1 Immune checkpoint inhibitors (ICIs) targeting cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1/ligand-1 (PD-1/PDL-1) have considerably improved the management of metastatic melanoma as well as many other cancers [1]. However, these new drugs may engender new adverse events (AEs), especially immune-related adverse events (irAEs) which have been shown to be relatively unpredictable [2].

2 In patients treated with ICIs, we have witnessed an emergence of AEs affecting various organs [3], with cutaneous side effects being the most common (43-45%). Gastro-intestinal [4, 5] and endocrine disorders (6-8%) [6, 7] were also found. IrAEs tend to be less common in patients treated with anti-PD-1 than with anti CTLA-4 [8].

3 Cardiac toxicities and side effects associated with chemotherapy are well-established [9]. In all of the first clinical trials involving anti-PD-1 treatment for advanced and metastatic melanoma, there were no reports of cardiac effects [10-16]. Those associated with ICIs and, particularly with anti-PD-1, have been recently reported in clinical practice. Regarding cardiac AEs, several cases of fulminant myocarditis have been described [17-19] as well as less frequent AEs such as heart rhythm disorders, heart failure, pericarditis and vasculitis [20-25]. Unlike other irAEs, cardiovascular AEs tend to occur early after ICI initiation, usually between 17 and 39 days [26-28].

4 Auto-immune myocarditis associated with ICIs have shown poor prognosis with a 40% mortality rate [20, 29, 30]. It has been shown that patients developing immune cardiomyopathy have a significantly higher risk of developing myositis, encephalitis and hepatitis [31]. In the literature, myocarditis prevalence has been found in 1% of patients treated with all ICIs (0.5% for anti-PD-1 alone) among whom 50% will develop severe cardiac toxicity [32].

5 Although most patients experience mild to moderate symptoms, such as dyspnoea, chest pain, palpitations or myalgia, patients can have a non-symptomatic presentation, making early diagnosis difficult [27]. In the first clinical trials involving anti-PD-1, there was no routine testing for myocarditis by either biochemical analysis or cardiac imaging, which may well explain the scarcity of cardiac effects reported.

6 There are no clear guidelines for how patients treated with anti-PD-1 should be monitored (electrocardiogram [ECG] and troponin levels). Nor is there any precise recommendation for the therapeutic options that might be considered. Most irAEs require ICI discontinuation along with the use of oral steroids, and the adjunction of other immunosuppressive drugs if needed [33].

7 Cardiac troponins are biomarkers with a major role in the diagnosis of cardiac injuries. The troponin regulatory complex includes troponin C and troponin I that inhibit actin-myosin coupling through the inhibition of ATPase activity along with troponin T (TnT) that binds to tropomyosin and stabilizes the complex on the actin filament. When disruption of the myocyte membrane occurs, the troponins in the cytosol pass into the bloodstream. Increased troponin levels correlate with the extent of cellular injury. Thus, elevated cardiac troponin levels have a strong correlation with adverse cardiovascular outcome whether coronary artery disease is present or not. Troponins are present in both skeletal and cardiac muscles, and their levels increase during myocarditis and heart rhythm disorders [34]. Cardiac troponin T is also detectable in the blood of patients with chronic kidney disease [35].

8 Serum troponin T levels could be an interesting and simple tool in the diagnosis of anti-PD-1-related myocarditis as they are abnormal in more than 90% of patients developing this side effect, even in those presenting few or no cardiac symptoms [36].

9 In this article, we report firstly the frequency of myocarditis in our cohort of patients, and subsequently we investigate whether troponin levels before and during anti-PD-1 therapy might help predict cardiac immune side effects.

Materials and methods

Results

Retrospective study

20 Between November 2017 and April 2020, among the 183 patients treated with anti-PD-1 therapy for advanced melanoma, four patients developed cardiac AEs during immunotherapy, with a prevalence of 2.2%. The characteristics of the four patients are described in table 1. There were three cases of myocarditis and one of myocardial infarction. All the diagnoses of myocarditis were confirmed by a cardiologist, and patients were referred to a cardiologic unit. The three cases of myocarditis were diagnosed without any clinical symptom and no ECG modification. Troponin T level was increased > 500 ng/L in one case and > 1,000 ng/L in two cases. The patient who experienced myocardial infarction had typical clinical symptoms including chest pain, dyspnoea and pulmonary oedema, along with a modified ECG with segment ST depression. The three patients with myocarditis were diagnosed within less than two months of anti-PD-1 therapy. Two of them had myocardial biopsies, one of whom did not show any inflammatory infiltrate, whereas the other had focal lesions of acute myocarditis with an histiocytic and lymphocytic infiltrate, and numerous CD8+ T-cells. All of the four cardiac ailments disappeared after treatment. The details are given in table 1.

