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Factors predicting poor outcomes of patients treated with tocilizumab for COVID‑19‑associated pneumonia: A retrospective study

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is a significant global issue that has major implications for the healthcare system. The mortality rates associated with SARS-CoV-2 infection vary according to the geographical region and are associated with age, comorbidities and vaccination status. Organ damage is caused by the cytokine release syndrome, which plays a crucial role in the course of coronavirus disease 2019 (COVID-19) infection. Innate and adaptive immune system stimulation in patients with COVID-19 results in inappropriate cytokine release. The anti-IL-6 receptor antagonist, tocilizumab, is used in the treatment of connective tissue diseases. The present single-center retrospective study on patients with COVID-19 admitted to hospital between September, 2020 and April, 2022 aimed to identify predictors of mortality and other unfavorable outcomes in patients treated with tocilizumab for COVID-19-associated pneumonia. Demographics, vaccination status against SARS-CoV-2, the Charlson comorbidity index (CCI), laboratory data and chest X-ray scores were recorded upon admission. In total, 174 subjects (121 males; mean age, 62.43±13.47 years) fulfilling the inclusion criteria were included. Among the 174 participants, 58 (33.3%) were intubated. The mortality rate was 35.1%. The non-survivors were older, mostly females, and had a higher CCI score. At the evaluation upon admission, the survivors presented with higher levels of alanine transferase and gamma glutamyl-transferase and with a greater number of platelets (PLTs), while patients that were intubated were also older, mostly females, and had a higher CCI score (P<0.05). Age was identified as the only independent factor predicting mortality in the Cox proportional hazards multivariate regression analysis. By performing a sub-analysis regarding sex, it was revealed that the value of PLTs was an independent factor predicting intubation and 90-day mortality in male patients, and the lymphocyte count was the only factor associated with intubation in female patients. On the whole, the data of the present study may be used to identify patient subpopulations responding to treatment with tocilizumab in prospective clinical trials.


Introduction
As of July 14, 2022, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused 6,356,812 deaths globally (1). The mortality rates associated with SARS-CoV-2 infection vary according to the geographical region and are associated with age, comorbidities and vaccination status (2). Organ damage is considered to be caused by the cytokine release syndrome, which is crucial during the course of coronavirus disease 2019 (COVID- 19) infection. Organ damage is also caused by septic shock, thrombosis and oxidative stress (3). Excessive cytokine release in patients with COVID-19 is induced by the stimulation of the innate and adaptive immune systems. An unbalanced immune response and excessive inflammation are key pathogenic factors in COVID-19 (4).
Interleukin (IL)-6) is secreted by macrophages in response to specific microbial molecules known as pathogen-associated molecular patterns. These patterns bind to a key type of innate immune system detection molecules known as pattern recognition receptors, which include Toll-like receptors. These are found on the cell surface and in intracellular compartments, and they initiate intracellular signaling cascades, resulting in the release of inflammatory cytokines (5). IL-6 has been found to be implicated in severe SARS-CoV-2 infection (6). IL-6 levels of 80 pg/ml suggest an increased risk of respiratory failure and mortality, and immunomodulatory therapy is an area of urgent research (6).
Tocilizumab is an anti-IL-6 receptor antagonist that is used in the treatment of connective tissue disorders, such as rheumatoid arthritis, giant cell arteritis, polyarticular juvenile idiopathic arthritis and systemic juvenile idiopathic arthritis (7). This agent has exhibited efficacy against COVID-19 (8)(9)(10)(11)(12). The RECOVERY trial reported a decrease in the mortality rate from 35 to 31% when corticosteroids were used simultaneously in hospitalized patients with moderate, severe or critical COVID-19 infection, and with evidence of inflammation (8). Another study demonstrated that patients who were critically ill with COVID-19 who received tocilizumab or sarilumab presented with a mortality rate of 27% in the REMAP-CAP trial, compared to 36% in the control group receiving only standard care (9). Furthermore, another three meta-analyses (10)(11)(12) all agreed that tocilizumab should be used in the treatment of patients with severe COVID-19 infection. The first revealed a pooled mortality rate of 19% in the tocilizumab group (10), the second revealed a lower 28-day mortality rate with 32 fewer individuals per 1,000 who succumbed when treated with tocilizumab plus standard care, compared with standard care alone or placebo (11), and the third revealed a 22% 28-day mortality rate (12).
On the other hand, there is evidence to indicate that tocilizumab is ineffective in some cases of COVID-19, as it has been hypothesized that early drug administration is probably more beneficial (13). The World Health Organization (WHO) recommends the use of tocilizumab if inflammation is evident and a patient has severe or critical COVID-19 (14). More specifically tocilizumab is recommended in patients who have rapidly increasing oxygen needs and systemic inflammation (14). Tocilizumab is a potent anti-inflammatory drug that has been shown to reduce C-reactive protein (CRP) levels, although not always with a therapeutic effect. Other clinical parameters, such as the degree of hypoxia, may be a crucial factor in the decision of whether to escalate treatment or in determining prognosis (15). The present study aimed to describe in detail the characteristics of patients who received this agent and to identify determinants of mortality and other unfavorable outcomes.

