Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.
References
[1]
Syrjanen KJ (2009) Histology, classification and natural history of cervical intraepithelial neoplasia (CIN). CME J Gynecol Oncol 14: 4–21.
[2]
Crum C. CIN II exists. United States and Canadian Academy of Pathology (USCAP) 2011 Annual Meeting. Specialty Conference Handout http://www.uscap.org/index.htm?educmaste？r.htm: Gynecologic Pathology.
[3]
Crum CP (2008) Laboratory management of CIN 2: The consensus is consensus. Am J Clin Pathol 130: 162–4. doi: 10.1309/gcvb5c4kfv7tfc7f
[4]
Stoler MH. CIN2 does exist. United States and Canadian Academy of Pathology (USCAP) 2011 Annual Meeting. Specialty Conference Handout http://www.uscap.org/index.htm?educmaste？r.htm: Gynecologic Pathology.
[5]
Waxman AG, Chelmow D, Darragh TM, Lawson H, Moscicki AB (2012) Revised terminology for cervical histopathology and its implications for management of high-grade squamous intraepithelial lesions of the cervix. Obstet Gynecol 120: 1465–71.
[6]
Darragh TM, Colgan TJ, Thomas Cox J, Heller DS, Henry MR, et al. (2013) For Members of the LAST Project Work Groups. The Lower Anogenital Squamous Terminology Standardization Project for HPV-associated Lesions: Background and Consensus Recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Int J Gynecol Pathol 32: 76–115. doi: 10.1097/pgp.0b013e31826916c7
[7]
Sadler L, Saftlas A, Wang W, Exeter M, Whittaker J, et al. (2004) Treatment for cervical intraepithelial neoplasia and risk of preterm delivery. JAMA 291: 2100–6. doi: 10.1097/01.ogx.0000141202.50796.00
[8]
Noehr B, Jensen A, Frederiksen K, Tabor A, Kjaer SK (2009) Depth of cervical cone removed by loop electrosurgical excision procedure and subsequent risk of spontaneous preterm delivery. Obstet Gynecol 114: 1232–8. doi: 10.1097/aog.0b013e3181bf1ef2
[9]
Zeng SY, Liang MR, Li LY, Wu YY (2012) Comparison of the efficacy and complications of different surgical methods for cervical intraepithelial neoplasia. Eur J Gynaecol Oncol 33: 257–60.
[10]
Wang F, Flanagan J, Su N, Wang LC, Bui S, et al. (2012) RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14: 22–9.
[11]
Bosch FX, Lorincz A, Mu？oz N, Meijer CJ, Shah KV (2002) The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 55: 244–65. doi: 10.1136/jcp.55.4.244
[12]
Doorbar J (2006) Molecular biology of human papillomavirus infection and cervical cancer Clin Sci (Lond). 110: 525–41. doi: 10.1042/cs20050369
[13]
Doorbar J (2007) Papillomavirus life cycle organization and biomarker selection. Dis Markers 23: 297–313. doi: 10.1155/2007/613150
[14]
Doorbar J, Quint W, Banks L, Bravo IG, Stoler M, et al. (2012) The biology and life-cycle of human papillomaviruses. Vaccine 30 Suppl 5F55–70. doi: 10.1016/j.vaccine.2012.06.083
[15]
Doeberitz Mv, Vinokurova S (2009) Host factors in HPV-related carcinogenesis: cellular mechanisms controlling HPV infections. Arch Med Res. 40: 435–42. doi: 10.1016/j.arcmed.2009.06.002
[16]
von Knebel Doeberitz M, Reuschenbach M, Schmidt D, Bergeron C (2012) Biomarkers for cervical cancer screening: the role of p16INK4a to highlight transforming HPV infections. Expert Rev Proteomics. 9: 149–63. doi: 10.1586/epr.12.13
[17]
Chaiwongkot A, Vinokurova S, Pientong C, Ekalaksananan T, Kongyingyoes B, et al. (2013) Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions. Int J Cancer 132: 2087–94. doi: 10.1002/ijc.27906
[18]
Solomon D, Schiffman M, Tarone R (2001) ALTS Study group (2001) Comparison of three management strategies for patients with atypical squamous cells of undetermined significance: baseline results from a randomized trial. J Natl Cancer Inst 93: 293–9. doi: 10.1093/jnci/93.4.293
[19]
Evans MF, Mount SL, Vacek PM, Cooper K (2003) High-risk human papillomavirus type does not predict grade of cervical intraepithelial neoplasia. Eur J Gynaecol Oncol 24: 373–6.
[20]
Evans MF, Adamson CS, Simmons-Arnold L, Cooper K (2005) Touchdown General Primer (GP5+/GP6+) PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus. BMC Clin Pathol 16 5: 10.
[21]
de Roda Husman AM, Snijders PJ, Stel HV, van den Brule AJ, Meijer CJ, et al. (1995) Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. Br J Cancer 72: 412–7.
