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Molecular Diagnostics

Application of liquid biopsy as multi-functional biomarkers in head and neck cancer

British Journal of Cancer volume 126, pages 361–370 (2022)Cite this article

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Abstract

Head and neck squamous cell carcinoma (HNSCC) is a molecularly heterogeneous disease, with a 5-year survival rate that still hovers at ~60% despite recent advancements. The advanced stage upon diagnosis, limited success with effective targeted therapy and lack of reliable biomarkers are among the key factors underlying the marginally improved survival rates over the decades. Prevention, early detection and biomarker-driven treatment adaptation are crucial for timely interventions and improved clinical outcomes. Liquid biopsy, analysis of tumour-specific biomarkers circulating in bodily fluids, is a rapidly evolving field that may play a striking role in optimising patient care. In recent years, significant progress has been made towards advancing liquid biopsies for non-invasive early cancer detection, prognosis, treatment adaptation, monitoring of residual disease and surveillance of recurrence. While these emerging technologies have immense potential to improve patient survival, numerous methodological and biological limitations must be overcome before their implementation into clinical practice. This review outlines the current state of knowledge on various types of liquid biopsies in HNSCC, and their potential applications for diagnosis, prognosis, grading treatment response and post-treatment surveillance. It also discusses challenges associated with the clinical applicability of liquid biopsies and prospects of the optimised approaches in the management of HNSCC.

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Fig. 1: Liquid biopsy biomarkers and clinical applications.

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References

  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–49.

    Article  PubMed  Google Scholar 

  2. Tumban E. A current update on human papillomavirus-associated head and neck cancers. Viruses. 2019;11:922.

    Article  CAS  PubMed Central  Google Scholar 

  3. Beynon RA, Lang S, Schimansky S, Penfold CM, Waylen A, Thomas SJ, et al. Tobacco smoking and alcohol drinking at diagnosis of head and neck cancer and all-cause mortality: Results from head and neck 5000, a prospective observational cohort of people with head and neck cancer. Int J Cancer. 2018;143:1114–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Johnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR. Head and neck squamous cell carcinoma. Nat Rev Dis Prim. 2020;6:92.

    Article  PubMed  Google Scholar 

  5. Elrefaey S, Massaro MA, Chiocca S, Chiesa F, Ansarin M. HPV in oropharyngeal cancer: the basics to know in clinical practice. Acta Otorhinolaryngol Ital. 2014;34:299–309.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006;354:567–78.

    Article  CAS  PubMed  Google Scholar 

  7. Astsaturov I, Cohen RB, Harari PM. EGFR-targeting monoclonal antibodies in head and neck cancer. Curr Cancer Drug Targets. 2006;6:691–710.

    Article  CAS  PubMed  Google Scholar 

  8. Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecular biology of head and neck cancer. Nat Rev Cancer. 2011;11:9–22.

    Article  CAS  PubMed  Google Scholar 

  9. Shanmugam A, Hariharan AK, Hasina R, Nair JR, Katragadda S, Irusappan S, et al. Ultrasensitive detection of tumor-specific mutations in saliva of patients with oral cavity squamous cell carcinoma. Cancer. 2021;127:1576–89.

    Article  CAS  PubMed  Google Scholar 

  10. Soda N, Rehm BHA, Sonar P, Nguyen NT, Shiddiky MJA. Advanced liquid biopsy technologies for circulating biomarker detection. J Mater Chem B. 2019;7:6670–704.

    Article  CAS  PubMed  Google Scholar 

  11. Egyud M, Sridhar P, Devaiah A, Yamada E, Saunders S, Stahlberg A, et al. Plasma circulating tumor DNA as a potential tool for disease monitoring in head and neck cancer. Head Neck. 2019;41:1351–58.

    Article  PubMed  Google Scholar 

  12. Wang Y, Springer S, Mulvey CL, Silliman N, Schaefer J, Sausen M, et al. Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas. Sci Transl Med. 2015;7:293ra104.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Agrawal N, Frederick MJ, Pickering CR, Bettegowda C, Chang K, Li RJ, et al. Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science. 2011;333:1154–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, et al. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;333:1157–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. The Cancer Genome Atlas Network. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 2015;517:576–82.

  16. Lawrence MS, Sougnez C, Lichtenstein L, Cibulskis K, Lander E, Gabriel SB, et al. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 2015;517:576–82.

    Article  CAS  Google Scholar 

  17. Boyle JO, Mao L, Brennan JA, Koch WM, Eisele DW, Saunders JR, et al. Gene mutations in saliva as molecular markers for head and neck squamous cell carcinomas. Am J Surg. 1994;168:429–32.

    Article  CAS  PubMed  Google Scholar 

  18. Pall AH, Jakobsen KK, Grønhøj C, von Buchwald C. Circulating tumour DNA alterations as biomarkers for head and neck cancer: a systematic review. Acta Oncol. 2020;59:845–50.

    Article  CAS  PubMed  Google Scholar 

  19. Liu MC, Oxnard GR, Klein EA, Swanton C, Seiden MV, Consortium C. Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA. Ann Oncol. 2020;31:745–59.

    Article  CAS  PubMed  Google Scholar 

  20. Liyanage C, Wathupola A, Muraleetharan S, Perera K, Punyadeera C, Udagama P. Promoter hypermethylation of tumor-suppressor genes p16(INK4a), RASSF1A, TIMP3, and PCQAP/MED15 in salivary DNA as a quadruple biomarker panel for early detection of oral and oropharyngeal cancers. Biomolecules. 2019;9:148.

