key: cord-0776840-t3p8srxe authors: Salvati, Lorenzo; Occhipinti, Mariaelena; Gori, Leonardo; Ciani, Luca; Mazzoni, Alessio; Maggi, Laura; Capone, Manuela; Parronchi, Paola; Liotta, Francesco; Miele, Vittorio; Annunziato, Francesco; Lavorini, Federico; Cosmi, Lorenzo title: Pulmonary vascular improvement in severe COVID-19 patients treated with tocilizumab date: 2020-11-05 journal: Immunol Lett DOI: 10.1016/j.imlet.2020.10.009 sha: c0eff5b929fcc036a6502f81abb3c133fc05e8bb doc_id: 776840 cord_uid: t3p8srxe As of October 2020 management of Coronavirus disease 2019 (COVID-19) is based on supportive care and off-label or compassionate-use therapies. On March 2020 tocilizumab - an anti-IL-6 receptor monoclonal antibody - was suggested as immunomodulatory treatment in severe COVID-19 because hyperinflammatory syndrome occurs in many patients similarly to the cytokine release syndrome that develops after CAR-T cell therapy. In our retrospective observational study, 20 severe COVID-19 patients were treated with tocilizumab in addition to standard-of-care therapy (SOC) and compared with 13 COVID-19 patients receiving only SOC. Clinical respiratory status, inflammatory markers and vascular radiologic score improved after one week from tocilizumab administration. On the contrary these parameters were stable or worsened in patients receiving only SOC. Despite major study limitations, improvement of alveolar-arterial oxygen gradient as well as vascular radiologic score after one week may account for improved pulmonary vascular perfusion and could explain the more rapid recovery of COVID-19 patients receiving tocilizumab compared to controls. Coronavirus disease 2019 , the illness caused by SARS-CoV-2 infection, has emerged as a novel complex disease with a variable clinical course from asymptomatic to life-threatening condition [1, 2] . Management of COVID-19 is currently based on supportive care and off-label or compassionate-use therapies. Many treatments are under investigation and so far those showing most promising results are remdesivir and dexamethasone [3, 4] . Among others, the Infectious Diseases Society of America (ISDA) and the National Institutes of Health are providing up-to-date recommendations for the treatment and management of COVID-19 patients that, particularly in critical cases, needs expertise [5, 6] . Patients with severe disease requiring intensive care often present an hyperinflammatory syndrome, with elevated serum interleukin-6 (IL-6) levels as well as increase of other pro-inflammatory cytokines [7] [8] [9] [10] . The "cytokine storm" described in COVID-19 patients shows some common features with chimeric antigen receptor (CAR) T cell-induced cytokine release syndrome (CRS), the most common adverse event following CAR-T cell infusion [11, 12] . In 2017 the Food and Drug Administration approved tocilizumab, a recombinant humanized anti-human IL-6 receptor monoclonal antibody, for the treatment of CAR-T cell-induced CRS [13] . Tocilizumab binds the IL-6 receptor with high affinity and prevents IL-6 from binding to the receptor, and had been already approved for the treatment of various inflammatory diseases (i.e. rheumatoid arthritis, systemic juvenile idiopathic arthritis, polyarticular juvenile idiopathic arthritis, and giant cell arteritis). At the beginning of March 2020, Xu et al. firstly reported the use of Tocilizumab in a case-series of 21 patients with severe or critical COVID-19 demonstrating the improvement of symptoms, arterial oxygen levels and lung opacities. Despite major limitations, the study came out very early in the COVD-19 pandemic and suggested the use of tocilizumab as a potential immunomodulatory treatment of severe and critical COVID-19 patients [14] . Then the following literature showed conflicting results, with the most recent study by the BACC Bay Tocilizumab Trial Investigators demonstrating that tocilizumab was not effective for preventing intubation or death in moderately ill hospitalized patients [15] . However, the