Fatal myocarditis and rhabdomyositis in a patient with stage IV melanoma treated with combined ipilimumab and nivolumab.

Combination immune checkpoint blockade with concurrent administration of the anti-ctla4 antibody ipilimumab and the anti-PD-1 antibody nivolumab has demonstrated impressive responses in patients with advanced melanoma and other diseases. That combination has also been associated with increased toxicity, including rare immune-related adverse events. Here we describe a case of fatal steroid-refractory myocarditis and panmyositis associated with the use of this combination in a patient with metastatic melanoma. Correlative studies indicated increased levels of serum interleukin 6 in this patient at the onset of toxicity, suggesting a possible role for anti-interleukin 6 receptor antibodies in the treatment of subsequent cases of this rare, but fatal, toxicity.


INTRODUCTION
Compared with either agent alone, combined administration of the anti-ctla4 antibody ipilimumab (ipi) and the anti-PD-1 antibody nivolumab (nivo) has been associated with improved response rates in patients with advanced melanoma (reviewed in Carlino and Long 1 ). However, the increased efficacy of the combination is accompanied by increased toxicity. Approximately 55% of patients treated with combined ipi-nivo in clinical trials experience grade 3 or 4 toxicities 2 . Most are immune-related adverse events (iraes).
The well-characterized iraes experienced by treated patients include colitis, hepatitis, thyroiditis, and various cutaneous reactions. As more patients are treated with the ipi-nivo regime, additional rare iraes are being encountered. Recently, 2 cases of lethal myocarditis with associated myositis were reported after a single dose of combination ipi-nivo 3 . Here, we describe an additional case of fulminant and fatal combined myocarditis and myositis after a single dose of ipi-nivo in a patient with metastatic melanoma.

CASE DESCRIPTION
The patient was a 67-year-old man with BRAF wild-type metastatic melanoma. This patient's past history was significant for hypercholesterolemia and hypertension, both of which were controlled. He had initially presented with stage iiic melanoma 1 year earlier and had been treated with resection and adjuvant radiation to the left axilla. At 9 months after his initial surgery, a left axillary nodal recurrence was re-treated with resection. Two months later, he developed left anterior chest wall subcutaneous lesions, confirmed to be melanoma, and new pulmonary nodules suspicious for metastases. He was then started on combined ipi-nivo.
At 16 days after his first dose of ipi-nivo, the patient presented to the emergency room with a 3-day history of increasing fatigue, weakness, and dyspnea. On presentation, he also complained of feeling pre-syncopal. In the emergency room, he was bradycardic (heart rate: 32 bpm), and an electrocardiogram demonstrated 3rd-degree block [ Figure 1(A)]. Troponin i and creatinine kinase were markedly elevated [ Figure 1(B)]. An echocardiogram demonstrated normal biventricular function, and no significant arterial occlusion was observed on coronary angiography. After cardiac catheterization, myocarditis was diagnosed, and high-dose methylprednisone was started (200 mg on day 1, then 1000 mg daily for 3 days).
The patient appeared to stabilize with steroid treatment. However, 3 days later, he developed increased respiratory distress and hypotension progressing to hypercapnic respiratory failure requiring inotropes and mechanical ventilation. He was given 1 dose of infliximab (5 mg/kg) and 2 doses of intravenous immunoglobulin. His condition worsened, requiring dialysis for acute kidney injury. He remained intubated and on dialysis for 7 more days. A lack of diaphragmatic activity was then confirmed. On day 18 of his admission, he became febrile and hypotensive and expired within 24 hours.
At autopsy, the cause of death was found to be multiple organ failure in the context of rhabdomyositis and myocarditis. Features of acute biventricular heart failure, including dilatation of all chambers, pulmonary edema, pleural effusions, and severe congestive hepatopathy were also noted. Grossly, the myocardium demonstrated patchy areas of pallor in the left ventricular wall, not corresponding to any vascular territory [ Figure 1 Staining of the myocardium for CD20 was negative (data not shown). A similar mononuclear leucocyte infiltrate was observed in skeletal muscle, including the biceps, thigh, and calf muscles and the diaphragm. Again, those cells were predominantly CD4+ and CD8+ T cells and CD68+ myeloid cells. The immune infiltrates were associated with rhabdomyolysis, muscle fibre necrosis, and focal calcification that was particularly severe in the diaphragm [ Figure 2(F)]. The severe diaphragmatic involvement was felt to have caused the patient's respiratory failure.
Post-mortem examination of the lung revealed 2 remaining foci of melanoma [ Figure 3(A)]. Histology demonstrated extensive tumour necrosis, with small remaining nodules of viable tumour surrounded by a brisk mononuclear infiltrate of CD4+ and CD8+ T lymphocytes and CD68+ myeloid cells [ Figure 3(B-F)]. Interestingly, melanoma was not identified in skin biopsies from the chest and back. A site of previously biopsy-proven melanoma overlying the left clavicle showed no residual disease. Thus, it was apparent from the post-mortem examination that the patient was simultaneously experiencing both a dramatic reduction in tumour burden in response to ipi-nivo and extensive autoimmune pathology.
Serum chemokine and cytokine levels were assessed before treatment with ipi-nivo and 21 days after the first  dose. Consistent with previous reports 4 , treatment with ipi-nivo resulted in a marked increase in serum levels of the chemokine interferon-γ-inducible protein 10 kDa (also called cxcl10) [ Figure 4(A)]. Of the cytokines tested, interleukin 6 (il-6) was observed to be markedly elevated after treatment, but no changes in serum levels of other inflammatory cytokines such as interleukin 1β, tumour necrosis α, or interferon γ were detected [ Figure 4(B)].

