Elucidating associations between technetium pyrophosphate scintigraphy, echocardiography and cardiac biomarkers in transthyretin cardiac amyloidosis

Abstract

Background

Transthyretin amyloidosis is an under-recognised systemic disease whereby misfolded transthyretin proteins form fibrils capable of depositing in various tissues and organs. Despite improvements to diagnostic modalities, the associations between imaging techniques and clinical laboratory metrics remain unclear.

Methods

A single-centre retrospective cohort study was performed including 183 patients aged 18 years or older diagnosed with transthyretin cardiac amyloidosis (ATTR-CA) in a tertiary care setting. Linear regression and multivariate proportional hazard models were used to examine the associations between established imaging modalities (ie, technetium-99m pyrophosphate (^99mTc-PYP) scintigraphy and echocardiography) and cardiac biomarkers (ie, cardiac troponin I and B-type natriuretic peptide (BNP)). The study included patients who visited the Toronto General Hospital Peter Munk Cardiac Centre between October 2012 and December 2022.

Results

Of the 183 patients included, 143 (78.1%) were male, with a median age (IQR) of 73.0 (66.0–79.0) years. Primary analyses revealed significant associations between positive ^99mTc-PYP grading and echocardiographic parameters, particularly increased left ventricular (LV) mass (β=111.21, p=0.009) and greater interventricular septal thickness at end-diastole (IVSd) (β=0.48, p=0.003) in patients with hereditary transthyretin amyloidosis (ATTRm). Additionally, positive ^99mTc-PYP grades correlated significantly with cardiac biomarkers, including log-transformed BNP (logBNP; β=1.99, p=0.002) in patients with ATTRm and log-transformed Troponin (logTroponin; β=1.68, p=0.007) in patients with wild type ATTR (ATTRwt). Conversely, the heart-to-contralateral lung ratio, a quantitative index derived from ^99mTc-PYP scintigraphy, did not show significant correlations with cardiac biomarkers (logBNP and logTroponin), but demonstrated significant associations with LV mass (β=134.52, p=0.001) and IVSd (β=0.46, p=0.002) in patients with ATTRm.

Conclusions

These data suggest strong associations exist between cardiac biomarkers, structural echocardiographic changes and ^99mTc-PYP scintigraphy, emphasising the importance of a multipronged diagnostic approach stratified by genotype.

WHAT IS ALREADY KNOWN ON THIS TOPIC?

WHAT THIS STUDY ADDS?

• In a cohort of 183 patients with confirmed ATTR-CA, we identified strong correlations between positive ^99mTc-PYP grades and elevated BNP, troponin levels and echocardiographic markers of cardiac remodelling (left ventricular mass, interventricular septal thickness at end-diastole, diastolic dysfunction).

• Genotype-specific diagnostic patterns emerged: BNP was more predictive in ATTRm while troponin correlated more with ATTRwt. Visual PYP grading outperformed the heart-to-contralateral lung ratio, which showed weaker and inconsistent associations across subtypes.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY?

• Supports the integration of routine, low-cost tools such as BNP, troponin and echocardiography into first-line screening protocols for ATTR-CA, especially in patients with unexplained heart failure with preserved ejection fraction.

• Highlights to healthcare providers the diagnostic variability by genotype, underscoring the need for tailored diagnostic thresholds, including indexed echocardiographic parameters for women and genotype-informed biomarker interpretation.

Introduction

Transthyretin cardiac amyloidosis (ATTR-CA) is a progressive, infiltrative cardiomyopathy that has undergone a remarkable transition from being perceived as a rare disorder to an increasingly recognised and clinically significant cause of heart failure.¹,⁵ This evolution in understanding is largely attributable to concurrent advances in non-invasive diagnostic imaging and a heightened index of clinical suspicion within the cardiology community.⁶ Consequently, ATTR-CA is no longer considered an esoteric ‘zebra’ but is now identified in a substantial proportion of older adults presenting with common cardiovascular phenotypes.⁴ Epidemiological data and screening studies have revealed that ATTR-CA accounts for approximately 12%–13% of patients with heart failure with preserved ejection fraction (EF) and 10%–15% of those with severe aortic stenosis undergoing valve replacement, underscoring its growing importance for the practising cardiologist.¹⁷,⁹ The disease is driven by the destabilisation of the transthyretin (TTR) protein, whose subsequent misfolding and aggregation into insoluble amyloid fibrils leads to their deposition within the myocardial interstitium.^{2 10} This infiltrative process imparts cardiac injury through a dual mechanism: (1) The physical disruption of myocardial architecture, which increases ventricular stiffness, and (2) A direct cytotoxic effect from soluble TTR precursors, culminating in progressive systolic and diastolic dysfunction.²

