Various studies have looked into the impact of the COVID-19 vaccine on large populations. However, very few studies have looked into the remote setting of hospitals where vaccination is challenging due to social structure, myths, and misconceptions. There is a consensus that elevated inflammatory markers such as CRP, ferritin, D-dimer correlate with increased severity of COVID-19 and are associated with worse outcomes. In the present study, through retrospective meta-analysis, we have looked into ~20 months of SARS-COV2 infected patients with known mortality status and identified predictors of mortality concerning their comorbidities, various clinical parameters, inflammatory markers, superimposed infections, length of hospitalization, length of mechanical ventilation and ICU stay. Studies with larger sample sizes have covered the outcomes through epidemiological, social, and survey-based analysis; however, most studies cover larger cohorts from tertiary medical centers. In the present study, we assessed the outcome of non-vaccinated COVID 19 patients in a remote setting for 20 months from January 1, 2020, to August 30, 2021, at CHI Mercy Health in Roseburg, Oregon. We also included two vaccinated patients from September 2021 to add to the power of our cohort. The study will provide a comprehensive methodology and deep insight into multi-dimensional data in the unvaccinated group, translational biomarkers of mortality, and state-of-art to conduct such studies in various remote hospitals.

Recently published whole exome sequencing studies in head and neck squamous cell carcinoma (HNSCC) tumors revealed that few had therapeutically targetable alterations using current strategies. This finding defines translational gap between genomics and HNSCC treatment. One potential targetable alteration is PIK3CA mutations. However, clinical trials testing PI3K/mTOR pathway inhibitors have had limited success and these inhibitors only lead to cell cycle arrest in PIK3CA mutant HNSCC cell lines. Thus, there is a critical need to identify therapeutic vulnerabilities for common mutation groups, including tumor suppressors, in HNSCC. One of these molecular subgroups is NOTCH1 which is the second most frequently mutated gene in HNSCC, with a 10-15% prevalence of inactivating mutations. Although there are several studies underscoring the importance of NOTCH1 as a tumor suppressor in HNSCC, none has identified a therapy that targets NOTCH1 mutant (mut) HNSCC. Our objective was to identify predictive biomarkers of sensitivity to PI3K/mTOR inhibitors by integrating drug and multiple-omics data. Cell viability with six PI3K/mTOR inhibitors in 68 HNSCC lines was measured by the CellTiter Glo assay. The peak plasma concentration of each drug was used as the cut-off to determine sensitivity. We observed a striking correlation between NOTCH1mut and sensitivity to PI3K/mTOR pathway inhibitors. When fisher's exact test was performed, NOTCH1mut lines were more sensitive to GSK2126458 (P<0.027), BYL719 (P<0.004) and PQR309 (P<0.014) than NOTCH1 wild type cell lines. NOTCH1 was also identified as an upstream regulator in sensitive cell lines by Ingenuity® Pathway Analysis. Basal NOTCH1 protein expression was higher in HNSCC lines resistant to PI3K/mTOR inhibition using unsupervised hierarchical clustering of Reverse Phase Protein Array data. NOTCH1mut lines underwent more apoptosis after GSK2126458 treatment compared to NOTCH1wt lines (PCI15B- 48.1 fold; P<0.05, HN31- 46.9 fold; P<0.05). There was also increased accumulation of cells in G1 after GSK2126458 treatment in NOTCH1mut lines (PCI15B-1.3 fold, P<0.05; HN31- 1.4 fold, P<0.05). To check if inhibition of NOTCH1 pathway inhibition sensitizes NOTCH1wt lines to PI3K/mTOR inhibition, resistant NOTCH1wt lines were treated with Gamma secretase inhibitors and GSK2126458. The combination led to significantly decreased cell viability (DAPT- 1.5 fold and YO010227- 1.7 fold). The combination studies will be further expanded to 38 NOTCH1wt lines. On-going studies include assessment of drug sensitivity in vivo, mechanistic studies and the effect of genetic manipulation of NOTCH1 signaling on sensitivity to PI3K/mTOR inhibitors. Our data suggests that loss of active NOTCH1 signaling confers sensitivity to PI3K/mTOR inhibition. If the combination of NOTCH1 and PI3K/mTOR inhibition leads to apoptosis, this combination could be translated into the clinic. Citation Format: Faye M. Johnson, Vaishnavi Sambandam, Li Shen, Ming Zhang, Rishi Saigal, Pan Tong, Tuhina Mazumdar, Lauren A. Byers, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick. NOTCH1 inactivating mutation mediates sensitivity to PI3K/mTOR inhibitors in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 393.

Background: HNSCC is the sixth most common cancer worldwide. To date, Cetuximab is the only approved targeted therapy for HNSCC treatment. Thus,there is an immediate need to discover effective targets. Two pharmacogenomic HTS studies, Cancer Genome Project (CGP) and Cancer Cell Line Encyclopedia (CCLE) provide a large repository of drug sensitivity data. The PI3K/mTOR pathway is one of the frequently activated signaling cascades in HNSCC. However, it is unclear which class of PI3K/mTOR inhibitors is most promising and which biomarkers may be used to predict sensitivity. The rationale for this study is to identify novel biomarkers to targets such as PI3K/mTOR pathway by data mining these public databases. The potential biomarkers will be characterized in vitro in 68 HNSCC cell lines.Methods:The landscape of drug sensitivity profiles in 23 HNSCC cell lines (CGP) was analyzed by boxplot illustrations. Drugs that induce growth inhibition at low doses (median≤10 μM) were considered “effective”. Chemotherapy drugs, drugs with missing values and unknown targets were excluded from analyses. Hierarchical clustering of cell lines was performed based on drug sensitivity using GOWER distance metric and Ward's linkage after normalization. Clustering of 140 drugs based on their sensitivity profiles was also done. In vitro, drug response to PI3K pathway inhibitors in 28 HNSCC cell lines was assessed by ATP based cell viability assay (CellTiter-Glo). Results: In the CGP datasets, we identified a set of effective drugs with median IC75<10 μM. These include drugs targeting HDAC, HSP, BCL2 and CHK1/2, and PI3K/mTOR pathway. When hierarchical clustering of drugs based on drug sensitivity was performed, 3 clusters were classified. Predictably, chemotherapy agents clustered together. Selective drugs that were effective in a subset of cell lines were also identified. To identify cell lines that were uniformly sensitive to inhibitors targeting the PI3K/mTOR pathway, diverse classes of inhibitors targeting PI3K pathway were selected and drug sensitivity was analyzed across 28 HNSCC cell lines. Notably, all cell lines were sensitive to the pan Class I PI3K inhibitor, BKM120 (IC75 <Cmax: 1.68 μM). We identified seven lines that were resistant and twelve lines that were sensitive to different PI3K/mTOR inhibitors. To identify novel biomarkers of sensitivity, we will use Reverse Phase Protein Array (RPPA), exome sequencing and gene expression data on cells that are uniformly sensitive and resistant to PI3K pathway inhibition. Conclusion: HNSCC cell lines were sensitive to a broad range of targeted therapies in in vitro screens including several inhibitors of the PI3K/mTOR pathway. We independently confirmed sensitivity to similar inhibitors and identified lines that were universally sensitive and resistant to this class of drug for biomarker development. Citation Format: Vaishnavi Sambandam, Li Shen, Ming Zhang, Rishi Saigal, Lauren A. Byers, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. Integrative drug sensitivity analysis of PI3K /mTOR pathway inhibitors in Head and Neck Squamous Cell Carcinoma (HNSCC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 417. doi:10.1158/1538-7445.AM2015-417