The p16^INK4A mRNA transcripts estimation in cervical smear with different degrees of cervical dysplasia

Czech version

Authors: V. Janušicová ^1,2; I. Kapustová ¹; P. Žúbor ¹; P. Slezák ³; K. Kajo ⁴; Z. Lasabová ²; L. Plank ⁴; J. Danko ¹
Authors‘ workplace: Gynekologicko-pôrodnícka klinika JLF UK a UNM, Martin, prednosta prof. MUDr. J. Danko, CSc. ¹; Ústav molekulovej biológie JLF UK a UNM, Martin, prednosta RNDr. Z. Lasabová, PhD. ²; Ústav normálnej a patologickej fyziológie, SAV, Bratislava, prednosta RNDr. O. Pecháňová, DrSc. ³; Ústav patologickej anatómie JLF UK a UNM, Martin, prednosta prof. MUDr. L. Plank, CSc. ⁴
Published in: Ceska Gynekol 2012; 77(3): 245-250

Overview

Objective:
To determine the presence of HPV infection and expression level of p16^INK4A mRNA transcripts in cervical smears as adjunct biomarker in detection of cervical intraepithelial neoplasia or cancer.

Design:
Prospective pilot clinical study assessing clinical utility and validity of ddCt method for qPCR mRNA expression of p16ink4a in comparison to immunohistochemistry.

Setting:
Department of Molecular Biology, Department of Obstetrics and Gynecology, Jessenius Medical Faculty, Commenius University, Martin, Slovak Republic.

Methods:
Cervical smears (OC) from patients with different cervical lesions (L-SIL, H-SIL, SCA; n=45) and from healthy controls (n=45) were tested for the presence of HPV infection and p16^INK4A mRNA transcripts using relative quantification (RQ). Results were compared to H&E and IHC histological findings from biopsies (conization, hysterectomy).

Results:
HPV 16 was the most frequent finding (53.3%) in the group of subjects with cervical dysplasia. The p16^INK4A mRNA expression analysis revealed the slightly reduced expression in L-SIL group, 4-fold increased expression in H-SIL and 10-fold increase in women with SCA when compared to controls. The p16^INK4A mRNA expression in OC was present in 30% of L-SIL, 75% of H-SIL and 85.7% of SCA samples, respectively. The test overall sensitivity was 81.48% (95% CI: 61.92–93.7) and specificity 60% (95% CI: 26.24- 87.84) with PPV of 84.62% and NPV of 54.55%. The likelihood ratio (LR) in case of test positivity was 2.04 and for negativity 0.31. The diagnostic accuracy of p16^INK4A expression by RQ method in OC smears for prediction of p16 positivity in cervical dysplasia was 66.7% for the L-SIL lesions, 59.5% for H-SIL lesions, and 100% for SCA (r=0.9897, p<0.0913) when compared to IHC p16 positive findings in surgically treated samples.

Conclusion:
The relative quantification is able to determine the level of p16^INK4A mRNA transcripts in cervical smear cells with active carcinogenesis nearly at the same level as IHC staining. The advance of biopsy sparing over IHC is qualifying this diagnostic approach for useful candidate in selective management of women with cervical dysplasia looking for cervix preservation or avoiding the unnecessary overtreatment.

Key words:
p16^INK4A, HR-HPV, H-SIL, L-SIL, relative quantification, immunohistochemistry.

Sources

1. Clifford, G., Franceschi, S., Diaz, M., et al. Chapter 3: HPV type-distribution in women with and without cervical neoplastic diseases. Vaccine, 2006, 24, Suppl 3, p. S26–S34.

2. Cuschieri, K., Wentzensen, N. Human papillomavirus mRNA and p16 detection as biomarkers for the improved diagnosis of cervical neoplasia. CEBP, 2008, 17, p. 2536–2545.

3. Cuzick, J., Mayrand, MH., Ronco, G., et al. Chapter 10: New dimensions in cervical cancer screening. Vaccine, 2006, 24, p. S90–S97.

4. Doorbar, J. The papillomavirus life cycle. J Clin Virol, 2005, 32, p. S7–S15.

5. Dussan, C., Zubor, P., Fernandez, M., et al. Spontaneous regression of a breast carcinoma: a case report. Gynecol Obstet Invest, 2008, 65, 3, p. 206–211.