    Article  CAS  PubMed Central  Google Scholar 

  21. Lim Y, Wan Y, Vagenas D, Ovchinnikov DA, Perry CF, Davis MJ, et al. Salivary DNA methylation panel to diagnose HPV-positive and HPV-negative head and neck cancers. BMC Cancer. 2016;16:749.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Rapado-Gonzalez O, Martinez-Reglero C, Salgado-Barreira A, Muinelo-Romay L, Muinelo-Lorenzo J, Lopez-Lopez R, et al. Salivary DNA methylation as an epigenetic biomarker for head and neck cancer. Part I: a diagnostic accuracy meta-analysis. J Pers Med. 2021;11:568.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Schrock A, Leisse A, de Vos L, Gevensleben H, Droge F, Franzen A, et al. Free-circulating methylated DNA in blood for diagnosis, staging, prognosis, and monitoring of head and neck squamous cell carcinoma patients: an observational prospective cohort study. Clin Chem. 2017;63:1288–96.

    Article  PubMed  Google Scholar 

  24. Langevin SM, Koestler DC, Christensen BC, Butler RA, Wiencke JK, Nelson HH, et al. Peripheral blood DNA methylation profiles are indicative of head and neck squamous cell carcinoma: an epigenome-wide association study. Epigenetics. 2012;7:291–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. de Jesus LM, Dos Reis MB, Carvalho RS, Scapulatempo Neto C, de Almeida GC, Laus AC, et al. Feasibility of methylated ctDNA detection in plasma samples of oropharyngeal squamous cell carcinoma patients. Head Neck. 2020;42:3307–15.

    Article  PubMed  Google Scholar 

  26. Challen C, Brown H, Cai C, Betts G, Paterson I, Sloan P, et al. Mitochondrial DNA mutations in head and neck cancer are infrequent and lack prognostic utility. Br J Cancer. 2011;104:1319–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Dasgupta S, Koch R, Westra WH, Califano JA, Ha PK, Sidransky D, et al. Mitochondrial DNA mutation in normal margins and tumors of recurrent head and neck squamous cell carcinoma patients. Cancer Prev Res. 2010;3:1205–11.

    Article  CAS  Google Scholar 

  28. Koc EC, Haciosmanoglu E, Claudio PP, Wolf A, Califano L, Friscia M, et al. Impaired mitochondrial protein synthesis in head and neck squamous cell carcinoma. Mitochondrion. 2015;24:113–21.

    Article  CAS  PubMed  Google Scholar 

  29. Sun W, Zhou S, Chang SS, McFate T, Verma A, Califano JA. Mitochondrial mutations contribute to HIF1alpha accumulation via increased reactive oxygen species and up-regulated pyruvate dehydrogenease kinase 2 in head and neck squamous cell carcinoma. Clin Cancer Res. 2009;15:476–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Mithani SK, Taube JM, Zhou S, Smith IM, Koch WM, Westra WH, et al. Mitochondrial mutations are a late event in the progression of head and neck squamous cell cancer. Clin Cancer Res. 2007;13:4331–5.

    Article  CAS  PubMed  Google Scholar 

  31. Schubert AD, Channah Broner E, Agrawal N, London N, Pearson A, Gupta A, et al. Somatic mitochondrial mutation discovery using ultra-deep sequencing of the mitochondrial genome reveals spatial tumor heterogeneity in head and neck squamous cell carcinoma. Cancer Lett. 2020;471:49–60.

    Article  CAS  PubMed  Google Scholar 

  32. Kim MM, Clinger JD, Masayesva BG, Ha PK, Zahurak ML, Westra WH, et al. Mitochondrial DNA quantity increases with histopathologic grade in premalignant and malignant head and neck lesions. Clin Cancer Res. 2004;10:8512–15.

    Article  CAS  PubMed  Google Scholar 

  33. Ha PK, Tong BC, Westra WH, Sanchez-Cespedes M, Parrella P, Zahurak M, et al. Mitochondrial C-tract alteration in premalignant lesions of the head and neck: a marker for progression and clonal proliferation. Clin Cancer Res. 2002;8:2260–5.

    CAS  PubMed  Google Scholar 

  34. Fliss MS, Usadel H, Caballero OL, Wu L, Buta MR, Eleff SM, et al. Facile detection of mitochondrial DNA mutations in tumors and bodily fluids. Science. 2000;287:2017–9.

    Article  CAS  PubMed  Google Scholar 

  35. Kumar M, Srivastava S, Singh SA, Das AK, Das GC, Dhar B, et al. Cell-free mitochondrial DNA copy number variation in head and neck squamous cell carcinoma: A study of non-invasive biomarker from Northeast India. Tumour Biol. 2017;39:1010428317736643.

    Article  PubMed  Google Scholar 

  36. Jiang WW, Masayesva B, Zahurak M, Carvalho AL, Rosenbaum E, Mambo E, et al. Increased mitochondrial DNA content in saliva associated with head and neck cancer. Clin Cancer Res. 2005;11:2486–91.

    Article  CAS  PubMed  Google Scholar 

  37. Wang X, Guo J, Yu P, Guo L, Mao X, Wang J, et al. The roles of extracellular vesicles in the development, microenvironment, anticancer drug resistance, and therapy of head and neck squamous cell carcinoma. J Exp Clin Cancer Res. 2021;40:35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Cao J, Zhang M, Xie F, Lou J, Zhou X, Zhang L, et al. Exosomes in head and neck cancer: Roles, mechanisms and applications. Cancer Lett. 2020;494:7–16.

    Article  CAS  PubMed  Google Scholar 

  39. Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL. Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. J Extracell Vesicles. 2016;5:29289.

