Carcinogenic HPV infection in the cervical squamo-columnar junction

Abstract

Recent studies have suggested the involvement of a unique population of cells at the cervical squamo-columnar junction (SCJ) in the pathogenesis of early (squamous intraepithelial lesion or SIL) and advanced (squamous cell and adeno-carcinomas) cervical neoplasia. However, there is little evidence to date showing that SCJ cells harbour carcinogenic HPV or are instrumental in the initial phases of neoplasia. This study was designed to (1) determine if normal-appearing SCJ cells contained evidence of carcinogenic HPV infection and (2) trace their transition to early SIL. Sections of cervix from high-risk reproductive age women were selected and SCJ cells were analysed by using several techniques which increasingly implicated HPV infection: HPV DNA (genotyping and in situ hybridization)/RNA (PCR), immunostaining for HPV16 E2 (an early marker of HPV infection), p16(ink4), Ki67, and HPV L1 protein. In 22 cases with a history of SIL and no evidence of preneoplastic lesion in the excision specimen, HPV DNA was isolated from eight of ten with visible SCJ cells, six of which were HPV16/18 DNA-positive. In five of these latter cases, the SCJ cells were positive for p16(ink4) and/or HPV E2. Transcriptionally active HPV infection (E6/E7 mRNAs) was also detected in microdissected SCJ cells. Early squamous atypia associated with the SCJ cells demonstrated in addition diffuse p16(ink4) immunoreactivity, elevated proliferative index, and rare L1 antigen positivity. We present for the first time direct evidence that normal-appearing SCJ cells can be infected by carcinogenic HPV. They initially express HPV E2 and their progression to SIL is heralded by an expanding metaplastic progeny with increased proliferation and p16(ink4) expression. Whether certain SCJs are more vulnerable than others to carcinogenic HPV genotypes and what variables determine transition to high-grade SIL remain unresolved, but the common event appears to be a vulnerable cell at the SCJ.

Keywords: HPV; cervical intraepithelial neoplasia; squamo-columnar junction.

Figure 1

HPV16 E2 immunoreactivity and viral oncogene expression (mRNA) are detected in normal-appearing SCJ cells from high-risk women. (A) SCJ cells stained for Krt7, HPV16 E2, p16^ink4 and Ki67. Note the absence of p16^ink4 immunoreactivity and the positive HPV16 E2 staining (an early marker of HPV infection). HPV DNA was not detected by in situ hybridization (ISH). (B) Target cell populations (squamous, columnar and junctional) were detected and microdissected. (C) Total RNA from each cell population was extracted, amplified and HPV16 E6*I, E6*II, E7 and L1 expression was analyzed by PCR. HPV E6 and E7 mRNAs were detected in SCJ cells suggesting a transcriptionally active infection in these cells. No HPV oncogene expression or immunohistochemical evidence of HPV infection was observed in squamous (ectocervix/TZ) or columnar (endocervix) cells immediately adjacent to the SCJ cells. Original magnifications: X40 (microdissection), X100 (immunostaining) or X200 (bracketed, H&E, ISH).

Figure 2

(A) p16^ink4/HPV16 E2 immunoreactivity in normal-appearing SCJ cells from high-risk women. Some rare Ki67-positive cells were also observed. HPV DNA was not detected by in situ hybridization (ISH). (B) Target cell populations (squamous, columnar and junctional) were detected and microdissected. (C) Total RNA from each cell population was extracted, amplified and HPV16 E6*I, E6*II, E7 and L1 expression was analyzed. HPV E6 and E7 mRNAs were detected in SCJ cells suggesting a transcriptionally active infection in these cells. Internal controls included adjacent squamous and columnar epithelia. Original magnifications: X40 (immunostaining and microdissection) or X200 (bracketed, H&E, ISH).

Figure 3

Characterization of the early steps of cervical neoplasia initiated within the SCJ. Early cervical neoplasia stained for Krt7, HPV16 E2, p16^ink4 and Ki67. The patches of p16^ink4/HPV E2 positive cells were significantly associated with increased proliferation. HPV DNA was detected by in situ hybridization (ISH) in both “early” SIL (A) and immature metaplastic lesions (C). After microdissection, mRNA extraction and amplification, HPV16 E6*I, E6*II, E7 and L1 expression was analyzed (B and D). Note the L1 mRNA expression in immature metaplastic SIL. Adjacent squamous and columnar epithelia do not express viral oncogenes and do not display immunohistochemical evidence of HPV infection. Original magnifications: X100 [immunostaining (A–C), H&E (C), ISH (C)] or X200 [bracketed, H&E (A) and ISH (A)].

Figure 4

(A) Some remaining SCJ cells in direct continuity with atypical epithelium are infected by HPV. Note the absence of both p16^ink4 and HPV L1 immunoreactivity in SCJ cells and the positive HPV16 E2 staining. (B) Schematic illustration of the HPV infection sequence in the cervical SCJ. SCJ cells are infected, initially display expression of HPV E2 and terminate with proliferation that signifies the earliest morphological evidence of SIL. Original magnifications: X40 (H&E and Krt7) or X100 (p16, HPV E2 and L1).