Cardiomuscular Biomarkers in the Diagnosis and Prognostication of Immune Checkpoint Inhibitor Myocarditis

Abstract

Background: Immune checkpoint inhibitors (ICIs) are approved for multiple cancers but can result in ICI-associated myocarditis, an infrequent but life-threatening condition. Elevations in cardiac biomarkers, specifically troponin-I (cTnI), troponin-T (cTnT), and creatine kinase (CK), are used for diagnosis. However, the association between temporal elevations of these biomarkers with disease trajectory and outcomes has not been established.

Methods: We analyzed the diagnostic accuracy and prognostic performances of cTnI, cTnT, and CK in patients with ICI myocarditis (n=60) through 1-year follow-up in 2 cardio-oncology units (APHP Sorbonne, Paris, France and Heidelberg, Germany). A total of 1751 (1 cTnT assay type), 920 (4 cTnI assay types), and 1191 CK sampling time points were available. Major adverse cardiomyotoxic events (MACE) were defined as heart failure, ventricular arrhythmia, atrioventricular or sinus block requiring pacemaker, respiratory muscle failure requiring mechanical ventilation, and sudden cardiac death. Diagnostic performance of cTnI and cTnT was also assessed in an international ICI myocarditis registry.

Results: Within 72 hours of admission, cTnT, cTnI, and CK were increased compared with upper reference limits (URLs) in 56 of 57 (98%), 37 of 42 ([88%] P=0.03 versus cTnT), and 43 of 57 ([75%] P<0.001 versus cTnT), respectively. This increased rate of positivity for cTnT (93%) versus cTnI ([64%] P<0.001) on admission was confirmed in 87 independent cases from an international registry. In the Franco-German cohort, 24 of 60 (40%) patients developed ≥1 MACE (total, 52; median time to first MACE, 5 [interquartile range, 2-16] days). The highest value of cTnT:URL within the first 72 hours of admission performed best in terms of association with MACE within 90 days (area under the curve, 0.84) than CK:URL (area under the curve, 0.70). A cTnT:URL ≥32 within 72 hours of admission was the best cut-off associated with MACE within 90 days (hazard ratio, 11.1 [95% CI, 3.2-38.0]; P<0.001), after adjustment for age and sex. cTnT was increased in all patients within 72 hours of the first MACE (23 of 23 [100%]), whereas cTnI and CK values were less than the URL in 2 of 19 (11%) and 6 of 22 (27%) of patients (P<0.001), respectively.

Conclusions: cTnT is associated with MACE and is sensitive for diagnosis and surveillance in patients with ICI myocarditis. A cTnT:URL ratio <32 within 72 hours of diagnosis is associated with a subgroup at low risk for MACE. Potential differences in diagnostic and prognostic performances between cTnT and cTnI as a function of the assays used deserve further evaluation in ICI myocarditis.

Keywords: biomarkers; drug-related side effects and adverse reactions; immune checkpoint inhibitors; myocarditis; pharmacology; troponins.

Figure 1:. Time course of troponins and creatine kinase (CK) after admission for ICI-myocarditis.

URL stands for 99^th percentile upper reference limit for troponins and 95^th percentile for CK. Evolution of maximal (A), minimal (B), and median (C) values (median, IQR in the boxplots) of cardiac troponin-T (cTnT)/URL, cardiac troponin-I (cTnI)/URL and CK/URL ratios over time after initial diagnosis of ICI-myocarditis within specific timeframes (x-axis) in follow-up. (D) Ratio of maximum cTnT/URL over cTnI /URL over time after initial diagnosis of ICI-myocarditis within specific timeframes (x-axis) in follow-up. Nonlinear mixed models p-values are shown (*<0.001, See Supplementary Table-2A). For D, n of patients available for each biomarker at each time frame is just above the x-axis. (E) Proportion of patients with biomarkers above URL over time after diagnosis are displayed, numbers indicate patients with abnormal values. Light grey area represents the proportion of patients with biomarker levels below URL. Minimal values within the indicated time period were used for figure E (d for days).

Figure 2.. LOESS (Locally Estimated Scatterplot Smoothing) of the mean (and standard-error) of the ratio of cTnT/URL over cTnI /URL (A) and cTnI/URL (B) over time after initial admission for ICI-myocarditis in a one-year follow-up as a function of cTnI assays.

Figure 3.. Cardiac biomarkers as predictors of MACE

(A) Maximal cardiac biomarkers values within 72h of ICI myocarditis diagnosis as a predictor of MACE within 90 days with receiver operating curve of cTnT/URL, and CK/URL. (B) MACE over a one-year time-course after diagnosis as a function of cTnT/URL value above and below 32 (n=57) using maximal cTnT values within 72h of ICI myocarditis diagnosis. (C) Proportion of patients with biomarkers above URL before first MACE are displayed in yellow (cTnT), blue (cTnI) and dark grey (CK). Light grey area represents the proportion of patients with biomarker levels below URL. Abbreviations: AUC, area under the curve; MACE, major adverse cardio-myotoxic event; fc, fold-change; URL, upper reference limit being upper 99^th percentile of normal values for troponins and 95^th for CK; ^95%CI, 95% confidence interval; HR, hazard ratio.

Figure 4:. Pathobiology of cardio-muscular biomarkers in ICI-myotoxicity patients.

Volcano plot showing the distribution of all differentially expressed genes (n=17070) in muscle samples from patients with ICI-myotoxicities (n=6, concomitant ICI-myocarditis and ICI-myositis in n=5 and one ICI myositis not screened for concomitant myocarditis) vs. normal skeletal muscle samples (n=6). Blue dots represent significantly upregulated and downregulated genes (at least >log2(∣2∣)), after adjustment for multiple testing. TNNT2 gene (coding for cTnT) is significantly overexpressed in ICI-myotoxicity patients (Log2 fold-change:5.2, adjusted-p:0.3x10⁻⁶; red dot) whereas TNNI3 (coding for cTnI) is not (Log2 fold-change:0.96, adjusted-p=0.2; black dot) (panel A). Immunostaining (immunochemistry identifying protein expression– hematoxylin counterstaining) showing transversal sections of skeletal muscle fibers positive for cTnT (brown) (100X, panel B; 400X, panel C) adjacent to inflammatory cells (cluster of nuclei with blue staining) from a representative patient with ICI-myotoxicity. Longitudinal section of a skeletal muscle sample showing a linear pattern of positive immunostaining for cTnT tracking the morphology of muscle fibers (200X, panel D).

Figure 5:. Anti-cTnI IgG/IgM antibodies and influence on cTnT/cTnI ratio.

Evolution of anti-cTnI IgG and IgM antibodies circulating levels (at least on detectable value >1/40 in each time frame) over time in ICI-myocarditis in the French discovery cohort (A). Influence of detectable levels of anti-cTnI IgG (B) or IgM (C) on cTnT/cTnI ratio over their respective URL (99^th percentile upper reference limit). No significant interaction between anti-cTnI IgG or IgM levels and cTnT/cTnI ratio over their respective URL was identified (Supplementary-Table-3B for detailed statistics).