effect on severely ill patients admitted to ICU remains unclear. Therefore, in this study we aimed at evaluating the J o u r n a l P r e -p r o o f clinical and imaging response after one week of treatment with tocilizumab in patients with severe COVID-19 requiring intensive care. This is a retrospective observational single-center study. Adult patients admitted to the Pneumology and Intensive Care Unit (ICU) of the Careggi University Hospital, Florence, Italy, from March 11th to April 28th 2020, for COVID-19 pneumonia were included either as controls if treated only with standard-of-care (SOC) or as cases if treated with tocilizumab in addition to SOC. Patients with evidence of bacterial sepsis, an absolute neutrophil count below 500 per mm 3 , thrombocytopenia (below 50000 platelets per mm 3 ), liver impairment (ALT above 2.5 times ULN), medical history positive for gastrointestinal perforation, and/or known hypersensitivity to tocilizumab were excluded from treatment with tocilizumab. SOC included supplemental oxygen therapy as needed, low-molecular-weight heparin (6000 UI q.d.), hydroxychloroquine 400 mg b.i.d., Organization interim guidance [16] . Tocilizumab was administered intravenously twice 12-24 hours apart at 8 mg/kg (up to 800 mg). The study was approved by the Careggi University Hospital Ethical Committee (protocol 16859) and conducted in compliance with the Declaration of Helsinki Good Clinical Practice guidelines. The study was not funded by sponsors. All recruited patients provided informed written consent for treatment with off-label drugs, as provided for by local protocol. Physical examination, arterial blood gases test, laboratory parameters, and chest-X-ray (CXR) on day 1 (baseline time) and day 8 (after one week from baseline) were retrieved. We considered as J o u r n a l P r e -p r o o f baseline the day before treatment with tocilizumab (within day 3 from ICU admission) for cases, and the second day after ICU admission for controls. Clinical and laboratory data were collected from hospital records and stored in a database after anonymization. Clinical and laboratory variables included gender, age, supplemental oxygen support, adverse events, outcome, hemoglobin levels, white blood cells, neutrophils, lymphocytes, and platelets counts, serum levels of IL-6, C-reactive protein (CRP), ferritin, fibrinogen, and Ddimer, fraction of inspired oxygen (FiO2), partial pressure of oxygen (PaO2), partial pressure of carbon dioxide (PaCO2), ratio of partial pressure of arterial oxygen to FiO2 (PaO2:FiO2), ratio of arterial oxygen saturation to FiO2 (SpO2:FiO2), and alveolar-arterial oxygen (A-a O2) gradient. Adverse events were collected according to the common terminology criteria for adverse event (CTCAE) version 5.0. Chest-X-rays (CXRs) were performed at patient bedside by using portable machines. CXRs were reviewed by two independent radiologists and scored by evaluating both lung parenchyma and hilar vessels. Lung parenchyma was evaluated by applying the scoring method recently reported by Wong et al. and specifically developed for grading COVID-19 pneumonia on: 0 for no involvement, 1 for less than 25%, 2 for 25 to 50%, 3 for 50-75%, 4 for more than 75% of parenchymal involvement CXRs [17] . Hilar vessels were scored by adapting the score previously used by Pistolesi et al. for acute respiratory distress syndrome (ARDS): 0 for normal, 1 for either increased density or dimensions, 2 for both increased density and dimensions [18, 19] . A final score was obtained by adding parenchymal and hilar scores (0 to 6), as summarized in Table 1 . Data were expressed as mean ± standard deviation. Paired two-tailed Student's t test was used for comparison of clinical, laboratory and imaging data obtained on baseline and day 7. Unpaired twotailed Student's t test was used for comparison of clinical and laboratory data of cases versus controls. A p value equal or less than 0.05 was considered as statistically significant. Intraclass J o u r n a l P r e -p r o o f