key: cord-255794-55ubow92
authors: Galván-Román, José María; Rodríguez-García, Sebastián C.; Roy-Vallejo, Emilia; Marcos-Jiménez, Ana; Sánchez-Alonso, Santiago; Fernández-Díaz, Carlos; Alcaraz-Serna, Ana; Mateu-Albero, Tamara; Rodríguez-Cortes, Pablo; Sánchez-Cerrillo, Ildefonso; Esparcia, Laura; Martínez-Fleta, Pedro; López-Sanz, Celia; Gabrie, Ligia; Guerola, Luciana del Campo; Suarez, Carmen; Ancochea, Julio; Canabal, Alfonso; Albert, Patricia; Rodríguez-Serrano, Diego A.; Aguilar, Juan Mariano; Arco, Carmen del; Santos, Ignacio de los; García-Fraile, Lucio; Camara, Rafael de la; Serra, José María; Ramírez, Esther; Alonso, Tamara; Landete, Pedro; Soriano, Joan B.; Martín-Gayo, Enrique; Torres, Arturo Fraile; Zurita Cruz, Nelly Daniela; García-Vicuña, Rosario; Cardeñoso, Laura; Sánchez-Madrid, Francisco; Alfranca, Arantzazu; Muñoz-Calleja, Cecilia; González-Álvaro, Isidoro
title: IL-6 serum levels predict severity and response to Tocilizumab in COVID-19: an observational study
date: 2020-09-30
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2020.09.018
sha: 
doc_id: 255794
cord_uid: 55ubow92

Background COVID-19 patients can develop a cytokine release syndrome that eventually leads to acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation (IMV). Since interleukin-6 (IL-6) is a relevant cytokine in ARDS, the blockade of its receptor with Tocilizumab (TCZ) could reduce mortality and/or morbidity in severe COVID-19. Objective To determine whether baseline IL-6 serum levels can predict the need for IMV and the response to TCZ. Methods Retrospective observational study performed in hospitalized patients diagnosed of COVID-19. Clinical information and laboratory findings, including IL-6 levels, were collected approximately 3 and 9 days after admission to be matched with pre- and post-administration of TCZ. Multivariable logistic and linear regressions, and survival analysis were performed depending on outcomes: need for IMV, evolution of arterial oxygen tension/fraction of inspired oxygen ratio (PaO2/FiO2) or mortality. Results One hundred and forty-six patients were studied, predominantly male (66%); median age was 63 years. Forty-four patients (30%) required IMV, and 58 patients (40%) received treatment with TCZ. IL-6 levels>30 pg/ml was the best predictor for IMV (OR:7.1; p<0.001). Early administration of TCZ was associated with improvement of oxygenation (PaO2/FiO2) in patients with high IL-6 (p=0.048). Patients with high IL-6 not treated with TCZ showed high mortality (HR: 4.6; p=0.003), as well as those with low IL-6 treated with TCZ (HR: 3.6; p=0.016). No relevant serious adverse events were observed in TCZ-treated patients. Conclusion Baseline IL-6>30 pg/ml predicts IMV requirement in patients with COVID-19 and contributes to establish an adequate indication for TCZ administration.

1 ABSTRACT 117 Background 118 COVID-19 patients can develop a cytokine release syndrome that eventually leads to acute 119 respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation (IMV). Since 120 interleukin-6 (IL-6) is a relevant cytokine in ARDS, the blockade of its receptor with 121 Tocilizumab (TCZ) could reduce mortality and/or morbidity in severe Objective 123 To determine whether baseline IL-6 serum levels can predict the need for IMV and the 124 response to TCZ. 125 