    Article  PubMed  Google Scholar 

  40. Li P, Kaslan M, Lee SH, Yao J, Gao ZQ. Progress in exosome isolation techniques. Theranostics. 2017;7:789–804.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Theodoraki MN, Yerneni SS, Brunner C, Theodorakis J, Hoffmann TK, Whiteside TL. Plasma-derived exosomes reverse epithelial-to-mesenchymal transition after photodynamic therapy of patients with head and neck cancer. Oncoscience. 2018;5:75–87.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Theodoraki MN, Yerneni S, Gooding WE, Ohr J, Clump DA, Bauman JE, et al. Circulating exosomes measure responses to therapy in head and neck cancer patients treated with cetuximab, ipilimumab, and IMRT. Oncoimmunology. 2019;8:1593805.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Langevin S, Kuhnell D, Parry T, Biesiada J, Huang S, Wise-Draper T, et al. Comprehensive microRNA-sequencing of exosomes derived from head and neck carcinoma cells in vitro reveals common secretion profiles and potential utility as salivary biomarkers. Oncotarget. 2017;8:82459–74.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Rabinowits G, Taylo CG, Kloecke GH, Pate A, Hall MB, Taylor DD. Exosomal-miRNA profiles as diagnostic and prognostic biomarkers in head and neck squamous cell carcinoma (HNSCC). J Clin Oncol. 2011;29:5515.

    Article  Google Scholar 

  45. Chang YA, Weng SL, Yang SF, Chou CH, Huang WC, Tu SJ, et al. A three-microRNA signature as a potential biomarker for the early detection of oral cancer. Int J Mol Sci. 2018;19:758.

    Article  PubMed Central  Google Scholar 

  46. Greither T, Vorwerk F, Kappler M, Bache M, Taubert H, Kuhnt T, et al. Salivary miR-93 and miR-200a as post-radiotherapy biomarkers in head and neck squamous cell carcinoma. Oncol Rep. 2017;38:1268–75.

    Article  CAS  PubMed  Google Scholar 

  47. Severino P, Oliveira LS, Andreghetto FM, Torres N, Curioni O, Cury PM, et al. Small RNAs in metastatic and non-metastatic oral squamous cell carcinoma. BMC Med Genomics. 2015;8:31.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Haas BJ, Dobin A, Stransky N, Li B, Yang X, Tickle T, et al. STAR-Fusion: fast and accurate fusion transcript detection from RNA-Seq. bioRxiv:120295 [Preprint] 2017. Available from: https://doi.org/10.1101/120295.

  49. Qu X, Li JW, Chan J, Meehan K. Extracellular vesicles in head and neck cancer: a potential new trend in diagnosis, prognosis, and treatment. Int J Mol Sci. 2020;21:8260.

    Article  CAS  PubMed Central  Google Scholar 

  50. Zhou S, Wang L, Zhang W, Liu F, Zhang Y, Jiang B, et al. Circulating tumor cells correlate with prognosis in head and neck squamous cell carcinoma. Technol Cancer Res Treat. 2021;20:1533033821990037.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rodrigues-Junior DM, Tan SS, de Souza Viana L, Carvalho AL, Lim SK, Iyer NG. et al. A preliminary investigation of circulating extracellular vesicles and biomarker discovery associated with treatment response in head and neck squamous cell carcinoma. BMC Cancer. 2019;19:373

    Article  PubMed  PubMed Central  Google Scholar 

  52. Lacombe J, Brooks C, Hu CC, Menashi E, Korn R, Yang F, et al. Analysis of saliva gene expression during head and neck cancer radiotherapy: a pilot study. Radiat Res. 2017;188:75–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Panta P, Venna VR. Salivary RNA signatures in oral cancer detection. Anal Cell Pathol. 2014;2014:450629.

    Article  Google Scholar 

  54. Li Y, St John MA, Zhou X, Kim Y, Sinha U, Jordan RC, et al. Salivary transcriptome diagnostics for oral cancer detection. Clin Cancer Res. 2004;10:8442–50.

    Article  CAS  PubMed  Google Scholar 

  55. Zanotti L, Paderno A, Piazza C, Pagan E, Bignotti E, Romani C, et al. Epidermal growth factor receptor detection in serum and saliva as a diagnostic and prognostic tool in oral cancer. Laryngoscope. 2017;127:E408–14.

    Article  CAS  PubMed  Google Scholar 

  56. Liu GH, Zeng XJ, Wu BL, Zhao J, Pan YB. RNA-Seq analysis of peripheral blood mononuclear cells reveals unique transcriptional signatures associated with radiotherapy response of nasopharyngeal carcinoma and prognosis of head and neck cancer. Cancer Biol Ther. 2020;21:139–46.

    Article  CAS  PubMed  Google Scholar 

  57. Yao Y, Chen XY, Lu S, Zhou C, Xu GL, Yan ZY, et al. Circulating long noncoding RNAs as biomarkers for predicting head and neck squamous cell carcinoma. Cell Physiol Biochem. 2018;50:1429–40.

    Article  CAS  PubMed  Google Scholar 

  58. Li Y, Elashoff D, Oh M, Sinha U, St John MAR, Zhou XF, et al. Serum circulating human mRNA profiling and its utility for oral cancer detection. J Clin Oncol. 2006;24:1754–60.

    Article  CAS  PubMed  Google Scholar 

  59. Diez-Fraile A, Ceulaer J, Derpoorter C, Spaas C, Backer T, Lamoral P, et al. Circulating non-coding RNAs in head and neck cancer: roles in diagnosis, prognosis, and therapy monitoring. Cells. 2020;10:48.

