key: cord-0814215-q04yqjfe
authors: Dimopoulos, George; de Mast, Quirijn; Markou, Nikolaos; Theodorakopoulou, Maria; Komnos, Apostolos; Mouktaroudi, Maria; Netea, Mihai G.; Spyridopoulos, Themistoklis; Verheggen, Rebecca J.; Hoogerwerf, Jacobien; Lachana, Alexandra; van de Veerdonk, Frank L.; Giamarellos-Bourboulis, Evangelos J.
title: FAVORABLE ANAKINRA RESPONSES IN SEVERE COVID-19 PATIENTS WITH SECONDARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS
date: 2020-05-14
journal: Cell Host Microbe
DOI: 10.1016/j.chom.2020.05.007
sha: c471590ba91d0a0a50fc0239f131221276918ae3
doc_id: 814215
cord_uid: q04yqjfe

SUMMARY Dysregulation of inflammation is hypothesized to play a crucial role in the severe complications of COVID-19, with IL-1/IL-6 pathway being central. Here, we report on the treatment of eight severe COVID-19 pneumonia patients-- seven hospitalized in intensive care units (ICUs) in Greece and one non-ICU patient in the Netherlands-- with the interleukin-1 receptor antagonist Anakinra. All patients scored positive for the hemophagocytosis score (HScore) and were diagnosed with secondary hemophagocytic lymphohistocytosis (sHLH) characterized by pancytopenia, hyper-coagulation, acute kidney injury and hepatobiliary dysfunction. At the end-of-treatment, ICU patients had less need for vasopressors, significantly improved respiratory function and lower HScore. Although three patients died, the mortality was lower than historical series of patients with sHLH in sepsis. These data suggest that administration of Anakinra may be beneficial for treating severe COVID-19 patients with sHLH as determined by the HScore and support the need for larger clinical studies to validate this concept.

The most fearsome complication of pneumonia caused by the novel coronavirus SARS-Cov-2 (COVID-19 illness) is severe respiratory failure leading to mechanical ventilation (MV). The mortality in patients with severe COVID-19 admitted in the intensive care units is reported to be between 50% and 65% (Bhatraju et al., 2020; . The cytokine storm described in these patients (Qin et al., 2020) introduced the concept of attenuation of the hyper-inflammation with agents targeting pro-inflammatory cytokines mainly interleukin (IL)-1β and IL-6. Several clinical trials on the efficacy of Anakinra, that targets IL-1β, and of Tocilizumab, Siltuximab and Sarilumab that target the IL-6 pathway are on-going.

Overproduction of IL-1β by tissue macrophages triggers secondary hemophagocytic lymphohistiocytosis (sHLH), also called macrophage activation syndrome, that is characterized by pancytopenia, hyper-coagulation, acute kidney injury and hepatobiliary dysfunction. In sepsis, sHLH is a complication leading independently to early death in the first 10 days (Kyriazopoulou et al., 2017) ; 28-day mortality is reaching 67%. Administration of Anakinra, that is the recombinant soluble receptor antagonist of IL-1β and IL-1α, in patients with signs of sHLH, reduced mortality by 30% (Shakoory et al., 2016) .

We wanted to assess whether administration of Anakinra would benefit severely ill COVID-19 patients. Since March 2020, seven patients in three Intensive Care Units (ICUs) in Greece and one non-ICU patient in the Radboud University Medical Center (Radboudumc) in the Netherlands with severe COVID-19 were given salvage treatment with Anakinra on the ground of sHLH diagnosed using the HScore (hemophagocytosis score). Promising indications on treatment efficacy are described here.

The seven patients in Greece were treated with Anakinra intravenously 200mg every eight hours for seven days. The dose regimen was selected since it was administered in patients with sepsis participating in one randomized clinical trial with the acronym PROVIDE (ClinicalTrials.gov NCT03332225). All patients were male and they suffered from severe comorbidities mainly coronary heart disease and arterial hypertension (Table 1) . All patients scored positive by the HScore, which is a classification system using nine different variables; any score more than or equal to 169 provides 93% sensitivity for the diagnosis of HLH (Fardet et al., 2004) . All treated patients had HScores more than or equal to 169 (Table 2) . Assays for the detection of cytomegalovirus (CMV) and Ebstein-Barr virus (EBV) in their plasma were negative.