Retrospective observational study performed in hospitalized patients diagnosed of Clinical information and laboratory findings, including IL-6 levels, were collected 128 approximately 3 and 9 days after admission to be matched with pre-and post-administration The recent exponential increase in cases of severe acute respiratory syndrome caused by 181 coronavirus 2 (SARS-CoV-2) has led the World Health Organization to declare a pandemic. The 182 disease, known as coronavirus disease 2019 , has pushed national health systems 183 to the brink of collapse, prompting national governments to impose complete population 184 lockdowns in an attempt to slow down the dynamics of infection (1, 2) 185 The spectrum of COVID-19 clinical manifestations varies widely, from mild to severe cases of 186 atypical pneumonia, some of them developing acute respiratory distress syndrome (ARDS), 187 which often requires invasive mechanical ventilation (IMV) and is the leading cause of death. 188 It is suggested that the severity of the respiratory disease caused by SARS-CoV-2 is largely due 189 to an exacerbated immune response against the virus (3, 4). This response has been observed 190 in previous respiratory virus outbreaks and can be also seen in patients treated with chimeric 191 antigen receptor (CAR) T cell therapy (5-7). Pro-inflammatory cytokines such as interleukin (IL) 192 1 and IL-6 are crucial mediators of this process (3, 6). In this regard, recent studies have 193 indicated the usefulness of IL-6 serum levels, lymphocyte count, fibrinogen or D-dimer to 194 evaluate the development of ARDS and its mortality (8-11). However, further evidence is 195 required to support these observations (12). 196 Tocilizumab (TCZ), an anti-IL-6 receptor (IL-6R) antibody, is the only drug currently licensed for 197 the treatment of the cytokine release syndrome (CRS) associated with CAR-T cell therapy (13). 198 Due to the virulence of the current outbreak, its use has been advised in severe cases of 199 . The rationale is to curb the deleterious effects of inflammation, thereby 205 This is a retrospective observational study including 146 consecutive patients with confirmed 206 detection of SARS-CoV-2 RNA, baseline IL-6 serum level measurement and admitted to the 207 Hospital Universitario La Princesa (HUP) with severe to critical COVID-19 (18), from February 208 24 th to March 23 rd 2020 (Flow chart in Suppl. Fig. 1 240 Surplus sera from laboratory routine determinations were used to assess IL-6 levels, which 241 were retrospectively quantified in duplicate with the Human IL-6 Quantikine high sensitivity 242 enzyme-immune assay from R&D Systems Europe Ltd. (Abingdon, UK). The intra-assay and 243 inter-assay variability were 3% and 5%, respectively. Therefore, the decision to treat with TCZ was based on the AEMPS criteria, excluding IL-6>40 261 pg/ml, since no information about IL-6 results was available to physicians during the study 262 period. After a preliminary analysis of data by the end of March, IL-6 measurement was 263 included in the baseline assessment of COVID-19 patients. 264 The administration schedule of TCZ at the time of the study was a first intravenous infusion of 265 8 mg/kg (maximum 800 mg) followed by a second one after 12 hours. To analyze whether IL-6 levels can predict disease severity, two main outcomes were 269 considered: need for IMV and all-cause mortality. 270 To determine the effect of TCZ we analyzed the evolution of PaO 2 /FiO 2 between both 271 evaluation times. In 160 out of 267 evaluations arterial oxygen tension (PaO 2 ) was 272 J o u r n a l P r e -p r o o f unavailable. So, in order to avoid missing data in these relevant outcomes, this parameter was 273 estimated from mean oxygen saturation (SatO 2 ) as proposed elsewhere (22). 274 IL-6 serum levels showed a heterogeneous distribution in patients and in healthy donors 275 (Suppl. Fig. 3A ). To improve its representation in figures, this variable was normalized through 276 natural logarithmic transformation (Suppl. Fig. 3B ). The procedure to determine the cut-off for 277 high baseline IL-6 is described below. represented as median and IQR and the Mann Whitney test was used to assess significant 283 differences. Variables with a normal distribution were described by mean±standard deviation 284 (SD) and differences between groups were assessed with Student's t-test. Qualitative 285 variables were described as counts and proportions and 2 or Fisher´s exact test was used for 286 comparisons. Correlation between quantitative variables was analyzed using the Pearson 287 correlation test. To estimate the 95% confidence interval of correlation coefficients we used 288 the ci2 command of Stata. 289 To determine whether IL-6 serum levels were able to discriminate between: i) COVID-19 290 patients vs. healthy donors; ii) patients requiring IMV vs. those that did not; or iii) patients 291 treated with TCZ vs. not treated, receiver operating characteristic (ROC) analysis was 292 performed using the roctab command. Each cut-off point was selected based on the best 293 trade-off values between sensitivity, specificity and the percentage of patients correctly 294 classified. Positive and negative likelihood ratios and ROC curves were also obtained.

