Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 7 July 2021 Article Transcriptomic Analysis for Prognostic Value in Head and Neck Squamous Cell Carcinoma Li-Hsing Chi 1,2,3 , Alexander TH Wu 1, Michael Hsiao 4* and Yu-Chuan (Jack) Li 1,5* 1 The Ph.D. Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei, Taiwan 2 Division of Oral and Maxillofacial Surgery, Department of Dentistry, Wan Fang Hospital, Taipei Medical University 3 Division of Oral and Maxillofacial Surgery, Department of Dentistry, Taipei Medical University Hospital, Taipei Medical University 4 Genomics Research Center, Academia Sinica, Taipei, Taiwan 5 Graduate Institute of Biomedical Informatics, College of Medical Science and Technology, Taipei Medical University, No.172-1, Sec. 2, Keelung Rd., Taipei 106, Taiwan * Correspondence: Hsiao: [email protected]; Li: [email protected] 1 Abstract: The survival analysis of the Cancer Genome Atlas (TCGA) dataset is a well-known 2 method to discover the gene expression-based prognostic biomarkers of head and neck squamous 3 cell carcinoma (HNSCC). A cutoff point is usually used in survival analysis for the patients’ 4 dichotomization in the continuous gene expression. There is some optimization software for cutoff 5 determination. However, the software’s predetermined cutoffs are usually set at the median or 6 quantiles of gene expression value to perform the analyses. There are also few clinicopathological 7 features available on their pre-processed data sets. We applied an in-house workflow, including 8 data retrieving and pre-processing, feature selection, sliding-window cutoff selection, Kaplan- 9 Meier survival analysis, and Cox proportional hazard modeling for biomarker discovery. In our 10 approach for the TCGA HNSCC cohort, we scanned human protein-coding genes to find optimal 11 cutoff values. After adjustment with confounders, the clinical tumor stage and the surgical margin 12 involvement are independent risk factors for patients’ prognosis. According to the resulting tables 13 with Bonferroni-adjusted P value under the optimal cutoff and the hazard ratio, three biomarker 14 candidates, CAMK2N1, CALML5, and FCGBP, are significantly associated with the patients’ 15 overall survival. We validated this discovery by using the other independent HNSCC dataset Citation: Chi, LH.; Wu, ATH.; Hsiao, 16 (GSE65858). Thus, we suggest the transcriptomic analysis could help for biomarker discovery. M.; Li, YCJ. Transcriptomic Analy- sis for Prognostic Value in Head and Neck Squamous Cell Carcinoma. J. 17 Keywords: Head and Neck Squamous Cell Carcinoma (HNSCC); the Cancer Genome Atlas Pers. Med. 2021, 1, 0. https://dx. 18 (TCGA); Transcriptomic Analysis; Survival Analysis; Optimal Cutoff; Effect Size; calcium/calmodulin doi.org/10.3390/jpm1010000 19 dependent protein kinase II inhibitor 1(CAMK2N1); calmodulin like 5(CALML5); Fc fragment of 20 IgG binding protein(FCGBP); Mindfulness Meditation Received: Accepted: Published: 21 1. Introduction Publisher’s Note: MDPI stays neu- 22 Head and neck squamous cell carcinoma (HNSCC), including oral, oropharyngeal, tral with regard to jurisdictional claims 23 and hypopharyngeal origin, is the fourth leading cancer causes of death for males in in published maps and institutional 24 Taiwan[1]. The age-standardized incidence rate of HNSCC in males is 42.43 per 100,000 affiliations. 25 persons[2]. The treatment strategies of HNSCC are surgery alone, systemic therapy 26 Copyright: © 2021 by the authors. with concurrent radiation therapy (systemic therapy/RT), or surgery with adjuvant Submitted to J. Pers. Med. for pos- 27 systemic therapy/RT (according to National Comprehensive Cancer Network, NCCN sible open access publication under 28 Clinical Practice Guidelines in HNSCC, Version 2.2020)[3]. Despite the improvement the terms and conditions of the Cre- 29 in those interventions, the survival of HNSCC has improved only marginally over the ative Commons Attribution (CC BY) 30 past decade worldwide[4]. The critical advancement of targeted therapy and immuno- license (https://creativecommons.org/ 31 oncology should benefit from emerging prognostic biomarkers guiding modern systemic licenses/by/4.0/). 