The diagnostic landscape for ATTR-CA has been revolutionised by this improved pathophysiological understanding, shifting from a reliance on invasive endomyocardial biopsy to a validated, non-invasive algorithm.^{1 2 11} Central to this new paradigm is nuclear scintigraphy with bone-avid radiotracers, most notably technetium-99m pyrophosphate (^99mTc-PYP). In patients presenting with clinical and imaging features suggestive of cardiac amyloidosis, a positive ^99mTc-PYP scan demonstrates exceptionally high diagnostic specificity and positive predictive value for ATTR-CA, without a concomitant monoclonal gammopathy (eg, amyloid light-chain (AL) amyloidosis).¹¹,¹³ This evolution towards a safer and more accessible diagnosis is of clinical importance, as it has coincided with the advent of effective disease-modifying therapies, including TTR stabilisers and gene silencers, that can slow disease progression and improve outcomes.^{3 14} The efficacy of these treatments is greatest when initiated early, making timely and accurate diagnosis essential to altering the disease’s natural history.^{3 14}

While ^99mTc-PYP scintigraphy is a cornerstone for diagnosing ATTR-CA, suspicion is often initially raised by conventional investigations like echocardiography and cardiac biomarkers.^{5 15} On an echocardiogram, increased interventricular septal thickness and a relative apical sparing pattern of global longitudinal strain often raise suspicion for the diagnosis.^{5 15} Concurrently, elevated levels of B-type natriuretic peptide (BNP) and high-sensitivity cardiac troponin, which are highly sensitive for myocardial stress and injury, are used in prognostic staging.^{5 15} However, the precise quantitative relationships between these widely available ‘red flag’ indicators and the findings of specialised scintigraphy remain incompletely characterised.^{12 16 17} It is not fully understood how well the degree of scintigraphic uptake, as assessed by the semiquantitative Perugini grade¹⁸ or the quantitative heart-to-contralateral lung (H/CL) ratio, reflects the severity of structural remodelling or the intensity of ongoing myocardial injury. Given these uncertainties, the present study aimed to elucidate the relationships between conventional cardiac imaging, laboratory biomarkers and specialised scintigraphic tools to clarify their prognostic relevance and enhance the integrated clinical approach to diagnosing and managing ATTR-CA.

Methods

Study design and population

This single-centre, retrospective cohort study was conducted at the Peter Munk Cardiac Centre, University Health Network in Toronto, Ontario. We identified all patients referred for suspected ATTR-CA between October 2012 and December 2022. The study protocol adhered to the principles of the Declaration of Helsinki II.¹⁹ Eligible participants were adults (≥18 years) with a confirmed diagnosis of ATTR-CA, established through one of the following validated pathways: (1) Histological confirmation of cardiac amyloidosis via endomyocardial biopsy showing Congo red-positive amyloid deposits with typing by mass spectrometry or immunohistochemistry (n=68); (2) Pathogenic or likely pathogenic TTR gene mutation with typical echocardiographic findings of cardiac amyloidosis (n=10); (3) Positive 99^mTc-PYP scintigraphy (Perugini grade 2 or 3)¹³ in conjunction with histological evidence of amyloid deposits in a non-cardiac tissue (n=89); or (4) Characteristic echocardiographic findings of amyloid infiltration in the absence of a monoclonal gammopathy (n=16). Genetic testing was used to validate findings, with patients having an identified pathogenic TTR mutation classified as having hereditary ATTR-CA (ATTRm), while those without a mutation were classified as having wild type ATTR-CA (ATTRwt). Additional exclusion criteria included a final diagnosis of an alternative form of amyloidosis (eg, AL amyloidosis) or clinical contraindications to requisite diagnostic imaging. Relevant demographic, clinical, therapeutic and outcomes data were obtained through a retrospective review of institutional electronic health records. Neither patients nor the public were involved in the design, conduct, reporting or dissemination plans of this research.

