Advances in Anatomic Pathology:
Laboratory Management of Cervical Intraepithelial Neoplasia: Proposing a New Paradigm
Herfs, Michael PhD*,†; Crum, Christopher P. MD*
*Department of Pathology, Division of Women’s and Perinatal Pathology, Brigham and Women’s Hospital, Boston, MA
†Department of Pathology, GIGA-Cancer, University of Liege, Liege, Belgium
The authors declare no conflict of interest or NIH funding sources.
All figures can be viewed online in color at http://www.anatomicpathology.com.
Reprints: Christopher P. Crum, MD, Department of Pathology, Amory 3, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115 (e-mail: email@example.com).
Since the discovery of human papillomavirus (HPV) type 16 in early 80s, the link between HPV and cervical cancer has been established with certainty, a function of the discovery and cloning of a range of HPV types associated with both cancer precursors (cervical intraepithelial neoplasia or CIN) and carcinomas and extensive epidemiologic, clinical, pathologic, and experimental data. These accumulated results have culminated in new paradigms of cancer prevention through screening and triage. Despite this, the management of women with CIN is still suboptimal and the overtreatment of these conditions still occurs, largely due to the lack of clarity regarding which precancerous lesions are most likely to progress in grade. Recently, a discrete population of cuboidal cells was discovered at the cervical squamocolumnar junction, the anatomic site where the large majority of HPV-related (pre)neoplastic lesions develop. These cells seem to be embryonic in nature and participate both in benign metaplasias and the initial phase of precancer development. This review summarizes the historical evolution of precursor management, assesses the potential role of this and other discoveries in segregating lower from higher-risk precursors, and examines their potential impact on the management of women with real or potential cervical cancer precursors.
Passed from one person to another through direct contact, human papillomavirus (HPV) infection is considered to be the most common sexually transmitted disease. HPV is so prevalent that at least 40% of sexually active men and women acquire genital HPV infection at some point in their lives.1,2 Although most (∼90%) HPV strains induce either asymptomatic infection or benign hyperproliferative lesions, chronic infection by some HPV “high-risk” (carcinogenic) genotypes, particularly types 16 and 18, is associated with the development of cervical cancer and its precursors.3,4 Numerous studies demonstrated that HPV is also an important risk factor for many other cancers. Carcinogenic HPV infection was found in the large majority of vaginal, vulvar, and anal preneoplastic lesions as well as in about half of all penile and oropharyngeal cancers.5–8 At the molecular level, the interactions between the major viral oncoproteins and host cellular factors such as cell-cycle and apoptosis proteins are absolutely required to initiate the malignant transformation and latter maintain the cancer phenotype of infected cells.9,10 Despite the general conservation of genetic structure among all 150 HPV types, the interaction of HPV E6 and E7 with target proteins and subsequent proteasome-mediated degradation seem to be type specific explaining why only 15 HPV strains are been labeled as high risk to cause cancer.11,12
Every 24 hours, an estimated 3700 women are newly diagnosed with low-grade cervical intraepithelial neoplasia (CIN1) in the United States.13 Although the large majority of these preneoplastic lesions will spontaneously regress to normal or ASCUS within 6 to 12 months,13–16 the American Cancer Society estimated that, in 2010, there were about 12,170 new cases of invasive cervical cancer, with a number of death just above 4100.17,18 The incidence of cervical cancer is, however, much higher in developing countries (South America, sub-Saharan Africa) mainly due to limited access to health services.19 Clinically, successful construction of papilloma virions in vitro has resulted in the development of bi/quadrivalent HPV vaccines that have recently demonstrated to have a great potential for the prevention of a large proportion of HPV-associated cancers, assuming adequate duration of protection.20–24 Despite the hope generated by the commercialization of HPV vaccines, with an incidence of 470,000, cervical cancer is still the second most common cancer in women and remains a leading cause of morbidity and mortality worldwide.8,25 Consequently, as recently mentioned,26 an improvement of our understanding of carcinogenic HPV types and how these viruses cause disease could continue to improve clinical management of a large number of women still at risk for this disease.
In the present paper, we will summarize the latest findings in cervical pathology and potential avenues for improving management of early cervical neoplasia.