To determine the variables associated with the need for IMV, we performed a multivariable 296 logistic regression analysis that was first modeled by adding all the variables with a p value 297 lower than 0.15 in the bivariable analysis, namely total lymphocyte count, D-dimer, LDH, 298 PaO 2 /FiO 2 , COPD, obesity, hypertension, C-reactive protein, and IL-6 (high vs low). The final 299 model was reached with backward stepwise removal of variables with p-value higher than 300 0.15, and using Wald tests to demonstrate that each model was better than its previous 301 iteration. 302 Next, we performed a multivariable analysis using generalized linear models nested by patient 303 and visit (xtgee command) in which the dependent variable was PaO 2 /FiO 2 . This approach 304 allowed us to identify which variables influenced the evolution of PaO 2 /FiO 2 . The first model 305 included all variables with a p value <0.15 in the bivariable analysis, namely hypertension, 306 baseline radiological pattern, LDH, total lymphocyte count, baseline C-reactive protein, IMV. 307 After that, through backward stepwise approach, we obtained the best model as described 308 above. Then, to assess the role of IL-6 as predictor of TCZ effect on PaO 2 /FiO 2, the composite 309 variable IL-6/TCZ (low IL-6/no TCZ, low IL-6/Early TCZ, low IL-6/Late TCZ, high IL-6/no TCZ, high 310 IL-6/Early TCZ and high IL-6/Late TCZ) was forced in the model. 329 One hundred and forty-six patients were included; their main demographic and clinical 330 characteristics are shown in Table 1 Fig. 3A and B). 346 No significant correlation was found between PaO 2 and IL-6 serum levels at baseline (r= -0.09 347 [95% CI: -0.270 to 0.085]; p=0.299; Figure 1A) , likely due to a higher oxygen supply in the most 348 severe cases; in fact, serum IL-6 levels showed a significant negative correlation with PaO 2 /FiO 2 ( Figure 1B ; r= -0.38 [95% CI: -0.526 to -0.218]; p<0.001), meaning that higher levels 350 of IL-6 at baseline were associated with lower PaO 2 /FiO 2 . 351 In this regard, forty-four patients (30%) required IMV at some point during their 352 hospitalization. As expected, these patients showed significantly worse PaO 2 /FiO 2 levels than 353 those not requiring IMV (p<0.001; Table 2 ). In addition, they showed increased leukocytes, 354 total lymphocytes, IL-6, C-reactive protein (CRP), and procalcitonin (PCT) showing a higher 355 inflammatory status than those not requiring IMV (p≤0.001 except p=0.003 for CRP and 356 p=0.029 for lymphocyte count; Table 2 ). 357 Furthermore, a baseline IL-6 above 30 pg/ml (henceforth high IL-6) discriminated patients 358 requiring IMV with 68% Se and 73% Sp. AUC was 0.725 ( Figure 1C ; LR+ 2.5, LR-0.4). A logistic 359 regression model, adjusted for COPD and baseline white blood cell count, also showed that 360 high baseline IL-6 was a predictive biomarker for IMV (OR:7.1; 95% CI: 3.0 to 16.6; Supp Table   361 2). 