    Article  PubMed Central  Google Scholar 

  60. Fadhil RS, Wei MQ, Nikolarakos D, Good D, Nair RG. Salivary microRNA miR-let-7a-5p and miR-3928 could be used as potential diagnostic bio-markers for head and neck squamous cell carcinoma. PLoS ONE. 2020;15:e0221779.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Song X, Yang X, Narayanan R, Shankar V, Ethiraj S, Wang X, et al. Oral squamous cell carcinoma diagnosed from saliva metabolic profiling. Proc Natl Acad Sci USA. 2020;117:16167–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Ishikawa S, Sugimoto M, Kitabatake K, Sugano A, Nakamura M, Kaneko M, et al. Identification of salivary metabolomic biomarkers for oral cancer screening. Sci Rep. 2016;6:31520.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Wojakowska A, Zebrowska A, Skowronek A, Rutkowski T, Polanski K, Widlak P, et al. Metabolic profiles of whole serum and serum-derived exosomes are different in head and neck cancer patients treated by radiotherapy. J Pers Med. 2020;10:229.

    Article  PubMed Central  Google Scholar 

  64. Mukherjee PK, Funchain P, Retuerto M, Jurevic RJ, Fowler N, Burkey B, et al. Metabolomic analysis identifies differentially produced oral metabolites, including the oncometabolite 2-hydroxyglutarate, in patients with head and neck squamous cell carcinoma. BBA Clin. 2017;7:8–15.

    Article  PubMed  Google Scholar 

  65. Xu J, Chen YH, Zhang RP, Song YM, Cao JZ, Bi N, et al. Global and targeted metabolomics of esophageal squamous cell carcinoma discovers potential diagnostic and therapeutic biomarkers. Mol Cell Proteom. 2013;12:1306–18.

    Article  CAS  Google Scholar 

  66. Shahid N, Iqbal A, Siddiqui AJ, Shoaib M, Musharraf SG. Plasma metabolite profiling and chemometric analyses of tobacco snuff dippers and patients with oral cancer: Relationship between metabolic signatures. Head Neck. 2019;41:291–300.

    CAS  PubMed  Google Scholar 

  67. Brondum L, Eriksen JG, Sorensen BS, Mortensen LS, Toustrup K, Overgaard J, et al. Plasma proteins as prognostic biomarkers in radiotherapy treated head and neck cancer patients. Clin Transl Radiat Oncol. 2017;2:46–52.

    Article  PubMed  PubMed Central  Google Scholar 

  68. Patel PS, Shah MH, Jha FP, Raval GN, Rawal RM, Patel MM, et al. Alterations in plasma lipid profile patterns in head and neck cancer and oral precancerous conditions. Indian J Cancer. 2004;41:25–31.

    CAS  PubMed  Google Scholar 

  69. Abdul Rahman M, Mohamad Haron DE, Hollows RJ, Abdul Ghani ZDF, Ali Mohd M, Chai WL, et al. Profiling lysophosphatidic acid levels in plasma from head and neck cancer patients. PeerJ. 2020;8:e9304.

    Article  PubMed  PubMed Central  Google Scholar 

  70. Hu S, Arellano M, Boontheung P, Wang J, Zhou H, Jiang J, et al. Salivary proteomics for oral cancer biomarker discovery. Clin Cancer Res. 2008;14:6246–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Vidotto A, Henrique T, Raposo LS, Maniglia JV, Tajara EH. Salivary and serum proteomics in head and neck carcinomas: before and after surgery and radiotherapy. Cancer Biomark. 2010;8:95–107.

    Article  CAS  PubMed  Google Scholar 

  72. Wang LN, Wang X, Li Y, Hou Y, Sun FY, Zhou S, et al. Plasma lipid profiling and diagnostic biomarkers for oral squamous cell carcinoma. Oncotarget. 2017;8:92324–32.

    Article  PubMed  PubMed Central  Google Scholar 

  73. Jou YJ, Lin CD, Lai CH, Chen CH, Kao JY, Chen SY, et al. Proteomic identification of salivary transferrin as a biomarker for early detection of oral cancer. Anal Chim Acta. 2010;681:41–8.

    Article  CAS  PubMed  Google Scholar 

  74. Garrel R, Mazel M, Perriard F, Vinches M, Cayrefourcq L, Guigay J, et al. Circulating tumor cells as a prognostic factor in recurrent or metastatic head and neck squamous cell carcinoma: the CIRCUTEC Prospective Study. Clin Chem. 2019;65:1267–75.

    Article  CAS  PubMed  Google Scholar 

  75. Grisanti S, Almici C, Consoli F, Buglione M, Verardi R, Bolzoni-Villaret A, et al. Circulating tumor cells in patients with recurrent or metastatic head and neck carcinoma: prognostic and predictive significance. PLoS ONE. 2014;9:e103918.

    Article  PubMed  PubMed Central  Google Scholar 

  76. Kulasinghe A, Hughes BGM, Kenny L, Punyadeera C. An update: circulating tumor cells in head and neck cancer. Expert Rev Mol Diagn. 2019;19:1109–15.

    Article  CAS  PubMed  Google Scholar 

  77. Perumal V, Corica T, Dharmarajan AM, Sun Z, Dhaliwal SS, Dass CR, et al. Circulating tumour cells (CTC), head and neck cancer and radiotherapy; future perspectives. Cancers. 2019;11:367.

    Article  CAS  PubMed Central  Google Scholar 

  78. Künzel J, Gribko A, Lu Q, Stauber RH, Wünsch D. Nanomedical detection and downstream analysis of circulating tumor cells in head and neck patients. Biol Chem. 2019;400:1465–79.

    Article  PubMed  Google Scholar 

  79. Rao VU, Arakeri G, Subash A, Bagadia RK, Thakur S, Kudpaje AS, et al. Circulating tumour cells in head and neck cancers: biological insights. J Oral Pathol Med. 2020;49:842–48.