DEVELOPMENT AND NORMAL CERVICAL REMODELING
In mammals, the entire female reproductive tract, from the oviduct to the vagina, develops from a pair of Müllerian ducts of mesodermal origin. Consequently, in response to organ-specific mesenchymal signals, the uniform Müllerian epithelium differentiates into several epithelial cell types with unique morphology (squamous, columnar, ciliated), function, and gene expression pattern.27 In recent studies, we showed that, at early stages of human fetal development (prior 16 wk), vagina and cervix were completely covered by an undifferentiated Müllerian epithelium expressing several markers such as keratin (Krt) 7, anterior gradient 2 (AGR2), guanine deaminase (GDA), matrix metalloproteinase 7 (MMP7), or CD63.28,29 At 17 to 18 weeks, an induction of early squamous markers krt5 and p63 was observed in the large majority of Müllerian cells, and this was followed by the emergence of a basal population of krt5/p63 cells from beneath the embryonic Müllerian epithelium. These newly formed basal squamous cells, still expressing the primitive biomarkers, then proliferated inducing both the stratification of the vaginal/ectocervical epithelium and the displacement of the primitive epithelial cells above the stratifying squamous epithelium. The superficial population of embryonic precursor cells was finally exfoliated several months after birth with the exception of some residual cells located at the squamocolumnar (SC) junction (Fig. 1).28,29 In adult, microarray and immunohistochemical analysis demonstrated that these cuboidal SC junction cells have a unique gene expression profile that is different from that of the adjacent ectocervical squamous and endocervical columnar epithelia.28
Usually observed within or in close proximity to the SC junction, epithelial metaplasia and microglandular hyperplasia are 2 frequent, benign cervical remodeling processes.30,31 The transition from a purely columnar to a squamous mucosa involves the presence of reserve cells in both conditions.32 The origin and the “stemness” properties of reserve cells are, however, still controversial. Theories have successively suggested that these cells were derived from the columnar endocervical cells,33 from the stromal cells34,35 and from the Müllerian epithelium at midgestional age.36 In addition, on the basis of morphologic considerations, endocervical reserve cells were proposed as the progenitors to both normal and neoplastic squamous and columnar epithelia.36,37 Recently, we analyzed adult cervical SC junction and observed that, in some cases, a high percentage of residual embryonic krt7-positive cuboidal cells expressed squamous markers (krt5, p63) without evidence of subjacent reserve or metaplastic cells. Similar that the findings during embryogenesis, the reserve cells then emerged from beneath the cuboidal cells, proliferated, and finally evolved to create a stratified epithelium (multilayered squamous metaplasia). This scenario was termed reverse or “top-down” differentiation because the evolution from progenitor cells to metaplastic progeny occurred downward or basal in orientation rather than upward and apical.29
DISCOVERY OF HUMAN PAPILLOMAVIRUS TARGET CELLS IN THE SQUAMOCOLUMNAR JUNCTION: A NEW CONCEPT
HPVs are small, double-stranded circular DNA viruses of approximately 8000 base pairs. Containing 8 open reading frames, HPV genome is surrounded by a capsid of roughly 55 nm in diameter composed by the 2 structural proteins L1 and L2.38 Like all other viruses, HPVs are intracellular parasites whose viral replication is totally dependent of the host cellular machinery. Although many details remain to be clarified, recent data suggest that host cell entry of HPV is initiated by binding of the mature viral capsid to heparan sulfate proteoglycans on the basement membrane. This interaction leads to the cleavage of L2 proteins by a convertase (furin) inducing a capsid conformation change. After this proteolytic cleavage, HPV capsid interacts with a still undetermined cell surface receptor, which then triggers endocytosis.39–41
