key: cord-0902189-x128akqk
authors: Canziani, Lorenzo M.; Trovati, Serena; Brunetta, Enrico; Testa, Amidio; De Santis, Maria; Bombardieri, Emilio; Guidelli, Giacomo; Albano, Giovanni; Folci, Marco; Squadroni, Michela; Beretta, Giordano D.; Ciccarelli, Michele; Castoldi, Massimo; Lleo, Ana; Aghemo, Alessio; Vernile, Laura; Malesci, Alberto; Omodei, Paolo; Angelini, Claudio; Badalamenti, Salvatore; Cecconi, Maurizio; Cremonesi, Alberto; Selmi, Carlo
title: Interleukin-6 receptor blocking with intravenous tocilizumab in COVID-19 severe acute respiratory distress syndrome: A retrospective case-control survival analysis of 128 patients
date: 2020-07-08
journal: J Autoimmun
DOI: 10.1016/j.jaut.2020.102511
sha: 18b9ae6ec938a2a81671d119d2e081d717cbde20
doc_id: 902189
cord_uid: x128akqk

In cases of COVID-19 acute respiratory distress syndrome, an excessive host inflammatory response has been reported, with elevated serum interleukin-6 levels. In this multicenter retrospective cohort study we included adult patients with COVID-19, need of respiratory support, and elevated C-reactive protein who received intravenous tocilizumab in addition to standard of care. Control patients not receiving tocilizumab were matched for sex, age and respiratory support. We selected survival as the primary endpoint, along with need for invasive ventilation, thrombosis, hemorrhage, and infections as secondary endpoints at 30 days. We included 64 patients with COVID-19 in the tocilizumab group and 64 matched controls. At baseline the tocilizumab group had longer symptom duration (13 ± 5 vs. 9 ± 5 days) and received hydroxychloroquine more often than controls (100% vs. 81%). The mortality rate was similar between groups (27% with tocilizumab vs. 38%) and at multivariable analysis risk of death was not significantly influenced by tocilizumab (hazard ratio 0.61, 95% confidence interval 0.33–1.15), while being associated with the use at baseline of non invasive mechanical or invasive ventilation, and the presence of comorbidities. Among secondary outcomes, tocilizumab was associated with a lower probability of requiring invasive ventilation (hazard ratio 0.36, 95% confidence interval 0.16–0.83; P = 0.017) but not with the risk of thrombosis, bleeding, or infections. The use of intravenous tocilizumab was not associated with changes in 30-day mortality in patients with COVID-19 severe respiratory impairment. Among the secondary outcomes there was less use of invasive ventilation in the tocilizumab group.

• Survival in ARDS from COVID-19 is significantly associated with age and comorbidities;

• The use of tocilizumab is not associated with difference in 30-day mortality;

• Patients treated with tocilizumab require invasive ventilation significantly less frequently than controls;

• No clear effect of tocilizumab was measured also on secondary outcome such as bleeding, thrombosis or infections;

• In patients with a survival exceeding 5 days, tocilizumab is associated with a significantly better survival compared to controls 3 Abstract.

In cases of COVID-19 acute respiratory distress syndrome, an excessive host inflammatory response has been reported, with elevated serum interleukin-6 levels. In this multicenter retrospective cohort study we included adult patients with COVID-19, need of respiratory support, and elevated C-reactive protein who received intravenous tocilizumab in addition to standard of care. Control patients not receiving tocilizumab were matched for sex, age and respiratory support.

We selected survival as the primary endpoint, along with need for invasive ventilation, thrombosis, hemorrhage, and infections as secondary endpoints at 30 days. We included 64 patients with COVID-19 in the tocilizumab group and 64 matched controls. At baseline the tocilizumab group had longer symptom duration (13±5 vs. 9±5 days) and received hydroxychloroquine more often than controls (100% vs. 81%). The mortality rate was similar between groups (27% with tocilizumab vs. 38%) and at multivariable analysis risk of death was not significantly influenced by tocilizumab (hazard ratio 0.61, 95% confidence interval 0.33-1. 15 ), while being associated with the use at baseline of non invasive mechanical or invasive ventilation, and the presence of comorbidities. Among secondary outcomes, tocilizumab was associated with a lower probability of requiring invasive ventilation (hazard ratio 0.36, 95% confidence interval 0.16-0.83; P=0.017) but not with the risk of thrombosis, bleeding, or infections. The use of intravenous tocilizumab was not associated with changes in 30-day mortality in patients with COVID-19 severe respiratory impairment. Among the secondary outcomes there was less use of invasive ventilation in the tocilizumab group.

