IMPORTANCE Therapies that improve survival in critically ill patients with coronavirus disease 2019 (COVID-19) are needed. Tocilizumab, a monoclonal antibody against the interleukin 6 receptor, may counteract the inflammatory cytokine release syndrome in patients with severe COVID-19 illness. OBJECTIVE To test whether tocilizumab decreases mortality in this population. DESIGN, SETTING, AND PARTICIPANTS The data for this study were derived from a multicenter cohort study of 4485 adults with COVID-19 admitted to participating intensive care units (ICUs) at 68 hospitals across the US from March 4 to May 10, 2020. Critically ill adults with COVID-19 were categorized according to whether they received or did not receive tocilizumab in the first 2 days of admission to the ICU. Data were collected retrospectively until June 12, 2020. A Cox regression model with inverse probability weighting was used to adjust for confounding. EXPOSURES Treatment with tocilizumab in the first 2 days of ICU admission. MAIN OUTCOMES AND MEASURES Time to death, compared via hazard ratios (HRs), and 30-day mortality, compared via risk differences. RESULTS Among the 3924 patients included in the analysis (2464 male [62.8%]; median age, 62 [interquartile range {IQR}, 52-71] years), 433 (11.0%) received tocilizumab in the first 2 days of ICU admission. Patients treated with tocilizumab were younger (median age, 58 [IQR, 48-65] vs 63 [IQR, 52-72] years) and had a higher prevalence of hypoxemia on ICU admission (205 of 433 [47.3%] vs 1322 of 3491 [37.9%] with mechanical ventilation and a ratio of partial pressure of arterial oxygen to fraction of inspired oxygen of <200 mm Hg) than patients not treated with tocilizumab. After applying inverse probability weighting, baseline and severity-of-illness characteristics were well balanced between groups. A total of 1544 patients (39.3%) died, including 125 (28.9%) treated with tocilizumab and 1419 (40.6%) not treated with tocilizumab. In the primary analysis, during a median follow-up of 27 (IQR, 14-37) days, patients treated with tocilizumab had a lower risk of death compared with those not treated with tocilizumab (HR, 0.71; 95% CI, 0.56-0.92). The estimated 30-day mortality was 27.5% (95% CI, 21.2%-33.8%) in the tocilizumab-treated patients and 37.1% (95% CI, 35.5%-38.7%) in the non-tocilizumab-treated patients (risk difference, 9.6%; 95% CI, 3.1%-16.0%). CONCLUSIONS AND RELEVANCE Among critically ill patients with COVID-19 in this cohort study, the risk of in-hospital mortality in this study was lower in patients treated with tocilizumab in the first 2 days of ICU admission compared with patients whose treatment did not include early use of tocilizumab. However, the findings may be susceptible to unmeasured confounding, and further research from randomized clinical trials is needed.

BackgroundCancer associated with smoking and drinking remains a serious health problem worldwide. The survival of patients is very poor due to the lack of effective early biomarkers. FOXM1 overexpression is linked to the majority of human cancers but its mechanism remains unclear in head and neck squamous cell carcinoma (HNSCC).Methodology/Principal FindingsFOXM1 mRNA and protein expressions were investigated in four independent cohorts (total 75 patients) consisting of normal, premalignant and HNSCC tissues and cells using quantitative PCR (qPCR), expression microarray, immunohistochemistry and immunocytochemistry. Effect of putative oral carcinogens on FOXM1 transcriptional activity was dose-dependently assayed and confirmed using a FOXM1-specific luciferase reporter system, qPCR, immunoblotting and short-hairpin RNA interference. Genome-wide single nucleotide polymorphism (SNP) array was used to ‘trace’ the genomic instability signature pattern in 8 clonal lines of FOXM1-induced malignant human oral keratinocytes. Furthermore, acute FOXM1 upregulation in primary oral keratinocytes directly induced genomic instability. We have shown for the first time that overexpression of FOXM1 precedes HNSCC malignancy. Screening putative carcinogens in human oral keratinocytes surprisingly showed that nicotine, which is not perceived to be a human carcinogen, directly induced FOXM1 mRNA, protein stabilisation and transcriptional activity at concentrations relevant to tobacco chewers. Importantly, nicotine also augmented FOXM1-induced transformation of human oral keratinocytes. A centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, both located within a consensus loci (10q23), were found to be novel targets of FOXM1 and their expression correlated tightly with HNSCC progression.Conclusions/SignificanceThis study cautions the potential co-carcinogenic effect of nicotine in tobacco replacement therapies. We hypothesise that aberrant upregulation of FOXM1 may be inducing genomic instability through a program of malignant transformation involving the activation of CEP55 and HELLS which may facilitate aberrant mitosis and epigenetic modifications. Our finding that FOXM1 is upregulated early during oral cancer progression renders FOXM1 an attractive diagnostic biomarker for early cancer detection and its candidate mechanistic targets, CEP55 and HELLS, as indicators of malignant conversion and progression.

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AbbreviationsHNSCC, head and neck squamous cell carcinoma; FOXM1, forkhead box M1; CEP55, centrosomal protein 55; HELLS, lymphoid specific helicase; qPCR, absolute real time reverse transcription quantitative polymerase chain reaction; NHOK, primary normal human oral keratinocytes; LnMet, lymph node metastasis; FFPE, formalin-fixed paraffin embedded. ABSTRACTWe recently showed that upregulation of a key oncogene FOXM1 precedes head and neck squamous cell carcinoma (HNSCC) malignancy. Furthermore, we also identified a centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, which are both downstream targets of FOXM1.In this study, we have investigated the expression profiles of CEP55 and HELLS using immunohistochemistry and quantified by digital densitometry in a tissue panel (20 samples) consisting of normal oral mucosa, dysplasias, HNSCC and lymph node metastasis (LnMet) samples.Furthermore, we corroborated our findings using absolute real-time PCR (qPCR) on a panel of 12 primary normal human oral keratinocytes, 5 dysplasia and 10 HNSCC cell lines. Finally, we validated our study using bioinformatics microarray analysis on an independent HNSCC patient cohort (4 normal and 16 tumours). In normal oral mucosa, CEP55 protein was detected at very low level within the upper differentiated layers. In contrast, CEP55 was highly expressed in oral dysplasia whereas only moderate expression was detected in HNSCC and LnMet. Low level of HELLS expression was detected in the basal cell layer of the normal oral mucosa, moderate level was seen in dysplasia and high levels in both HNSCC and LnMet. These expression patterns were consistent with both qPCR data from the cell line panel and microarray data analysis of TNM-stage defined HNSCC samples confirming the progressive expression pattern of CEP55 and HELLS. To our knowledge, this is the first pilot study demonstrating that both CEP55 and HELLS mRNA and protein expression positively correlate with pre-malignancy and HNSCC progression. This study provides strong evidence that CEP55 and HELLS may be used in conjunction with FOXM1 as a biomarker set for early cancer detection and indicators of malignant conversion and progression.

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