All treated patients had acute respiratory failure since all had presented with diffuse lung infiltrates in chest X-rays and all had ratio of partial oxygen pressure to the fraction of inspired oxygen (pO 2 /FiO 2 ) lower than 100 ( Figure 1 ). Only one patient had acute kidney injury.

Seven of the described patients started Anakinra treatment after initiation of MV. Three patients died by day 28. All seven patients received treatment with hydroxychloroquine and azithromycin and broad-spectrum antibiotics ( Table 1) . As shown in Figure 1 , lung infiltrates were decreased by the end of treatment (EOT) in five patients and this was accompanied by increase of the pO 2 /FiO 2 ratio. The end of 7-day treatment was accompanied by decrease of the dose of administered vasopressors in six patients (Table 1) .

Although treatment with Anakinra seemed to improve the majority of laboratory findings of the patients (Figures 2A to 2H ) by EOT which were also maintained two days later, significant decreases from the baseline were mainly found for the pO 2 /FiO 2 ratio which is an index of the respiratory function; this was increased in five patients by EOT and in all seven patients two days later ( Figure 2I ). This increase ranged between 15% and 117% ( Figure 2J ). The HScore was also significantly decreased at the EOT ( Figure 2K ). No association between the time interval from start of symptoms until start of MV and survival time was found ( Figure   2L ).

The first patient died after 12 days by refractory shock. Although one strain of of Acinetobacter baumannii resistant to carbapenems and colistin was isolated from the tip of the central vein catheter, blood cultures were sterile. The second patient died after nine days by refractory shock. He had severe hypoxemia accompanied by increase of inflammatory markers and elevated core temperature; blood cultures were sterile. The fourth patient died after 19 days due to refractory shock aggravated by acute kidney injury and anuria; blood cultures were sterile.

A patient case at the Radboud University Medical Center shows that Anakinra in SARS-Cov-2 infected patients with features of sHLH may prevent progression of respiratory failure and the need of MV. The patient was a 71-year old female with a recurrent ethmoidal adenoid cystic carcinoma and rheumatoid arthritis treated with hydroxychloroquine. She was hospitalized 14 days after the third cycle of palliative chemotherapy (cyclophosphamide, doxorubicin and carboplatin) with COVID-19.

Chest CT showed bilateral ground glass opacities with consolidations (data not shown). Anakinra was administered at day nine of hospitalization because of clinical deterioration with increasing oxygen demand in combination with persistent pancytopenia and rapidly increasing concentrations of serum ferritin (maximum concentration 12,670 ng/ml) and of hepatic aminotransferases. Plasma EBV PCR showed a low load (200 IU/ml), whereas CMV DNA was undetectable. Anakinra (300mg once daily intravenously for 4 days, followed by 100mg once daily) resulted in a rapid improvement in clinical condition within 24 hours with decrease in oxygen need and decrease in sHLH blood parameters ( Figure 3 ). The HScore (presence of organomegaly and hemophagocytosis in bone marrow not scored) decreased from 198 points at baseline to 71 points at EOT and the patient was discharged home at day nine of start of Anakinra.

Immunomodulation remains a promising strategy for the management of severe COVID-19 since it aims to attenuate the exaggerated inflammatory response of the host. The case-series of 7 patients coming from Greece and 1 from the Netherlands reported here supports the concept that Anakinra treatment may improve the respiratory function of patients who have sHLH at the EOT and at followup. Patients with severe COVID-19 are reported to experience hyper-coagulation and hyper-inflammation evidenced by increased D-dimers, ferritin, C-reactive protein (CRP) and procalcitonin (PCT). They also experience cardiomyopathy with increased Troponin I (Guan et al., 2020; Huang et al., 2020) . All these markers decreased with Anakinra treatment.

All seven patients under MV were male and this could be consistent with reports that the MV complication of COVID-19 is more frequent in male patients (Bhatraju et al. 2020) , also this may also result by chance. The case that sHLH is more common complication of COVID-19 in males cannot be excluded.