correlation coefficient (ICC) was used to test the inter-reader agreement agreement for CXR evaluation. In this retrospective study, we included 33 critical patients with SARS-CoV-2 infection who received SOC treatment. Twenty out of 33 (61%) patients received tocilizumab in addition to SOC and 13 (39%) only SOC treatment (control group). The main clinical features are summarized in Table 2 . The PaO2:FiO2 and SpO2:FiO2 ratio were significantly lower, while the A-a O2 gradient was significantly higher in the tocilizumab group compared with controls ( Table 2 ). At baseline patients presented more commonly with low hemoglobin level, normal white blood cells, normal neutrophils, lymphocytopenia, and normal platelets counts. High serum levels of IL-6, CRP, ferritin, fibrinogen, or D-dimer were detected in all the patients at baseline (Table 2) . Among the 20 patients who received tocilizumab, a total of 14 (70%) were discharged home at 35 (±26) days after treatment, 5 (25%) deceased at 23 (±18) days, while 1 (5%) was lost to follow-up. Among patients who received only SOC, 6 (46%) were discharged home at 62 (±27) days after evaluation and 7 (54%) deceased at 16 8(±18) days. Hospitalization time from ICU admission to discharge home or death was 33 (±24) days for patients treated with tocilizumab and 38 (±33) days for patients of the control group. Before treatment with tocilizumab, 15/20 (75%) patients received invasive ventilation and only 5/20 (25%) non-invasive ventilation ( Table 2 ). An improvement on oxygen-support class was observed PaO2:FiO2 after one week from treatment ( Figure 1) . Likewise, in this group CRP, ferritin, and fibrinogen significantly decreased at one week ( Figure 2 ). D-dimer showed only a trend towards reduction ( Figure 2 ). No significant changes in inflammatory markers were observed in the control group after one week from baseline ( Figure 2 ). The total radiographic score and the vascular score were significantly lower at one week after treatment with tocilizumab, while the lung parenchymal score remained unchanged ( Figure 3A ). Figure 3B shows the CXR before and after treatment with tocilizumab in a severe COVID-19 patient. In controls the total score and the lung parenchymal score significantly increased after one week, whereas the vascular score remained stable over time ( Figure 3A ). Inter-reader agreement for CXR scoring was excellent for the vascular (ICC: 0.8; 95%CI: 0.6-0.9) and the lung parenchymal scores (ICC: 0.8; 95% CI: 0.6 to 0.9), and good for the total radiographic score (ICC: 0.7; 95% CI: 0.4-0.8). COVID-19 is a biphasic disease. The first stage is that of viral replication, but then SARS-CoV-2 infection may lead to an aberrant hyperinflammatory response that overcomes the anti-viral immune response and seems to be critical in the pathogenesis [20] . Patients with severe COVID-19 may need intensive care and mechanical ventilation because of the acute onset of bilateral pulmonary infiltrates, severe hypoxemia, and lung edema in the context of ARDS as well as for the progression towards multi-organ failure [1] . These conditions are characterized by increase of biochemical markers of systemic inflammation and sustained by the release of pro-inflammatory cytokines [21] . These latter include elevated IL-6 levels typically found in patients with severe disease requiring intensive care who also show reduced frequency of granzyme A-expressing NK J o u r n a l P r e -p r o o f cells [7] . On this base, tocilizumab has been proposed as immunomodulatory therapy to mitigate the hyperinflammatory response associated with severe or critical COVID-19 [14, 22] . In autopsied lungs of deceased COVID-19 patients, severe endothelial injury, diffuse vascular thrombosis with microangiopathy and occlusion of alveolar capillaries, together with angiogenesis are present in addition to diffuse alveolar damage with edema, hemorrhage, and infiltrating perivascular lymphocytes [23, 24] . In line with these pathological observations, chest computed