The cell of origin for cervical cancer has been subject to speculation for long time but has traditionally been linked to the proliferating basal layer of the ectocervical/ transformation zone (TZ) squamous epithelium.30,42,43 It is thought that microtrauma or abrasions to the stratified mucosa exposed the basal cells of the epithelium to HPV infection.44,45 Although basal keratinocyte infection is likely to result in productive infections and subsequent HPV-related lesions developing in the TZ, ectocervix, and other mucosal sites, this theory is inconsistent with the well-known observation that cervical cancer and its precursors develop mainly in the SC junction.46,47 Subsequent the recent discovery of residual embryonic SC junction cells, we stained a large number of low-grade and high-grade CINs, cervical squamous cell carcinoma and adenocarcinoma with SC junction-specific antibodies to ascertain their relationship to junctional cells. We showed that a high percentage (>90%) of CIN2/3, squamous, and glandular neoplasms were located in the SC junction and displayed the immunologic markers common to the SC junction-specific cells (Fig. 2).28 In contrast, only ∼25% of CIN1 stained positive for SC junction markers.28 To address the possibility that infection of basal keratinocytes by carcinogenic HPV could induce the expression of SC junction-specific genes and mimic the SC junction immunophenotype, primary foreskin keratinocytes were stably transfected with HPV16 E6 and E7 proteins. As revealed by Western blot, the 2 major viral oncoproteins did not upregulate SC junction markers suggesting that the expression observed in cervical lesions was related to the gene expression pattern displayed by the HPV target cells.28 In agreement with our results, an overexpression of cystic fibrosis transmembrane conductance regulator, another SC junction-specific gene (accordingly to microarray analysis), were recently observed in 15% and 90% of CIN1 and cancers, respectively.48 According to these authors, the expression of this protein was also associated with cervical cancer progression, aggressive behaviors, and poorer prognosis. Although squamous cell carcinomas of various origins (head and neck, esophagus, vagina) were negative for the SC junction marker Krt7, Chu et al49 also showed a strong expression of Krt7 in the majority (87%) of cases of cervical cancer. Taking together, all these results support that cervical cancer and its high-risk precursors (CIN2/3) originate from the HPV infection of the SC junction cell population.
THE EVOLVING LABORATORY MANAGEMENT OF CERVICAL INTRAEPITHELIAL NEOPLASIA
Conventionally, cytology-based screening (PAP test) is linked to treatment through an intermediary diagnostic step using colposcopy, followed by confirmatory biopsy when indicated. Endocervical curettage can be used to sample the endocervical canal. Pathology assessment of the tissue samples obtained by biopsy confirms the presence or absence of HPV-related lesions. This algorithm has been in place for over 40 years, and the manner in which information is used for patient management has evolved as a function of greater understanding of the pathogenesis of disease. The reader is forewarned that not all aspects of this evolution have been beneficial. For example, the approach to women with cervical abnormalities in the 60s, which typically was to observe lesions classified as mild to moderate dysplasia and excise severe dysplasia and carcinoma in situ, is surprisingly similar to current strategies. The next few paragraphs will critically examine these periods in history and the parameters that underscored conventional thinking.
The Morphology Era
The morphology era had 2 phases. Publications between 1960 and 1980 focused on those precancers considered to be at risk for progressing to malignancy. Because these predated the HPV era, texts and monographs almost exclusively concentrated on lesions that would be classified as moderate dysplasia to carcinoma in situ. In the late 1960s, Richart introduced the term cervical intraepithelial neoplasia to encompass the entire group, and in retrospect CIN—defined as a full-thickness population of abnormal cells that was graded as a function of maturity—can be seen, at least in principle if not in practice, as a continuum of changes within an aneuploid epithelium.50,51 The second phase of the morphology era could be seen as one with 2 additional complications. The first was that the transmission of this concept to pathologists would not be consistent. For example, minor atypias of the cervix would be overdiagnosed as mild dysplasias (CIN1), polluting the lower end of the CIN spectrum with innocuous conditions. The second complication was the discovery of koilocytotic atypia and its association with HPV.52,53 Koilocytosis, being a superficial karyomegaly with cytoplasmic halos, was not invariably associated with full-thickness atypia. Thus, it was separate from the CIN group. This distinction is still maintained by some authors who prefer to separate flat condyloma from CIN1, ultimately creating a 4-grade classification. In the opinion of these authors, it is biologically appropriate but difficult to accomplish in practice.