Biomarker analysis

All serum biomarker measurements were performed at a centralised core laboratory. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation, consistent with standard clinical practice in Canada.²⁰

^99mTc-PYP scintigraphy

The primary analysis, which was based on myocardial tracer uptake, used the two established methods: (1) Semiquantitative visual scoring of cardiac retention by a qualified nuclear cardiology specialist (0=absent cardiac uptake, 1=mild uptake less than bone, 2=moderate uptake equal to bone, 3=high uptake greater than bone), and (2) Quantitative analysis of H/CL ratio of uptake by drawing circular target regions of interest (ROI) over the heart on the planar images and subsequent mirroring over the contralateral chest to normalise against spillover from the ribs. Total and absolute mean counts were measured in each ROI correcting for background counts, and the fraction of mean counts in the heart ROI-to-contralateral chest ROI was calculated and reported as the H/CL ratio. A positive PYP grading includes an H/CL ratio of >1.5 and a PYP grade of 2 or 3.¹³

Echocardiography

Echocardiographic variables were retrospectively obtained from electronic patient records. All measurements were performed by a single experienced cardiologist, who was blinded to clinical data, using a validated imaging analysis software (Philips). Inter-rater variability for echocardiographic measurements has been previously assessed at our institution, although in a different patient population, and demonstrated excellent reproducibility.²¹ Functional calculations were conducted by trained cardiologists in accordance with the American Society of Echocardiography guidelines.²² Specifically, EF and left atrial volume were calculated using the biplane Simpson method from volumes acquired during the apical two-chamber and four-chamber views. Left ventricular (LV) mass was calculated through the Cube formula, and relative wall thickness was assessed as the ratio of 2 × posterior wall thickness at end diastole over LV end-diastolic diameter with the threshold for concentric hypertrophy diagnosis being >0.42 and LV mass index >115 g/m². Indexing to the body surface area estimated by the LV cavity dimensions and wall thickness at end diastole provided the metrics by which LV Mass Index was calculated. Right ventricular wall thickness was measured from subcostal views using a wall thickness >5 mm to be indicative of hypertrophy.

Statistical analysis

Descriptive analyses were performed to characterise baseline demographic, laboratory and imaging characteristics. Continuous variables were summarised as medians with IQRs, and categorical variables as frequencies with percentages. Between-group differences (ATTRwt-CA vs ATTRm-CA) were evaluated using the Wilcoxon rank-sum test for continuous data and either χ² or Fisher’s exact tests for categorical data, as appropriate, based on cell counts. Linear regression models were used to examine associations between PYP grade and selected cardiac biomarkers, as well as interventricular septal thickness. The normality of continuous variables was verified using graphical methods (eg, histograms, Q-Q plots) and formal statistical assessments, including the Shapiro–Wilk test, informing the selection of parametrical or non-parametrical tests accordingly. Analyses were performed using SAS V.9.4 (SAS Institute, Cary, North Carolina, USA) and GraphPad Prism V.8 (GraphPad Software, La Jolla, California). A two-tailed value of p<0.05 was considered statistically significant for all analyses.

Results

Baseline characteristics

A total of 183 patients with confirmed ATTR-CA were included in the analysis, of whom 112 (61.2%) had ATTRwt and 71 (38.8%) had ATTRm disease (table 1). The median age for the overall cohort was 73.0 years (IQR, 66.0–79.0), and patients were predominantly male (78.1%). Significant demographic differences were noted between the two ATTR subtypes at baseline. Specifically, patients with ATTRwt were significantly older than those with ATTRm (median 74.5 vs 69.0 years, p<0.001) and were more likely to be male (83.9% vs 69.0%, p=0.017). Conversely, a family history of amyloidosis was more prevalent in the ATTRm group (26.8% vs 3.6%, p<0.001). Patients predominantly presented with cardiac symptoms; however, neuropathic symptoms were significantly more common in the ATTRm cohort (14.1% vs 1.8%, p<0.001). This was further reflected by significantly higher rates of sensory peripheral neuropathy (41.4% vs 12.5%, p<0.001) and autonomic dysfunction (27.1% vs 10.7%, p=0.004) in patients with ATTRm. Most patients presented in New York Heart Association functional classes II (38.9%) or III (35.4%), with no significant differences between the ATTRwt and ATTRm groups.

