Simultaneous Detection of the HPV L1 Gene and the Human β-Globin Gene in the Blood Components of Cervical Cancer Patients Living in Yakutia

ORIGINAL ARTICLE
Irina V. Kononova, Sargylana N. Mamaeva, V.A. Alekseev, Nadezhda A. Nikolaeva, L.N. Afanasyeva, Petr V. Nikiforov, N.A. Vasilyeva, I.V. Vasiliev, Georgy V. Maximov

 
International Journal of Biomedicine. 2022;12(1):109-114.
DOI: 10.21103/Article12(1)_OA10
Originally published March 10, 2022

Abstract: 

Background: To create a test for the early detection of cervical cancer (CC), we conducted exploratory studies on detecting HPV genes and genes of the human β-globin locus in plasma (Pl) and RBC suspension (RBCsus) samples from patients with newly detected CC (NDCC).
Methods and Results: Smears of venous blood containing K3-EDTA from five anonymous patients aged from 45 to 55 years (residents of Yakutia), with NDCC were obtained. Three types of blood component samples were prepared – plasma (Pl), red blood cell (RBC) suspension (RBCsus), and the erythrocyte fraction treated with trypsin (RBCsus-Try). To detect circulating cell-free DNA (cfDNA) in NDCC patients, we studied the presence of genes corresponding to the HPV L1 protein region and genes of the human β-globin locus by real-time PCR (qPCR) using appropriate primers.
The genes of β-globin locus and HPV L1 were detected in Pl of NDCC patients in 20% of cases, and in RBCsus in 60% of cases. The amplified gene products using primers were present in RBCsus-Try in only one patient (20% of cases).
In patients with uncertain amplification, electrophoresis showed the absence of amplified products in Pl and their presence in RBCsus.
Conclusion: In NDCC patients, HPV L1 and β-globin genes can be detected in both Pl and RBCsus. In addition, in RBC samples, these genes were detected more often than in plasma samples, and no absolute absence of amplification products was observed in RBC samples. The research needs to be continued.

