td></tr><tr><td>Coronary artery disease, n (%)</td><td>88 (34)</td><td>59 (37)</td><td>29 (30)</td><td>0.28</td></tr><tr><td>Stroke, n (%)</td><td>31 (12)</td><td>18 (11)</td><td>13 (13)</td><td>0.69</td></tr><tr><td>Neurological involvement, n (%)</td><td>101 (39)</td><td>61 (38)</td><td>40 (41)</td><td>0.69</td></tr><tr><td>Supraventricular arrhythmia, n (%)</td><td>182 (71)</td><td>98 (62)</td><td>84 (86)</td><td>0.0001</td></tr><tr><td>Pacemaker, n (%)</td><td>83 (32)</td><td>51 (32)</td><td>32 (33)</td><td>0.93</td></tr><tr><td>Laboratory data</td><td></td><td></td><td></td><td></td></tr><tr><td>eGFR (CKD-EPI), ml/min</td><td>53 [41–70]</td><td>56 [41–72]</td><td>53 [41–67]</td><td>0.35</td></tr><tr><td>NT-proBNP, pg/ml</td><td>2039<br>[1027–3610]</td><td>1549<br>[769–3039]</td><td>2822<br>[1872–4646]</td><td>&lt; 0.0001</td></tr><tr><td>Troponin, ng/l</td><td>47 [33–74]</td><td>44 [29–64]</td><td>62 [39–84]</td><td>0.0001</td></tr><tr><td>AST, UI/l</td><td>26 [22–32]</td><td>25 [20–30]</td><td>28 [24–36]</td><td>0.0001</td></tr><tr><td>ALT, UI/l</td><td>23 [17–29]</td><td>22 [17–27]</td><td>25 [18–31]</td><td>0.02</td></tr><tr><td>GGT, UI/l</td><td>52 [28–108]</td><td>37 [23–59]</td><td>93 [57–189]</td><td>&lt; 0.0001</td></tr><tr><td>ALP, UI/l</td><td>81 [62–106]</td><td>75 [58–93]</td><td>95 [73–135]</td><td>&lt; 0.0001</td></tr><tr><td>Bilirubin, mmol/l</td><td>9.3 [7–13.3]</td><td>8.8<br>[6.5–11.5]</td><td>11.9<br>[8.5–16.2]</td><td>&lt; 0.0001</td></tr><tr><td>Albumin, g/l</td><td>41 [38–42]</td><td>41 [39–43]</td><td>40 [38–42]</td><td>0.53</td></tr><tr><td>Echocardiographic parameters</td><td></td><td></td><td></td><td></td></tr><tr><td>LVEF, %</td><td>55 [46–62]</td><td>57 [50–63]</td><td>52 [43–60]</td><td>0.008</td></tr><tr><td>GLS, %, n = 250</td><td>-12.1 ± 3.9</td><td>-12.8 ± 3.9</td><td>-10.9 ± 3.6</td><td>0.0001</td></tr><tr><td>TR &gt; 1/5, n (%)</td><td>10 (3)</td><td>3 (2)</td><td>7 (7)</td><td>0.045</td></tr><tr><td>IVS, mm</td><td>15 [14–17]</td><td>15 [14–17]</td><td>16 [14–18]</td><td>0.10</td></tr><tr><td>E/e' ratio, n = 255</td><td>12.9<br>[10.0–16.7]</td><td>12.2<br>[9.1–16.3]</td><td>14.3<br>[10.8–18]</td><td>0.01</td></tr><tr><td>Estimated sPAP, mmHg</td><td>37 [30–45]</td><td>34 [28–41]</td><td>43 [35–51]</td><td>&lt; 0.0001</td></tr><tr><td>Elevated RAP, n(%)</td><td>85 (33)</td><td>26 (16)</td><td>59 (60)</td><td>&lt; 0.0001</td></tr><tr><td>Treatments</td><td></td><td></td><td></td><td></td></tr><tr><td>Tafamidis, n (%)</td><td>254 (98)</td><td>159 (99)</td><td>95 (97)</td><td>0.16</td></tr><tr><td>Anticoagulant, n (%)</td><td>184 (72)</td><td>101 (64)</td><td>83 (85)</td><td>0.0006</td></tr><tr><td>Antiarrhythmic, n (%)</td><td>62 (24)</td><td>41 (26)</td><td>21 (21)</td><td>0.55</td></tr><tr><td>ACEI, n (%)</td><td>108 (42)</td><td>71 (45)</td><td>37 (38)</td><td>0.19</td></tr><tr><td>MRA, n (%)</td><td>76 (29)</td><td>36 (22)</td><td>40 (40)</td><td>0.002</td></tr><tr><td>Furosemide, n (%)</td><td>207 (80)</td><td>119 (75)</td><td>88 (90)</td><td>0.003</td></tr><tr><td>Furosemide dose, mg</td><td>80 [40–125]</td><td>40 [40–120]</td><td>80 [40–250]</td><td>&lt; 0.0001</td></tr></tbody></table>

