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 analyzed 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), p16ink4, 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 8 of 10 with visible SCJ cells, 6 of which were HPV16/18 DNA positive. In 5 of these latter cases, the SCJ cells were positive for p16ink4 and/or HPV E2. Transcriptionally active HPV infection (E6/E7 mRNAs) was also detected in micro-dissected SCJ cells. Early squamous atypia associated with the SCJ cells demonstrated in addition diffuse p16ink4 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 p16ink4 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, Squamocolumnar junction, cervical intraepithelial neoplasia
Introduction
Human papillomavirus (HPV) infection causes cervical cancer and its precursor lesions (squamous intraepithelial lesion or SIL), specifically at the squamo-columnar junction (SCJ) [1,2]. Recent studies of the gastro-oesophageal and ecto-endocervical junctions have unearthed residual embryonic cell populations with both a capacity to differentiate and a vulnerability to neoplastic transformation [3,4]. The cervical SCJ cells share an identical immuno-phenotype [including strong staining for Keratin 7 (Krt7)] with over 90% of high-grade SILs (HSIL) and cervical carcinomas, supporting their role in the initiation of this carcinogenic sequence [3,5]. The predilection of carcinogenic HPV infection for these cells provides one explanation why the number of new cervical cancers yearly worldwide is nearly 20-fold that of carcinomas in well developed lower genital tract squamous epithelium [6]. Despite the proximity of the SCJ to cervical neoplasia and their shared immunophenotype, there is little information directly linking SCJ cells to HPV infection. This issue is significant for several reasons. First, the histological link between SCJ infection and SIL development has not been clearly established, apart from limited immunohistochemical and in situ hybridization studies that have placed the SCJ cells in close proximity to HSIL [7]. Second, despite the fact that the concept of “latent” HPV infection has been in play for over three decades, the actual cells harbou1ring this form of HPV infection have yet to be revealed. This study focused on SCJ cells in high-risk reproductive age women; the main goal being to determine if transcriptionally active HPV infection could be observed in these junctional cells. Herein we provide evidence that the SCJ cells can be infected by HPV and serve as a nidus for early lesion development.
Materials and Methods
Tissue samples
The protocol was approved by the Ethics Committee of the University Hospital of Liege (Liege, Belgium) and by the institutional review board at Brigham and Women’s Hospital (Boston, MA, USA). First, we searched in our databases (Brigham and Women’s Hospital, Boston, MA, USA and University Hospital of Liege, Liege, Belgium) for patients with a history of SIL who underwent excision procedure [loop electrosurgical excision procedure (LEEP)] during the previous year (July 2013–July 2014) but had no evidence of preneoplastic lesion in the LEEP specimen. Twenty-two patients were selected. Second, 31 HPV16 positive paraffin-embedded specimens involving the SCJ [10 immature metaplastic LSIL and 21 “classical” lesions (7 LSIL and 14 HSIL)] were also obtained. All cases stained positive for krt7 and were re-examined by experienced pathologists.
Immunohistochemistry
Immunohistochemical analyses were performed as previously described [8,9]. Antibodies anti-keratin 7 (Clone RCK 105, Thermo Scientific, Waltham, MA, USA), anti-Ki67 (Clone SP6, Abcam, Cambridge, MA, USA), anti-involucrin (Clone SY5, Novocastra, Newcastle, UK), anti-p16ink4 (Clone JC8, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HPV L1 (Clone K1H8, Dako, Glostrup, Denmark) and anti-HPV16 E2 (kindly provided by Dr S. Bellanger, Institute of Medical Biology, Singapore) were selected. This latter antibody detects HPV16 E2 in immunohistochemical experiments (Supplemental Figure 1). A cross-reaction with HPV18 E2 was also previously reported by Western blots [10] and confirmed by immunohistochemistry (Supplemental Figure 1). Signal detection was achieved using the LSAB2 or Envision kit (Dako) according to the supplier’s recommendations. Positive cells were visualized using a 3, 3′-diaminobenzidine (DAB) substrate and the sections were counterstained with haematoxylin. Mouse and rabbit control IgG (Santa Cruz Biotechnology) were used as negative control.
Immunohistochemical assessment
The procedure is detailed in Supplementary Materials and Methods.
In situ hybridization
As previously described [7], HPV genotypes were detected by in situ hybridization (ISH) using the Ventana INFORM HPV III family 16 probe (Ventana Medical Systems, Tucson, AZ, USA) according to the supplier’s recommendations. This kit contains labeled probes allowing the detection of 12 carcinogenic HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66). Visualization of hybridized DNA was performed using Red Counterstain II (Ventana Medical Systems).