Table 1. Baseline clinical and demographic characteristics of patients with transthyretin amyloid cardiomyopathy, stratified by subtype.

Variable	Patients with ATTR-CA, No. (%)			P value
	Overall (n=183)	ATTRwt (n=112)	ATTRm (n=71)
Age, years	73.0 (66.0–79.0)	74.5 (69.0–81.0)	69.0 (59.0–77.0)	<0.001
Sex				0.017
Male	143 (78.1%)	94 (83.9%)	49 (69.0%)
Female	40 (21.9%)	18 (16.1%)	22 (31.0%)
Follow-up duration (years)	1.36 (0.48–2.97)	1.05 (0.43–2.86)	1.51 (0.66–3.10)	0.196
Death	46 (25.3%)	30 (27.0%)	16 (22.5%)	0.496
Method of diagnosis				0.043
PYP scan	88 (48.1%)	55 (49.1%)	33 (46.5%)
Biopsy	68 (37.2%)	46 (41.1%)	22 (31.0%)
Imaging*	16 (8.7%)	8 (7.1%)	8 (11.3%)
Genetics only	10 (5.5%)	2 (1.8%)	8 (11.3%)
Family history	23 (12.6%)	4 (3.6%)	19 (26.8%)	<0.001
Symptoms at diagnosis				<0.001
No symptoms	19 (10.4%)	6 (5.4%)	13 (18.3%)
Cardiac	140 (76.5%)	99 (88.4%)	41 (57.8%)
Neuropathic	12 (6.6%)	2 (1.8%)	10 (14.1%)
Mixed	11 (6.0%)	5 (4.5%)	6 (8.5%)
Other†	1 (0.6%)	0 (0%)	1 (1.4%)
Comorbidities
Dyslipidaemia	81 (51.9%)	54 (56.8%)	34 (55.7%)	0.125
Diabetes	29 (17.5%)	15 (14.9%)	14 (21.5%)	0.269
Hypertension	95 (56.9%)	56 (54.9%)	39 (60.0%)	0.517
Carpal tunnel syndrome	55 (30.2%)	35 (31.2%)	20 (28.6%)	0.702
NYHA class	175	108	67	0.141
Asymptomatic‡	9 (5.1%)	2 (1.9%)	7 (10.5%)
Class 1	26 (14.9%)	15 (13.9%)	11 (16.4%)
Class 2	68 (38.9%)	45 (41.7%)	23 (34.3%)
Class 3	62 (35.4%)	40 (37.0%)	22 (32.8%)
Class 4	10 (5.7%)	6 (5.6%)	4 (6.0%)
Transplantation				0.064
Liver	4 (2.2%)	1 (0.9%)	3 (4.3%)
Heart	2 (1.1%)	0 (0%)	2 (2.9%)
None	174 (96.7%)	109 (99.1%)	65 (92.9%)
Systems dysfunction
Multiorgan involvement*	21 (11.6%)	7 (6.3%)*	14 (20.0%)*	0.005
Autonomic dysfunction	31 (17.0%)	12 (10.7%)	19 (27.1%)	0.004
Sensory peripheral neuropathy	43 (23.6%)	14 (12.5%)	29 (41.4%)	<0.001
Motor neuropathy	21 (11.5%)	7 (6.3%)	14 (20.0%)	0.005
Neuropathy symptoms	87 (47.8%)	46 (41.2%)	41 (58.6%)	0.022
Laboratory measurements
Creatinine, µmol/L	122.79±66.75	126.37±54.17	117.41±82.26	0.019
eGFR, mL/min	58.35±24.15	53.8±21.1	65.03±26.83	0.009
BNP, pg/mL	469.82±463.7	524.29±513.46	397.51±380.31	0.093
cTn, pg/mL	309.77±1243.07	173.52±486.55	475.75±1766.92	0.975
Echocardiogram parameters
IVSd, mm	1.52±0.39	1.55±0.36	1.48±0.43	0.321
LVIDd, mm	4.37±0.57	4.43±0.57	4.3±0.58	0.384
LVIDs, mm	3.33±0.69	3.34±0.73	3.32±0.64	0.869
LVPWd, mm	1.45±0.4	1.52±0.38	1.36±0.4	0.029
LV mass, g	269.68±109.71	287.29±110.82	246.32±104.73	0.024
LVEF, %	51.26±12.69	52.18±11.24	49.99±14.45	0.391
99mTc‐PYP scintigraphy parameters
H/CL ratio	1.90±0.47	1.92±0.47	1.86±0.47	0.660
Medications
Diuretic	125 (68.3%)	82 (73.2%)	43 (60.6%)	0.073
Beta blocker	83 (45.4%)	55 (49.1%)	28 (39.4%)	0.200
ACEi	50 (27.3%)	35 (31.2%)	15 (21.1%)	0.134
ARB	31 (16.9%)	17 (15.2%)	14 (19.7%)	0.425
Digoxin	7 (3.8%)	5 (4.5%)	2 (2.8%)	0.571
MCRA	57 (31.2%)	41 (36.6%)	16 (22.5%)	0.045
Diflunisal	10 (5.5%)	6 (5.4%)	4 (5.6%)	0.936
Other§	67 (36.6%)	37 (33.0%)	30 (42.3%)	0.207