Table 1

Case reports of severe cardiac AEs.

Patient n°	1	2	3	4
Type of cardiac AE	Myocarditis	Myocarditis	Myocarditis	Myocardial infarction
Baseline LVEF	> 50%	–	> 50%	> 50%
Prior cardiac medical history	None	Cerebrovascular accident, hypertension, aortic aneurysm	Myocardial infarction with quadruple coronary bypass	Myocardial infarction, triple coronary bypass diabetes
Baseline troponin level	8 ng/L	10 ng/L	28 ng/L	30 ng/L
Troponin peak (normal <13 ng/L)	1004 ng/L	584 ng/L	1500 ng/L	1860 ng/L
Clinical symptoms	None	None	None	Acute pulmonary oedema, dyspnoea, chest pain
CTCAE grade	3	3	3	3
Delay after initiation of anti-PD-1 therapy	36 days	28 days	28 days	127 days
Echocardiography during cardiac AE	Stable	Normal LVEF Dilatation of the right cavities	LVEF 50%	LVEF 60-65 %, stable.
Coronarography	Normal	–	Significant coronary stenosis No sign of acute coronary lesion	Triple vessel coronary disease Significant right artery stenosis Permeable bypasses
ECG	Regular sinusal rhythm, 53 bpm, narrow QRS, no repolarization disorders. No change on ECG	Regular sinusal rhythm, 85 bpm, no repolarization disorders. No change on ECG	Regular sinusal rhythm, 77 bpm, right bundle branch block, no repolarization disorders. No change on ECG	Regular sinusal rhythm, 70 bpm, ST segment depression, QT prolongation 140 ms.
Cardiac magnetic resonance imagery	Normal	–	Doubt between ischaemic disorder and myocarditis	No sign of myocarditis Signs of ischaemic heart disease
Myocardial histology	Focal lesions of acute myocarditis with a histiocytic and lymphocytic infiltrate, numerous CD8+ T-cells.	–	No inflammatory infiltrate	–
Discontinuation of anti-PD-1	Yes	Yes	Yes	No
Treatment	IV and oral steroids 1.5 mg/kg/d with progressive decrease Abatacept 500mg/14d, 6 cycles	IV and oral steroids 2 mg/kg/d with progressive decrease	IV steroids 1000 mg/d 3 days Oral steroids 1,5mg/kg/d Plasmatic exchange, 2 cycles Abatacept 20 mg/kg, 3 cycles	Double platelet anti-aggregant
AE evolution	Resolution	Resolution	Resolution	Resolution
Other irAEs	Thyroiditis Neuropathy	Hepatitis (mycofenolate mofetil therapy initiated)	None	Nephropathy
Melanoma response to anti-PD-1 therapy	Progression	Complete response	–	Complete response

Case reports of severe cardiac AEs.

–: no data; bpm: beats per minute; ms: milliseconds

Follow-up

26 Fifty-one patients were followed with an ECG and monthly T troponin serum measurements. Follow-up was not possible for one patient who died two weeks after anti-PD-1 initiation. Eight patients developed non-cardiological irAEs. An increase in troponin T level was reported in 14 patients (27%) (table 2). During the follow-up period, an increase in troponin T level was noted in six patients among the 14, among whom two patients had clinical symptoms. In only one patient, the increase in troponin T level was associated with ECG modification, which was a segment ST depression. The mean delay for developing a troponin T increase after treatment initiation for the six patients was 93 days (within a range of 30 to 145 days). Four of six (67%) of these patients had a cardiovascular history and abnormal baseline ECG. The rise in troponin T levels was between 10 and 1,830 ng/L, with a median of 18.5 ng/L.

27 For the two patients who had clinical cardiac signs associated with the increase in troponin T level, one developed an acute pulmonary oedema secondary to myocardial infarction (troponin: 1,860 ng/L), occurring five months after treatment initiation. This patient had a severe cardiovascular history with several prior myocardial infarctions. Two platelet aggregation inhibitors were initiated which permitted clinical improvement. This was the only patient who experienced a second irAE, corresponding to immune-related kidney disease, which occurred several months later. Anti-PD-1 therapy was not discontinued, and there was no recurrence of the cardiac event and the therapy resulted in complete remission of the melanoma. For the second patient, the increase in troponin T level (84 ng/L with a baseline level of 43 ng/L) was not associated with a cardiac problem but rather septic shock which was treated with antibiotherapy. When the acute attack resolved, troponin T levels decreased to baseline levels and the anti-PD-1 therapy was pursued (table 3).

Table 3

Characteristics of patients with significant troponin increase.

