Extracardiac Biopsy Sensitivity in Transthyretin Amyloidosis Cardiomyopathy Patients With Positive ^{99 m}Tc-Labeled Pyrophosphate Scintigraphy Findings

Abstract

Background: The accurate sensitivity of amyloid deposition in extracardiac tissue (subcutaneous tissue and gastrointestinal tract) has not been evaluated in transthyretin amyloidosis cardiomyopathy (ATTR-CM) patients. This study aimed to evaluate the sensitivity of amyloid deposition in obtained endomyocardial and extracardiac biopsies.

Methods and Results: This study retrospectively evaluated 175 consecutive ATTR-CM patients (wild-type [ATTRwt]: 134, hereditary [ATTRv]: 41) who had positive findings on ^{99 m}Tc-labeled pyrophosphate (^{99 m}Tc-PYP) scintigraphy and underwent tissue biopsy of at least one organ (subcutaneous tissue, gastrointestinal tract, and endomyocardium). Amyloid deposition was observed in the subcutaneous tissue of 57/150 patients (38%), gastrointestinal tract of 80/131 patients (61%), and endomyocardium of 108/109 patients (99%). Compared to patients with ATTRv, ATTRwt had significantly lower sensitivity in subcutaneous tissue (73% vs. 25%, P<0.01) and tended to be lower in the gastrointestinal tract (74% vs. 57%, P=0.08) biopsies. Among 124 patients who underwent both subcutaneous tissue and gastrointestinal tract biopsies, amyloid was detected in at least 1 specimen in 91 (73%) patients. The sensitivity of the combination of extracardiac biopsies was 66% and 94% in ATTRwt-CM and ATTRv-CM, respectively. Multivariate analysis reveals that ATTRv was the only significant predictor of amyloid deposition in the subcutaneous tissue.

Conclusions: Subcutaneous tissue and gastrointestinal tract biopsy sensitivity are inadequate, especially in patients with ATTRwt; however, the combination of these extracardiac biopsies contributes to increased sensitivity in patients with positive ^{99 m}Tc-PYP scintigraphy findings.

Cardiac amyloidosis is a secondary cardiomyopathy characterized by hypertrophic appearance due to the extracellular deposition of misfolded amyloid fibrils. There are 3 main types of cardiac amyloidosis: acquired monoclonal immunoglobulin light chain amyloidosis (AL amyloidosis), hereditary transthyretin (TTR) amyloidosis (ATTRv), and wild-type transthyretin amyloidosis (ATTRwt).^1,2 Transthyretin amyloid cardiomyopathy (ATTR-CM) is gaining recognition because of its high prevalence in elderly patients who have heart failure with preserved ejection fraction and severe aortic stenosis, the development of imaging modalities leading to a non-invasive diagnosis of ATTR-CM, and the potential benefit of novel disease-specific drugs: TTR stabilizers (e.g., tafamidis), small-interfering RNA (patisiran), and antisense oligodeoxynucleotide (inotersen).^3–9

Recently, the diagnostic use of bone scintigraphy for ATTR-CM diagnosis has been proposed by using a semi-quantitative visual scoring of positive cardiac uptake of a tracer without a monoclonal protein by serum and urine testing.^7,8 ATTR-CM can be identified non-invasively.^10,11 However, AL cardiac amyloidosis can lead to positive findings on bone scintigraphy (positive rate: 21–27%)¹⁰ and the coexistence of an unrelated monoclonal gammopathy is common in ATTR-CM (up to 40–50% of cases).¹² Therefore, pathological and immunohistochemical confirmation of amyloid precursor protein is essential to provide a precise diagnosis and allow appropriate therapeutic strategies to be used.

The gold standard for the detection of amyloid deposition is endomyocardial biopsy (EMB) in cardiac amyloidosis; however, this procedure is associated with a low risk of serious complications (∼1%).¹³ Therefore, EMB cannot be routinely performed. In contrast, extracardiac biopsies, such as abdominal fat aspiration, subcutaneous tissue, and gastrointestinal tract, are simple and safe compared to EMB, and their diagnostic use has been recently reported.^14–16 However, the accurate sensitivity of amyloid deposition in the subcutaneous tissue and gastrointestinal tract has not been fully evaluated in a sufficient number of patients who have positive bone scintigraphy findings.