This period in time coincided with the introduction of cryotherapy54–56 and laser ablation,57 both of which could be done in the office setting. Typically CIN1 or CIN2 were treated with ablation, whereas CIN3 was treated with cone biopsy.58,59
The Human Papillomavirus Era
The HPV era brought with it numerous studies that showed a strong link between HPV16 and CIN2/3. This dichotomy, between CIN1 and CIN2/3, was initially supported by molecular studies using mainly DNA hybridization techniques,60–64 but it soon became clear that many CIN1s were also associated with cancer-associated HPVs. Nevertheless, given the historical data supporting a much lower risk of progression for CIN1 plus the strong association of high-risk HPVs with moderated/severe (pre)neoplastic lesions, the impetus was to create a new 2-grade cytologic classification. Introducing low-grade (HPV infections+CIN1) and high-grade (CIN2 to carcinoma in situ) squamous intraepithelial lesions (SIL), this translated into CIN1 versus CIN2-CIN3.65,66 A formal switch to histology was not made, but the message conveyed was that CIN1 was different from CIN2. This would have a profound impact in the next era.
The LEEP Era
In early 90s, the LEEP era coincided with the promotion of large loop excision of the transformation zone.67,68 This necessitated positioning the various lesion grades in a therapeutic model. What transpired was to follow CIN1 and remove (excise) CIN2 or CIN3. In practice, it exploited the vulnerability of the pathologist who was now forced to tell the difference between CIN1 and CIN2, in as much as it would determine whether the woman received a surgical procedure. Two misconceptions fueled this unfortunate period. The first was that CIN1 could be consistently distinguished from CIN2. The second was that CIN2, even when unequivocal, signified a lesion with a high rate of persistence and progression to malignancy.
The Biomarker Era
The biomarker era started in 2000 after the discovery of p16ink4, a protein that is strongly upregulated in association with infections by high-risk HPVs.69–72 P16ink4 was a powerful marker described in numerous publications. Related to the degradation of pRB by the E7 viral oncoprotein, an intense nuclear and cytoplasmic p16ink4 staining is observed in neoplastic squamous epithelium.70 This marker has been investigated for its utility in 2 settings. The most useful aspect of p16ink4 staining is its ability to distinguish a (pre)neoplastic lesion (whatever the grade) from either low-risk HPV infection or non–HPV-related epithelium, both synonymous with benign mucosa.73 That p16ink4 has utility as a marker to discriminate inflammatory or reactive atypias from high-grade CIN is not disputed. The second setting has proven more controversial, which is to use p16ink4 to adjudicate grading conundrums and predict the cancer progression.74,75 Central to this debate are 3 findings:
Continuous p16ink4 Staining is Often Present in Low-grade Squamous Intraepithelial Lesion
Several studies have verified both the association of p16ink4 staining with low-grade squamous intraepithelial lesion (LSIL) and with high-risk HPV types. Up to 70% of LSILs exhibited uninterrupted stainings that occupied at least the lower (basal) third of the infected epithelium.28,76,77
Predicting “Progression” is Difficult
In determining whether a lesion merits a diagnosis of high-grade squamous intraepithelial lesion (HSIL) or contains properties that increase the risk of HSIL on follow-up, the diagnostic criteria must be both unassailable and denote a process that carries increased risk. An important study initiated by the National Cancer Institute (ASCUS-LSIL Triage study or ALTS) showed that approximately 13% of women with a diagnosis of LSIL will develop an HSIL in 3 years.13,14 However, it does not clarify whether the HSIL outcome is directly linked to the original LSIL. Studies tracking the distribution of HPV types over time in women undergoing follow-up after an HSIL diagnosis have demonstrated considerable variability, implying that new HPV infections can be superimposed on existing ones.78 A second factor is the overdiagnosis of a follow-up biopsy, leading to the false impression that progression occurred when in fact the same or a similar low-grade process was present. Sampling is another issue. All of these point to the difficulties in confirming “progression,” an issue that must be approached critically when ascertaining whether a given biomarker will actually predict an HSIL in the future.
A High Percentage of Cervical Intraepithelial Neoplasia-2 Will Regress
Up to 67% of HSILs will regress in young women, including those who were diagnosed by the agreement of 2 or more pathologists.79,80 This fact underscores the difficulty of predicting outcome.
A recent task force recommended the use of p16ink4 immunostaining to assist in adjudicating cases in which the differential diagnosis was CIN2.81 Given the rather high frequency of diagnostic uncertainty for lesions in the CIN1-CIN2 range, this may lead to several scenarios including:
Blanket use of biomarker staining in all cases, at considerable expense.