Patient	10	16	30	32	38	41
Age at treatment onset	63	78	87	70	72	74
Troponin baseline	30	34	58	3	3	43
Troponin peak	1860	44	68	24	19	84
CTCAE grade	3	1	1	1	1	1
ECG modification at troponin elevation	ST segment depression	None	None	None	None	None, complete atrio-ventricular block, stable.
Clinical symptoms	Dyspnoea, OMI, chest pain	None	None	None	None	Septic shock
Delay in troponin increase (days)	127	136	140	30	56	55
Cardiovascular history	Myocardial infarction, diabetes, chronic renal failure	Aortic stenosis, hypertension, chronic renal failure	Atrial fibrillation, hypertension, chronic renal failure	None	None	atrioventricular block 3, hypertension
Other irAEs	Nephropathy	None	None	None	None	None
Best response to anti-PD-1	Complete response	Partial response	Progressive disease	Complete response	Progressive disease	Partial response
Cardiac investigation	Ischaemic heart disease on cardiac RMI, artery stenosis or coronarography	None	None	None	None	None
Treatment	Double platelet anti-aggregant.	None	None	None	None	Antibiotics for septic shock

Characteristics of patients with significant troponin increase.

28 The four other patients with an increase in T troponin level did not experience any symptoms or abnormality on ECG. The mean increase in serum troponin T levels was 14.3 +/- 5.3 ng/L. For these patients, anti-PD-1 therapy was continued without any cardiovascular event during follow-up.

29 In summary, in the prospective cohort of 52 patients, only one patient developed a high level of troponin T resulting in a myocardial infarction.

30 During this follow-up period, three factors were also significantly associated with increased troponin T level: diabetes (p = 0.007), previous myocardial infarction (p = 0.016), and long duration of anti-PD-1 therapy (p = 0.01).

Discussion

31 The aims of this study were first to assess the prevalence of patients with an adverse cardiac outcome in our retrospective cohort of patients treated with anti-PD-1. The second part was a prospective study of 52 patients to define whether troponin T levels might be a good marker to detect a cardiovascular AEs in patients treated with anti-PD-1 drugs, before and during treatment.

32 Concerning our retrospective study, the prevalence of severe cardiac AEs in patients treated with anti-PD-1 was low; only 2.2% (4/183 patients). However, this prevalence is higher than that in the literature. Clinical trials involving anti-PD-1 therapies for melanoma (such as KEYNOTE and CHECKMATE pivotal studies [5, 10, 11, 39-41]) have, as yet, not revealed any Stage 3 or 4 cardiac-related adverse events such as heart attack or myocarditis. However, based on our two trials, it is not possible to affirm whether Stage 1 or 2 adverse cardiac events can be formally eliminated.

33 AEs related to other anti-PD-1-treated cancers rarely involved cardiac events that were not deemed to be linked to the treatment. The prevalence of myocarditis was reported to be 0.09% based on the Bristol Myers Squibb safety database for nivolumab and ipilimumab in combination, and 0.06% with nivolumab as a single therapy [26]. Retrospective studies showed a higher prevalence of cardiac events in patients treated with ICIs; 0.4% and 0.5% in two studies involving patients receiving anti-PD-1 therapy [3, 32]. This might suggest that patients in real-life settings with previous cardiac events are more likely to experience adverse cardiac events due to anti-PD-1 therapy than patients selected in clinical studies who present fewer comorbidities and are often excluded from testing.

34 Concerning the four cases of our retrospective study, three of them experienced these adverse events shortly after drug initiation (28 to 36 days) in accordance with the literature which reports a mean delay of between 17 and 39 days, and more than 80% within the three months after starting anti-PD-1 [26, 29, 36, 42].

35 An important point on the practical level is that, in three patients, the increase in troponin T level was reported without any clinical sign, but lead to the diagnosis of myocarditis and the discontinuation of the anti-PD-1 drug. These three patients were given intravenous steroids, then oral steroids (1 to 2 mg/kg/d) with progressive tapering. Two of them showed a relapse with a decrease in oral steroids, and were treated successfully with abatacept. One patient developed a myocardial infarction which did not need any immunosuppressive agent for resolution.

36 The main strategy used to treat ICI-associated cardiotoxicity targets the T-cell response. Glucocorticoid therapy is considered as the first line of treatment, at an initial dose of 1-2 mg/kg. However, for some patients, alternative treatment is necessary [43]. Many treatments, such as plasmapheresis, intravenous immunoglobulins, antithymocyte globulin, mycofenolate mofetil, tacrolimus, infliximab, and abatacept [25], have been offered, but for the moment, no consensus has been obtained and guidelines are lacking [33].