HPV genotyping
The Abbott Real Time high-risk HPV test (Abbott, Wiesbaden, Germany) was used to simultaneously genotype HPV16 and 18 and detected 12 other HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Although this assay has been shown to be highly sensitive and specific for the genotyping of HPV16 and 18 in paraffin-embedded samples [11], the collected results were confirmed by real time PCR as previously described [12].
Laser capture microdissection, mRNA extraction and amplification
Seven serial sections (6 μm thick) of each cervical specimen (immunostained and HPV typed as described above) were microdissected using a Leica LMD7000 system (Leica) and each cell population (squamous, columnar or SCJ cells) from different slides but from the same patient were pooled. Total RNA was extracted using the Nucleospin RNA XS kit (Macherey-Nagel, Düren, Germany). cDNA library synthesis and amplification was then performed using the complete whole transcriptome amplification (WTA2) Kit (Sigma Aldrich, Saint-Louis, MO) which is optimized to amplify RNA from paraffin-embedded and other damaged samples. Before cDNA synthesis, DNase treatment was performed in order to remove contaminating genomic DNA and avoid the detection of HPV DNA by PCR (Supplemental Figures 2 and 3).
Polymerase chain reaction analysis
HPV16 E6*I, E6*II, E7 and L1 mRNAs as well as HPV18 E6 and E7 transcripts were detected in microdissected samples. Primer sequences and detailed procedure are described in Supplemental Materials and Methods. Samples were loaded on 2% agarose gels containing ethidium bromide and visualized with an UV transilluminator (Bio-Rad, Hercules, CA, USA).
Results
Detection of HPV in normal-appearing SCJ cells from high-risk women
The goal was to determine where in the transformation zone HPV could be isolated in women with a recent history of cervical neoplasia. We focused on the SCJ, squamous metaplasia and mature columnar epithelium (Figures 1 and 2). Twenty-two patients with a prior history of SIL on histological examination but no evidence of HPV-related lesion in the LEEP specimen were identified. We targeted SCJ cells based on location (interface of mature squamous and endocervical mucosa), morphology (cuboidal or low columnar phenotype) and strong Krt7 immunoreactivity. In ten patients, the SCJ region was visible in serial sections and contained normal-appearing SCJ cells not associated with reserve or metaplastic cells. HPV DNA was detected in 8 patients. HPV16 and HPV18 were isolated from 5 and 1 of the 8 positive cases, respectively. The exact genotype could not be determined in two samples. Similar HPV genotypes were detected in both the diagnostic biopsies and excision specimens. To pinpoint the specific cells infected by HPV, we immunostained for p16ink4, HPV E2 and Ki67. In four cases, discrete foci of SCJ cells stained strongly for both p16ink4 and HPV E2 (Figure 2A and Supplemental Figure 4). Some rare Ki67-positive cells were also observed. In one case, HPV E2 immunoreactivity was observed in krt7-positive SCJ cells without evidence of p16ink4 expression (Figure 1A). Although HPV DNA was not detected by ISH (which is known to have a limited sensitivity when the copy number of HPV DNA is low [13]), the presence of HPV was confirmed by PCR (Figures 1C and 2C). When total mRNA was recovered from micro-dissected SCJ cells and amplified, HPV16 E6*I, E6*II and E7 were detected in 3 of 5 cases. HPV E6 and E7 transcripts were also detected in the HPV18-infected specimen (Supplemental Figure 4). Altogether, these results were interpreted as evidence for a transcriptionally active infection in these cells. No HPV or immunohistochemical evidence of HPV infection was detected in the control mature squamous or endocervical cells immediately adjacent to the SCJ cells (Figures 1C and 2C). The results obtained for each HPV-positive cervical specimen tested are summarized in Supplemental Table 1.
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, p16ink4 and Ki67. Note the absence of p16ink4 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) p16ink4/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).
HPV infection in both SCJ cells and mild squamous atypia (also known as early CIN)
A high percentage of HSILs express SCJ related biomarkers [3,5]. To clarify the histological link between SCJ infection and SIL development, cervical biopsies with SCJ cells accompanied by an underlying squamous atypia were identified (Figure 3A). Significantly, the patches of p16ink4/HPV E2 positive cells were associated with an increased proliferation. Both HPV DNA (ISH) and mRNAs (PCR) were also detected (Figure 3A-B). However, epithelial differentiation/stratification seemed insufficient to produce virions as suggested by the absence of HPV L1 expression in “early” SIL (Figure 3B).
Figure 3.
Characterization of the early steps of cervical neoplasia initiated within the SCJ. Early cervical neoplasia stained for Krt7, HPV16 E2, p16ink4 and Ki67. The patches of p16ink4/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)].