Data are presented as median (IQR) for continuous variables and n (%) for categorical variables. Due to missing observations, the number of patients included in the analysis is specified (ie, overall total) for variables where it differs from the total. P values were calculated using the Wilcoxon rank-sum test for continuous variables and the χ2 or Fisher’s exact test for categorical variables.

^*Imaging includes CMR and/or transthoracic echocardiography as the primary imaging modality for diagnosis.

^†'Other’ symptoms include those related to specific comorbid conditions that led to hospitalisation preceding the formal diagnosis of amyloidosis.

^‡'Asymptomatic’ under NYHA class refers to patients with amyloid deposits identified in non-cardiac tissue without clearly defined clinical cardiac disease at the time of assessment.

^§'Other’ medications include disease-modifying therapies for transthyretin amyloidosis (eg, patisiran, inotersen, tafamidis) and other classes of cardiac medications not otherwise specified in the table.

ACEi, ACE inhibitor; ARB, angiotensin-receptor blocker; ATTR-CA, transthyretin amyloid cardiomyopathy; ATTRm, hereditary transthyretin amyloidosis; ATTRwt, wild type transthyretin amyloidosis; BNP, B type natriuretic peptide; CMR, cardiovascular magnetic resonance; cTn, cardiac troponin; eGFR, estimated glomerular filtration rate; H/CL, heart-to-contralateral lung (ratio); IVSd, interventricular septal thickness at end-diastole; LV, left ventricular; LVEF, left ventricular ejection fraction; LVIDd, left ventricular internal diameter at end-diastole; LVIDs, left ventricular internal diameter at end-systole; LVPWd, left ventricular posterior wall thickness at end-diastole; MCRA, mineralocorticoid receptor antagonist; 99mTc-PYP, technetium-99m pyrophosphate; NYHA, New York Heart Association; PYP, pyrophosphate.

Laboratory and echocardiographic findings also varied between subtypes. Patients with ATTRw exhibited significantly impaired renal function, evidenced by a lower eGFR (53.8 vs 65.0 mL/min; p=0.009). Echocardiography indicated that the ATTRwt group had significantly increased LV posterior wall thickness (1.52 vs 1.36 mm, p=0.029) and LV mass (287.3 vs 246.3 g, p=0.024). However, interventricular septal thickness and EF did not significantly differ between groups. Medication assessment revealed a significantly higher usage of mineralocorticoid receptor antagonists in the ATTRwt cohort (36.6% vs 22.5%, p=0.045). The median follow-up duration for the cohort was 4.9 years (IQR 3.9–7.8), with comparable follow-up durations between ATTRwt (4.7 years) and ATTRm (5.0 years) subgroups (p=0.14). During follow-up, 46 patients (25.3%) died, with mortality rates similar between the ATTRwt and ATTRm cohorts (27.0% vs 22.5%, p=0.496).

