Over expression of p16ink4a associated with high grade lesions of (CIN) and cervical cancer (study applied on women infected with HPV in Basrah, Iraq)


CIN lesions and cervix cancer is a consequence for the infection with HPV, molecular interaction between the virus and cellular proteins leads to an increase in expression of p16INK4a and inhibitor for CDK, so p16INK4a expression is a biomarker for HR-HPV. CIN grading and SCC according to p16INK4a expression pattern and the associated infection with HR-HPV was analysed in. Biopsy samples were collected from 52 women’s cervix with an age ranged from (15) to (>75) years exhibited abnormal histopathological changes for period between 1/2/2018 to 1/2/2019 from Basrah Hospital for women and children in Basrah, Iraq. The HPV prevalence detection was carried out by PCR using the general Primer My09/My11. cDNA s from the same samples were used as templates for gene expression analysis of E6, E7 mRNA in HR-HPV types using qPCR. The same samples used also to evaluate the P16INK4a expression by IHC. The PCR results showed 45 (86.54%) samples were infected with HPV with highest detection about (36%) in age group (35-45) years and 7 (13.46%) samples were negative to HPV. Gene expression of oncogenes E6 & E7 showed high percintage for HPV-16 (36%). Immunohistochemistry staining showed an increase in p16INK4a expression and intensity staining in the high grade of CIN and carcinoma and (P< 0.001). in our study, p16INK4a shows overexpression for HR-HPV infected cases with high grade of CIN and carcinoma.