    Article  Google Scholar 

  80. Curtin J, Choi SW, Thomson PJ, Lam AK. Characterization and clinicopathological significance of circulating tumour cells in patients with oral squamous cell carcinoma. Int J Oral Maxillofac Surg. 2021. https://doi.org/10.1016/j.ijom.2021.05.020.

  81. Buglione M, Grisanti S, Almici C, Mangoni M, Polli C, Consoli F, et al. Circulating tumour cells in locally advanced head and neck cancer: preliminary report about their possible role in predicting response to non-surgical treatment and survival. Eur J Cancer. 2012;48:3019–26.

    Article  PubMed  Google Scholar 

  82. Tada H, Takahashi H, Ida S, Nagata Y, Chikamatsu K. Epithelial-mesenchymal transition status of circulating tumor cells is associated with tumor relapse in head and neck squamous cell carcinoma. Anticancer Res. 2020;40:3559–64.

    Article  CAS  PubMed  Google Scholar 

  83. Hsieh JC, Lin HC, Huang CY, Hsu HL, Wu TM, Lee CL, et al. Prognostic value of circulating tumor cells with podoplanin expression in patients with locally advanced or metastatic head and neck squamous cell carcinoma. Head Neck. 2015;37:1448–55.

    Article  PubMed  Google Scholar 

  84. Tinhofer I, Konschak R, Stromberger C, Raguse JD, Dreyer JH, Johrens K, et al. Detection of circulating tumor cells for prediction of recurrence after adjuvant chemoradiation in locally advanced squamous cell carcinoma of the head and neck. Ann Oncol. 2014;25:2042–7.

    Article  CAS  PubMed  Google Scholar 

  85. Worden FP, Kumar B, Lee JS, Wolf GT, Cordell KG, Taylor JM, et al. Chemoselection as a strategy for organ preservation in advanced oropharynx cancer: response and survival positively associated with HPV16 copy number. J Clin Oncol. 2008;26:3138–46.

    Article  CAS  PubMed  Google Scholar 

  86. Lassen P, Eriksen JG, Hamilton-Dutoit S, Tramm T, Alsner J, Overgaard J. Effect of HPV-associated p16INK4A expression on response to radiotherapy and survival in squamous cell carcinoma of the head and neck. J Clin Oncol. 2009;27:1992–8.

    Article  CAS  PubMed  Google Scholar 

  87. Ang KK, Harris J, Wheeler R, Weber R, Rosenthal DI, Nguyen-Tan PF, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med. 2010;363:24–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Gillison M. HPV and its effect on head and neck cancer prognosis. Clin Adv Hematol Oncol. 2010;8:680–2.

    PubMed  Google Scholar 

  89. Cao H, Banh A, Kwok S, Shi X, Wu S, Krakow T, et al. Quantitation of human papillomavirus DNA in plasma of oropharyngeal carcinoma patients. Int J Radiat Oncol. 2012;82:e351–8.

    Article  CAS  Google Scholar 

  90. Ahn SM, Chan JY, Zhang Z, Wang H, Khan Z, Bishop JA, et al. Saliva and plasma quantitative polymerase chain reaction-based detection and surveillance of human papillomavirus-related head and neck cancer. JAMA Otolaryngol Head Neck Surg. 2014;140:846–54.

    Article  PubMed  PubMed Central  Google Scholar 

  91. Dahlstrom KR, Li G, Hussey CS, Vo JT, Wei Q, Zhao C, et al. Circulating human papillomavirus DNA as a marker for disease extent and recurrence among patients with oropharyngeal cancer. Cancer. 2015;121:3455–64.

    Article  CAS  PubMed  Google Scholar 

  92. Arvia R, Sollai M, Pierucci F, Urso C, Massi D, Zakrzewska K. Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies. J Virol Methods. 2017;246:15–20.

    Article  CAS  PubMed  Google Scholar 

  93. Chera BS, Kumar S, Beaty BT, Marron D, Jefferys S, Green R, et al. Rapid Clearance profile of plasma circulating tumor HPV type 16 DNA during chemoradiotherapy correlates with disease control in HPV-associated oropharyngeal cancer. Clin Cancer Res. 2019;25:4682–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Kacew AJ, Hanna GJ. Value and unmet needs in non-invasive human papillomavirus (HPV) testing for oropharyngeal cancer. Cancers. 2021;13:562.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Veyer D, Wack M, Mandavit M, Garrigou S, Hans S, Bonfils P, et al. HPV circulating tumoral DNA quantification by droplet-based digital PCR: a promising predictive and prognostic biomarker for HPV-associated oropharyngeal cancers. Int J Cancer. 2020;147:1222–7.

    Article  CAS  PubMed  Google Scholar 

  96. Isaac A, Kostiuk M, Zhang H, Lindsay C, Makki F, O’Connell DA, et al. Ultrasensitive detection of oncogenic human papillomavirus in oropharyngeal tissue swabs. J Otolaryngol Head Neck Surg. 2017;46:5.

    Article  PubMed  PubMed Central  Google Scholar 

  97. Yi X, Zou J, Xu J, Liu T, Liu T, Hua S, et al. Development and validation of a new HPV genotyping assay based on next-generation sequencing. Am J Clin Pathol. 2014;141:796–804.

    Article  PubMed  Google Scholar 

  98. Sloane H, Izumchenko E, Mattox A, Hasina R, Patel A, Jones F, et al. Ultra-sensitive detection and quantification of HPV DNA in the plasma of patients with oropharyngeal squamous cell carcinoma (OPSCC) enrolled in the OPTIMA 2 treatment de-escalation trial. J Clin Oncol. 2021;39:6048.