Therefore, this study aimed to evaluate the sensitivity of amyloid deposition in extracardiac tissue (subcutaneous and gastrointestinal tract) and EMB in patients with ATTR-CM who have positive findings on ^{99 m}Tc-labeled pyrophosphate (^{99 m}Tc-PYP) scintigraphy.

Methods

Study Patients

We enrolled 231 consecutive patients with positive findings on ^{99 m}Tc-PYP scintigraphy between June 2002 and April 2021 at Kumamoto University Hospital. Among them, 201 patients underwent tissue biopsy from at least one organ (subcutaneous tissue, gastrointestinal tract, and myocardium), and the histological findings were evaluated. We excluded patients who did not undergo TTR gene testing. The site for the extracardiac biopsy and indication of EMB were decided by the attending physician and upon the preference of the patients. If there was no amyloid deposition in the extracardiac biopsy, we recommended an EMB. Among almost all patients with ATTRv-CM, the TTR mutation type was known at the time of histological evaluation. Conversely, genetic testing and histological evaluation were performed at the same time in patients with ATTRwt-CM; therefore, their genetic background was unknown at the time of histological evaluation.

The study conformed to the principles outlined in the Declaration of Helsinki and was approved by the institutional review board and ethics committee of Kumamoto University (No. 2334). However, the requirement for informed consent was waived because of the low-risk nature of this retrospective study and the inability to obtain consent directly from all subjects. Instead, we extensively announced this study protocol at Kumamoto University Hospital and on our website (http://www.kumadai-junnai.com) and gave patients the opportunity to withdraw from the study.

^{99 m}Tc-PYP Scintigraphy

^{99 m}Tc-PYP scintigraphy was performed using a GE Discovery 670 dual-headed single-photon emission computed tomography/computed tomography/computed tomography camera with low-energy, high-resolution collimators (GE Healthcare, Waukesha, WI, USA). We intravenously administered 555–740 MBq of ^{99 m}Tc-PYP (FUJIFILM, RI, Pharma, Tokyo, Japan). Anterior and lateral planar views of the heart were obtained 3 h after the administration of the radiotracer. The image acquisition time was 5 min, the matrix size was 256×256, and the energy window was set at 140 keV (±10%). Following planar imaging, single-photon emission computed tomography images were acquired for 7 min using a 128×128 matrix size. Scans were performed and interpreted by experienced nuclear cardiologists who were blinded to the subjects’ cohort assignments. Radiotracer accumulation in the myocardium was assessed using both planar and single-photon emission computed tomography to rule out false positive results secondary to radiotracer retention in the left-ventricular blood pool. We evaluated the cardiac uptake of ^{99 m}Tc-PYP using a semi-quantitative visual scoring method in relation to bone (rib) uptake: grade 0: no uptake and normal bone uptake; grade 1: uptake less than rib uptake; grade 2: uptake equal to rib uptake; grade 3: uptake greater than rib uptake with mild or absent rib uptake. Based on previously published results,^{17 99 m}Tc-PYP positivity was defined by a visual score of 2 or 3. Quantitative analysis of cardiac retention was assessed by the heart-to-contralateral (H/CL) ratio, which was calculated as the total counts in a region of interest over the heart divided by the background counts in a region of identical size over the contralateral chest, including soft tissue, ribs, and blood pool.¹¹

Subcutaneous Tissue Biopsy

Subcutaneous tissue evaluation was performed as follows. After disinfection and local anesthesia with 1% lidocaine injected intradermally using a small needle, a fingertip-sized piece of skin that included the epidermis, dermis, and sufficient subcutaneous adipose tissue were obtained using a scalpel from the abdominal wall or lower leg by a dermatologist.