As of May 10 th , 2020 over 4 million cases of SARS-coronavirus-2 disease (COVID-19) have been reported worldwide with variable mortality rates and 218.000 cases in Italy, one of the most impacted Countries [1]. Current treatments proposed for COVID-19 are merely supportive as the main cause of death is the development of severe acute respiratory distress syndrome (ARDS) [2] with biochemical features resembling macrophage activation syndrome. These include the progressive increase in C reactive protein, ferritin, and D-dimer levels with reduced lymphocyte count [3] . Furthermore, increased levels of inflammatory cytokines, such as IL-1β, IL-6, GM-CSF, and TNF-α have been reported in the peripheral blood of hospitalized patients with COVID-19, with higher levels in those admitted to Intensive Care Units and an association between IL-6 levels and the probability of survival [4, 5] , as confirmed by a recent meta-analysis [6] . Previous reports suggested a beneficial effect of tocilizumab (TCZ), a humanized monoclonal antibody directed at the IL-6 receptor, in COVID-19 [7] [8] [9] [10] . Similarly, proposed agents to treat COVID-19 include glucocorticoids [11] , hydroxychloroquine [12] , anakinra [13, 14] , baricitinib [15] , and direct antivirals [16] , with negative or inconclusive results reported to date largely from uncontrolled studies.

We report herein data from our retrospective study of death and other 30-day clinical outcomes in 128 patients treated for COVID-19-related ARDS and hyperinflammation with or without intravenous tocilizumab.

We conducted this study in two affiliated general hospitals in the Lombardy region of Northern Italy; these hospitals are located in two of the highest-impact cities (Milan and Bergamo) in Northern Italy, where COVID-19 was most incident. In both cases, hospitals were completely transformed over a two-week period, and wards fully dedicated to the care of patients with COVID-19 pneumonia [17] . Between February 23 rd and May 9 th , 2020, the two hospitals admitted over 2000 patients with such infection. In both hospitals, local recommendations were issued and updated regularly by a dedicated clinical and scientific committee. According to such recommendations, (i) all admitted patients were treated with subcutaneous enoxaparin at prophylactic dosage, (ii) direct antivirals including first lopinavir 400mg + ritonavir 100mg twice daily and subsequently, when this became unavailable, darunavir 800mg + cobicistat 150mg once a day were used in patients without contraindications, (iii) hydroxychloroquine 200mg twice a day was used in all patients who could take oral treatments and had no contraindications (i.e. prolonged QT interval, retinopathies, advanced renal failure, known hypersensitivity). The same committee provided the criteria that candidates had to fulfill to be eligible for tocilizumab: (i) clinical worsening in the previous 24 hours with increasing need for oxygen or ventilatory support, (ii) absence of clinical or biochemical signs of an active bacterial infection, (iii) elevated C reactive protein, (iv) a higher risk for mortality at blood tests, based on the odds ratios reported elsewhere [18] and including lymphocyte count, ferritin, creatine kinase, alanine aminotransferase, and D-dimer. Late intubation (over 24 hours) was considered an exclusion criterion. The process of eligibility evaluation lasted a maximum of 2 days.

Of major relevance for the design of this study, not all patients fulfilling these criteria could be treated based on the insufficient availability of the drug during the weeks of highest demand.

6 Demographic, clinical, imaging, and laboratory data were obtained from the Humanitas clinical data warehouse which includes the information available on all inpatient admitted at Istituto Clinico Humanitas (Rozzano, Milan) or Cliniche Humanitas Gavazzeni (Bergamo). The extracted data included the patient demographic details, hourly vital signs, laboratory test results, medication administration dose and frequency, oxygen support needed, and outcome. The presence of comorbidities was manually abstracted from the electronic medical record or charts.

For each of the 64 patients treated with tocilizumab an extraction based on age, sex, and enrolling center led to 2-5 potential controls which had a proven COVID-19 pneumonia and were subsequently matched according to the respiratory support, as well as the presence or absence of exclusion criteria. To allow a uniform matching for all tocilizumab-treated patients, a 1:1 control:case ratio was ultimately chosen leading to the identification of 64 patients in the control group. As mentioned, these patients would have also been treated with tocilizumab if the drug had been widely available.

Variables assessed.

Using the approach described above, we obtained for each patient at the time of tocilizumab administration or control matching: age, sex, daily recorded vital signs, the daily partial pressure of arterial oxygen to the fraction of inspired oxygen (PaO2:FiO2), estimated with the use of methods described elsewhere [19, 20] , required oxygen support which was grouped into low-medium flows (nasal canulae and facial masks with or without reservoir), high flows (non-invasive mechanical ventilation -NIMV) and invasive ventilation in the ICU, body mass index and smoking status, past and current diagnoses that were evaluated singularly and grouped into the Charlson comorbidity index [21] , and laboratory tests.