Associations between cardiac biomarkers and echocardiographic parameters in the ATTRwt subgroup

To better understand the associations between basic cardiac investigations, the total ATTR-CA population was stratified by ATTR subtype. In the ATTRwt-CA subgroup (table 2), logBNP showed no significant association with either interventricular septal thickness at end-diastole (IVSd) or LV mass. In contrast, logTroponin was significantly associated with IVSd (β=1.33, 95% CI 0.46 to 2.19, p=0.003) but not with LV mass (p=0.089). A positive ⁹⁹ᵐTc-PYP grade was significantly associated with higher logTroponin (β=1.68, 95% CI 0.49 to 2.87, p=0.007) and approached statistical significance with logBNP (β=1.07, 95% CI −0.04 to 2.18, p=0.058). No significant associations were observed between the H/CL ratio and either biomarker.

Table 2. Associations between cardiac laboratory values with echocardiography and ^99mTc-PYP scintigraphy parameters.

Variable	Group	logBNP estimate (95% CI)	P value	logTroponin estimate (95% CI)	P value
LV mass	ATTRwt	0.002 (−0.001 to 0.004)	0.190	0.003 (−0.001 to 0.007)	0.089
	ATTRm	0.005 (0.002 to 0.009)	0.005	0.009 (0.003 to 0.019)	0.002
IVSd	ATTRwt	0.51 (−0.18 to 1.19)	0.140	1.33 (0.46 to 2.19)	0.003
	ATTRm	1.12 (0.25 to 1.98)	0.013	2.17 (1.05 to 3.29)	<0.001
Positive PYP*	ATTRwt	1.07 (−0.04 to 2.18)	0.058	1.68 (0.49 to 2.87)	0.007
	ATTRm	1.99 (0.76 to 3.23)	0.002	1.30 (−0.46 to 3.06)	0.140
H/CL ratio	ATTRwt	0.16 (−0.52 to 0.83)	0.650	0.074 (−0.63 to 0.78)	0.840
	ATTRm	1.12 (0.02 to 2.22)	0.047	1.32 (−0.061 to 2.71)	0.060

^*Positive PYP includes results yielding Grade two or 3.

ATTRm, hereditary transthyretin amyloidosis; ATTRwt, wild type transthyretin amyloidosis; BNP, B-type natriuretic peptide; H/CL, heart-to-contralateral lung (ratio); IVSd, interventricular septal thickness at end-diastole; LV, left ventricular; 99mTc-PYP, technetium-99m pyrophosphate; PYP, pyrophosphate.

Among the imaging findings for the ATTRwt subgroup (table 3), a positive ⁹⁹ᵐTc-PYP grade was associated with increased IVSd (β=0.339, 95% CI 0.028 to 0.650, p=0.034), but not with LV mass, LV Mass Index or left ventricular posterior wall thickness at end-diastole (LVPWd). The H/CL ratio demonstrated significant associations with LV mass (β=71.19, 95% CI 18.94 to 123.43, p=0.009), IVSd (β=1.95, 95% CI 1.26 to 2.63, p=0.001), LV Mass Index (β=36.11, 95% CI 14.33 to 57.89, p=0.002) and LVPWd (β=0.34, 95% CI 0.18 to 0.50, p=0.001).

Table 3. Associations between echocardiography and ^99mTc-PYP scintigraphy parameters.