ACEI: angiotensin converting enzyme inhibitor, ALP: alkaline phosphatase, ALT: alanine transaminase, AST: aspartate transaminase, CAP: controlled attenuation parameter, eGFR: estimated glomerular filtration rate, GGT: gamma glutamyl
transferase, GLS: global longitudinal strain, IVS: interventricular septum, LVEF: left ventricular ejection fraction, MRA: mineralocorticoid antagonist, RAP: right atrial pressure, sPAP: systolic pulmonary artery pressure, TR: tricuspid regurgitation.

showed higher systolic pulmonary artery pressure and right atrial pressure. Considering treatments, patients from the HLSM group were more often treated with mineralocorticoid antagonists and furosemide. In furosemide users, doses were higher in HLSM patients (p = 0.001).

Liver stiffness was 6.1 kPa [4.9–7.6] and 16.4 kPa [13.2–21.5], for LLSM and HLSM respectively, liver viscosity was 1.9 Pa/s [1.7–2.2] and 2.6 Pa/s [2.4–2.9], respectively (p < 0.0001), CAP values were 250 dB/m [204–286] and 217 dB/m [189–247], respectively (p = 0.04) and FIB-4 score was 2.13 [1.73–2.77] and 2.58 [1.92–3.45], respectively (p = 0.005).

While NYHA status did not significantly differ according to liver stiffness, six-minutes walking test was lower in the HLSM group compared to the LLSM group, with 300 m [200–375] vs. 370 m [255–429], respectively (p = 0.001). Peak oxygen consumption did not differ between groups but tended to be lower in the HLSM group (13.7 vs. 15.8 ml/min/kg, p = 0.054).

### 3.3. Liver stiffness and prognostic characteristics in asymptomatic patients

Among the 47 ATTRwt patients with NYHA stage 1, HLSM was found in 13 (28 %) patients. In this subgroup, median NT-proBNP levels were 859 pg/ml [477–2115], median eGFR was 70 ml/min [52–80] and troponin levels were 33 ng/l [22–48]. Ten patients (33 %) had an estimated pulmonary artery pressure over 35 mmHg over the 30 estimations available. Seven patients (15 %) had elevated right atrial pressure, four of them having HLSM.

### 3.4. Histology

Liver biopsies were performed on 7 patients who presented with HLSM after discussion between cardiologist and hepatologist. All had ATTRwt, median LSM was 20.3 kPa (range: 12.8–38.3). NT-proBNP, troponin and eGFR median were 2223 pg/mL [2054–2822], 51 ng/l [34–99] and 61 ml/min [50–70], respectively. All patients were classified stage I or II of Gillmore as well as Grogan staging systems. All biopsies showed liver fibrosis with 4 (57 %) scoring F1 and 3 (43 %) scoring F2. Four (57 %) biopsies showed congestion, and one (14 %) was positive for amyloid deposition. None of the sample exhibited signs inflammation.

### 3.5. Liver stiffness and viscosity among staging systems

Classification of patients according to Gillmore and Grogan staging systems are presented in Table 2, as well as liver stiffness, viscosity, CAP and FIB-4 score. When applying the Gillmore staging system over the wild-type cohort, 134 (52 %) patients were classified as stage I, 84 (33 %) of them as stage II and 40 (15 %) at stage III. Prevalence of HLSM was 33 %, 42 % and 48 % for stage I, II and III. Regarding Grogan staging system, 117 (45 %) patients were stage I, 108 (42 %) of them stage II and 33 (13 %) stage III, with a prevalence of HLSM 28 %, 42 % and 61 %, respectively.

Liver stiffness was significantly higher in more advanced stages in both cases. Viscosity and FIB-4 score differed between stages only according to the Grogan staging system. CAP values showed a trend toward lower values without reaching statistical significance.