Patients and methods
Study design. The present study was a single-center retrospective study on patients with COVID-19 admitted to the Department of Infectious Diseases-COVID-19 Unit of Laiko General Hospital (Athens, Greece) between September 21, 2020 and April 15, 2022. The study was conducted in line with the Declaration of Helsinki and obtained approval by the Institutional Review Board of Laiko General Hospital (protocol no. 765/12-2021). Written informed was obtained from all patients. The following criteria were required for inclusion in the study: A polymerase chain reaction diagnosis of COVID-19, a WHO clinical progression scale score ≥5, and tocilizumab treatment in accordance with the WHO recommendations (13). Some of the participants had a follow-up appointment 3 months after their admission to the post-COVID-19 outpatient clinic of Laiko General Hospital, and if that was not possible, a telephone call was made to determine the 90-day mortality rate. The exclusion criteria were an age <18 years and a lack of available data on survival at 3 months post-diagnosis.
Investigations. Demographics, vaccination status against SARS-CoV-2 and the Charlson comorbidity index (CCI) were recorded. Hemoglobin levels, white blood cell (WBC) count, blood neutrophil, lymphocyte and immature granulocyte counts, neutrophil-to-lymphocyte ratio, the number of platelets (PLTs), platelet-to-lymphocyte ratio, CRP and serum albumin levels, CRP-to-albumin ratio (CAR), serum lactate dehydrogenase (LDH), d-dimer, ferritin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase and gamma glutamyl-transferase (GGT) levels were recorded upon admission. In addition, the Modified Chest X-ray Scoring System was calculated for chest X-ray upon admission by two experienced radiologists, as previously described (16). Charts were evaluated for the implementation of intubation and all-cause mortality rates at 90 days.
Statistical analysis. Continuous variables are presented as the mean (standard deviation). The assessment of the normal distribution of variables was performed with the use the Kolmogorov-Smirnov test. The comparison of normally distributed variables was performed using an independent samples Student's t-test on variables with two groups and not normally distributed variables were examined using an unpaired non-parametric two-tailed Mann-Whitney test.  Table I. The non-survivors were older, mostly females and had a higher CCI score. At the evaluation upon admission to the hospital unit, the survivors presented with higher levels of ALT and GGT and with a greater number of PLTs (P<0.05; Table II). The patients that were intubated were also older, mostly females, and had a higher CCI score (P<0.05; Table III).
All parameters with significant differences in the univariate analysis were analyzed using the Cox proportional hazards multivariate regression analysis. The outcome was all-cause mortality, and cases were censored at 90 days. The only independent predictor of mortality found was age (P<0.05; Table IV).
In addition, age was also found to be a significant predictor of mortality using ROC analysis (Fig. 1). An age >64.5 years predicted mortality with 72.1% sensitivity and 71.7% specificity (AUC, 0.784). Kaplan-Meier survival analysis based on cut-off values for age (>64.5 years and ≤64.5 years) revealed a worse survival in subjects with an age >64.5 years (log-rank test for trend, P<0.05; Fig. 2). Furthermore, Cox proportional hazards multivariate regression analysis with intubation as the outcome did not identify any independent factors predicting intubation (Table V).
Of note, a sub-analysis regarding sex was performed, which revealed that the male survivors were younger, had lower chest X-ray scores, greater PLTs values and serum albumin, and lower values of CCI and creatinine (P<0.05; Table VI).
Cox proportional hazards multivariate regression analysis with mortality as the outcome identified PLTs as an independent factor predicting mortality in males (Table VII). In addition, the male patients that were intubated were older, had higher values of CCI, creatinine and ferritin and lower values of platelets (P<0.05; Table VI). Moreover, Cox proportional hazards multivariate regression analysis with intubation as the     outcome, identified PLTs as an independent factor predicting intubation in male patients (Table VII). As regards the female patients, the survivors were younger, had lower values of CCI, and greater values of GGT and CAR compared to the non-survivors (P<0.05). In addition, female patients that were intubated had lower values of lymphocytes compared to those that were not intubated (P<0.05; Table VIII). Furthermore, Cox proportional hazards multivariate regression analysis with mortality as the outcome did not identify any independent factor predicting mortality in female patients (Table IX).

Discussion
The mortality rate found in the present study was relatively higher compared to the rates demonstrated in randomized controlled trials of tocilizumab and in meta-analyses mentioning a pooled mortality prevalence of 20-30% (17)(18)(19)(20)(21). One of the main findings of the present study was that among all patients treated with tocilizumab for COVID-19-associated pneumonia, age was the only independent factor predicting 90-day mortality.