Gastrointestinal Tract Biopsy

An endoscopist performed an upper gastrointestinal endoscopy in patients under conscious sedation. Biopsy samples are obtained from at least 2 specimens from the stomach and duodenum as deep as possible because TTR amyloids are likely deposited in the muscularis mucosae.¹⁸

Endomyocardial Biopsy

We performed an EMB from the right ventricle via the right femoral vein approach. After a long 7-Fr preformed sheath with a primary distal curve was placed into the right ventricle, a long flexible bioptome was advanced through the sheath into the right ventricle. Finally, biopsies were obtained from the ventricular septum. Samples were obtained from at least 3 specimens.

Histological Evaluation, Diagnosis of ATTR-CM, and Analysis of Monoclonal Protein

The diagnosis of amyloid deposition was confirmed by Congo red staining and apple-green birefringence examination under cross-polarized light microscopy. Immunohistochemistry and DNA analyses were used to determine the subtypes of cardiac amyloidosis. ATTR-CM was diagnosed by: (1) presence of TTR deposition in the myocardium; or (2) presence of TTR deposition in extracardiac tissue with positive findings on ^{99 m}Tc-PYP scintigraphy; or (3) positive findings on ^{99 m}Tc-PYP scintigraphy without confirmation of pathological TTR deposition and excluded AL amyloidosis by confirming the absence of monoclonal protein. ATTRwt was diagnosed based on the absence of mutations in the TTR gene. ATTRv was diagnosed based on documented TTR mutations in the DNA analysis. Serum and urine protein electrophoresis by immunofixation electrophoresis and free light chain (FLC; Freelite, The Binding Site Group Ltd., UK) were used to evaluate the presence of monoclonal proteins.

Statistical Analysis

Normally distributed parameters are expressed as mean±SD, whereas variables with skewed distribution data are expressed as medians with interquartile ranges. Differences between groups were examined by using Student’s t-test or the Mann-Whitney U-test for unpaired data. Categorical values are presented as numbers (percentage) and compared using the chi-squared test or Fisher’s exact test, as appropriate. Univariate logistic regression analysis was performed to identify the significant parameters related to amyloid deposition in the extracardiac biopsy. Multivariate logistic regression analysis was performed using the forced inclusion model. All statistical analyses were performed using IBM SPSS Statistics version 25 (IBM Corp., Armonk, USA).

Results

Clinical Characteristics

We included 231 consecutive patients with positive findings on ^{99 m}Tc-PYP scintigraphy. Thirty patients (mean age at diagnosis: 83.2±5.7 years) did not undergo any histological evaluation at the discretion of the attending physician or due to their own preference. The remaining 201 patients underwent tissue biopsy of at least one organ with ^{99 m}Tc-PYP positivity. Among them, 3 patients were diagnosed with AL amyloidosis with its deposition on extracardiac biopsy. A total of 23 patients did not undergo TTR gene testing; the remaining patients (n=175) were diagnosed with ATTRwt-CM (n=134) and ATTRv-CM (n=41). The details of the gene mutations in ATTRv-CM cases are as follows: Val30Met (pVal50Met) (n=20), Glu89Lys (p.Glu109Lys), Gly47Arg (p.Gly67Arg), Gly47Val (p.Gly67Val), Ser50Ile (p.Ser70Ile), Thr49Ser (p.Thr69Ser) (n=2), Ala25Thr (p.Ala45Thr), Ala36Asp (p.Ala56Asp), Glu54Leu (p.Glu74Leu), Glu61Lys (p.Glu81Lys), Ile107Val (p.Ile127Val), Leu55Pro (p.Leu75Pro), Phe33Val (p.Phe53Val), Ser50Arg (p.Ser70Arg), Thr49Ile (p.Thr69Ile), and Thr60Ala (p.Thr80Ala) (n=1). One patient had acquired ATTR-CM and underwent a domino liver transplant from a donor who had ATTRv (mutation unknown).