37 The second part of our study was based on a prospective study revealing that before any treatment with anti-PD-1, patients may have had an increase in troponin T baseline level. Based on a meta-analysis of several studies of patients with abnormal troponin levels above 14 ng/L, 3.4 to 19% had abnormal troponin T levels [44].

38 In our study this applied to 27% of the patients. This basal increase in troponin T level, before anti-PD-1 treatment, could be linked to events such as physical exercise or chronic kidney failure [45, 46]. Among our 14 patients with basal increase in troponin T, seven (50%) had renal dysfunction (glomerular filtration rate <60). The rate of false positives in our hospital was estimated at less than 3% and close to 0 for values that were two-fold higher than normal values. In all cases, ECG and echography are useful tools to rule out false positives. CPK may also be of interest for the detection of false positives, but unfortunately we did not perform this biological test.

39 Another explanation of this non-symptomatic troponin elevation could be linked to subclinical inflammatory processes, such as subclinical myocarditis, however, all patients underwent ECG and cardiac echography before starting treatment. Thus, these results show that it is important to know the basal troponin T level for the follow-up of patients. Only one other prospective study has been performed on lung cancer in 2018 [47] with a similar number of patients (n = 59). The authors reported a lower percentage (5.1%) of elevated troponin I baseline levels before anti-PD-1 treatment.

40 During the follow-up period of our cohort, we noted an increase introponin T level in six patients (11.5%) with the detection of a myocardial infarction in one patient and septic shock in another. Thus, a low increase in T troponin level (less than two-fold) with spontaneous normalization does not seem to be a contraindication to continuing anti-PD-1 treatment.

41 Among the six patients with an increase in T troponin level, none showed new associated ECG abnormalities. Furthermore, in the retrospective cohort, in the three patients diagnosed with myocarditis, the same phenomenon was noted. This suggests that troponin T levels may well be more sensitive than ECG for the detection of cardiac adverse events.

42 A comparison between groups at baseline showed that older age, cardiac or vascular history and diabetes were more often associated with a subnormal baseline level of troponin T.

43 During the follow-up period under anti-PD-1 treatment, diabetes and cardiac history were still associated with increased troponin T levels, together with a long duration of anti-PD-1 therapy, thus indicating that the cardiac follow up should be stricter with a long prescription of anti-PD-1.

44 Concerning the other prospective cohort with lung cancer, 14 patients had an increase in troponin I level during follow-up (11 with a normal baseline level). Thus, our results are in accordance with these previous results, although the troponin subtypes were different [47]. Both T and I subtypes seem to be linked to cardiovascular events; troponin I seems to be more related to coronary diseases [48]. Regarding mortality, troponin T seems to be more related to overall mortality, whereas troponin I is more related to cardiovascular mortality [49].

45 At the pathophysiological level, animal studies have demonstrated that PD-1 deletion causes inflammation of the myocardium [50]. In PD-1 deficient mice, Okazaki et al. showed that autoantibodies against cardiac troponin I were responsible for dilated cardiomyopathy [51]. ICI-associated myocarditis might be initiated by the release of cardiac antigens such as troponin into the circulation. This antigen release might be the consequence of a deleterious process, such as viral myocardial injury [47]. The primary inflammation, exacerbated by ICI-enhanced immunity, could initiate a worsening cycle due to further cardiac antigen release [30]. Some patients may be less sensitive to ICI-intensified autoimmunity and a more serious primary defect may be necessary to cause the inflammation to spread and lead to a symptomatic irAE. In our cohort, at baseline, we identified 10/14 in whom C-reactive protein (CRP) had been measured. Among them, three had increased CRP above 20 ng/L.

46 In our study, 26 patients had received previous treatments. In both groups of patients with or without abnormal troponin T baseline levels, the number of patients with previous therapeutics for melanoma was the same, including those who already received ICIs such as anti-CTLA-4. In addition, targeted therapies with BRAF and MEK inhibitors are known to decrease LVEF, but not increase troponin levels [52].

47 In addition to troponin, another marker of interest could be NT-proBNP, often used in cases of cardiac irAEs associated with ICIs [53].

48 In conclusion, our study suggests that the prevalence of cardiac events might be higher in real-life settings than in clinical trials. Monitoring could allow us to detect adverse events related to anti-PD-1 therapy earlier, as the increase in troponin T seems to be an early event before cardiac manifestation. However, the interpretation of troponin T level requires knowledge of basal dosage established before any treatment with immunotherapy. A mild increase is not a contraindication to anti-PD-1 if ECG and cardiac echography are normal. Other biological investigations, such as testing for troponin I, NT-proBNP and CPK, could be interesting to consider in future studies which may confirm these results.

Disclosure

49 Funding sources: none. Conflicts of interest: none.