    Article  Google Scholar 

  99. Fung SY, Lam JW, Chan KC. Clinical utility of circulating Epstein-Barr virus DNA analysis for the management of nasopharyngeal carcinoma. Chin Clin Oncol. 2016;5:18.

    Article  PubMed  Google Scholar 

  100. Chen Y, Zhao W, Lin L, Xiao X, Zhou X, Ming H, et al. Nasopharyngeal Epstein-Barr virus load: an efficient supplementary method for population-based nasopharyngeal carcinoma screening. PLoS ONE. 2015;10:e0132669.

    Article  PubMed  PubMed Central  Google Scholar 

  101. Wang WY, Twu CW, Chen HH, Jiang RS, Wu CT, Liang KL, et al. Long-term survival analysis of nasopharyngeal carcinoma by plasma Epstein-Barr virus DNA levels. Cancer. 2013;119:963–70.

    Article  CAS  PubMed  Google Scholar 

  102. Liu TB, Zheng ZH, Pan J, Pan LL, Chen LH. Prognostic role of plasma Epstein-Barr virus DNA load for nasopharyngeal carcinoma: a meta-analysis. Clin Investig Med. 2017;40:E1–12.

    Article  CAS  Google Scholar 

  103. Sengar M, Chorghe S, Jadhav K, Singh S, Laskar SG, Pai P, et al. Cell-free Epstein-Barr virus-DNA in patients with nasopharyngeal carcinoma: plasma versus urine. Head Neck. 2016;38:E1666–73.

    Article  PubMed  Google Scholar 

  104. Lam WKJ, Jiang P, Chan KCA, Cheng SH, Zhang H, Peng W, et al. Sequencing-based counting and size profiling of plasma Epstein-Barr virus DNA enhance population screening of nasopharyngeal carcinoma. Proc Natl Acad Sci USA. 2018;115:E5115–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Chan KCA, Woo JKS, King A, Zee BCY, Lam WKJ, Chan SL, et al. Analysis of plasma Epstein-Barr Virus DNA to screen for nasopharyngeal cancer. N Engl J Med. 2017;377:513–22.

    Article  CAS  PubMed  Google Scholar 

  106. Lam WKJ, Jiang P, Chan KCA, Peng W, Shang H, Heung MMS, et al. Methylation analysis of plasma DNA informs etiologies of Epstein-Barr virus-associated diseases. Nat Commun. 2019;10:3256.

    Article  PubMed  PubMed Central  Google Scholar 

  107. Tan LP, Tan GW, Sivanesan VM, Goh SL, Ng XJ, Lim CS, et al. Systematic comparison of plasma EBV DNA, anti-EBV antibodies and miRNA levels for early detection and prognosis of nasopharyngeal carcinoma. Int J Cancer. 2020;146:2336–47.

    Article  CAS  PubMed  Google Scholar 

  108. Sethi S, Benninger MS, Lu M, Havard S, Worsham MJ. Noninvasive molecular detection of head and neck squamous cell carcinoma: an exploratory analysis. Diagn Mol Pathol. 2009;18:81–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Liu K, Chen N, Wei J, Ma L, Yang S, Zhang X. Clinical significance of circulating tumor cells in patients with locally advanced head and neck squamous cell carcinoma. Oncol Rep. 2020;43:1525–35.

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Wu XL, Tu Q, Faure G, Gallet P, Kohler C, Bittencourt Mde C. Diagnostic and prognostic value of circulating tumor cells in head and neck squamous cell carcinoma: a systematic review and meta-analysis. Sci Rep. 2016;6:20210.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Sun T, Zou K, Yuan Z, Yang C, Lin X, Xiong B. Clinicopathological and prognostic significance of circulating tumor cells in patients with head and neck cancer: a meta-analysis. Onco Targets Ther. 2017;10:3907–16.

    Article  PubMed  PubMed Central  Google Scholar 

  112. Wang HM, Wu MH, Chang PH, Lin HC, Liao CD, Wu SM, et al. The change in circulating tumor cells before and during concurrent chemoradiotherapy is associated with survival in patients with locally advanced head and neck cancer. Head Neck. 2019;41:2676–87.

    PubMed  Google Scholar 

  113. Jatana KR, Lang JC, Chalmers JJ. Identification of circulating tumor cells: a prognostic marker in squamous cell carcinoma of the head and neck? Fut Oncol. 2011;7:481–4.

    Article  CAS  Google Scholar 

  114. Gröbe A, Blessmann M, Hanken H, Friedrich RE, Schön G, Wikner J, et al. Prognostic relevance of circulating tumor cells in blood and disseminated tumor cells in bone marrow of patients with squamous cell carcinoma of the oral cavity. Clin Cancer Res. 2014;20:425–33.

    Article  PubMed  Google Scholar 

  115. Chan KC, Hung EC, Woo JK, Chan PK, Leung SF, Lai FP, et al. Early detection of nasopharyngeal carcinoma by plasma Epstein-Barr virus DNA analysis in a surveillance program. Cancer. 2013;119:1838–44.

    Article  CAS  PubMed  Google Scholar 

  116. Anh VNQ, Van Ba N, Anh DT, Ung ND, Hiep NH, Ly VT, et al. Validation of a highly sensitive qPCR assay for the detection of plasma cell-free Epstein-Barr virus DNA in nasopharyngeal carcinoma diagnosis. Cancer Control. 2020;27:1073274820944286.

    Article  PubMed  PubMed Central  Google Scholar 

  117. Damerla RR, Lee NY, You D, Soni R, Shah R, Reyngold M, et al. Detection of early human papillomavirus–associated cancers by liquid biopsy. JCO Precis Oncol. 2019;1–17. https://doi.org/10.1200/PO.18.00276.

