Cervical Intraepithelial Neoplasia: History and Detection

Hippocrates and Galen described invasive cancers of the cervix, but the existence of asymptomatic neoplasms within the cervical epithelium was not recognized until early in the last century, and the preinvasive nature of these lesions has been clarified only in the last few decades. With the development of techniques that allow molecular biologists to explore genomic changes in dysplastic cells the fundamental biology of cervical intraepithelial neoplasia (CIN) has begun to emerge.

After defining CIN and briefly reviewing the history of the scientific advances leading to modern practice, this chapter describes the natural history of CIN, strategies for screening, and techniques for diagnosis. Other chapters discuss the epidemiology of cervical cancer, which is identical to that of these precursor lesions; the natural history of CIN; the pathologic characteristics of CIN and cervical cancer; and the biologic makeup of human papillomavirus (HPV), which is fundamental to understanding CIN. Although a glandular precursor lesion has been identified in the endocervix (adenocarcinoma in situ), this chapter focuses on the common squamous abnormalities.

Cervical intraepithelial neoplasms are atypical proliferations of immature squamous epithelium that do not penetrate the basement membrane of the epithelium. Mitotic figures, both normal and tripolar or even tetrapolar, are seen above their usual position among the reserve cells at the base of the epithelium. Nuclear abnormalities are characteristic and include a coarse chromatin pattern, abnormal chromatin distribution, pleomorphism, and hyperchromaticity. The nuclear-cytoplasmic ratio is increased. Progressive squamous differentiation is diminished, being restricted to the upper levels of the epithelium; in the most advanced lesions, differentiation is absent, and the full thickness of the epithelium is composed entirely of neoplastic cells.

The systems for classification of CIN that have been used to understand cervical carcinogenesis have evolved with our understanding of this disease process. The modified Papanicolaou system was developed to distinguish cancer and carcinoma in situ (CIS) from other lesions and now is obsolete. The dysplasia and CIN systems were standard until recently and remain the histologic descriptive terms of choice. However, it is the Bethesda system which is used most commonly for cytopathologic description and is most appropriate. First developed in 1988, the Bethesda system was revised in 2001; it offers a comprehensive terminology for the reporting of cervical cytology, and is discussed in greater detail in the section on 'Screening' below.

Although cervical CIS was described in the early 1900s,¹ the clinical importance of these lesions was not appreciated until useful means for detecting these asymptomatic, invisible lesions were developed. Beforehand, cervical cancer detection relied on inspection and palpation, with biopsy of obvious invasive cancers.² Schiller developed a technique for iodine staining as a gross means for detecting areas of abnormal epithelium,³ but this test could not distinguish metaplastic from neoplastic areas of the cervix and could not distinguish small areas of invasion present in a field of diffuse nonstaining epithelium. In this era, the nature of intraepithelial lesions was controversial, often described at the margin of invasive lesions but at times noted as a precursor to invasion.^{3, 4} Nevertheless, the description in the 1920s and 1930s of what came to be known as CIN provided the foundation for the development of cytologic study.

Although cytologic examination of exfoliated cervical epithelial cells was first described by Babes in 1928 in the French literature,⁵ only with the appearance in 1941 of the findings of Papanicolaou and Traut did this technique enter clinical practice as a means for the early diagnosis of cervical cancer.⁶ Like any epithelium, the cervical mucosa is constantly being sloughed and regenerated. When examined as thin smears, neoplastic cells from the cervical surface retain characteristic microscopic abnormalities that allow them to be distinguished after staining from normal squames, leukocytes, and glandular cells. Papanicolaou described a technique for aspiration of cells from the posterior vaginal pool, fixation, and cytologic staining that remains the foundation of current screening strategies. Whereas others identified unsuspected neoplasms after abnormal smears and suggested that CIS was a cancer precursor,^{7, 8, 9, 10, 11} Papanicolaou's findings generated significant controversy,¹² and his technique required modifications. Over time, vaginal pool aspiration was dropped, to be replaced by spatula collection, as developed by Ayre to facilitate the sampling of cells directly from the ectocervix.¹³ Augmented with a cotton-tipped applicator for endocervical cell collection,¹⁴ this instrument remained the standard collection instrument until the development of endocervical brushes and one-step samplers during the 1980s.