Table 1 shows the clinical characteristics of the patients with ATTR-CM. Compared to ATTRv-CM, ATTRwt-CM patients were older and predominantly male. In addition, history of hypertension, prior heart failure hospitalization, atrial fibrillation, increased plasma B-type natriuretic peptide levels and serum high-sensitivity cardiac troponin T, impaired renal function, and increased FLC ratio were more frequently observed in patients with ATTRwt-CM. There were 25% (34 of 139) of cases in which the FLC ratio was outside the normal range (0.26–1.65) in this study.

Table 1. Patient Characteristics

Variables	All (n=175)	ATTRwt (n=134)	ATTRv (n=41)	P value
Age (years)	72.5±10.4	76.0±6.1	61.0±13.1	<0.001
Male	149 (85%)	121 (90%)	28 (68%)	0.001
Hypertension	76 (43%)	68 (51%)	8 (20%)	<0.001
Diabetes mellitus	34 (19%)	30 (22%)	4 (10%)	0.074
Dyslipidemia	43 (25%)	37 (28%)	6 (15%)	0.091
Prior HF hospitalization	52 (30%)	45 (34%)	7 (17%)	0.043
Atrial fibrillation	72 (41%)	68 (50%)	4 (10%)	<0.001
Laboratory data
BNP (pg/mL)	191 [100–390]	240 [143–410]	71 [37–158]	<0.001
hs-cTnT (ng/mL)	0.0456 [0.0300–0.0711]	0.0517 [0.0356–0.0826]	0.0277 [0.0156–0.0455]	<0.001
Hemoglobin (g/dL)	13.5±1.8	13.7±1.9	12.9±1.3	0.018
eGFR (ml/min/1.73 m2)	58.7±21.2	53.5±14.5	75.8±29.5	<0.001
FLC κ (mg/L)	27.1 [20.5–38.8] (n=139)	29.1 [21.3–41.5] (n=114)	22.5 [16.5–29.2] (n=25)	0.002
FLC λ (mg/L)	22.2 [17.7–26.8] (n=139)	22.5 [18.1–27.0] (n=114)	20.4 [15.1–26.1] (n=25)	0.188
FLC ratio	1.34 [1.09–1.61] (n=139)	1.39 [1.15–1.67] (n=114)	1.10 [0.95–1.44] (n=25)	0.004
Serum protein electrophoresis; positive	10/98 (10%)	10/98 (10%)	–	–
Urine protein electrophoresis; positive	6/124 (5%)	6/124 (5%)	–	–
Echocardiography
LVDd (mm)	41.5±5.8	41.7±6.3	40.8±3.6	0.374
LVDs (mm)	30.6±6.0	31.4±6.2	27.7±4.5	0.001
IVSd (mm)	15.6±2.9	15.9±2.6	14.9±3.6	0.065
PWd (mm)	15.7±3.0	16.1±2.7	14.3±3.6	0.001
LVEF (%)	53.6±10.4	51.9±10.3	59.0±8.6	<0.001
E/A ratio	1.70±1.28	1.97±1.39	1.09±0.71	<0.001
E/e’ ratio	19.8±7.5	20.8±7.6	16.5±6.1	0.001
Imaging findings
H/CL	1.96±0.32	1.94±0.32	2.02±0.29	0.148

ATTRv, hereditary transthyretin amyloid; ATTRwt, wild-type transthyretin amyloid; BNP, B-type natriuretic peptide; eGFR, estimated glomerular filtration rate; FLC, free light chain; H/CL, heart-to-contralateral ratio; HF, heart failure; hs-cTnT, high-sensitivity cardiac troponin T; IVSd, interventricular septum diameter; LVDd, left ventricular diastolic diameter; LVDs, left ventricular systolic diameter; LVEF, left ventricular ejection fraction; PWd, posterior wall diameter.