Even before physicians were aware of the potential value of IL-6 serum levels as a predictor of 375 severe disease, those patients with high IL-6 were more frequently treated with TCZ ( Figure   376 1D; AUC 0.634; 30 pg/ml as cut-off showed Se 57%, Sp 69%, LR+ 1.9, LR-0.7), although with 377 less accuracy than for IMV. The median time from the beginning of symptoms to TCZ 378 treatment was 11 days (IQR: 8-12.5). Therefore, we considered early TCZ when the treatment 379 was applied before 11 days of disease duration and late TCZ after this cut-off. 380 As a consequence of IL-6R blockade with TCZ, a significant trend toward higher IL-6 serum 381 levels after administration of the drug was observed ( Since relevant differences were observed between patients treated and not treated with TCZ, 393 we used a multivariable analysis to determine which variables influenced the evolution of 394 PaO 2 /FiO 2 at the short-term. Baseline PaO 2 /FiO 2 and radiological pattern, HTA, LDH and CRP 395 levels and total lymphocyte blood count significantly explained variation in PaO 2 /FiO 2 (Suppl 396   Table 4 ). Adjusted by these confounders, the best PaO 2 /FiO 2 evolution was achieved in 397 J o u r n a l P r e -p r o o f patients with low IL-6 not requiring TCZ treatment ( Figure 3C first dot on the left; reference 398 group in analysis showed in Suppl Table 4 ), likely because they were the less severe patients. 399 Patients with low IL-6 that due to their bad evolution were prescribed with TCZ showed a 400 significant worsening of PaO 2 /FiO 2 ( Figure 3C , 2nd and 3rd dots; Suppl Table 4 ). A similar 401 evolution was observed in patients with high IL-6 and late TCZ treatment, whereas those with 402 high IL-6 not treated or treated early with TCZ showed no significant differences compared 403 with the reference group ( Figure 3C ; Suppl Table 4 ). role in the regulation of D-dimer production; or ii) more likely, D-dimer production has a 471 slower kinetics than CRP or other acute phase reactants, since in patients not treated with TCZ 472 a similar rise in D-dimer levels was observed (data not shown ). 473 Apart from the novelty and the immediate clinical utility of these findings, a drawback of our 474 study is its retrospective and observational nature involving mainly very severe cases in the 475 group of treatment with TCZ. A stricter selection of a control group through a propensity score 476 strategy was unfeasible, since once the physicians were aware of IL-6 measurement they 477 focused their efforts on treating with TCZ those patients with the highest IL-6 levels. 478 Therefore, prospective studies should be carried out to confirm our observations. 479 In addition, there is some controversy about the reliability of PaO 2 /FiO 2 as an outcome for 480 improvement of lung damage, especially in patients with IMV. However, our population was a 481 mix of patients with IMV and non-IMV, so despite the many factors that could interfere with 482 PaO 2 /FiO 2 , we considered it the most objective outcome for patients' assessment. and p5 (line below the box) before (grey boxes) and after (white boxes) treatment with TCZ. In 694 non-treated patients PRE and POST mean first and second evaluation, respectively. Statistical

Rapid and Severe Covid-19 Pneumonia with Severe Acute Chest Syndrome in a Sickle Cell 586

Patient Successfully Treated with Tocilizumab

First case of COVID-19 in a patient 589 with multiple myeloma successfully treated with tocilizumab

Cytokine release syndrome in severe COVID-592 19: Interleukin-6 receptor (IL-6R) antagonist Tocilizumab may be the key to reduce the 593 mortality

Characteristics of and Important Lessons From the Coronavirus 599 Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases From the 600

Chinese Center for Disease Control and Prevention. Jama. 2020

Detection of SARS-CoV-2 in Different 603 Types of Clinical Specimens

Genomic characterisation and 23

In the Eye of the Storm: Immune-mediated Toxicities Associated With CAR-T Cell 618

Monocyte-620 derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and 621 neurotoxicity due to CAR T cells

Expression of acute-phase cytokines, surfactant proteins, and epithelial apoptosis in small 625 airways of human acute respiratory distress syndrome

Acute 628 respiratory distress syndrome

Mechanisms and 634 pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after 635 administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid 636 arthritis and Castleman disease

Roche provides an update on the phase III COVACTA trial of

Accesed September 3, 2020. 642 30. Sanofi-Genzyme. Sanofi provides update on Kevzara® (sarilumab) Phase 3 trial in 643 severe and critically ill COVID-19 patients outside the

Pilot prospective open, 647 single-arm multicentre study on off-label use of tocilizumab in patients with severe COVID-19

Tocilizumab treatment in COVID-19: A single center 650 experience

All categorical variables are expressed as number (%) and quantitative variables as median

PaO 2 /FiO 2 : arterial oxygen tension -fraction of inspired oxygen ratio; LDH: Lactate Dehydrogenase; IL-669 6: Interleukin-6; CRP: C-reactive protein; PCT: Procalcitonin. significance was determined with the Mann-Whitney test. (C) The graph represents the 696 predicted mean (dots) with 95% confidence intervals (bars) of PaO 2 /FiO 2 according to baseline 697 IL-6 levels and early or late TCZ treatment. Data were obtained with the command 698 marginsplot of Stata

Lactic dehidrogenase and C-reactive protein levels, lymphocyte blood count 700 and need for IMV, according to the multivariable analysis displayed in Supplementary Table 2 701 (see Methods for further information)

Survival curves of COVID-19 patients grouped according to baseline IL-6 levels and 703 TCZ treatment. Statistical significance was established with log-rank test