Initially, in the United States, histologic evaluation of abnormal findings on cervical cytologic smears was achieved through blind biopsy of four quadrants of the cervix or through biopsy directed by the Schiller test.^{15, 16, 17, 18} However, although such biopsies were useful when results were positive, the inherent high negative predictive value of this random procedure in identifying lesions invisible to the naked eye soon was recognized.^{18, 19} Cone biopsy became and remains the definitive procedure for histologic evaluation of the cervical transformation zone, punch biopsy being reserved for patients with gross lesions or lesions detected colposcopically.^{17, 18, 20, 21, 22, 23} This procedure, which involves the excision of the cervical transformation zone, requires anesthesia in an operative suite and carries a significant risk of complications, whereas later studies showed disconcertingly high rates of negative findings in cone biopsies from patients with cytologic tests showing abnormalities of lesser severity than CIS or invasive cancer.¹⁸ Alternative methods of diagnosis were sought.

Colpomicroscopy was proposed as an alternative offering magnification as high as 480 times, but technical factors precluded its wide acceptance.^{24, 25} Hinselmann had described colposcopy in 1925, and the technique had become incorporated into clinical care in other countries, but only in the 1950s was intensive training in colposcopic techniques promoted in the United States.^{26, 27, 28, 29, 30} Initially, a role for colposcopy as a screening modality for CIN was proposed,^{26, 28, 29} and it still is used as such in some parts of Europe.  However, the simplicity and wider availability of cytologic screening led to retention of cytologic examination as the standard screening test in the United States, with colposcopy retained as the study of choice for evaluation of women with abnormal cytologic findings. The value of colposcopy for excluding invasive cancer or CIS in women with cytologic results consistent with dysplasia was established,^{31, 32} and studies comparing colposcopy with cone biopsy showed acceptable rates of correlation.^{33, 34} The American Society for Colposcopy and Cervical Pathology, dedicated to promoting skills in colposcopy through research and education, was founded in 1965 and, as a member of the International Federation of Cervical Pathology and Colposcopy, remains a major force for the development of strategies for screening, diagnosis, and management of CIN.

Several technologic enhancements to colposcopy have appeared in the past few decades. Videocolposcopy allows for image recording in real time, with enhanced visualization and opportunities for patient and trainee education. Systems for videocolposcopy include those added to traditional optical colposcopes as well as video-only systems. A comparison study of optical and video systems shows that use of video systems by even experienced colposcopists requires a learning phase but that similar findings result.³⁵ Computerized image analysis and image storage yields the potential to minimize subjective analysis of lesions and offer the option of serial follow-up with quantification of changes in lesion size and character.^{36, 37, 38} In addition, computerized colposcopy allows the integration of nonvisible spectra and tissue fluorescence into image analysis.³⁹

An understanding of the natural history of CIN has developed progressively. The preinvasive nature of CIS was the subject of prolonged debate,^{3, 40, 41} resolved once the ability of CIS to progress or to regress after biopsy was recognized. After this, the importance of lesser degrees of neoplastic change was investigated. Both the graded severity of lesions of the cervical epithelium and the sometimes prolonged interval between diagnosis of dysplasia and development of invasive cervical cancer came to be understood.^{42, 43, 44, 45, 46} The concept of cervical dysplasia as a continuum of disease was elaborated in the 1950s,^{47, 48} and the term dysplasia was agreed on at the first International Congress of Exfoliated Cytology in 1962⁴⁹ (Fig. 1). Recognizing that pathologic distinctions between severe dysplasia and CIS are difficult to reproduce,^{50, 51} Richart developed the CIN classification system currently used for description of histologic specimens, describing CIN as a continuum of neoplastic change with progressively increasing risk of invasion.⁵²