### 3.6. Prognostic impact of liver LSM among ATTRwt patients

Median follow-up time among ATTRwt patients was 26 months [15–36]. Thirty-three (13 %) patients were hospitalized for heart failure:

<caption>Table 2</caption>

- Non-invasive testing of liver tissue properties according to prognostic stages of ATTRwt patients.

<table><thead><tr><th>Population</th><th>Stage I</th><th>Stage II</th><th>Stage III</th><th>p value</th></tr></thead><tbody><tr><td>Gillmore staging</td><td>n = 134</td><td>n = 84</td><td>n = 40</td><td></td></tr><tr><td>Liver stiffness, kPa</td><td>6.9 [5.3–11.9]</td><td>9.0 [5.8–16.6]</td><td>9.4 [6.7–16.9]</td><td>0.006</td></tr><tr><td>Liver viscosity, Pa/s</td><td>2.1 [1.8–2.5]<br>253<br>[202–307]<br>2.27</td><td>2.4 [1.8–2.7]<br>238<br>[205–255]<br>2.17</td><td>2.3 [2.1–2.6]<br>225<br>[153–253]<br>2.5</td><td>0.17<br>0.18<br>0.07</td></tr><tr><td>CAP, dB/m</td><td>[1.75–2.73]</td><td>[1.74–3.15]</td><td>[1.86–4.04]</td><td></td></tr><tr><td>FIB-4 score</td><td>n = 117</td><td>n = 108</td><td>n = 33</td><td></td></tr><tr><td>Grogan staging</td><td></td><td></td><td>12.1</td><td></td></tr><tr><td>Liver stiffness, kPa</td><td>6.6 [5.2–10.6]</td><td>9.1 [6.1–16.2]</td><td>[7.4–21.4]</td><td>0.0001</td></tr><tr><td>Liver viscosity, Pa/s</td><td>2.0 [1.7–2.4]<br>253</td><td>2.4 [2.0–2.6]<br>228</td><td>2.6 [2.3–3.0]<br>225</td><td>0.0001</td></tr><tr><td>CAP, dB/m</td><td>[212–305]<br>2.15</td><td>[195–274]<br>2.45</td><td>[205–247]<br>2.47</td><td>0.18</td></tr><tr><td>FIB-4 score</td><td>[1.62–2.69]</td><td>[1.81–3.15]</td><td>[1.83–3.57]</td><td>0.03</td></tr></tbody></table>

CAP: controlled attenuation parameter.

13 (8 %) among the LLSM group and 20 (20 %) among the HLSM group. Thirty-eight (15 %) patients died during follow up, 16 (10 %) in HLSM and 22 (22 %) in HLSM group. Results of event-free survival for hospitalization and all-cause death are presented in Fig. 1.

HLSM was associated with an increased risk of hospitalization for heart failure: HR of 3.00 [1.41–6.41] (p = 0.004), and death: HR 2.50 [1.30–4.79] (p = 0.006). Other variables associated with hospitalization for heart failure were age, liver viscosity, FIB-4 score, NT-proBNP >3000 pg/ml, troponin >50 ng/l, eGFR <45 ml/min, higher estimated systolic pulmonary artery pressure, elevated right atrial pressure, anticoagulant treatment and furosemide dose (Table 3). Same variables were associated with all-cause mortality, except for liver viscosity, and to which were added NYHA stage, supraventricular arrhythmia, alkaline phosphatase >129 UI/l and left ventricular ejection fraction. (Table 4). After adjusting for age, NT-proBNP, eGFR and troponin, HLSM was still associated to an increased risk of hospitalization for heart failure, with a HR of 2.41 [1.05–5.55] (p = 0.04), but no longer for death, with a HR of 1.67 [0.83–3.35], (p = 0.15).

The AUC of LSM with a 10 kPa threshold to predict hospitalization for heart failure was 0.67 [0.56–0.79] with a specificity of 65 % and a sensitivity of 61 %. The AUC to predict death was 0.62 [0.51–0.72] with a specificity of 65 % specificity and a sensitivity of 58 %.

### 3.7. Integration of elevated RAP in the analysis

Elevated RAP was also found to be associated with hospitalization for heart failure as well as death, with respective HR of 3.15 [1.46–6.78, p = 0.003] and 2.78 [1.45–5.33, p = 0.002] (Tables 3 and 4). After adding elevated RAP in the multivariate analysis, LSM and elevated RAP were no longer associated with the risk of hospitalization for heart failure (2.05 [0.80–5.25], p = 0.13; and 1.52 [0.60–3.87], p = 0.37, respectively) or death (1.41 [0.66–3.04], p = 0.37; and 1.50 [0.71–3.15], p = 0.28, respectively).