Overdiagnosis of CIN2.
Excessive treatment of LSILs simply because they were diffusely p16ink4 positive and were thus classified as HSIL.
Counter to this is the fact that:
Most CIN2s in young women regress and therefore could be safely followed if patients were reliable and returned for examination.79,80
Guidelines exist for managing CIN2 in young women with repeat examination, at least on the short term.82
Beside p16ink4, which is the only universally used marker in the context of cervical carcinogenesis, other proteins have also been proposed as biomarkers to predict the progression of preneoplastic lesions into invasive cancer. Member of insulin-like growth factor mRNA-binding proteins, IMP3 expression was recently found in 18% of CIN3 and associated with cell migration in vitro.83 On the basis of these preliminary results, the authors of this study proposed to use IMP3 at the time of initial diagnosis of CIN to identify a subgroup of patients with an increased risk of developing invasive squamous cell carcinoma. Some data also indicated that an upregulation of HLA-DR and CD99 may be related to the evolution of cervical cancer.84 However, the expression of these proteins was not associated with clinicopathologic parameters. To conclude this nonexhaustive list, recent evidence indicated that methylation of HPV16 genome was potentially associated with CIN3 outcome and, thus, may constitute a predictive biomarker for risk of cervical cancer.85,86 Even if these results are confirmed and validated in further studies, the procedure (DNA extraction, treatment with bisulfite, pyrosequencing analysis) is long, complex, and expensive and, consequently, hardly feasible routinely.
The Cell of Origin Era
Historically, anatomic site has seemed critical for imposing risk of squamous carcinoma in the lower female genital tract. The uterine cervix is the principle site and the incidence of cervical cancer relative to vaginal or vulvar carcinoma is approximately 12:1.8,25 As discussed earlier in this article, this risk is likely a function of the SC junction that harbors a population that, although not the exclusive target of high-risk HPVs, eventuates in the majority of the derived malignancies.28
Both SC junction positive and SC junction-negative (pre)neoplastic lesions contain high-risk HPVs and can be diffusely p16ink4 positive.28 The ratio of SC junction-positive/SC junction-negative HSILs is about 20:1 (M. Herfs and C. Crum, 2012; unpublished data), in keeping with the historical ratio of cervix to vaginal/vulvar squamous neoplasia. Interestingly, the ratio of SC junction-positive/SC junction-negative LSILs is approximately 1:4,28 indicating that the majority of LSILs occur in metaplastic or ectocervical epithelium. As discussed above, metaplastic epithelium observed in the transformation zone is presumed to derive from the residual SC junction cells and infection of these keratinocytes usually leads to lower-grade precursors.29 Several observations pertaining to this site specificity are noteworthy.
In our experience, in a small number of cases, LSILs detected after cone biopsy stained negative for the SC junction-specific markers (unpublished data). This implies that removal of the SC junction might reduce the risk of developing more biologically aggressive precursors.
Historical anecdotes of postpartum cervical cautery have reported exceedingly low rates of subsequent cervical neoplasia.87 Such studies have not been conducted in a systematic way, but it is plausible that ablation of the SC junction might significantly reduce the risk of cervical cancer. The confounding parameter is exposure to HPV; women are immunized by their infecting HPV and would be protected anyway. There are no studies of young women followed after prophylactic SC junction ablation.
Women with SC junction-negative LSILs have an exceedingly low rate (approaching 0) of HSIL on follow-up (unpublished data). This suggests that ectocervical/metaplastic epithelial infection by HPV immunizes the patient to some degree from subsequent SC junction infection, at least for the index HPV infection.
SC junction-positive LSILs are more likely to contain HPV16, be diffusely p16ink4 positive, and be confused with HSIL (unpublished data). This implies that 2 factors are in play with LSILs occurring in the SC junction. (A) Some are not really LSILs but are either undersampled HSILs or LSILs more likely to progress to HSIL. (B) True SC junction-positive LSILs exist but may have a more worrisome appearance. Resolving these 2 concepts remains a work in progress. However, this new information has permitted the construction of a new model by which the pathologists can discriminate more from less problematic preneoplastic lesions in the cervix.