tomography (CT) demonstrated abnormal perfusion with proximal and distal pulmonary vessel dilatation in patients with COVID-19 [25, 26] . Vessel enlargement could be the result of an altered process of vaso-regulation leading to significant ventilation/perfusion (V/Q) mismatch [26] . The A-a O2 gradient as measuring the difference between the alveolar and the arterial oxygen concentration increases in conditions of V/Q mismatch, right-to-left shunt or alveolar hypoventilation [27] [28] [29] . Hypoxemia in COVID-19 patients is usually associated with increased A-a O2 gradient, meaning either V/Q mismatch or intrapulmonary shunting [30] . In our study on severe COVID-19 patients admitted to the ICU and treated with tocilizumab, one week after treatment a significant decrease of both the A-a O2 gradient and the vascular radiographic score was observed, without any modification of the lung parenchymal score. IL-6 has a well-known role in mediating endothelial dysfunction as well as in promoting haemostasis and coagulation thus contributing to a prothrombotic state [31] [32] . IL-6 increases megakaryocyte maturation and proliferation resulting in increased platelet production and enhanced platelet activation [33] . Excessive platelet activation has a central role in the immunothrombotic dysregulation that Nicolai et al. described in patients with severe COVID-19 [34] . Vascular injury and inflammation stimulate IL-6 synthesis by endothelial cells that in turn are activated by IL-6 [33] . In addition, IL-6 exerts pro-angiogenic effects that may account for new vessel formation, as described in COVID-19 pneumonia [23, 24, 35] . Thus, considering the role of IL-6 in promoting coagulation, the block of its receptor may be responsible of the rapid pulmonary vascular improvement in severe COVID-19 patients, while the parallel improvement of A-a O2 gradient and vascular score on CXR may account for improved V/Q mismatch or intrapulmonary shunting. Viceversa the lack of the lung parenchymal J o u r n a l P r e -p r o o f improvement on CXRs may be due to the fact that SARS-CoV-2 related pneumonia is often a migrant organizing pneumonia that needs at least more than a week to resolve [36] . To date among patients who have been admitted to the hospital with COVID-19, the IDSA guideline panel suggests against the routine use of tocilizumab [5, 37] . A double-blinded placebocontrolled randomized clinical phase 3 trial investigating the efficacy and safety of tocilizumab in patients with severe COVID-19 pneumonia found no difference in clinical status or mortality at day 28 between patients receiving tocilizumab versus placebo in addition to SOC, despite that median time to hospital discharge and duration of ICU stay were 8 days and 5.8 days shorter respectively in the tocilizumab compared to the placebo group [37] . The discrepancies between these results and our data could be due to various reasons. First, at variance with our patients, steroids were used in a large proportion of subjects recruited by Rosas et al. particularly in the placebo group, and this may account for a better outcome. Secondarily, our patients were selected by elevated levels of inflammatory markers, particularly IL-6 and this could have contributed to the results [37] . Finally, at variance with Rosas et al. [37] a double dose of tocilizumab was used in our group of patients. On the other hand, our study is a retrospective observational single-center study and the risk of bias in selecting the control group cannot be excluded. More recently the study of the BACC Bay Tocilizumab Trial Investigators demonstrated that tocilizumab was not effective for preventing intubation or death in moderately ill hospitalized patients [15] , ruling out an exclusive role of IL-6 in COVID-19 immunopathogenesis at the initial stage. Nonetheless, on severely ill patients, like in our study, tocilizumab might be beneficial at least by dampening the exuberant inflammatory response, occurring at a lower level in the moderately ill patients. In this study we unveiled a potential effect of tocilizumab on vascular pulmonary pathology in COVID-19 patients that could be responsible of more rapid recovery in patients treated with tocilizumab than those not receiving it, but that do not account for improved survival. In conclusion, we showed that in a subset of patients with severe COVID-19 presenting with systemic hyperinflammation, tocilizumab improved the A-a O2 gradient and the pulmonary vascular radiologic score, thus promoting early vascular pulmonary recovery. 