The AUCs of elevated RAP to predict hospitalization for heart failure and death were 0.59 [0.50–0.68] and 0.63 [0.54–0.72], respectively, without difference with LSM (p for difference = 0.11 and 0.77, respectively).

### 3.8. Integration of liver stiffness assessment in current staging system

Kaplan-Meier curves of hospitalization for heart failure and death depending on LSM and according to Gillmore staging are presented in Fig. 2. Across the different Gillmore stages, HLSM was significantly
<figure><img src="image_5.png" /><figcaption>Fig. 1. - Survival analysis of ATTRwt patients according to LSM.</figcaption></figure>

A: Kaplan Meier curves of hospitalization for heart failure, B: Kaplan Meier curves of all-cause mortality.

LSM: low liver stiffness measurement, HLSM: high liver stiffness measurement.

associated with hospitalizations for heart failure in stage 1 patients only with HR 7.13 [1.93–26.34, *p* = 0.003]. HR in stage 2 patients was 1.37 [0.36–5.29, *p* = 0.64], and 1.70 [0.41–7.10, *p* = 0.47] in stage 3 patients. HLSM was also statistically associated with death in stage 1 patients only with HR 4.00 [1.27–12.66, *p* = 0.02]. In stage 2 and stage 3 patients, HLSM was not significantly associated with death with respective HRs of 2.86 [0.85–9.59, *p* = 0.09] and 0.88 [0.29–2.64, *p* = 0.82].

Kaplan-Meier curves of hospitalization for heart failure and death integrating HLSM to NT-proBNP >3000 pg/ml and eGFR <45 ml/min thresholds, defining a four-stratum staging system depending on the number of pejorative criteria met, are presented in Fig. 3. The survival model of such staging with adjustment for age was found to be overall significant for both hospitalization and death.

Regarding hospitalization for heart failure, and taking stage 1 as the reference, stage 2, 3 and 4 provided an HR of 3.58 [1.03–12.44], (p = 0.04); 4.18 [1.16–15.00], (p = 0.03) and 6.14 [1.48–25.56], (p = 0.01), respectively. Regarding death, and taking stage 1 as the reference, stage 2, 3 and 4 provided an HR of 2.13 [0.73–6.15], (p = 0.16); 4.58 [1.65–12.68], (p = 0.003) and 3.79 [1.12–12.83], (p = 0.03), respectively.

## 4. Discussion

This study finds a prevalence of HLSM of 36 % in a population 284 patients with ATTR-CA patients, with a higher prevalence among patients ATTRwt compared to ATTRv (38 % vs 15 %, *p* = 0.02). ATTRv patient show better prognosis parameters, and 78 % of them are classified as stage 1 following the Gillmore staging system. ATTRwt patients with HLSM have higher global longitudinal strain, NT-pro BNP and troponin levels. HLSM (as well as elevated RAP) is associated with an increased risk of hospitalization for heart failure and death. After adjustment for age, eGFR, troponin and NT-proBNP thresholds, only hospitalization for heart failure remains associated with HLSM. However, once elevated RAP is included in the multivariate analysis, LSM does no longer predict hospitalization for heart failure or death.

In this study, the few liver biopsies taken as part of routine care systematically showed fibrosis, however histological fibrosis score was lower than expected from LSM. This discrepancy suggests that a different factor than fibrosis contributes to and enhances LSM results. The strong association of LSM and elevated RAP with hospitalization for heart failure or death suggests that, in this population, LSM is not a marker of liver fibrosis but of congestion, which could be related to heart failure. This hypothesis is concordant with higher prevalence of moderate or higher tricuspid regurgitation, the higher NT-proBNP levels in the HLSM group, as well as the frequently elevated right atrial pressure and estimated systolic pulmonary artery pressure. This hypothesis could also explain the lower LSM values in the ATTRv patient population, who were younger and had less advanced heart disease than ATTRwt patients. This is consistent with previous studies showing that LSM is increased in case of volume overload linked to heart failure [17,18], with an absence of correlation with histologic liber fibrosis. LSM is also found to reflect increased central venous pressure and liver congestion in patients presenting with heart failure [18]. Chronic high venous pressure could result in intestinal congestion and therefore lead to malabsorption and malnutrition. Although there was no difference in terms of albumin levels, patients from the HLSM presented significantly lower body mass index, which might be related to malnutrition in such population. Interactions between liver and cardiac function are numerous [18], and studies are supportive of this hypothesis, the origin of cachexia in heart failure being possibly related to intestinal congestion [20]. In our study, among the patients with NYHA stage 1, 28 % show HLSM consistent with higher NT-proBNP levels suggesting congestion despite the absence of heart failure symptoms. Finally, in patients with ATTR-CA, LSM could be an useful tool to better assess congestive state; NT-proBNP is known to be an imperfect biomarker in the disease, being elevated not only in relation to high ventricular filling pressure but also due to myocyte damage in relation to the extracellular deposits of amyloid fibrils [21].