Exposure to tocilizumab and other treatments.

After providing written informed consent for the off-label use of tocilizumab, patients in the tocilizumab group received one intravenous infusion of 8mg/kg tocilizumab, followed by a second dose 24 hours later if no clinical worsening had occurred between infusions; 61/64 (95%) patients in the tocilizumab group received 2 infusions. The patients treated with tocilizumab were also included in the comparator arm of a National trial (ClinicalTrials.gov Identifier: NCT04317092), as for other previously reported series [8, 9] . Other medications included hydroxychloroquine, direct antivirals (lopinavir and ritonavir, darunavir and cobicistat), antibiotics (ceftriaxone, azithromycin, piperacillin and tazobactam), glucocorticoids (IV methylprednisolone 1-2 mg/kg/day), prophylactic enoxaparin were used based on the internal guidelines.

The primary endpoint of the study was to compare mortality rates between the tocilizumab and control groups. Secondary endpoints included differences between groups in the incidence of invasive ventilation (only for patients who were not already being invasively ventilated at baseline), thromboembolic events (including pulmonary embolism and deep vein thrombosis), hemorrhagic events (defined as bleeding with the need for a therapeutic or diagnostic measure), and bacterial or fungal infection (defined as clinical evidence or serum procalcitonin >1 ng/ml).

Descriptive statistics included categorical variables reported as number (percentage) and continuous variables as mean (standard deviation). For missing data, we used listwise deletion for univariable and multivariable analysis. Chi-square test and Mann-Whitney test were used accordingly to the type of variable. We used time-to-event (survival) methods for censored observations to identify risk factors associated with different outcomes. Time of exposure was defined as time from first day of tocilizumab infusion (for treatment group) or first day of clinical worsening (for control group) to 8 the day of event. For the primary outcome, the event was death; for secondary outcome, the events were intubation, thrombosis, clinically relevant bleeding, bacterial or fungal infection. The only cause of right censoring was the transfer to a different hospital; in case of discharge from hospital due to clinical recovery, we implied no more events for the remaining time of exposure.

Kaplan-Meier estimates were used to draw the cumulative incidence curves, compared by log-rank tests. Furthermore, we utilized the univariable and multivariable Cox proportional hazard model of relevant prognostic factors for the primary outcome.

For multivariable survival analysis, variables were selected if the rate of missing values was very low (<5%) and proved significant in the univariable Cox analysis (P value <0.1). After fitting the model, the PH assumption was examined on the basis of Schoenfeld residuals. Hazard ratios (HR)

were presented with the 95% confidence intervals (CI) and respective P values. Adverse events were analyzed by treatment group and in survival analysis. P values lower than 0.05 were considered statistically significant; data were analyzed with STATA version 16.0 (StataCorp LLC, College Station, TX).

Characteristics of the cohort.

Between March 15 and April 22, 2020, 64 patients were treated with tocilizumab at the two participating hospitals (52 at Humanitas Gavazzeni Bergamo and 12 at Humanitas Milan). For each patient in the tocilizumab group, one patient was extracted from the data warehouse of the same hospital to compose the control group of 64 patients (52 at Humanitas Gavazzeni Bergamo and 12 at Humanitas Milan). Table 1 and Table 2 Hemoglobin was significantly lower in the tocilizumab group, but mean values remained above 12 g/dl in both groups; platelet and lymphocyte counts did not differ between groups. Laboratory tests at baseline demonstrated higher serum aspartate aminotransferase (AST), creatine kinase (CK), and creatinine levels in the control group without reaching statistical significance.

We performed univariable and multivariable analyses using death as the primary endpoint and HR with 95% CI observed for candidate variables are illustrated in Table 3 .

In the univariable analysis, patient mortality was significantly associated with age (HR 1.06, 95% CI 1.01-1. 10 Clinically relevant secondary outcomes, including the need for invasive ventilation, thrombotic events, major bleeding, and bacterial or fungal infections are illustrated in Table 4 in terms of both incidence over 30 days in the two groups and HR (95% CI) for tocilizumab vs. controls. The use of tocilizumab was associated with a lower risk to require invasive ventilation in patients who were 11 not receiving this respiratory support at baseline (HR 0.36, 95% CI 0.16-0.83; P=0.017) while not modifying the probability of thrombotic events, bleeding, or infections.