[22]
Evans MF, Mount SL, Beatty BG, Cooper K (2002) Biotinyl-tyramide-based in situ hybridization signal patterns distinguish human papillomavirus type and grade of cervical intraepithelial neoplasia. Mod Pathol 15: 1339–47. doi: 10.1038/modpathol.3880698
[23]
Cooper K, Herrington CS, Graham AK, Evans MF, McGee JO (1991) In situ human papillomavirus (HPV) genotyping of cervical intraepithelial neoplasia in South African and British patients: evidence for putative HPV integration in vivo. J Clin Pathol 44: 400–5. doi: 10.1136/jcp.44.5.400
[24]
Stoler MH, Broker TR (1986) In situ hybridization detection of human papillomavirus DNAs and messenger RNAs in genital condylomas and a cervical carcinoma. Hum Pathol 17: 1250–8.
[25]
Higgins GD, Uzelin DM, Phillips GE, Pieterse AS, Burrell CJ (1991) Differing characteristics of human papillomavirus RNA-positive and RNA-negative anal carcinomas. Cancer 68: 561–7. doi: 10.1002/1097-0142(19910801)68:3<561::aid-cncr2820680320>3.0.co;2-r
[26]
Higgins GD, Uzelin DM, Phillips GE, Burrell CJ (1991) Presence and distribution of human papillomavirus sense and antisense RNA transcripts in genital cancers. J Gen Virol 72: 885–95. doi: 10.1099/0022-1317-72-4-885
[27]
Stoler MH, Rhodes CR, Whitbeck A, Wolinsky SM, Chow LT, et al. (1992) Human papillomavirus type 16 and 18 gene expression in cervical neoplasias. Hum Pathol 23: 117–28.
[28]
Dürst M, Glitz D, Schneider A, zur Hausen H (1992) Human papillomavirus type 16 (HPV 16) gene expression and DNA replication in cervical neoplasia: analysis by in situ hybridization. Virology 189: 132–40. doi: 10.1016/0042-6822(92)90688-l
[29]
Higgins GD, Uzelin DM, Phillips GE, McEvoy P, Marin R, et al. (1992) Transcription patterns of human papillomavirus type 16 in genital intraepithelial neoplasia: evidence for promoter usage within the E7 open reading frame during epithelial differentiation. J Gen Virol. 1992 Aug;73 (Pt 8): 2047–57. doi: 10.1099/0022-1317-73-8-2047
[30]
Higgins GD, Phillips GE, Smith LA, Uzelin DM, Burrell CJ (1992) High prevalence of human papillomavirus transcripts in all grades of cervical intraepithelial glandular neoplasia. Cancer 70: 136–46. doi: 10.1002/1097-0142(19920701)70:1<136::aid-cncr2820700123>3.0.co;2-2
[31]
Vormwald-Dogan V, Fischer B, Bludau H, Freese UK, Gissmann L, et al. (1992) Sense and antisense transcripts of human papillomavirus type 16 in cervical cancers. J Gen Virol 73: 1833–8. doi: 10.1099/0022-1317-73-7-1833
[32]
Galgano MT, Castle PE, Atkins KA, Brix WK, Nassau SR, et al. (2010) Using biomarkers as objective standards in the diagnosis of cervical biopsies. Am J Surg Pathol 34: 1077–87. doi: 10.1097/pas.0b013e3181e8b2c4
[33]
Moscicki AB, Ma Y, Wibbelsman C, Darragh TM, Powers A, et al. (2010) Rate of and risks for regression of cervical intraepithelial neoplasia 2 in adolescents and young women. Obstet Gynecol 116: 1373–80. doi: 10.1097/aog.0b013e3181fe777f
[34]
McAllum B, Sykes PH, Sadler L, Macnab H, Simcock BJ, et al. (2011) Is the treatment of CIN 2 always necessary in women under 25 years old? Am J Obstet Gynecol 205: 478.e1–7. doi: 10.1016/j.ajog.2011.06.069
[35]
Syrj？nen K, Shabalova I, Sarian L, Naud P, Longatto-Filho A, et al. (2012) Covariates of high-risk human papillomavirus (HPV) infections are distinct for incident CIN1, CIN2 and CIN3 as disclosed by competing-risks regression models. Eur J Gynaecol Oncol 33: 5–14.
[36]
Fukuchi E, Fetterman B, Poitras N, Kinney W, Lorey T, et al. (2013) Risk of cervical precancer and cancer in women with cervical intraepithelial neoplasia grade 1 on endocervical curettage. J Low Genit Tract Dis 17: 255–60. doi: 10.1097/lgt.0b013e31826ca4d9
[37]
De Marchi Triglia R, Metze K, Zeferino LC, Lucci De Angelo Andrade LA (2009) HPV in situ hybridization signal patterns as a marker for cervical intraepithelial neoplasia progression. Gynecol Oncol 112: 114–8. doi: 10.1016/j.ygyno.2008.09.047