Sensitivity of Cardiac and Extracardiac Biopsy

Figure 1 shows the breakdown of biopsy location and the number of study patients. EMB, subcutaneous and gastrointestinal tract biopsies were performed in 109, 150, and 131 patients, respectively. The number of subcutaneous biopsy sites was 1 in all cases of ATTRwt-CM. Among ATTRv-CM patients, the number of subcutaneous biopsy sites was 1 in 17 cases, 2 in 22 cases, and 3 in 2 cases (mean: 1.6 sites).

Figure 1.

Breakdown of biopsy location and the number of study patients. Endomyocardial, subcutaneous, and gastrointestinal tract biopsies were performed in 109, 150, and 131 patients, respectively.

Table 2 shows the sensitivity of amyloid deposition for each tissue biopsy. Amyloid deposition is detected in the subcutaneous tissue (57 of 150, 38%), gastrointestinal tract (80 of 131, 61%), and myocardium (108 of 109, 99%). Compared to ATTRv-CM patients, ATTRwt-CM patients have a significantly lower sensitivity to amyloid deposition in subcutaneous tissue (73% vs. 25%; P<0.001), and this tended to be lower in the gastrointestinal tract (74% vs. 57%, P=0.08) biopsy. The sensitivity of the first subcutaneous biopsy in patients with ATTRv-CM was 61% (25 of 41); this was significantly higher than that in patients with ATTRwt-CM (P<0.001). The per-sample sensitivity of subcutaneous biopsy was 63% (42 of 67) in patients with ATTRv-CM. A comparison of cases with the Val30Met (pVal50Met) and non-Val30Met mutations revealed that the sensitivities of subcutaneous biopsy (65% vs. 80%; P=0.288) and gastrointestinal tract biopsy (75% vs. 77%; P=0.922) did not differ significantly between the 2 mutations.

Table 2. Sensitivity of Tissue Biopsy

Tissue	All (n=175)	ATTRwt (n=134)	ATTRv (n=41)
Heart	108/109 (99%)	97/98 (99%)	11/11 (100%)
Subcutaneous tissue	57/150 (38%)	27/109 (25%)	30/41 (73%)A
First subcutaneous biopsy	52/150 (35%)	27/109 (25%)	25/41 (61%)
Gastrointestinal tract	80/131 (61%)	55/97 (57%)	25/34 (74%)

^AMean number of biopsy sites: 1.6 sites. ATTRv, hereditary transthyretin amyloid; ATTRwt, wild-type transthyretin amyloid.

TTR amyloid deposition was confirmed in 166 patients (95%) in any biopsy specimen. Among them, the number of biopsies performed until confirmation of amyloid deposits was as follows: 1, 118 cases (71%); 2, 31 cases (19%); 3, 16 cases (10%); and 4, 1 cases (1%).

Figure 2 shows the use of a combination of subcutaneous tissue and gastrointestinal tract biopsies. Among 124 patients who underwent both subcutaneous tissue and gastrointestinal tract biopsy, amyloid deposition is detected in at least 1 specimen in 73% (91 of 124). The sensitivity of a combination of subcutaneous tissue and gastrointestinal tract biopsies was significantly higher than the sensitivity of single-site biopsies (subcutaneous tissue biopsy [P<0.001] and gastrointestinal tract biopsy [P=0.036]). In the examination divided into ATTRwt-CM and ATTRv-CM specimens, the sensitivity was 66% and 94%, respectively.

Figure 2.

Sensitivity of the combination of subcutaneous tissue and gastrointestinal tract biopsy. ATTRv, hereditary transthyretin amyloid; ATTRwt, wild-type transthyretin amyloid; GI, gastrointestinal tract; n.d., not done; Sub., subcutaneous tissue.

No complications required additional treatment in this study, such as infection, bleeding, ventricular or gastrointestinal perforation.