Previous research has indicated that age is a major risk factor for mortality in SARS-CoV-2 infection (22)(23)(24). As a result of age-related hematopoietic mosaic chromosomal changes that decrease immunity, age has been revealed to be the main risk factor for infections and the accompanying mortality (25). Moreover, differences in lung structure, muscular atrophy, poor airway clearance, diminished lung reserve, diminished resistance to infections, the increased expression of angiotensin converting enzyme (ACE)-2, particularly among the elderly receiving ACE inhibitors and angiotensin II receptor blockers, along with prior exposure to circulating coronaviruses with reduced neutralizing capacity to SARS-CoV-2, may all contribute to an increased vulnerability of elderly individuals to this infection and poor outcomes (26). Similar to the findings of the present study, age has been reported as a predictor of mortality in patients receiving tocilizumab for SARS-CoV-2 infection in other studies (27)(28)(29)(30)(31)(32)(33).
Another notable finding of the present study was that the mortality rate was higher among female patients compared to male patients. However, sex was not identified as an independent factor predicting mortality according to the multivariate analysis, as has been shown in previous research (34). It has been reported that males have significantly higher rates of adverse events and mortality due to COVID-19 (35). Biological sex differences manifest as differences in the balance between inflammation and tissue healing following the resolution of infection, differences in the time of pathogenesis, differences in innate viral control and adaptive immune responses, and a difference in vulnerability to infection (36). These disparities in sex are most likely pathogen-specific and complex in nature. Thus far, changes in immune function linked with the X chromosome, the impact of sex hormones and sex-related behavioral and   (37). Several studies have reported laboratory parameters, such as eosinophils, lymphocytes, PLTs, immature granulocytes, ferritin and liver enzymes as biomarkers of poor outcomes in patients with COVID-19 (36,(38)(39)(40)(41)(42). Of note, only a few studies have evaluated laboratory data as biomarkers of poor outcomes in tocilizumab-treated patients with COVID-19 (29)(30)(31)(32)(33)(34)(43)(44)(45). Some studies have examined the role of laboratory parameters on specific days following the tocilizumab administration as potential markers of mortality (31,45). According to the aforementioned studies, d-dimer levels (29), the WBC count (30), LDH levels (32,45), procalcitonin levels (33), ferritin (43), AST levels (44), CRP levels (34), lymphocyte count (44,45) and the number of PLTs (31,34,44), have all been identified as predictors of poor outcomes in tocilizumab-treated patients with COVID-19. Of note, the value of PTLs upon admission and following the tocilizumab administration was found to be        (31). The present study did not identify any independent laboratory factors predicting intubation or mortality in the population examined. However, when analyzing females and males who had a difference in mortality rate separately, it was identified that the value of PLTs was the only independent factor predicting intubation and 90-day mortality in male patients, and the lymphocyte count was the only factor associated with intubation in female patients. Several clinical studies have found that increased platelet activation leads to platelet deposition in injured pulmonary blood arteries, and thrombocytopenia is a common characteristic of SARS-CoV-2 infection (46,47). Thrombocytopenia is a major predictor of a poor prognosis. PLTs in patients with SARS-CoV-2 are inversely linked with soluble vascular cell adhesion molecule-1 (sVCAM-1) levels. sVCAM-1 is involved in adhesion and chemotaxis, and it leads to early vascular damage and T-cell inhibition. The poor outcome observed may be explained by vascular damage or immunosuppression (48).
The present study is one of a handful of studies evaluating laboratory data as biomarkers of poor outcomes in tocilizumab-treated patients with COVID-19 and, to the best of our knowledge, the first to mention PLTs as an independent factor predicting intubation and 90-day mortality in male patients treated with tocilizumab and the lymphocyte count as the only factor associated with intubation in female patients treated with tocilizumab.
The present study has certain limitations which should be mentioned. It was of a retrospective design, and there was no control group. Furthermore, it is possible that the negative results obtained in the present study (all-cause mortality) may be attributable to other etiologies in addition to severe COVID-19 (thromboembolism, sepsis, or coexisting diseases). The advantages of the present study were the relatively large number of tocilizumab-treated patients, the reliable follow-up data and the availability of 90-day data. Another strong point of the study was that participants were patients with COVID-19 admitted between September 21, 2020 and April 15, 2022, covering the periods of alpha, delta and omicron variant predominance.
In conclusion, in the present retrospective study, mortality occurred in 35.1% of the tocilizumab-treated COVID-19 patients, with a greater rate of mortality observed among females. The only independent prognosticator of mortality in the study population was age. In addition, the value of PLTs was an independent factor predicting intubation and 90-day mortality in male patients treated with tocilizumab, and the lymphocyte count was the only factor associated with intubation in female patients treated with tocilizumab. These data may be used to identify patient subpopulations responding to therapy in prospective clinical trials investigating the efficacy of treatment with tocilizumab.    Values in bold font indicate statistically significant differences (P<0.05). ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCI, Charlson comorbidity index; CRP, C-reactive protein; CAR, CRP-to-albumin ratio; GGT, gamma glutamyl-transferase; Hb, hemoglobin; IGs, immature granulocytes; LDH, lactate dehydrogenase; NLR, neutrophil-to-lymphocyte ratio; PLTs, platelets; PLR, platelet-to-lymphocyte ratio; WBC, white blood cell.