32 therapy. Version July 6, 2021 submitted to J. Pers. Med. https://www.mdpi.com/journal/jpm © 2021 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 7 July 2021 Version July 6, 2021 submitted to J. Pers. Med. 2 of 25 33 Accumulative knowledge shows that some biomarkers have prognostic significance 34 in HNSCC. For example, node-negative HNSCC patients with p53 overexpression were 35 found to hold lower survival[5]. Overexpression of hypoxia-inducible factor(HIF)-1 36 alpha[6] or Ki-67[7] was found to be correlated with poor response to radiotherapy of 37 HNSCC. The epidermal growth factor receptor(EGFR)[ 8][9] and matrix metallopro- 38 teinase(MMP)[ 10] were found to be overexpressed to promote invasion and metastasis 39 of HNSCC. From 2000 to 2006, the first anti-EGFR antibody-drug (cetuximab) has been 40 developed and combined with radiotherapy, known as bio-RT, to increase survival 41 of unresectable locoregionally advanced disease[11]. The systemic therapy of cetux- 42 imab plus platinum-fluorouracil chemotherapy (EXTREME regimen) improves overall 43 survival when given as first-line treatment in patients with recurrent or metastatic 44 HNSCC[12][13]. It has been approved by theUS Food and Drug Administration( 45 FDA) since 2008. In advance, the bio-RT could be proceeded with docetaxel, cisplatin, 46 and 5-fluorouracil(Tax-PF) induction chemotherapy to overcome radio-resistance of 47 HNSCC[14]. 48 However, Rampias and his colleagues[15] suggested that Harvey rat sarcoma viral 49 oncoprotein(HRAS) mutations could mediate cetuximab resistance in systemic therapy 50 of HNSCC via the EGFR/rat sarcoma(RAS)/extracellular signal-regulated kinases(ERK) 51 signaling pathway. After that, the EGFR tyrosine kinase inhibitor(TKI) was introduced 52 to help cetuximab in 2018. The anti-tumor activity was observed in a phase 1 trial for 53 HNSCC patients using cetuximab and afatinib, a TKI of EGFR, human epidermal growth 54 factor receptor(HER)2, and HER4[ 16]. Other EGFR TKIs, such as gefitinib, erlotinib, 55 osimertinib, were also developed to treat advanced HNSCC. Although 90% of HNSCC 56 has overexpression of EGFR, cetuximab has only 10% to 20% response rate on those 57 patients. Before the year 2019, cetuximab is still the only drug of choice with proven 58 efficacy, which has targeted the selected HNSCC patients[17]. 59 Until the immuno-oncology era, immune-checkpoint inhibitor(ICI) was introduced 60 since 2014 for treating HNSCC[18][19]. The ICI works on immune checkpoint molecules, 61 including programmed death 1(PD-1), cytotoxic T lymphocyte antigen 4(CTLA-4), 62 T-cell immunoglobulin mucin protein 3(TIM-3), lymphocyte activation gene 3(LAG-3), 63 T cell immunoglobin and immunoreceptor tyrosine-based inhibitory motif(TIGIT), 64 glucocorticoid-induced tumor necrosis factor receptor(GITR), and V-domain Ig suppres- 65 sor of T-cell activation(VISTA)[ 20]. TheUS FDA has approved the anti-PD-1 agents (e.g. 66 pembrolizumab and nivolumab) as a monotherapy for the platinum-treated patients 67 with recurrent or metastatic HNSCC[21]. According to the phase 3 KEYNOTE-048 study, 68 PD-L1 is a validated biomarker used in clinical guidance for candidate selection of 69 pembrolizumab[22][23]. However, due to the complexity of immune-tumor interaction, 70 ICI has 20% response rate to programmed death ligand 1(PD-L1) expressed patients 71 (over 50% in immunohistochemistry, IHC staining of HNSCC)[19][23]. 72 According to our previous proteomic study from 2010 to 2017, thymosin beta-4 73 X-linked(TMSB4X) was reported to be related to tumor growth and metastasis of 74 HNSCC[24]. It was also reported by the subsequent investigations that TMSB4X has 75 engaged in tumor aggressiveness through epithelial-mesenchymal-transition(EMT) 76 on pancreatic[25], gastric[26], colorectal[27], lung[28], ovarian[29], and melanoma[30] 77 cancers. Thus, it might be suggested that TMSB4X is a candidate for tumor type-agnostic 78 therapy[31] as a common biomarker crossing several types of cancer. 79 The Cancer Genome Atlas (TCGA) profiled HNSCC (528 participants) clinical and 80 genomic data, which were standardized and are available at a unified data portal, Ge- 81 nomic Data Commons(GDC) of the the National Cancer Institute(NCI). The highlight of 82 the advantages of applying the TCGA data for cancer biomarker identification includes: 83 • To the best of our knowledge, the TCGA database is the largest collection (in both 84 cancer types and cohort size, especially in HNSCC) of comprehensive genomics 85 with survival data available in the field of cancer research so far. The whole- 86 genome sequencing data were harmonized across all Genome Data Analysis Center Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 7 July 2021 Version July 6, 2021 submitted to J. Pers. Med. 3 of 25 87 (GDAC)s. Many databases adopt the essential demographic data from TCGA since 88 it has comprehensive physical and social features of patients, such as exposure to 89 alcohol, asbestos, radioactive radon, tobacco smoking, or cigarettes. 90 • TCGA has a remarkable advantage for computational and life scientists who study 91 cancer since useful web-based tools and Application Programmable