Predictive Features in Amyloid Deposition in Extracardiac Biopsy

Table 3 shows the results of the univariate and multivariate analyses of subcutaneous tissue and gastrointestinal tract biopsy positivity in study patients. In univariate analyses, the significant predictors for subcutaneous tissue positivity are often associated with patients’ characteristics of ATTRv, such as younger age, multisite biopsy, low B-type natriuretic peptide, and high-sensitivity cardiac troponin T levels, and preserved ejection fraction. Multivariate analysis reveals that ATTRv is the only significant predictor of amyloid deposition in the subcutaneous tissue (odds ratio: 12.326, 95% confidence interval: 2.458–61.817; P=0.002). Being female and ATTRv tend to be more frequently associated with gastrointestinal tract positivity in univariate analysis; however, they are not significant predictors in multivariate analysis.

Table 3. Univariate and Multivariate Analyses of (A) Subcutaneous Tissue and (B) Gastrointestinal Tract Biopsy Positivity

Variables	Univariable analysis			Multivariable analysis
	OR	95% CI	P value	OR	95% CI	P value
(A) Subcutaneous tissue biopsy
Clinical profiles
Age (years)	0.950	0.919–0.983	0.003	0.997	0.953–1.043	0.899
Female	2.541	0.955–6.762	0.062
Prior HF hospitalization	0.804	0.372–1.736	0.578
Atrial fibrillation	0.454	0.224–0.920	0.029	0.931	0.390–2.219	0.871
ATTRv	8.283	3.661–18.737	<0.001	12.326	2.458–61.817	0.002
Multisite biopsy	4.388	1.663–11.575	0.003	0.574	0.117–2.817	0.494
Laboratory data
Log BNP (pg/mL)	0.639	0.444–0.919	0.016	1.165	0.630–2.151	0.627
Log hs-cTnT (ng/mL)	0.532	0.307–0.923	0.025	1.184	0.496–2.827	0.704
Echocardiography
IVS (mm)	0.971	0.864–1.090	0.618
LVEF (%)	1.061	1.022–1.102	0.002	1.038	0.992–1.086	0.106
E/e’ ratio	1.008	0.964–1.053	0.731
Imaging findings
H/CL	1.022	0.356–2.934	0.968
(B) Gastrointestinal tract biopsy
Clinical profiles
Age (years)	0.998	0.966–1.031	0.897
Female	3.104	0.839–11.494	0.090	2.412	0.610–9.530	0.209
Prior HF hospitalization	1.109	0.499–2.464	0.800
Atrial fibrillation	0.728	0.352–1.503	0.390
ATTRv	2.121	0.896–5.019	0.087	1.732	0.697–4.300	0.237
Laboratory data
Log BNP (pg/mL)	0.835	0.565–1.234	0.365
Log hs-cTnT (ng/mL)	1.019	0.604–1.717	0.944
Echocardiography
IVS (mm)	1.053	0.931–1.191	0.412
LVEF (%)	1.000	0.966–1.034	0.987
E/e’ ratio	1.031	0.980–1.086	0.237
Imaging findings
H/CL	0.666	0.210–2.113	0.490

CI, confidence interval; OR, odds ratio. Other abbreviations are as per Table 1.

Biomarkers and imaging findings associated with the progression of cardiac amyloidosis (e.g., left ventricular hypertrophy, increased B-type natriuretic peptide levels, high-sensitivity cardiac troponin T levels, and H/CL ratio) are not predictors, even when they are examined separately for ATTRwt-CM and ATTRv (Supplementary Tables 1 and 2). Being female and increased E/e’ are considered predictors of subcutaneous tissue positivity in patients with ATTRwt-CM, and reduced left ventricular ejection fraction and increased E/e’ are predictors of gastrointestinal tract positivity in patients with ATTRv-CM.

Discussion

Our study revealed major findings in the sensitivity of amyloid deposition in endomyocardial and extracardiac biopsy from ATTR-CM patients with positive ^{99 m}Tc-PYP scintigraphy. First, the pathological sensitivity of amyloid deposition was 99% (ATTRwt: 99%; ATTRv: 100%) in the myocardium, 38% (ATTRwt: 25%; ATTRv: 73%) in subcutaneous tissue, and 61% (ATTRwt: 57%; ATTRv: 74%) in the gastrointestinal tract. Second, combining the subcutaneous tissue and gastrointestinal tract biopsies can increase the positive rate up to 73% (ATTRwt: 66%; ATTRv: 94%). Finally, the sensitivity of extracardiac biopsy was higher in ATTRv-CM than in ATTRwt-CM patients. To the best of our knowledge, this is the first report to evaluate the sensitivity of subcutaneous tissue and the gastrointestinal tract and the diagnostic use of a combination of these extracardiac biopsies in an adequate number of cases with ATTR-CM who had positive findings on ^{99 m}Tc-PYP scintigraphy.