6. Franekova, M., Zubor, P., Stanclova, A., et al. Association of p53 polymorphisms with breast cancer: a case-control study in Slovak population. Neoplasma, 2007, 54, 2, p. 155–161.

7. Gravitt, PE., Peyton, CL., Alessi, TQ., et al. Improved amplification of genital human papillomaviruses. J Clin Microbiol, 2000, 38, p. 357–361.

8. Kang, S., Kim, J., Kim, HB., et al. Methylation of p16ink4a is a non-rare event in cervical intraepithelial neoplasia. Diagn Mol Pathol, 2006, 15, 2, p. 74–82.

9. Khan, MJ., Castle, PE., Lorincz, AT., et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst, 2005, 97, 14, p. 1072–1079.

10. Liaw, KL., Hildesheim, A., Burk, RD., et al. A prospective study of human papillomavirus (HPV) type 16 DNA detection by polymerase chain reaction and its association with acquisition and persistence of other HPV types. J Inf Dis, 2001, 183, p. 8–15.

11. Murphy, N., Ring, M., Killalea, AG., et al. p16INK4A as a marker for cervical dyskaryosis: CIN and cGIN in cervical biopsies and ThinPrep smears. J Clin Pathol, 2003, 56, p. 56–63.

12. Negri, G., Bellisano, G., Zannoni, GF., et al. p16 ink4a and HPV L1 immunohistochemistry is helpful for estimating the behavior of low-grade dysplastic lesions of the cervix uteri. Am J Surg Pathol, 2008, 32, 11, p. 1715–1720.

13. Ondrusova, M., Zubor, P., Ondrus, D. Time trends in cervical cancer epidemiology in the Slovak Republic: reflection on the non-implementation of screening with international comparisons. Neoplasma. 2012, 59, 2, p. 121–128.

14. Įvestad, IT., Gudlaugsson, E., Skaland, I., et al. Local immune response in the microenvironment of CIN2-3 with and without spontaneous regression. Mod Pathol, 2010, 23, 9, p. 1231–1240.

15. Ozgul, N., Cil, AP., Bozdayi, G., et al. Staining characteristics of p16INK4a: Is there a correlation with lesion grade or high-risk human papilloma virus positivity? J Obst Gyn Res, 2008, 34, 5, p. 865–871.

16. Paavonen, J. Human papillomavirus infection and the development of cervical cancer and related genital neoplasias. Int J Inf Dis, 2007, 11, Suppl 2, p. S3–S9.

17. Pfaffl, MW. A new mathematical model for relative quantification real time PCR. Nucleic Acids Res, 2001, 29, 9, p. 2002–2007.

18. Scheurer, ME., Tortolero-Luna, G., Adler-Storthz, K. Human papillomavirus infection: biology, epidemiology, and prevention. Int J Gyn Cancer, 2005, 15, 5, p. 727–746.

19. Steinau, M., Rajeevan, MS., Unger, ER. DNA and RNA references for qRT-PCR assays in exfoliated cervical cells. J Mol Diagn. 2006, 8, 1, p. 113–118.

20. Weaver, BA. Epidemiology nad natural history of genital human papillomavirus infection. JAOA, 2006, 106, 3, Sup. 1, p. S2–S7.

21. World Health Organization: Comprehensive cervical cancer control: a guide to essential practice [on-line]. Geneva, Switzerland, WHO. [cit. 2006-06-24] Dostupný z WWW: <http://www.who.int/reproductive-health/publications/cervical_cancer_gep/index.htm>.

22. Yildiz, IZ., Usubutun, A., Firat, P., et al. Efficiency of immunohistochemical p16 expression and HPV typing in cervical squamous intraepithelial lesion grading and review of the p16 literature. Pathol Res Pract, 2007, 203, p. 445–449.

23. Zubor, P., Danko, J., Kajo, K., Szunyogh, N. Low affordability may limit the effect of cervical cancer vaccination in central and eastern European countries. J Clin Oncol, 2007, 25, 34, p. 5534–5537.