A recent position paper suggested measuring HScore in patients with severe COVID-19 encouraging the administration of Anakinra when HScore was diagnostic of sHLH (Mehta et al., 2020) . The reported mortality for the three out of seven Greek patients under MV appears high. However, it needs to be taken into consideration that sHLH is accompanied by substantial mortality as high as 67% in the case of sHLH developing in patients with sepsis (Kyriazopoulou et al., 2017; Shakoory et al., 2016) . This is not the first time when favourable Anakinra responses are shown in sHLH in adults. Several case-series report similar responses of Anakinra treatment in patients with HLH secondary to adult onset Still's disease (Lenert et al., 2016) and systemic infections (Kumar et al., 2017; Wohlfarth et al., 2019) . Anakinra was administered subcutaneously and at lower doses than those used herein. It was also co-administered with corticosteroids, antibiotics and antivirals. These authors highly recommend to measure the HScore when the patient is deteriorating and to administer treatment with Anakinra when sHLH is diagnosed.

Recently 29 patients with severe COVID-19 the majority of which had pO 2 /FiO 2 less than 100mmHg and treated intravenously with Anakinra were retrospectively analysed. Their mortality by day 21 was 10% and it was significantly lower than that of 16 comparators receiving standard-of-care treatment which was 44% (Cavalli et al., 2020) . These patients were less severe than those described herein since they were not under MV and they had lower ferritin levels. Although HScore was not criterion for treatment, these published outcomes support the results of our case-series showing favourable responses with Anakinra in the respiratory function of patients with COVID-19 who scored positive for the HScore. The one case of the non-ICU patient generates consideration for an early time window before the need of MV where immunomodulation may benefit.

The main study limitations are the lack of randomized design and the absence of untreated or placebo-treated comparators, both of which would allow better understanding of the magnitude of the survival benefit coming from Anakinra treatment. Considering these limitations, the presented data in patients with severe COVID-19 and sHLH warrant larger clinical trials to validate these results and demonstrate the usefulness of anti-IL-1 therapy.

The study was funded in part by the Horizon 2020 grant ImmunoSep (#847422) and in part by the Hellenic Institute for the Study of Sepsis. MGN was supported by an ERC Advanced grant (#833247) and a Spinoza grant of the Netherlands Organization for Scientific Research. Below each X-ray a brief diagnosis is provided along with the ratio of the partial oxygen pressure to the fraction of inspired oxygen (pO 2 /FiO 2 ) of that day. The presence (+) or absence (-) of acute kidney injury (AKI) is also noted.

Abbreviations: ↓: decrease; ↑: increase 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Evangelos J. Giamarellos-Bourboulis (egiamarel@med.uoa.gr)

This study did not generate new unique reagents. concentrations of triglycerides, fibrinogen, ferritin, aspartate aminotransferase; history of immunosuppression; and bone marrow hemophagocytosis. HScore more than or equal to 169 are considered diagnostic for sHLH since they are associated with 93% diagnostic sensitivity (Fardet et al. 2014) . Anakinra was administered as off-label salvage treatment in accordance to treatment suggestions for severe COVID-19 and the procedure suggested by the National Public Health Organization (NPHO) of Greece (www.eody.gr). No randomization to experimental groups was needed according to the study design.

The following information was recorded: past-history; co-administered drugs;

laboratory data and blood gases for the seven days of treatment and for two more follow-up days; and survival. Complete blood cell counts, serum or plasma concentrations of C-reactive protein, PCT, ferritin, troponin I and D-dimers were measured by automatic analyzers (Abbott Diagnostics). Blood gases were measured by automatic analyzers. Viral load of CMV and EBV was measured in plasma of these patients when sHLH was diagnozed. Viral DNA was isolated using the QIAamp DNA mini kit (QIAGEN) and this was followed by PCR reactions using the Genesig PCR CMV kit (Genesig) and the GeneProof EBV PCR kit (QIAgen). The lower detection limit was 2.77 copies/μl.

Results were presented as line graphs separately for every patient. Paired comparisons were done by the Wilcoxon's rank-signed test. Any value of p below 0.05 was considered significant.

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