Gillmore et al reported that EMB confirmed TTR amyloid deposition in 238 of 253 (94%) patients who were positive for bone scintigraphy,¹⁰ which was the same result found in this study. Thus, EMB is highly sensitive to amyloid deposition.

Regarding the sensitivity of amyloid deposition from abdominal fat aspiration in patients with ATTR-CM, Quarta et al¹⁴ reported a sensitivity of 15% (42 of 271) for ATTRwt-CM and 45% (51 of 113) for ATTRv-CM, which was higher than that of ATTRwt patients. This finding was consistent with our subcutaneous tissue biopsy results. Our study revealed that the sensitivity of subcutaneous tissue biopsy was higher than that of abdominal fat aspiration. This may be explained by the fact that subcutaneous tissue biopsy can obtain deep and large tissue, including the dermis and blood vessels, in addition to adipose tissue by surgical incision,^16,19 and allows for multiple examinations among patients with ATTRv in our study. The sensitivity of subcutaneous tissue biopsy has been reported to be approximately 63–73% in ATTRwt-CM patients.^16,19 These results were highly sensitive compared to our results and those of fat aspiration. This discrepancy may be caused by the low number of cases (approximately 10 cases) in previous reports. The sensitivity of subcutaneous tissue biopsy was higher than that of abdominal fat aspiration, but the sensitivity was still low, particularly in ATTRwt-CM patients.

Regarding the sensitivity of gastrointestinal tract biopsy, Ueda et al¹⁵ reported that the sensitivity of the duodenum in the autopsy ATTRwt-CM case was 3 out of 8 (38%). Thus, reliable and clinically useful data regarding the sensitivity of gastrointestinal biopsy for ATTR-CM patients is lacking. Our study provided accurate sensitivity of extracardiac biopsy based on an adequate number of patients compared to previous reports. In addition, the combination of subcutaneous tissue and gastrointestinal tract biopsy can increase the sensitivity of amyloid deposition with minimal invasiveness. This knowledge was novel and useful for clinical practice when treating patients who must have histological confirmation of amyloid deposition and a definition of precursor protein.

According to the non-biopsy criteria proposed by Gillmore et al,¹⁰ ATTR-CM can be diagnosed if there are positive findings on bone scintigraphy without monoclonal protein presence. However, the presence of monoclonal proteins is not rare in the elderly. Abnormalities in the FLC ratio were observed in 25% of our study patients with ATTRwt-CM, and histological diagnosis is required in these cases. In addition, histological confirmation of TTR deposition in the myocardium or extracardiac tissue is required for tafamidis use in Japan.²⁰ Skilled operators can safely perform EMB in specialized centers without a diagnostic delay;²¹ however, there are few high-volume centers for EMB, and attending physicians may be hesitant due to patients’ wishes, frailty, and difficulty in specialized hospital access, especially for the elderly. In contrast, subcutaneous tissue and gastrointestinal tract biopsies can be performed even in regional facilities where an EMB cannot be performed, and the diagnostic approach can be processed without delay. These findings will contribute to the early and accurate diagnosis and therapy of ATTR-CM patients, which has been regarded as important in recent years.