Keywords: 
cervical cancer • human papillomavirus • circulating cell-free DNA • quantitative PCR
References: 
  1. World Health Organization. 73rd World Health Assembly Decisions. Available from: https://www.who.int/news-room/feature-stories/detail/73rd-world-health-assembly-decisions
  2. The World Bank. Available from: https://datahelpdesk.worldbank.org/knowledgebase/articles/906519
  3. Kononova IV, Kirillina MP, Sofronova SI, Illarionova NA, Mamaeva SN,
    1. Arzhakova LI, et al. [Disparities of cervical cancer incidence and mortality in the republics located in Siberia and all over Russia in the period from 2007 to 2019]. Yakut Medical Journal. 2021;73(1):50-53. doi: 10.25789/YMJ.2021.73.14 [Article in Russian].
  4. Liu K, Tong H, Li T, Chen Y, Mao X. Potential value of circulating tumor DNA in gynecological tumors. Am J Transl Res. 2020 Jul 15;12(7):3225-3233.
  5. Pornthanakasem W, Shotelersuk K, Termrungruanglert W, Voravud N, Niruthisard S, Mutirangura A. Human papillomavirus DNA in plasma of patients with cervical cancer. BMC Cancer. 2001;1:2. doi: 10.1186/1471-2407-1-2. 
  6. Oshiro C, Kagara N, Naoi Y, Shimoda M, Shimomura A, Maruyama N, Shimazu K, Kim SJ, Noguchi S. PIK3CA mutations in serum DNA are predictive of recurrence in primary breast cancer patients. Breast Cancer Res Treat. 2015 Apr;150(2):299-307. doi: 10.1007/s10549-015-3322-6. 
  7. Newman AM, Bratman SV, To J, Wynne JF, Eclov NC, Modlin LA, Liu CL, Neal JW, Wakelee HA, Merritt RE, Shrager JB, Loo BW Jr, Alizadeh AA, Diehn M. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med. 2014 May;20(5):548-54. doi: 10.1038/nm.3519. 
  8. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, Bartlett BR, Wang H, Luber B, Alani RM, Antonarakis ES, Azad NS, Bardelli A, Brem H, Cameron JL, Lee CC, Fecher LA, Gallia GL, Gibbs P, Le D, Giuntoli RL, Goggins M, Hogarty MD, Holdhoff M, Hong SM, Jiao Y, Juhl HH, Kim JJ, Siravegna G, Laheru DA, Lauricella C, Lim M, Lipson EJ, Marie SK, Netto GJ, Oliner KS, Olivi A, Olsson L, Riggins GJ, Sartore-Bianchi A, Schmidt K, Shih lM, Oba-Shinjo SM, Siena S, Theodorescu D, Tie J, Harkins TT, Veronese S, Wang TL, Weingart JD, Wolfgang CL, Wood LD, Xing D, Hruban RH, Wu J, Allen PJ, Schmidt CM, Choti MA, Velculescu VE, Kinzler KW, Vogelstein B, Papadopoulos N, Diaz LA Jr. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014 Feb 19;6(224):224ra24. doi: 10.1126/scitranslmed.3007094. 
  9. Gu Y, Wan C, Qiu J, Cui Y, Jiang T, Zhuang Z. Circulating HPV cDNA in the blood as a reliable biomarker for cervical cancer: A meta-analysis. PLoS One. 2020 Feb 6;15(2):e0224001. doi: 10.1371/journal.pone.0224001.
  10. Li X, Wu Z, Wang Y, Mei Q, Fu X, Han W. Characterization of adult α- and β-globin elevated by hydrogen peroxide in cervical cancer cells that play a cytoprotective role against oxidative insults. PLoS One. 2013;8(1):e54342. doi: 10.1371/journal.pone.0054342.
  11. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 2011 Jun;11(6):426-37. doi: 10.1038/nrc3066.
  12. Kamat AA, Sood AK, Dang D, Gershenson DM, Simpson JL, Bischoff FZ. Quantification of total plasma cell-free DNA in ovarian cancer using real-time PCR. Ann N Y Acad Sci. 2006 Sep;1075:230-4. doi: 10.1196/annals.1368.031.
  13. NanoPhotometer® P-Class User Manual P 300 / P 330 / P 360. Available from: https://www.implen.de/wp-content/uploads/2015/04/NanoPhotometer-P-Class-...
  14. Evrogen. Available from: https://evrogen.ru/kit-user-manuals/qPCRmix-HS-SYBR-LowROX_PK156.pdf  [in Russian]
  15. Quellhorst G, Rulli S. A systematic guideline for developing the best real-time PCR primers. Lessons learned from designing assays for more than 14,000 genes. Qiagen. 2012;1–9. Available from: file:///C:/Users/Admin/Downloads/1073958_A_systematic_guideline_for_developing_the_best_real-time_PCR_primers.pdf
  16. Mazurek AM, Fiszer-Kierzkowska A, Rutkowski T, Składowski K, Pierzyna M, Scieglińska D, Woźniak G, Głowacki G, Kawczyński R, Małusecka E. Optimization of circulating cell-free DNA recovery for KRAS mutation and HPV detection in plasma. Cancer Biomark. 2013;13(5):385-94. doi: 10.3233/CBM-130371.
  17. Takakura M, Matsumoto T, Nakamura M, Mizumoto Y, Myojyo S, Yamazaki R, Iwadare J, Bono Y, Orisaka S, Obata T, Iizuka T, Kagami K, Nakayama K, Hayakawa H, Sakurai F, Mizuguchi H, Urata Y, Fujiwara T, Kyo S, Sasagawa T, Fujiwara H. Detection of circulating tumor cells in cervical cancer using a conditionally replicative adenovirus targeting telomerase-positive cells. Cancer Sci. 2018 Jan;109(1):231-240. doi: 10.1111/cas.13449. 
  18. Yim EK, Park JS. The role of HPV E6 and E7 oncoproteins in HPV-associated cervical carcinogenesis. Cancer Res Treat. 2005 Dec;37(6):319-24. doi: 10.4143/crt.2005.37.6.319. 
  19. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Human papillomaviruses. IARC Monogr Eval Carcinog Risks Hum. 2007;90:1-636.
