Carcinogenic HPV infection in the cervical squamo-columnar junction

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1 Journal of Pathology J Pathol 2015; 236: Published online 27 April 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: /path.4533 BRIEF DEFINITIVE REPORT Carcinogenic HPV infection in the cervical squamo-columnar junction Jelena Mirkovic, 1 Brooke E Howitt, 1 Patrick Roncarati, 2 Stephanie Demoulin, 2 Meggy Suarez-Carmona, 2 Pascale Hubert, 2 Frank D McKeon, 3 Wa Xian, 3 Anita Li, 1 Philippe Delvenne, 2 Christopher P Crum 1, *, and Michael Herfs 2, *, 1 Department of Pathology, Division of Women s and Perinatal Pathology, Brigham and Women s Hospital, Boston, MA, USA 2 Laboratory of Experimental Pathology, GIGA-Cancer, University of Liège, Liège, Belgium 3 Jackson Laboratory for Genomic Medicine, Farmington, CT, USA *Correspondence to: M Herfs, PhD, Laboratory of Experimental Pathology, GIGA-Cancer, University of Liège, 4000 Liège, Belgium. M.Herfs@ulg.ac.be Or Christopher P Crum, MD, Division of Women s and Perinatal Pathology, Department of Pathology, Brigham and Women s Hospital, Boston, MA 02115, USA. ccrum@partners.org CPC and MH contributed equally as senior authors. 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. Copyright 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: HPV; squamo-columnar junction; cervical intraepithelial neoplasia Received 13 January 2015; Revised 4 March 2015; Accepted 14 March 2015 No conflicts of interest were declared. 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 undergo neoplastic transformation [3,4]. The cervical SCJ cells share an identical immunophenotype [including strong staining for keratin 7 (Krt7)] with over 90% of high-grade SILs (HSILs) 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

2 266 J Mirkovic et al 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 harbouring 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 whether 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 Liège (Liège, 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 and University Hospital of Liège) for patients with a history of SIL who underwent an 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 LSILs and 21 classical lesions (7 LSILs and 14 HSILs)] 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-p16 ink4 (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 (Supplementary Figure 1). A cross-reaction with HPV18 E2 was also previously reported by western blots [10] and confirmed by immunohistochemistry (Supplementary 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 a negative control. Immunohistochemical assessment The procedure is described in detail in the 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 labelled 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 RealTime 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 was pooled. Total RNA was extracted using the Nucleospin RNA XS kit (Macherey-Nagel, Düren, Germany). cdna library synthesis and amplification were then performed using the Complete Whole Transcriptome Amplification (WTA2) Kit (Sigma Aldrich, St Louis, MO, USA), 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 to avoid the detection of HPV DNA by PCR (Supplementary 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 the Supplementary materials and methods. Samples were loaded on 2% agarose gels containing ethidium bromide and visualized with a UV transilluminator (Bio-Rad, Hercules, CA, USA).

3 Carcinogenic HPV infection in the cervical SCJ 267 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 analysed 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: 40 (microdissection), 100 (immunostaining) or 200 (insets, H&E, ISH). 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 eight patients. HPV16 and HPV18 were isolated from five and one of the eight positive cases, Copyright 2015 Pathological Society of Great Britain and Ireland. respectively. The exact genotype could not be determined in two samples. Similar HPV genotypes were detected in both the diagnostic biopsies and the 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 Supplementary 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 microdissected SCJ cells and amplified, HPV16 E6*I, E6*II, and E7 were detected in three of five cases. HPV E6 and E7 transcripts were also detected in the HPV18-infected specimen (Supplementary Figure 4). Altogether, these results were interpreted as evidence for a transcriptionally active infection in J Pathol 2015; 236:

4 268 J Mirkovic et al 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 analysed. 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: 40 (immunostaining and microdissection) or 200 (insets, H&E, ISH). 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 Supplementary Table 1. 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 p16 ink4 /HPV E2-positive cells were associated with an increased proliferation. Both HPV DNA (ISH) and mrnas (PCR) were also detected (Figures 3A and 3B). However, epithelial differentiation/stratification seemed insufficient to produce virions, as suggested by the absence of HPV L1 expression in early SIL (Figure 3B). 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 analysed lesions stained positive for keratin 7, p16 ink4, HPV E2, and Ki67 (Figure 3C). Interestingly, focal apical L1 immunoreactivity was also detected in five of ten cases (Supplementary Figure 5) and confirmed at the mrna level (Figure 3D). However, L1 expression was weak compared with that observed in mature ectocervical/tz SIL (Supplementary 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) p16 ink4 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).