  118. Capone RB, Pai SI, Koch WM, Gillison ML, Danish HN, Westra WH, et al. Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV-associated head and neck squamous cell carcinoma. Clin Cancer Res. 2000;6:4171–5.

    CAS  PubMed  Google Scholar 

  119. Zhao M, Rosenbaum E, Carvalho AL, Koch W, Jiang W, Sidransky D, et al. Feasibility of quantitative PCR-based saliva rinse screening of HPV for head and neck cancer. Int J Cancer. 2005;117:605–10.

    Article  CAS  PubMed  Google Scholar 

  120. Tang KD, Vasani S, Menezes L, Taheri T, Walsh LJ, Hughes BGM, et al. Oral HPV16 DNA as a screening tool to detect early oropharyngeal squamous cell carcinoma. Cancer Sci. 2020;111:3854–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Tang KD, Vasani S, Taheri T, Walsh LJ, Hughes BGM, Kenny L, et al. An occult HPV-driven oropharyngeal squamous cell carcinoma discovered through a saliva test. Front Oncol. 2020;10:408.

    Article  PubMed  PubMed Central  Google Scholar 

  122. Hilke FJ, Muyas F, Admard J, Kootz B, Nann D, Welz S, et al. Dynamics of cell-free tumour DNA correlate with treatment response of head and neck cancer patients receiving radiochemotherapy. Radiother Oncol. 2020;151:182–89.

    Article  CAS  PubMed  Google Scholar 

  123. Bai J, Zhang S, Li X, Zhang Y, Liu X. Next-generation sequencing of plasma cell-free DNA for treatment monitoring in advanced head and neck cancer patients. Clin Lab. 2020;66.

  124. Meng Y, Bian L, Zhang M, Bo F, Lu X, Li D. Liquid biopsy and their application progress in head and neck cancer: focus on biomarkers CTCs, cfDNA, ctDNA and EVs. Biomark Med. 2020;14:1393–1404.

    Article  CAS  PubMed  Google Scholar 

  125. Khandelwal AR, Greer AH, Hamiter M, Fermin JM, McMullen T, Moore-Medlin T, et al. Comparing cell-free circulating tumor DNA mutational profiles of disease-free and nonresponders patients with oropharyngeal squamous cell carcinoma. Laryngoscope Investig Otolaryngol. 2020;5:868–78.

    Article  PubMed  PubMed Central  Google Scholar 

  126. Chera BS, Kumar S, Shen C, Amdur R, Dagan R, Green R, et al. Plasma circulating tumor HPV DNA for the surveillance of cancer recurrence in HPV-associated oropharyngeal cancer. J Clin Oncol. 2020;38:1050–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Schirmer MA, Beck J, Leu M, Oellerich M, Rave-Frank M, Walson PD, et al. Cell-free plasma DNA for disease stratification and prognosis in head and neck cancer. Clin Chem. 2018;64:959–70.

    Article  CAS  PubMed  Google Scholar 

  128. Braig F, Voigtlaender M, Schieferdecker A, Busch CJ, Laban S, Grob T, et al. Liquid biopsy monitoring uncovers acquired RAS-mediated resistance to cetuximab in a substantial proportion of patients with head and neck squamous cell carcinoma. Oncotarget. 2016;7:42988–95.

    Article  PubMed  PubMed Central  Google Scholar 

  129. Abbott CW, Bedi N, Zhang SV, Northcott J, Li R, Pyke RM, et al. Abstract 555: longitudinal exome-scale liquid biopsy monitoring of evolving therapeutic resistance mechanisms in head and neck squamous cell carcinoma patients receiving anti-PD-1 therapy. Cancer Res. 2021;81:555.

    Article  Google Scholar 

  130. Cho JK, Lee GJ, Kim HD, Moon UY, Kim MJ, Kim S, et al. Differential impact of circulating tumor cells on disease recurrence and survivals in patients with head and neck squamous cell carcinomas: an updated meta-analysis. PLoS ONE. 2018;13:e0203758.

    Article  PubMed  PubMed Central  Google Scholar 

  131. Kulasinghe A, Perry C, Kenny L, Warkiani ME, Nelson C, Punyadeera C. PD-L1 expressing circulating tumour cells in head and neck cancers. BMC Cancer. 2017;17:333.

    Article  PubMed  PubMed Central  Google Scholar 

  132. Kulasinghe A, Kenny L, Punyadeera C. Circulating tumour cell PD-L1 test for head and neck cancers. Oral Oncol. 2017;75:6–7.

    Article  CAS  PubMed  Google Scholar 

  133. Kulasinghe A, Kapeleris J, Kimberley R, Mattarollo SR, Thompson EW, Thiery JP, et al. The prognostic significance of circulating tumor cells in head and neck and non-small-cell lung cancer. Cancer Med. 2018;7:5910–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Chikamatsu K, Tada H, Takahashi H, Kuwabara-Yokobori Y, Ishii H, Ida S, et al. Expression of immune-regulatory molecules in circulating tumor cells derived from patients with head and neck squamous cell carcinoma. Oral Oncol. 2019;89:34–9.

    Article  CAS  PubMed  Google Scholar 

  135. Tada H, Takahashi H, Kuwabara-Yokobori Y, Shino M, Chikamatsu K. Molecular profiling of circulating tumor cells predicts clinical outcome in head and neck squamous cell carcinoma. Oral Oncol. 2020;102:104558.