  20. Rungkamoltip P, Temisak S, Piboonprai K, Japrung D, Thangsunan P, Chanpanitkitchot S, Chaowawanit W, Chandeying N, Tangjitgamol S, Iempridee T. Rapid and ultrasensitive detection of circulating human papillomavirus E7 cell-free DNA as a cervical cancer biomarker. Exp Biol Med (Maywood). 2021 Mar;246(6):654-666. doi: 10.1177/1535370220978899. 
  21. Jeannot E, Becette V, Campitelli M, Calméjane MA, Lappartient E, Ruff E, Saada S, Holmes A, Bellet D, Sastre-Garau X. Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma. J Pathol Clin Res. 2016 Jun 28;2(4):201-209. doi: 10.1002/cjp2.47. 
  22. Campitelli M, Jeannot E, Peter M, Lappartient E, Saada S, de la Rochefordière A, Fourchotte V, Alran S, Petrow P, Cottu P, Pierga JY, Lantz O, Couturier J, Sastre-Garau X. Human papillomavirus mutational insertion: specific marker of circulating tumor DNA in cervical cancer patients. PLoS One. 2012;7(8):e43393. doi: 10.1371/journal.pone.0043393. 
  23. Jeannot E, Latouche A, Bonneau C, Calméjane MA, Beaufort C, Ruigrok-Ritstier K, Bataillon G, Larbi Chérif L, Dupain C, Lecerf C, Popovic M, de la Rochefordière A, Lecuru F, Fourchotte V, Jordanova ES, von der Leyen H, Tran-Perennou C, Legrier ME, Dureau S, Raizonville L, Bello Roufai D, Le Tourneau C, Bièche I, Rouzier R, Berns EMJJ, Kamal M, Scholl S. Circulating HPV DNA as a Marker for Early Detection of Relapse in Patients with Cervical Cancer. Clin Cancer Res. 2021 Nov 1;27(21):5869-5877. doi: 10.1158/1078-0432.CCR-21-0625. 
  24. Buck CB, Day PM, Trus BL. The papillomavirus major capsid protein L1. Virology. 2013 Oct;445(1-2):169-74. doi: 10.1016/j.virol.2013.05.038. 
  25. Chen AC, Keleher A, Kedda MA, Spurdle AB, McMillan NA, Antonsson A. Human papillomavirus DNA detected in peripheral blood samples from healthy Australian male blood donors. J Med Virol. 2009 Oct;81(10):1792-6. doi: 10.1002/jmv.21592.
  26. Cao H, Banh A, Kwok S, Shi X, Wu S, Krakow T, Khong B, Bavan B, Bala R, Pinsky BA, Colevas D, Pourmand N, Koong AC, Kong CS, Le QT. Quantitation of human papillomavirus DNA in plasma of oropharyngeal carcinoma patients. Int J Radiat Oncol Biol Phys. 2012 Mar 1;82(3):e351-8. doi: 10.1016/j.ijrobp.2011.05.061.
  27. Cocuzza CE, Martinelli M, Sina F, Piana A, Sotgiu G, Dell'Anna T, Musumeci R. Human papillomavirus DNA detection in plasma and cervical samples of women with a recent history of low grade or precancerous cervical dysplasia. PLoS One. 2017 Nov 28;12(11):e0188592. doi: 10.1371/journal.pone.0188592. 
  28. Graham SV. The human papillomavirus replication cycle, and its links to cancer progression: a comprehensive review. Clin Sci (Lond). 2017 Aug 10;131(17):2201-2221. doi: 10.1042/CS20160786. 
  29. Middleton K, Peh W, Southern S, Griffin H, Sotlar K, Nakahara T, El-Sherif A, Morris L, Seth R, Hibma M, Jenkins D, Lambert P, Coleman N, Doorbar J. Organization of human papillomavirus productive cycle during neoplastic progression provides a basis for selection of diagnostic markers. J Virol. 2003 Oct;77(19):10186-201. doi: 10.1128/jvi.77.19.10186-10201.2003. 
  30. Roden RB, Hubbert NL, Kirnbauer R, Breitburd F, Lowy DR, Schiller JT. Papillomavirus L1 capsids agglutinate mouse erythrocytes through a proteinaceous receptor. J Virol. 1995 Aug;69(8):5147-51. doi: 10.1128/JVI.69.8.5147-5151.1995. 
  31. Vogt AM, Winter G, Wahlgren M, Spillmann D. Heparan sulphate identified on human erythrocytes: a Plasmodium falciparum receptor. Biochem J. 2004 Aug 1;381(Pt 3):593-7. doi: 10.1042/BJ20040762.
  32. Mamaeva SN, Kononova IV, Alekseev VA, Nikolaevа NA, Pavlov AN, Semenova MN, et al. Determination of Blood Parameters using Scanning Electron Microscopy as a Prototype Model for Evaluating the Effectiveness of Radiation Therapy for Cervical Cancer.  International Journal of Biomedicine.2021;11(1):32-38. doi:10.21103/Article11(1)_OA6
  33. Doyle LM, Wang MZ. Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis. Cells. 2019 Jul 15;8(7):727. doi: 10.3390/cells8070727. 
  34. Tamkovich S, Laktionov P. Cell-surface-bound circulating DNA in the blood: Biology and clinical application. IUBMB Life. 2019 Sep;71(9):1201-1210. doi: 10.1002/iub.2070.
  35. Merker JD, Oxnard GR, Compton C, Diehn M, Hurley P, Lazar AJ, Lindeman N, Lockwood CM, Rai AJ, Schilsky RL, Tsimberidou AM, Vasalos P, Billman BL, Oliver TK, Bruinooge SS, Hayes DF, Turner NC. Circulating Tumor DNA Analysis in Patients With Cancer: American Society of Clinical Oncology and College of American Pathologists Joint Review. J Clin Oncol. 2018 Jun 1;36(16):1631-1641. doi: 10.1200/JCO.2017.76.8671. 
  36. Yilmaz FT, Demirel G. The relationship between body privacy and anxiety in women having gynecological examination. J Obstet Gynaecol. 2021 Oct;41(7):1112-1115. doi: 10.1080/01443615.2020.1835845. 
  37. Deacon B, Abramowitz J. Fear of needles and vasovagal reactions among phlebotomy patients. J Anxiety Disord. 2006;20(7):946-60. doi: 10.1016/j.janxdis.2006.01.004. 

Download Article
Received November 22, 2021.
Accepted February 25, 2022.
©2022 International Medical Research and Development Corporation.