Variable	Group	Positive PYP* (95% CI)	P value	H/CL ratio (95% CI)	P value
LV mass	ATTRwt	60.96 (−35.68 to 157.59)	0.210	71.19 (18.94 to 123.43)	0.009
	ATTRm	111.21 (30.51 to 191.91)	0.009	134.52 (63.90 to 205.13)	0.001
IVSd	ATTRwt	0.339 (0.028 to 0.650)	0.034	1.95 (1.26 to 2.63)	0.001
	ATTRm	0.48 (0.18 to 0.77)	0.003	0.46 (0.18 to 0.73)	0.002
LV Mass Index	ATTRwt	29.69 (−14.53 to 73.91)	0.200	36.11 (14.33 to 57.89)	0.002
	ATTRm	64.77 (26.92 to 102.62)	0.002	67.81 (37.33 to 98.29)	0.001
LVPWd	ATTRwt	0.27 (−0.04 to 0.58)	0.100	0.34 (0.18 to 0.50)	0.001
	ATTRm	0.49 (0.23 to 0.75)	0.001	0.49 (0.25 to 0.73)	0.001

^*Positive PYP includes results yielding grade 2 or 3.

ATTRm, hereditary transthyretin amyloidosis; ATTRwt, wild type transthyretin amyloidosis; H/CL, heart-to-contralateral lung (ratio); IVSd, interventricular septal thickness at end-diastole; LV, left ventricular; LVPWd, left ventricular posterior wall thickness at end-diastole; 99mTc-PYP, technetium-99m pyrophosphate; PYP, pyrophosphate.

Associations between cardiac biomarkers and echocardiographic parameters in the ATTRm subgroup

In the ATTRm-CA subgroup (table 2), both logBNP and logTroponin were significantly associated with measures of cardiac structure. LogBNP was associated with LV mass (β=0.005, 95% CI 0.002 to 0.009, p=0.005) and IVSd (β=1.12, 95% CI 0.25 to 1.98, p=0.013). LogTroponin was similarly associated with LV mass (β=0.009, 95% CI 0.003 to 0.019, p=0.002) and IVSd (β=2.17, 95% CI 1.05 to 3.29, p<0.001). A positive ⁹⁹ᵐTc-PYP grade was significantly associated with logBNP (β=1.99, 95% CI 0.76 to 3.23, p=0.002), but not with logTroponin (p=0.14). The H/CL ratio was also significantly associated with logBNP (β=1.12, 95% CI 0.02 to 2.22, p=0.047) and approached statistical significance with logTroponin (p=0.060).

Assessing the imaging parameters for the ATTRm subgroup (table 3), it could be seen that a positive ⁹⁹ᵐTc-PYP grade was significantly associated with LV mass (β=111.21, 95% CI 30.51 to 191.91, p=0.009), IVSd (β=0.48, 95% CI 0.18 to 0.77, p=0.003), LV Mass Index (β=64.77, 95% CI 26.92 to 102.62, p=0.002) and LVPWd (β=0.49, 95% CI 0.25 to 0.73, p=0.001). Additionally, the H/CL ratio was strongly associated with all measured structural parameters: LV mass (β=134.52, 95% CI 63.90 to 205.13, p=0.0005), IVSd (β=0.46, 95% CI 0.18 to 0.73, p=0.002), LV Mass Index (β=67.81, 95% CI 37.33 to 98.29, p=0.001) and LVPWd (β=0.49, 95% CI 0.25 to 0.73, p=0.001).

Discussion

This study provides a comprehensive analysis of the associations between cardiac biomarkers, advanced imaging and clinical parameters in a large, well-characterised cohort of 183 patients with ATTR-CA. Stratifying analyses by ATTR subtype (wild type vs hereditary), we identified distinct pathophysiological profiles for ATTRwt and ATTRm, clarifying the role of commonly used diagnostic tests in this complex condition. Notably, prior investigations examining these relationships were often limited by smaller sample sizes,^{16 23} underscoring the value of our larger cohort. To our knowledge, this is one of the largest studies to systematically evaluate these multimodal associations. We demonstrate that while ATTRwt and ATTRm can present with similar cardiac phenotypes, their underlying biomarker signatures and imaging associations differ meaningfully, which has important implications for diagnosis and monitoring.