5 Carcinogenic HPV infection in the cervical SCJ 269 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 analysed (B, 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: 100 [immunostaining (A C), H&E (C), ISH (C)] or 200 [insets, H&E (A) and ISH (A)]. Discussion We have previously shown that cells with an SCJ immunophenotype can be found on the surface of HSIL present at or near the SCJ and share both HPV DNA (ISH) and p16 ink4 immunoreactivity with these lesions [7]. Moreover, we showed that most lesions overexpressing 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, p16 ink4, 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) (Figures 1 and 2). p16 ink4 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 p16 ink4 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,

6 270 J Mirkovic et al 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: 40 (H&E and Krt7) or 100 (p16, HPV E2, and L1). 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

7 Carcinogenic HPV infection in the cervical SCJ 271 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 pre-emptive SCJ ablation on the subsequent acquisition or persistence of HPV infection. Acknowledgments We thank our clinical colleagues at the University of Liège and Brigham and Women s Hospital for their cooperation. This work was supported in part by a grant from the National Cancer Institute (5R21CA 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. We 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. Author 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. References 1. Marsh M. Original site of cervical carcinoma; topographical relationship of carcinoma of the cervix to the external os and to the squamocolumnar junction. Obstet Gynecol 1956; 7: Richart RM. Cervical intraepithelial neoplasia. Pathol Annu 1973;8: Herfs M, Yamamoto Y, Laury A, et al. A discrete population of squamocolumnar junction cells implicated in the pathogenesis of cervical cancer. Proc Natl Acad Sci U S A 2012; 109: Wang X, Ouyang H, Yamamoto Y, et al. Residual embryonic cells as precursors of a Barrett s-like metaplasia. Cell 2011;145: Herfs M, Parra-Herran C, Howitt BE, et al. Cervical squamocolumnar junction-specific markers define distinct, clinically relevant subsets of low-grade squamous intraepithelial lesions. Am J Surg Pathol 2013; 37: Chaturvedi AK. Beyond cervical cancer: burden of other HPV-related cancers among men and women. J Adolesc Health 2010; 46: S20-S Herfs M, Vargas SO, Yamamoto Y, et al. A novel blueprint for top down differentiation defines the cervical squamocolumnar junction during development, reproductive life, and neoplasia. J Pathol 2013; 229: Herfs M, Hubert P, Poirrier AL, et al. Proinflammatory cytokines induce bronchial hyperplasia and squamous metaplasia in smokers: implications for chronic obstructive pulmonary disease therapy. Am J Respir Cell Mol Biol 2012; 47: Hubert P, Herman L, Roncarati P, et al. Altered α-defensin 5 expression in cervical squamocolumnar junction: implication in the formation of a viral/tumour permissive microenvironment. J Pathol 2014; 234: Xue Y, Bellanger S, Zhang W, et al. HPV16 E2 is an immediate early marker of viral infection, preceding E7 expression in precursor structures of cervical carcinoma. Cancer Res 2010; 70: Kocjan BJ, Seme K, Poljak M. Comparison of the Abbott Real- Time High Risk HPV test and INNO-LiPA HPV Genotyping Extra test for the detection of human papillomaviruses in formalin-fixed, paraffin-embedded cervical cancer specimens. J Virol Methods 2011; 175: Ernoux-Neufcoeur P, Arafa M, Decaestecker C, et al. Combined analysis of HPV DNA, p16, p21 and p53 to predict prognosis in patients with stage IV hypopharyngeal carcinoma. J Cancer Res Clin Oncol 2011; 137: Kong CS, Balzer BL, Troxell ML, et al. p16ink4a immunohistochemistry is superior to HPV in situ hybridization for the detection of high-risk HPV in atypical squamous metaplasia. Am J Surg Pathol 2007; 31: van der Marel J, van Baars R, Alonso I, et al. Oncogenic human papillomavirus-infected immature metaplastic cells and cervical neoplasia. Am J Surg Pathol 2014; 38: Mitao M, Nagai N, Levine RU, et al. Human papillomavirus type 16 infection: a morphological spectrum with evidence for late gene expression. Int J Gynecol Pathol 1986; 5: Taylor S, Wang C, Wright TC, et al. Reduced acquisition and reactivation of human papillomavirus infections among older women treated with cryotherapy: results from a randomized trial in South Africa. BMC Med 2010; 8: Moscicki AB, Ma Y, Wibbelsman C, et al. Rate of and risks for regression of cervical intraepithelial neoplasia 2 in adolescents and young women. Obstet Gynecol 2010; 116: SUPPORTING INFORMATION ON THE INTERNET The following supporting information may be found in the online version of this article: Supplementary materials and methods. Figure S1. Anti-HPV E2 antibody efficiently detects E2 protein of both HPV16 and HPV18 in immunohistochemical experiments. Figure S2. Control experiments for the detection of HPV16 E6*I, E6*II, E7, andl1 mrnas. Figure S3. Control experiments for the detection of HPV18 E6 and E7 transcripts. Figure S4. (A) p16 ink4 /HPV E2 immunoreactivity in normal-appearing SCJ cells from HPV18-infected women. (B) Target cell populations (squamous, columnar, and junctional) were detected and microdissected. Figure S5. Focal apical L1 immunoreactivity was detected in immature metaplastic lesions (A). However, L1 expression was weak compared with that observed in mature ectocervical/tz SIL (B). Table S1. Characteristics of each HPV-positive cervical specimen tested (no evidence of CIN in the LEEP specimen).