    Article  CAS  PubMed  Google Scholar 

  136. Tada H, Takahashi H, Kawabata-Iwakawa R, Nagata Y, Uchida M, Shino M, et al. Molecular phenotypes of circulating tumor cells and efficacy of nivolumab treatment in patients with head and neck squamous cell carcinoma. Sci Rep. 2020;10:21573.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. Strati A, Koutsodontis G, Papaxoinis G, Angelidis I, Zavridou M, Economopoulou P, et al. Prognostic significance of PD-L1 expression on circulating tumor cells in patients with head and neck squamous cell carcinoma. Ann Oncol. 2017;28:1923–33.

    Article  CAS  PubMed  Google Scholar 

  138. Economopoulou P, Kladi-Skandali A, Strati A, Koytsodontis G, Kirodimos E, Giotakis E, et al. Prognostic impact of indoleamine 2,3-dioxygenase 1 (IDO1) mRNA expression on circulating tumour cells of patients with head and neck squamous cell carcinoma. ESMO Open. 2020;5:e000646.

    Article  PubMed  PubMed Central  Google Scholar 

  139. Chaturvedi AK, Engels EA, Pfeiffer RM, Hernandez BY, Xiao W, Kim E, et al. Human papillomavirus and rising oropharyngeal cancer incidence in the United States. J Clin Oncol. 2011;29:4294–301.

  140. Wuerdemann N, Jain R, Adams A, Speel EJM, Wagner S, Joosse SA, et al. Cell-free HPV-DNA as a Biomarker for oropharyngeal squamous cell carcinoma—a step towards personalized medicine? Cancers. 2020;12:2997.

    Article  CAS  PubMed Central  Google Scholar 

  141. Reder H, Taferner VF, Wittekindt C, Brauninger A, Speel EM, Gattenlohner S, et al. Plasma cell-free human papillomavirus oncogene E6 and E7 DNA predicts outcome in oropharyngeal squamous cell carcinoma. J Mol Diagn. 2020. https://doi.org/10.1016/j.jmoldx.2020.08.002.

  142. Hanna GJ, Supplee JG, Kuang Y, Mahmood U, Lau CJ, Haddad RI, et al. Plasma HPV cell-free DNA monitoring in advanced HPV-associated oropharyngeal cancer. Ann Oncol. 2018;29:1980–6.

    Article  CAS  PubMed  Google Scholar 

  143. Chera BS, Kumar S, Shen C, Amdur R, Dagan R, Green R, et al. Plasma circulating tumor HPV DNA for the surveillance of cancer recurrence in HPV-associated oropharyngeal cancer. J Clin Oncol. 2020. https://doi.org/10.1200/JCO.19.02444.

  144. Seiwert TY, Salama JK, Vokes EE. The concurrent chemoradiation paradigm-general principles. Nat Clin Pract Oncol. 2007;4:86–100.

    Article  CAS  PubMed  Google Scholar 

  145. Rosenberg AJ, Vokes EE. Optimizing treatment de-escalation in head and neck cancer: current and future perspectives. Oncologist. 2020. https://doi.org/10.1634/theoncologist.2020-0303.

  146. Vokes EE, De Souza JA, Hannigan N, Brisson RJ, Seiwert TY, Villaflor VM, et al. Response-adapted volume de-escalation (RAVD) in locally advanced head and neck cancer. Ann Oncol. 2016;27:908–13.

    Article  PubMed  Google Scholar 

  147. Seiwert TY, Foster CC, Blair EA, Karrison TG, Agrawal N, Melotek JM, et al. OPTIMA: a phase II dose and volume de-escalation trial for human papillomavirus-positive oropharyngeal cancer. Ann Oncol. 2019;30:1673.

    Article  CAS  PubMed  Google Scholar 

  148. Chuang AY, Chuang TC, Chang S, Zhou SY, Begum S, Westra WH, et al. Presence of HPV DNA in convalescent salivary rinses is an adverse prognostic marker in head and neck squamous cell carcinoma. Oral Oncol. 2008;44:915–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This study was supported by the US National Institutes of Health (NIH) grants R01DE027809 to EI, R01DE028674 to NA and MWL and U01CA230691 to NA.

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Authors and Affiliations

  1. Department of Medicine, Section of Hematology and Oncology, University of Chicago, Chicago, IL, USA

    Vasudha Mishra, Alka Singh, Xiangying Chen, Ari J. Rosenberg, Alexander T. Pearson & Evgeny Izumchenko

  2. InSilico Medicine Hong Kong Ltd., Pak Shek Kok, Hong Kong

    Alex Zhavoronkov

  3. Department of Pathology, University of Chicago, Chicago, IL, USA

    Peter A. Savage & Mark W. Lingen

  4. Department of Surgery, Section of Otolaryngology-Head and Neck Surgery, University of Chicago, Chicago, IL, USA

    Nishant Agrawal

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  1. Vasudha Mishra
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  2. Alka Singh
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  3. Xiangying Chen
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  4. Ari J. Rosenberg
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  5. Alexander T. Pearson
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  6. Alex Zhavoronkov
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  7. Peter A. Savage
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  9. Nishant Agrawal
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  10. Evgeny Izumchenko
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Contributions

VM, EI, AS and NA performed a literature search, analysed the data and wrote the original draft. VM, EI, NA, AS, XC, AJR, ATP, PAS, AZ and MWL developed study concepts, performed data interpretation, manuscript writing, editing and review. EI, NA and MWL acquired funding. All authors reviewed and edited the final manuscript.

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Correspondence to Nishant Agrawal or Evgeny Izumchenko.

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Mishra, V., Singh, A., Chen, X. et al. Application of liquid biopsy as multi-functional biomarkers in head and neck cancer. Br J Cancer 126, 361–370 (2022). https://doi.org/10.1038/s41416-021-01626-0

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  • DOI: https://doi.org/10.1038/s41416-021-01626-0

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