In addition to higher congestive state, patients with HLSM appear to present more advanced ATTR-CA and poorer prognosis. They are more frequently hospitalized for heart failure, death rate is higher, and they
<figure><img src="image_6.png" /><figcaption>Fig. 2. – Survival of ATTRwt population upon stratification over NAC stage and high or low LSM.</figcaption></figure>

A: Kaplan Meier curves of hospitalization for heart failure, B: Kaplan Meier curves of all-cause mortality.

HLSM: high liver stiffness measurement, LLSM: low liver stiffness measurement, NAC: National Amyloidosis Center stage.

<figure><img src="image_7.png" /><figcaption>Fig. 3. – Survival of ATTRwt population upon stratification over NT-proBNP, eGFR and LSM.</figcaption></figure>

A: Kaplan Meier curves of hospitalization for heart failure, B: Kaplan Meier curves of all-cause mortality.

Stages 1, 2, 3 and 4 are calculated on the basis of the absence (stage 1) or presence of one (stage 2), two (stage 3) or 3 (stage 4) parameters exceeding the positivity threshold.

eGFR: estimated glomerular filtration rate, LSM: liver stiffness measurement.

congestive state can be assessed by estimating elevated RAP based on vena cava diameter, LSM measurement offers the advantage of a rapid, non invasive and less expensive than echocardiography quantitative measurement, achievable at the patient's bedside.

## 4.1. Limitations

This study presents all the limitations associated with retrospective, single-site studies. Two different techniques were used to perform LSM, which could induce biased result. The modest number of patients included lead to a lack of power, and we could not perform adjustment with more than a few variables considering the few numbers of hospitalization and death among population. Moreover, the threshold of 10

kPa is the standard value usually used in hepatology, consistent with advanced liver fibrosis in chronic diseases. This threshold would probably require to be reassessed in the light of the population explored and the pathophysiological interpretation of the test results. Finally, the ATTRv population could not be studied for prognosis due to the small number of patients, dedicated studies would be interesting to investigate prognosis in patients with increased LSM in this population. LSM might be a useful value to integrate in a more global prognosis score, allowing for personalized follow-up and estimation of individual risk.

## 5. Conclusion

Anomalies of liver stiffness measurements are frequent among
patients with ATTR-CA. Values over 10 kPa are associated with advanced stage of ATTR-CA and increase the risk of hospitalization for heart failure and death among patients with ATTRwt. Liver stiffness assessment in patients known for ATTR-CA could be a useful tool in order to better assess congestion, and distinguish patients of poorer prognosis and needing increased surveillance. Further studies are needed to explore the origin of such liver stiffness, for confirmation of prognosis significance as well as exploration of physiological consequences of such anomalies.

**Disclosure statements, Competing interest:**

None of the authors has competing interests in this study.

**Data sharing statement**

The data that support the findings of this study are available from the
corresponding author, O. Lairez, upon reasonable request.

**Funding**

This work was not supported by any funding.

**CRediT authorship contribution statement**

**Emmanuelle Lointier:** Writing – original draft, Investigation. **Eve Cariou:** Supervision. **Maxime Beneyto:** Formal analysis. **Pauline Fournier:** Writing – review & editing, Methodology. **Yoan Lavie-Badie:** Writing – review & editing, Validation. **Damien Eyharts:** Writing – review & editing, Validation. **Christophe Bureau:** Writing – review & editing, Conceptualization. **Olivier Lairez:** Writing – review & editing, Validation.

**Declaration of competing interest**

None of the authors has competing interests in this study.

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