20% of cases develop severe respiratory symptoms and possibly need ventilatory support with variable mortality rates, in Italy currently estimated to be nearly 13% [1], depending on numerous individual factors [22] . Immunological alterations associated with different stages of COVID-19 have been described since the earliest reports, which included the observed significant decrease in the lymphocyte count or the association of IL6 levels with disease outcome, suggesting the onset of a cytokine release syndrome [23, 24] . SARS-coronavirus-2 in a subgroup of patients, mainly in aged subjects, trigger a vigorous inflammatory response, as represented by IL-6 levels, unbalanced by a rapid type I and III interferon, which would be crucial for virus clearance and is genetically determined [25] [26] [27] . These observations have prompted the use of available anti-rheumatic drugs, particularly tocilizumab, to treat COVID-19 [28] . Tocilizumab is a humanized monoclonal antibody directed at the interleukin-6 receptor and is widely used to treat rheumatoid arthritis and giant-cell arteritis; in the case of COVID-19, it was first used in 21 Chinese patients in critical conditions with remarkable improvements [29] . Since these first reports, IL-6 blockade strategy has been applied to treat other COVID-19 patients, including Italian patients in different areas of the Country, with conflicting results which led to a multicenter phase II clinical trial (ClinicalTrials.gov Identifier: NCT04317092) designed without a randomization and with broader inclusion criteria compared to the present study and a retrospective case-control study from Campochiaro and Colleagues failed to identify an advantage in survival [10] .

Our data shows that the use of tocilizumab was not associated with a significant change the mortality rate at 30 days of patients with severe COVID-19 acute respiratory distress syndrome with hyperinflammation, after correction for pre-existing comorbidities and the need for respiratory support. These results are similar using different methods (i.e. survival analysis). Furthermore, a post hoc survival analysis between day 6 and day 30, following the identical mortality rates between groups until day 5 (when we also observed the nadir of CRP levels in the tocilizumab group), suggested that patients may significantly benefit from tocilizumab. We should note that these observations follow reports from single-center smaller studies with a shorter observation period and often with a limited use of controls [7] [8] [9] .

We are aware of the strengths and weaknesses of the present study. Among the former, this is the first controlled study on a large number of patients with COVID-19 either receiving or potentially eligible for tocilizumab based on well-defined criteria for acute respiratory distress syndrome and signs of inflammation, with a significantly larger number of treated patients and matched controls compared to the published reports [8, 9] . The matching criteria, furthermore, allowed to identify a control group which is comparable to the tocilizumab group and which could have been treated with tocilizumab if this had been widely available. Third, the study was performed in two Centers belonging to the same group, thus allowing a more uniform clinical management of patients in terms of indication for other medical treatments and ventilatory support. Fourth, the baseline differences between the two groups, which may have influenced the results, were either of negligible clinical relevance, as in the case of hemoglobin levels, or failed to prove significant in our univariable analysis, such as symptom duration. In this latter case, we should also note that patients receiving TCZ had a 2-day lag due to the delay in drug supply thus reducing the average differente in disease duration between groups.

Among weaknesses, the retrospective design of the study is predominant, but we should also be aware that the numerosity of the study population and the rigorous matching criteria allow to support our conclusions. Second, patients in the two groups differed for the use of hydroxychloroquine, but the univariable analysis demonstrated that no medical therapy correlated with mortality. Third, we may also hypothesize that the patients included in this study may have been too compromised to benefit from IL-6 modulation and we could not determine whether the 14 limited occurrence of invasive ventilation may have been secondary to the allocation of the limited intensive care resources. It will be crucial to determine the immunological timeline of COVID-19 to ascertain the likely narrow window of opportunity for immunomodulatory treatments and achieve a more personalized treatment, as numerous authors have proposed that COVID-19 results from different immune mechanisms [30, 31] . Fourth, our study did not account for potential genetic factors pointing at a different response to tocilizumab [27, 32] despite the most recent genome-wide association data not yielding suggestive results [33] , nor investigated a broader spectrum of immune activation markers or cytokines [34] .

In conclusion, our data from a retrospective controlled study support that tocilizumab is not 

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• Survival in ARDS from COVID-19 is significantly associated with age and comorbidities;• The use of tocilizumab is not associated with difference in 30-day mortality;• Patients treated with tocilizumab require invasive ventilation significantly less frequently than controls;• No clear effect of tocilizumab was measured also on secondary outcome such as bleeding, thrombosis or infections;• In patients with a survival exceeding 5 days, tocilizumab is associated with a significantly better survival compared to controls Declarations of interest: none relevant to this manuscript.