A PROPOSED CONCEPTUAL MODEL FOR SUBCLASSIFYING CERVICAL INTRAEPITHELIAL NEOPLASIA
On the basis of a recent consensus conference,81 CIN will now formally be classified as LSIL (CIN1) and HSIL (CIN2-3). This system has been in place in many publications for the past 20 years. Irrespective of the terminology used, the primary decision point is between following the patient and performing an excisional or ablative procedure. This decision is often determined by whether the diagnosis is CIN1 or CIN2. The latter diagnosis has been “decriminalized” somewhat to provide options for follow-up rather than immediate excision.82 The question will be whether maintaining the term of CIN2 will result in overtreatment while the field adapts to new recommendations. One alternative is to have 3 grades: LSIL, HSIL, and “SIL of uncertain/questionable grade [Questionable grade Squamous Intraepithelial lesion (QSIL)].” The latter diagnosis would be managed with a 6-month follow-up visit to determine whether the lesion had persisted, after which therapy could be tailored to the findings.
What is the SIL group most likely to fall into the “QSIL” category? In a recent survey of over 200 cases of SIL, we found that 2 groups (HSILs and SC junction-negative LSILs) were interpreted with a high level of concordance between a panel of 2 experts and the original diagnosis by one or several pathologists in a practice (unpublished data). In other words, if either of the expert panel members classified lesions into the above 2 groups (in blind review), there was a high level of agreement by the other expert and the original pathologist(s). Examples of such cases are illustrated in Figure 3. Note the percentage of cases with diffuse positive p16ink4 staining (Figs. 3, 4). Diffuse staining is higher in the HSIL group, but it is still substantial in the SC junction-negative LSILs (Figs. 3, 4).28
The third category, and one that is more likely to be diagnostically problematic, is that of the SC junction-positive LSILs. Diagnostic concordance for this entity was lower within the panel and approximately half of these SC junction-positive LSILs were classified as HSIL by the original pathologists (unpublished paper). Predictably, some had an HSIL outcome on LEEP excision. Moreover, as shown in the Figures 3 and 4 and recently demonstrated,28 SC junction-positive “LSILs” are more likely to manifest with strong, diffuse p16ink4 immunostaining and contain HPV16, similar to their HSIL counterparts. On the basis of these observations, SC junction-positive LSILs might comprise a group with greater likelihood of diagnostic disagreement or risk of an HSIL outcome, justifying the term QSIL. Lesions in this category are illustrated in Figure 3.
TRANSLATION TO PRACTICE: RISKS AND BENEFITS
On the basis of these recent discoveries, and in keeping with the new recommendations, the utility of p16ink4 immunostaining lies principally in its occasional value in discriminating immature reactive proliferations from HSIL.73 Its value in distinguishing whether a HPV-related lesion is LSIL or HSIL is limited if based on “positive” staining alone. Accordingly, the recommendation is that p16ink4 only be used if a diagnosis of CIN2 is under consideration.81 It is clear that HSILs will be almost universally p16ink4 positive, but there are 2 pitfalls in using this biomarker to determine grade. First, up to 70% of LSILs will score positive; second, the threshold for “considering” HSIL may vary considerably, depending on the pathologist. Moreover, if p16ink4 staining is billable when HSIL is “considered,” there is the risk that this test will be performed as a reflex for all cervical biopsies, adding costly weight to an already ponderous algorithm for managing women with SIL, a high percentage of whom have little risk of developing cervical cancer.15,16
An alternative to the above approach would be to avoid the decision endpoint of “CIN2”—a somewhat ephemeral entity that is politically and emotionally charged—and classify lesions with 3 options (HSIL, LSIL, QSIL) governed by the level of certainty and influenced by site of origin. It remains to be determined whether additional studies will establish that SC junction-positive HSILs and SC junction-negative LSILs are reproducible entities and easily distinguished or will show that SC junction-positive LSILs (QSILs) are problematic in both diagnosis and outcome. If they do, pathologists might be able to quickly recognize SC junction LSILs (QSILs) and designate them for 6-month follow-up. With this strategy, uncertainty would be an endpoint, leading to follow-up, rather than a trigger for a test that might lead to an inappropriate excisional procedure.
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squamocolumnar junction; cervical intraepithelial neoplasia; clinical management
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