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Comparison with computed tomography The trinity of COVID-19: immunity, inflammation and intervention When You Come Out of the Storm, You Won't Be the Same Person Who Walked in" Pharmacologic Treatments for Coronavirus Disease 2019 (COVID-19): A Review Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19 Pulmonary post-mortem findings in a series of COVID-19 cases from northern Italy: a two-centre descriptive study Hypoxaemia related to COVID-19: vascular and perfusion abnormalities on dual-energy CT Pulmonary Vascular Manifestations of COVID-19 Pneumonia The abbreviated alveolar air equation Use of the alveolar-arterial oxygen gradient in the diagnosis of pulmonary embolism Differentiating COVID-19 Pneumonia From Acute Respiratory Distress Syndrome and High Altitude Pulmonary Edema: Therapeutic Implications Basing Respiratory Management of COVID-19 on Physiological Principles IL-6: from its discovery to clinical applications Roles of IL-6-gp130 Signaling in Vascular Inflammation Interleukin 6 and haemostasis Interleukin-6: an angiogenic target in solid tumours Time Course of Lung Changes at Chest CT during Recovery from Coronavirus Disease 2019 (COVID-19) Tocilizumab in Hospitalized Patients With IL-6 serum levels predict severity and response to Tocilizumab in COVID-19: an observational study The authors declare no conflict of interest with the submitted work. IV TCZ 3 70 M 13,3 8620 7810 510 138 307 57,7 1058 846 28426 142 34 100 142 99 151 IV TCZ 4 78 F 13,9 7570 5600 1250 287 46 18,9 1013 510 1076 103 40 80 129 123 216 NIV TCZ 5 85 F 8,4 8970 8390 260 257 151 251,3 936 755 1176 147 43 80 184 124 410 IV TCZ 6 85 M 11,1 11100 10260 460 224 78 4,4 468 567 68582 122 63 70 174 140 249 IV TCZ 7 1177 127 42 80 159 124 196 NIV TCZ 11 58 M 10,1 5510 4850 350 185 223 32,7 700 619 109 83,8 37 60 140 162 141 IV TCZ 12 58 M 13 9700 8620 610 298 364 72,3 1369 729 587 69,4 29 100 69 94 242 IV TCZ 13 75 M 11,9 6960 6090 590 231 186 161,4 1610 918 26033 182 45 85 214 116 164 IV TCZ 14 86 M 10,6 7410 6710 540 79 260 171,9 2740 390 82917 97,4 58 100 97 96 223 IV TCZ 15 62 M 13 11000 9450 1140 402 166 3,5 751 651 899 88 56 55 160 173 192 IV TCZ 16 58 M 12,2 4780 4380 240 193 104 21,8 3150 552 977 103 34 50 206 196 151 NIV TCZ 17 64 M 12,9 11900 10460 710 430 264 80 1518 537 110200 121 44 100 121 98 216 IV TCZ M 10 26400 25400 80 280 335 61,1 1131 881 1855 90 67 50 180 192 183 IV SOC 3 74 M 8,9 11800 10960 500 -171 35,6 2367 423 64595 124 48 65 191 151 280 IV SOC 4 68 M 14,2 8230 7350 530 413 78 17,3 6166 523 294 103 41 45 229 216 167 IV SOC 5 72 F 9,2 7990 5200 2260 265 0 -599 507 1693 100 40 24 417 410 21 NIV SOC 6 45 F 7,2 20 10 10 10 286 200,1 3523 619 4033 115 29 40 288 249 134 IV J o u r n a l P r e -p r o o f SOC 7 80 M 12,1 9050 8222 500 237 110 81,9 2895 563 829 86 36 30 287 320 83 IV SOC 8 60 M 7,7 12400 11420 430 92 107 -4305 192 11790 113 54 50 226 196 176 no SOC 9 69 M 8,6 5600 4790 470 179 170 143 2844 601 3334 106 59 100 106 96 533 IV SOC 10 65 M 9,7 5330 4650 440 188 36 -3033 452 674 103 51 50 206 196 190 IV SOC 11 58 F 11,5 11600 10520 650 168 267 223 171 390 1817 130 39 70 186 141 320 NIV SOC 12 77 M 9 4900 4030 540 176 118 23,4 849 -3479 93 43 40 231 242 138 no SOC13 73 M 10,4 8230 7490 610 196 103 37,8 5855 659 7349 121 31 60 202 164 268 IV Mean 68 -10 10027 8736 670 200 153 91 2731 517 8479 106 44 51 228 215 205 -SD 9 -2 6683 6210 619 104 99 78 1894 171 17973 14 11 19 73 81