A central finding is the differential relationship between cardiac biomarkers and cardiac structure across subtypes. In ATTRm, both logBNP and logTroponin were strongly associated with measures of cardiac hypertrophy, including LV mass and IVSd. In contrast, for ATTRwt, logBNP showed no significant association with cardiac mass or septal thickness, while logTroponin correlated with IVSd. These findings suggest that in ATTRwt, myocyte injury (as reflected by troponin elevation) may be more directly linked to septal infiltration, whereas BNP elevation may arise from factors beyond simple myocardial mass, such as advanced diastolic dysfunction or direct myocardial toxicity from wild type amyloid fibrils. This interpretation is supported by prior observations that troponin T in ATTR-CA correlates better than BNP with the extent of amyloid burden and structural abnormalities.¹⁶ Our findings therefore reinforce that troponin may be a more direct indicator of amyloid-related myocardial injury in ATTRwt, while BNP is a more sensitive indicator of cardiac overload and dysfunction that is not strictly proportional to amyloid mass.¹⁶ This divergence in biomarker behaviour between subtypes reinforces the need for subtype-specific interpretation of cardiac biomarkers in ATTR-CA.

Our analysis of ^99mTc-PYP scintigraphy further illuminates the interplay between amyloid burden, structural change and cellular stress. Across the cohort, positive PYP grades and higher H/CL ratios were associated with increased cardiac mass and septal thickness, underscoring their value in detecting anatomical severity. The H/CL ratio, in particular, showed stronger and more consistent associations with structural parameters than Perugini grade, suggesting its use as a more sensitive marker of infiltrative burden. Notably, consistent with prior reports (eg, Castano et al), we observed no significant relationship between H/CL ratio and either BNP or troponin levels.²⁴ This supports the notion that while the H/CL ratio reflects cumulative amyloid deposition, biomarkers capture more dynamic, real-time cardiomyocyte stress and injury.²⁵Given many existing studies are cross-sectional in nature, serial evaluations in ATTR-CA could further clarify the temporal and causal relationships among biomarkers, amyloid deposition and cardiac structural changes.

Limitations

Our study, the largest Canadian analysis to date of both wild type and hereditary ATTR-CA, is constrained by its single-centre, retrospective design, a modest sample size that heightens the risk of overfitting in our multivariate models, and inconsistencies in historical clinical records. The relative rarity of ATTR-CA required us to span many years of patient accrual, during which diagnostic criteria, imaging protocols and management strategies evolved, introducing heterogeneity that complicates direct longitudinal comparisons. Important comorbidities, notably chronic kidney disease, which both frequently co-occurs with amyloid deposition and significantly alters circulating biomarker levels through impaired clearance, were not uniformly characterised, limiting our ability to disentangle disease-specific signals from renal effects. While most ^99mTc-PYP scintigraphy was performed on site using a consistent protocol, a subset of external studies employed differing semiquantitative grading methods, adding analytical variability. Furthermore, our reliance on planar scintigraphy without routine single photon emission CT, cardiovascular magnetic resonance or echocardiographic correlation restricted our capacity to validate and enrich imaging–biomarker associations. As a tertiary referral centre, we also disproportionately see advanced or overtly symptomatic cases, and our hereditary cohort is dominated by the Val142Ile variant (64.8%), which may limit the generalisability of our findings to patients with mixed or predominantly neuropathic ATTRm genotypes and those with earlier stages of disease. Future efforts should therefore focus on multicentre, prospective cohorts with standardised imaging and biomarker protocols, comprehensive comorbidity profiling and broader representation of genotypical and phenotypical variants to enhance external validity and uncover more nuanced insights into ATTR-CA pathophysiology and management. Finally, multiple statistical analyses were conducted without experiment-wide adjustments for multiple comparisons. We recognise that this approach introduces a potential risk of type 1 error (false-positive findings). We therefore underscore the necessity for independent validation of our findings in prospective cohorts to ensure robustness and reliability.

Conclusions

Our study highlights that ATTRwt and ATTRm, while phenotypically similar, represent distinct biological entities. The relationship between BNP and cardiac mass appears specific to hereditary disease, whereas quantitative scintigraphy (via H/CL ratio) is a robust marker of structural severity in both forms. Although ^99mTc-PYP scintigraphy remains essential for diagnosis, traditional markers of cardiorenal stress continue to drive prognostication. These results emphasise the need for subtype-specific interpretation of diagnostic tests to refine risk stratification and guide management. Future prospective studies should validate these findings, explore sex-specific diagnostic criteria and evaluate novel biomarkers that may further improve care for patients with ATTR-CA.

Data availability statement

No data are available.