We proposed a diagnostic strategy to confirm amyloid deposition in patients with suspected cardiac amyloidosis (Figure 3). EMB is recommended if it is performed by a skilled operator at specialized centers or if an immediate histological diagnosis is required. An extracardiac biopsy is preferred for patients with frailty, TTR mutations, those with difficulty in accessing a specialized hospital, those who are of advanced age, and those who refuse an EMB. However, if extracardiac biopsy could not confirm amyloid deposition, an EMB should be considered to avoid diagnostic delay. In terms of AL amyloidosis, the sensitivity of amyloid deposition detected by extracardiac biopsy is higher (~80%) than in cases of ATTR-CM;^14,22 therefore, extracardiac biopsy should be considered first to minimize complications in patients with extremely abnormal FLC ratios.

Figure 3.

Diagnostic strategy to confirm amyloid deposition in patients with suspected cardiac amyloidosis. AL, light chain amyloid; ATTR-CM, transthyretin amyloid cardiomyopathy; ATTRv, hereditary transthyretin amyloid; ATTRwt, wild-type transthyretin amyloid; EMB, endomyocardial biopsy; FLC, free light chain; ^{99 m}Tc-PYP, ^{99 m}Tc-labeled pyrophosphate; TTR, transthyretin.

In this study, no factors other than TTR mutations were found to be related to positive extracardiac biopsy. In amyloidosis, disease progression is thought to be caused by increased amyloid deposits, and total body amyloid burden associated with diagnostic sensitivity in fat aspiration in AL amyloidosis.²³ In ATTR-CM patients, it is expected that the amount of total body amyloid burden and the sensitivity of extracardiac biopsy will increase as ATTR-CM advances. However, the H/CL ratio, cardiac biomarkers, and cardiac hypertrophy and function, which are thought to be related to the progression of cardiac amyloidosis,^24–26 had no clear correlation with the sensitivity of extracardiac biopsy. Thus, several factors have not yet been clarified regarding the mechanisms of amyloid deposition and tissue tropism in ATTR.

Study Limitations

This study has some limitations. First, this was a single-centered study that evaluated Japanese patients who commonly had a Val30Met (pVal50Met) mutation in ATTRv. Future large studies in several countries, with different races and genotypes are warranted to validate our results. Second, genetic testing was not performed in 23 patients (11%) among those with ^{99 m}Tc-PYP positivity, and these patients were excluded from the analysis. Among them, 19 patients (83%) did not have a histological diagnosis confirmed by extracardiac biopsy. Therefore, the sensitivity of an extracardiac biopsy would be decreased if these patients were included (subcutaneous tissue: 36% and gastrointestinal tract: 59%). Third, known TTR mutations affected the choice and number of extracardiac biopsy sites, and multiple biopsies may have influenced the high sensitivity of subcutaneous tissue biopsy in ATTRv-CM patients. The use of multiple subcutaneous tissue biopsies could not be determined in our study. Finally, subcutaneous tissue biopsy and gastrointestinal tract biopsy procedures are standardized and common procedures; however, differences in the technique used by different examiners could exist. We encourage obtaining samples as deeply as possible in gastrointestinal biopsies from the stomach and duodenum because ATTR amyloids are likely to deposit submucosally.

Conclusions

A recent non-invasive diagnostic algorithm for cardiac amyloidosis is very useful; however, histological confirmation of amyloid deposition and precursor protein are required in patients with the presence of monoclonal protein. Our findings on the sensitivity of extracardiac biopsy show that extracardiac biopsy and the combination of these extracardiac biopsies can be a safe alternative to EMB and provide important insights for the histological diagnosis of cardiac amyloidosis.

Acknowledgments

We would like to thank Editage (www.editage.com) for English-language editing.

This work was supported by the Japan Agency for Medical Research and Development (grant number: 20ek0109466 h0001) and JSPS KAKENHI (grant number: 21K08131).

Disclosures

S.T. and K.T. have received remuneration for lectures from Pfizer Japan Inc. M.U. has received remuneration for lectures from Pfizer Japan Inc and Alnylam, and research funding from Pfizer Japan Inc.

K.T. is a member of Circulation Journal’s Editorial Team.

IRB Information

The Kumamoto University Hospital (Reference number: 2334) approved this study.

Data Availability

The deidentified participant data will not be shared.

Supplementary Files

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-22-0118

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