DISCUSSION
We report a case of fulminant fatal myocarditis and myositis in a patient with metastatic melanoma treated with a single dose of combination ipi-nivo, at 3 mg/kg and 1 mg/kg respectively. At 16 days after treatment with the first ipi-nivo infusion, the patient presented with high-degree heart block and biochemical evidence of significant myocarditis and myositis. Despite treatment with high-dose intravenous steroids, an anti-tumour necrosis α agent, and intravenous immunoglobulin, the patient deteriorated progressively and succumbed to multiple organ failure.
This case is strikingly similar to two other recently described cases 3 , which together illustrate the presentation of a highly lethal irae with combination ipi-nivo.
Thankfully, this irae appears to be extremely rare. Upon reviewing the Bristol-Myers Squibb corporate safety database, Johnson et al. 3 found that, as of April 2016, myocarditis was reported in only 8 of 2974 patients treated with ipi-nivo (0.27%). That observation agrees with a data review from published phase iii studies of patients treated with either combined or single-agent checkpoint blockade, which found the total incidence of all adverse  cardiovascular events to be only 0.19% 5 . Despite the rarity of myocarditis as an irae, 5 of the 8 reported cases of in the Bristol-Myers Squibb database were fatal, including all cases with concomitant myositis. Another case of fatal myocarditis was recently reported in a patient receiving PD-1 blockade after previous ipi-nivo 6 . No current biomarker is predictive for this rare toxicity, and no effective therapies to prevent its lethality have been found.
Increased understanding of the immunologic mechanisms underlying this fulminant myocarditis and myositis might identify both predictive biomarkers and novel therapeutic approaches. In agreement with the findings of Johnson et al. 3 , examination of myocardium and skeletal muscle revealed CD3+ mononuclear cell infiltration into areas associated with cardiomyolysis and rhabdomyolysis, suggesting a T cell-mediated immunopathology (Figure 2). We also found brisk T cell infiltration into the tumour and a striking response to therapy (Figure 3). Interestingly, increased serum levels of cxcl10, a chemokine known to recruit activated T cells both to sites of active inflammation and to the tumour microenvironment 7 , were also observed. It is tempting to speculate that cxcl10 plays a role in driving both the observed autoimmunity and the antitumour response. However, the utility of cxcl10 as a potential biomarker or therapeutic target seems limited. Elevated serum cxcl10 has previously been described in patients treated with combination ipi-nivo who did not develop severe autoimmunity 4 . Thus, although cxcl10 might have contributed to the autoimmune pathology in our patient, its elevation is not specific to patients who develop this rare irae.
Serum levels of the inflammatory cytokine il-6 were also increased in our patient. The role of il-6 in mediating the response to combination ipi-nivo has not been defined. In small patient cohorts treated with ipi alone, elevated serum il-6 was associated with reduced response rates and lower overall survival 8,9 . With respect to PD-1 blockade, elevated serum il-6 during treatment has been linked to cutaneous toxicity 10 . There is also a case report of concurrent administration of pembrolizumab (an anti-PD-1 antibody) and tocilizumab [an anti-il-6 receptor (il-6r) antibody] in a patient with metastatic melanoma and refractory Crohn disease 11 . A durable antitumour response and relative quiescence of the Crohn disease were observed. Further investigation into the role of il-6 signalling in driving autoimmunity in patients who develop ipi-nivo-induced myocarditis and myositis is clearly required. Additionally, we propose treating other patients that present with this rare irae with tocilizumab or other anti-il-6/il-6r agents. Based on the observation of elevated serum il-6 in cytokine release syndrome, tocilizumab has been successfully used to treat the immune-mediated cytokine release syndrome associated with the use of chimeric antigen receptor T cells to treat hematologic malignancy 12 .

SUMMARY
Our patient, and the 2 patients reported by Johnson et al. 3 , succumbed despite aggressive treatment with steroids and anti-tumour necrosis α agents. Clearly, additional therapies are required to prevent mortality from this rare, but lethal, autoimmune toxicity. We propose prompt treatment with tocilizumab in addition to corticosteroids for patients presenting with fulminant myocarditis and rhabdomyositis associated with ipi-nivo.