SCJ cells appear to give rise to reserve cells capable of squamous differentiation, seen during embryogenesis and transformation zone development [7]. We observed cuboidal cells on the surface of mild metaplastic atypias suggesting early SIL development. All analyzed lesions stained positive for keratin 7, p16ink4, HPV E2 and Ki67 (Figure 3C). Interestingly, focal apical L1 immunoreactivity was also detected in 5 of 10 cases (Supplemental Figure 5) and confirmed at the mRNA level (Figure 3D). However, L1 expression was weak compared to that observed in “mature” ectocervical/TZ SIL (Supplemental Figure 5) and this result was not interpreted as evidence confirming virion production.
Based on morphology and krt7 positivity, we identified remaining SCJ cells in direct continuity with atypical epithelium in 10/21 (47.6%) SILs seen near the SCJ. In contrast to the strong and diffuse (basal or full thickness) p16ink4 immunostaining observed in all (pre)neoplastic lesions, no contiguous SCJ cells expressed this marker. However, in three cases, these normal-appearing SCJ cells displayed HPV E2 immunoreactivity in keeping with HPV16 infection (Figure 4A).
Figure 4.
(A) Some remaining SCJ cells in direct continuity with atypical epithelium are infected by HPV. Note the absence of both p16ink4 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).
Discussion
We have previously shown that cells with an SCJ immuno-phenotype can be found on the surface of HSIL present at or near the SCJ and share both HPV DNA (ISH) and p16ink4 immunoreactivity with these lesions [7]. Moreover, we showed that most lesions over-expressing SCJ related biomarkers were HPV16 positive, irrespective of their lesion grade [5]. In a subsequent study published recently, a spectrum of immature metaplastic lesions near the SCJ ranging from bland appearing metaplasia to HSIL were highly associated with HPV16 or 18 and stained positive for Krt7 [14]. The implication from these studies was that SCJ infection by HPV initiated this progressive sequence from immature metaplasia through HSIL. The current study is the first to focus specifically on the SCJ as an initial site of infection and a potential reservoir of HPV nucleic acids prior to the development of SIL.
We selected several techniques that were intended to highlight early and late HPV infection: HPV DNA (ISH), HPV mRNAs (PCR) and immunohistochemical detection of HPV16 E2, p16ink4 and HPV L1, with isolated positivity for HPV E2 signifying the earliest phase of infection as shown previously [10]. Predictably, the earliest sign of infection in normal SCJ cells was manifested by positive E2 immunoreactivity and HPV detection (by PCR) (Figure 1 and 2). P16ink4 staining was negative or variable and proliferative activity (Ki67 staining) was absent or weak. In contrast, when a squamous lesion developed, HPV DNA (ISH) and diffuse p16ink4 staining were also detected, accompanied by an increase in proliferative index (Figure 3). An intriguing finding was the presence of L1 mRNA signal in these latter immature lesions accompanied by rare immunostaining suggesting L1 protein. It is impossible to confirm that virions were present without ultrastructural confirmation. However, the presence of these signals is of interest given the rarity of immunohistochemical evidence of L1 antigen detection in HPV16 associated lesions in the lower genital tract [15].
Figure 4B proposes a model for an HPV infection sequence in the SCJ, whereby the SCJ cells are infected and initially display HPV E2 expression, followed by metaplastic outgrowth that signifies the earliest morphological evidence of SIL. This model is supported by the observations in this study, but several fundamental questions remain. The first is whether the SCJ is the universal site of “latent HPV”. This would seem unlikely given the geographic range of HPV infection in the lower genital tract. However, given its exposure level as a cuboidal surface population, the SCJ could be viewed as a preferred site of initial HPV infection. One study showed that ablation of this area by cryotherapy reduced the detection rate of cervical HPV by 50% [16]. It would be important to know if this reduction was SCJ related and specifically a preferential reduction in HPV16 infection. Second and third questions would be what the trigger is that initiates lesion development in the infected SCJ cells and whether the HPV16 positive immature proliferations that ensue are destined to become persistent high grade SIL as implied in some reports [5,14]. Given the high percentage of CIN2s that disappear spontaneously, it is highly likely that certain patient-specific factors favour the progression of a subset of these lesions to a persistent CIN3 [17]. This raises the additional question of whether certain individuals are uniquely prone to developing HSIL through some facet of SCJ biology or immune surveillance. Addressing these questions could improve our understanding of the characteristics that place such a small number of women at the greatest risk. It would also provide further insight into the possible effects of preemptive SCJ ablation on the subsequent acquisition or persistence of HPV infection.