  • Agoff SN, Lin P, Morihara J, Mao C, Kiviat NB, Koutsky LA (2003) P16INK4a expression correlates with degree of cervical neoplasia: A comparison with Ki-67 expression and detection of high-risk HPV types. Mod Pathol, 16: 665-673.
  • Ambühl LMM, Villadsen AB, Baandrup U, Dybkær K, Sørensen S (2017) HPV16 E6 and E7 Upregulate Interferon-Induced Antiviral Response Genes ISG15 and IFIT1 in Human Trophoblast Cells. Pathogenes. https://doi.org/10.3390/pathogens6030040.V6(3)
  • Barine KKD, Victor N (2016) The Particle Size and Thermal Properties of Flour from three Plantain (Musa Paradisiaca) Cultivars Grown in Nigeria. Journal of Food Technology Research, 3(1): 23-27.
  • Bonner JA, Mesia R, Giralt J, Psyrri A, Keilholz U, Rosenthal TI, et al. (2017) p16, HPV, and Cetuximab: What Is the Evidence? Oncologist, 22(7): 811-822. https://doi.org/10.1634/theoncologist.2016-0433
  • Brown CA, Bogers J, Sahebali S, Depuydet CE, De Prins F, Malinowski DP (2012) Role of Protein Biomarkers in the Detection of High-Grade Disease in Cervical Cancer Screening Programs. J Oncol: 289315. https://doi.org/10.1155/2012/289315
  • Butterworth CE, Hatch KD, Macaluso M. Folate (1992) Deficiency and cervical dysplasia Am Med Assoc, 267: 528.
  • Cioffi-Lavina M, Chapman-Fredricks J, Gomez-Fernandez C, Ganjei-Azar P, Manoharan M, Jorda M (2010) P16 expression in squamous cell carcinomas of cervix and bladder. Applied Immunohistochemistry & Molecular Morphology. 18(4): 344-347.
  • Darragh TM (2015) The LAST Project and the diagnostic bottom line. Cytopathology. 26(6): 343-345. https://doi.org/10.1111/cyt.12299
  • Gao G, Chernock RD, Gay HA, Thorstad WL, Zhang TR, Wang H, Ma X, Luo Y, Lewis JS Jr, Wang X (2013) A novel RT-PCR method for quantification of human papilloma virus transcripts in archived tissues and its application in oropharyngeal cancer prognosis. Int J Cancer, 132(4): 882-890. https://doi.org/10.1002/ijc.27739
  • Goncalves JES, de Andrade CV, Russomano FB, Nuovo GJ, Filho SMA, Carvalho MOO, Nico AF (2017) The role of p16 as putative biomarker for cervical neoplasia: A controversial issue? MedicalExpress (Sao Paulo, online), 4(6): M170601.
  • Hwang S, Shroyer K (2012) Biomarker of cervical dysplasia and carcinoma. J Oncol: 1-9
  • Izadi-Mood N, Asadi K, Shojaei H, Sarmadi S, Ahmadi SA, Sani S, et al. (2012). Potential diagnostic value of P16 expression in premalignant and malignant cervical lesions. J Res Med Sci, 17: 428-433.
  • Karcheva M, Popovska S, Nachev R (2007) Immunohistochemical investigations of P16 ink 4A expression in carcinomas and high grade cervical lesions. Journal of IMAB - Annual Proceeding (Scientific Papers), 13(1).
  • Keating JT, Cviko A, Riethdorf S, Riethdorf L, Quade BJ, Sun D, et al, (2001). Ki-67, Cyclin E, and P16INK4a are complementary surrogate biomarkers for human papillomavirus-¬ related cervical neoplasia. American journal of surgical pathology. 25: 884-891.
  • Kjaer SK, van den Brule AJ, Paull G, Svare EI, Sherman ME, Thomsen BL, et al. (2002) Type specific persistence of high risk human papillomavirus (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: population based prospective follow up study. BMJ, 325(7364): 572.
  • Klaes R, Friedrich T, Spitkovsky D, Ridder R, Rudy W, Petry U, Dallenbach-Hellweg G, Schmidt D, Von Knebel Doeberitz M, (2001). Overexpression of P16INK4a as a specific marker for dysplastic and neoplastic epithelial cells and of the cervix uteri. Int J. cancer, 92:276-284.
  • Kumar VS, Sagunthala P, Ravi S, Premalatha S (2016) Analysis of immunohistochemical expression of P16INK4a pre neoplastic squamous cell lesions of cervix. IOSR-JDMS; 15(3): 46-51.
  • Leniskova I, Lidang M, Hamilton-Dutoit S, Koch J (2009) P16 as a diagnostic marker of cervical neoplasia: ASSS tissue microarray study of 769 archival specimens. Diagnostic pathology. 4: 22. https://doi.org/10.1186/1746-1596-4-22
  • Liggett WH, Sidransky D (1998) Role of P16 tumor suppressor gene in cancer. Journal of clinical oncology. 16(3): 1197-1206. https://doi.org/10.1200/JCO.16.3.1197
  • Ma T, Su Z, Chen L, Liu S, Zhu N, Wen L, Yuan Y, Lv L, Chen X, Huang J, Chen H (2012). Human Papillomavirus Type 18 E6 and E7 Genes Integrate into Human Hepatoma Derived Cell Line Hep G2. PLoS One, 7(5): e37964. https://doi.org/10.1371/journal.pone.0037964
  • Mahajan A (2016) Practical issues in the application of p16 immunohistochemistry in diagnostic pathology. Hum Pathol, 51: 64-74.
  • Manos MM, Ting Y, Wright DK, Lewis AJ, Broker TR, Wolinsky SM (1989) Use of polymerase chain reaction amplification for the detection of genital human papilloma virus. Cancer cells: 209-213.
  • Murphy N, Ring M, Killalea AG, et al. (2003) P16INK4a as a marker for cervical dyskaryosis: CIN and cGIN in cervical bio psies and thin PrepTM smears. Journal of clinical pathology. 56(1): 56-63.
  • Na K, Sung JY, Kim HS (2017) Stromal p16 Overexpression in Adult Granulosa Cell Tumors of the Ovary. Anticancer Res, 37(5): 2437-2444. https://doi.org/10.21873/anticanres.11583
  • Nam EJ, Kim JW, Hong JW, et al, (2008). Expression of the P16INK4a and K-67 in relation to the grade of cervical intraepithelial neoplasia and high-risk human papillomavirus infection. Journal of Gynecologic oncology. 19(3):162-168. https://doi.org/10.3802/jgo.19.3.162
  • Nicol AF, Andrade CV, Russomano FB, Rodrigues LL, Oliveira NS, Provance DW Jr (2016) HPV vaccines: a controversial issue? Braz J Med Biol Res, 49(5): e5060. https://doi.org/10.1590/1414-431X20155060
  • O’Neill C, McCluggage WG (2006) P16 Expression in the Female Genital Tract and Its Value in Diagnosis. Advances in Anatomic Pathology: 13(1): 8-15.
  • Paes EF, de Assis AM, Teixeira CSC, Aoki FH, Teixeira JC (2015) Development of a Multiplex PCR Test with Automated Genotyping Targeting E7 for Detection of Six High-Risk Human Papillomaviruses, PLoS One, 10(6): e0130226.
  • Patil S, Rao RS, Amrutha N, Sanketh DS (2014) Analysis of human papillomavirus in oral squamous cell carcinoma using P16INK4a: An immunohistochemical study. Journal of international society of preventive & community dentistry, 4(1): 61-66. https://doi.org/10.4103/2231-0762.131269
  • Sari Aslani F, Safaei A, Pourjabali M, Momtahan M, (2013). Evaluation of Ki67, p16 and CK 17 markers in differentiating cervical intraepithelial neoplasia and benign lesions. Iran J Med Sci; 38: 15-21.
  • Serrano M, Hannon GJ, Beach D (1993) A new regulatory motif in cell- cycle control causing specific inhibition of cyclin D/CDK4. Nature 366(6456): 704-707. https://doi.org/10.1038/366704a0 PMID:8259215
  • Underbrink MP, Dupuis C, Tyring SK (2016) E6 proteins from Low-risk human papillomavirus types 6 and 11 are able to protect keratinocytes from apoptosis via Bak degradation. J Gen Virol, 97(Pt3): 715-724.
  • Vestergaard AL, Knudsen UB, Munk T, Rosbach H, Martensen PM (2011) Transcriptional expression of type I interferon response Genes and stability of housekeeping genes in the human endometrium and endometriosis. Mol Hum Reprod, 17: 243-254.
  • Volgareva G, Zavalishinina L, Andreeva Y, Frank G, Krutikova E, Golovina D, et al. (2004) Protein as a marker of dysplastic and neoplastic alterations in cervical epithelial cells.BMC Cancer, 4: 58.
  • Von Knebel Doeberitz M, Syrjanen KJ (2006) Molecular markers: how to apply in practice. Gynecol Oncol, 103:18-20.
  • WHO (2019) Cervical Cancer. https://www.who.int/cancer/prevention/diagnosis-screening/cervical-cancer/en/
  • Zur Hausen H (2002) Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2: 342-50.


This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.