Supplementary Material
Suppl. Figure 1. Anti-HPV E2 antibody efficiently detects E2 protein of both HPV16 and HPV18 in immunohistochemical experiments. This specificity was previously demonstrated in both Western blotting and immunofluorescence experiments [9]. In the present tissue specimens, the exact HPV genotypes (HPV16, 18 and 66) were determined using the Linear Array HPV genotyping assay. Original magnification: X100.
Suppl. Figure 2. Control experiments for the detection of HPV16 E6*I, E6*II, E7 and L1 mRNAs. SiHa cells were embedded in paraffin, genomic DNA and mRNA were then extracted. E6*I and E6*II primers specifically amplify E6 splice variant mRNAs. E7 and L1 primers detect both HPV DNA and RNA. DNase treatment efficiently removed contaminating genomic DNA.
Suppl. Figure 3. Control experiments for the detection of HPV18 E6 and E7 transcripts. C-4II cells were embedded in paraffin. Genomic DNA and mRNA were then extracted. The primers detected both HPV DNA and RNA.
Suppl. Figure 4. (A) p16ink4/HPV E2 immunoreactivity in normal-appearing SCJ cells from HPV18-infected women. (B) Target cell populations (squamous, columnar and junctional) were detected and microdissected. Total RNA from each cell population was extracted and amplified, and HPV18 E6 and E7 expression was then analyzed by PCR. HPV18 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: X100 (Krt7) or X200 (p16 and HPV E2).
Suppl. Figure 5. Focal apical L1 immunoreactivity was detected in immature metaplastic lesions (A). However, L1 expression was weak compared to that observed in “mature” ectocervical/TZ SIL (B). Anti-involucrin immunoreactivity was useful in confirming the degree of maturity/differentiation of SIL. Original magnification: X100.
Characteristics of each HPV-positive cervical specimen tested (no evidence of CIN in the LEEP specimen).
Acknowledgments
The authors thank their clinical colleagues at the University of Liege and Brigham and Women’s Hospital for their cooperation. This work was supported in part by a grant from the National Cancer Institute (5R21CA173190-02 to CPC), by the Belgian Fund for Medical Scientific Research (FNRS), by the Centre Anti-Cancereux près l’Université de Liège, by the Faculty of Medicine of the University of Liège and by the Fonds Léon Frédéricq. The authors thank Estelle Dortu for her technical assistance. We are also grateful to Dr. Sophie Bellanger (Institute of Medical Biology, Singapore) for kindly providing us with the anti-HPV E2 antibody.
Footnotes
The authors have no conflict of interest to declare
Authors’ contributions
JM, CPC and MH designed the study. PR, MSC and MH performed experiments. JM, BH, AL, PD and CPC collected the tissue samples. JM, CPC and PD reviewed all cases. PH, FM, WX, CPC and MH interpreted the data. SD and MH generated figures. CPC and MH wrote the manuscript. All authors had final approval of the submitted manuscript.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Suppl. Figure 1. Anti-HPV E2 antibody efficiently detects E2 protein of both HPV16 and HPV18 in immunohistochemical experiments. This specificity was previously demonstrated in both Western blotting and immunofluorescence experiments [9]. In the present tissue specimens, the exact HPV genotypes (HPV16, 18 and 66) were determined using the Linear Array HPV genotyping assay. Original magnification: X100.
Suppl. Figure 2. Control experiments for the detection of HPV16 E6*I, E6*II, E7 and L1 mRNAs. SiHa cells were embedded in paraffin, genomic DNA and mRNA were then extracted. E6*I and E6*II primers specifically amplify E6 splice variant mRNAs. E7 and L1 primers detect both HPV DNA and RNA. DNase treatment efficiently removed contaminating genomic DNA.
Suppl. Figure 3. Control experiments for the detection of HPV18 E6 and E7 transcripts. C-4II cells were embedded in paraffin. Genomic DNA and mRNA were then extracted. The primers detected both HPV DNA and RNA.
Suppl. Figure 4. (A) p16ink4/HPV E2 immunoreactivity in normal-appearing SCJ cells from HPV18-infected women. (B) Target cell populations (squamous, columnar and junctional) were detected and microdissected. Total RNA from each cell population was extracted and amplified, and HPV18 E6 and E7 expression was then analyzed by PCR. HPV18 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: X100 (Krt7) or X200 (p16 and HPV E2).
Suppl. Figure 5. Focal apical L1 immunoreactivity was detected in immature metaplastic lesions (A). However, L1 expression was weak compared to that observed in “mature” ectocervical/TZ SIL (B). Anti-involucrin immunoreactivity was useful in confirming the degree of maturity/differentiation of SIL. Original magnification: X100.
Characteristics of each HPV-positive cervical specimen tested (no evidence of CIN in the LEEP specimen).