Once CIS was linked to invasive cancer, principles of management of CIS were derived from those established for cervical cancer, with radical and modified radical hysterectomy recommended for women with CIS.^{16, 22, 51, 53} This probably was appropriate when no method for thoroughly evaluating the cervical transformation zone existed short of cone biopsy and when cone biopsies were not universally performed or exhaustively sectioned because numerous patients undergoing hysterectomy for CIS were found to have foci of invasive cancer.^{15, 54} Once lesser dysplasias were recognized as cancer precursors, hysterectomy also was recommended for these lesions.⁵⁵ However, data accumulated to show that intraepithelial neoplasms did not metastasize and that hysterectomy could be avoided when invasion was excluded by meticulous examination of cone biopsy specimens.^{56, 57, 58, 59, 60} Conservative office techniques for treatment of CIN were developed. These include, in order of their incorporation into practice, electrocautery, cryotherapy, carbon dioxide laser ablation and conization, and loop excision.^{61, 62, 63, 64, 65, 66, 67} Studies of natural history suggest that some patients with early CIN may have spontaneous regression of lesions and may require no treatment.^{68, 69}

Cytologic and clinical observations led Ayre to the concept that viral infection of “halo cells” represents the first stage of cervical carcinogenesis.⁷⁰ This concept was validated when Zur Hausen defined the link between HPV and cervical dysplasia in 1977,⁷¹ and the concept of high- and low-risk HPV subtypes was recognized in the years after.^{72, 73, 74} Through the 1980s, as HPV testing became more available, substantial epidemiologic and biochemical evidence accumulated supporting the role of HPVs as necessary but not sufficient factors for cervical oncogenesis. A full discussion of these and other areas in HPV research is presented in a separate chapter.

By the late 1980s this quickly expanding body of knowledge led to confusion among clinicians regarding the proper management of women with abnormal findings on cytopathology reports, especially those consistent with early lesions.  One attempted solution was the development of automated cytologic systems based on image recognition software.⁷⁵ Adjunctive measures, such as HPV typing, and new instruments were developed to improve the sensitivity of cytologic study as a screening test for cervical cancer, especially for women with borderline abnormalities.^{76, 77, 78, 79, 80}

The National Institutes of Health addressed the issue through consensus conferences charged with the development of standards for diagnosis and management of abnormal cytologic findings.^{81, 82, 83} First, the Bethesda system for cervical cytology interpretation was promulgated. Afterward, interim guidelines for management were devised. Recently, criteria for diagnosis have been published to standardize use of the various categories of the Bethesda system. The results of this group have been widely accepted, but not without criticism.^{84, 85, 86}

Finally, in the last few decades, molecular biologists have explored the changes that lead cells in the cervical epithelium to become neoplastic. In addition to the inactivation of tumor suppressors from the p53 and retinoblastoma genes by HPV proteins, investigators have identified alterations in the expression of the epidermal growth factor receptor and the ras and myc oncogenes.^{87, 88, 89, 90, 91} Allelic loss follows the induction of tetraploidy and aneuploidy by p53 inactivation.^{92, 93} Rates of apoptosis fall as proapoptotic proteins are down-regulated.^{94, 95} Loss of heterozygosity analyses suggest that other important tumor suppressor genes may be deleted in preneoplastic cervical epithelium,^{96, 97} including alterations in expression of the fragile histidine triad gene on chromosome 3.^{98, 99} Angiogenesis is activated, and matrix interactions are altered as lesions advance.^{100, 101} Despite these advances, however, the precise sequence of genetic alterations after HPV infection that leads to neoplastic transformation in the cervix remains to be elucidated.

The sole rationale for the identification of cervical intraepithelial neoplasms is their risk for progression to invasive cancer. These lesions often have no symptoms and cause no morbidity. Considerable attention has been devoted to determining the potential of CIN to progress, and varying recommendations for intervention or observation are based on differing interpretations of these data. Considerable differences exist among estimates of progression and regression rates for CIN, but large series have found that CIN I can progress to invasive cancer and that even CIS sometimes may regress.

Existing studies of the natural history of CIN are limited by several factors, which have been reviewed.¹⁰² First, confirmation and definitive classification of CIN requires biopsy. However, biopsy can alter the natural history of CIN, perhaps by inciting a local inflammatory reaction. Some studies have avoided this by diagnosing CIN through cytologic examination, but even when combined with colposcopy, this approach risks both overdiagnosis and underdiagnosis, with consequent overestimation or underestimation of progressive potential. A second limitation to studies of the natural history of CIN is the duration of follow-up. Progression to invasive cancer requires years, especially for early lesions, and many patients in most studies have been lost, leaving their status in question. A third factor confounding interpretation of natural history studies is the heterogeneity of some CIN lesions. Colposcopy with directed biopsy may miss significant lesions, and sampling errors may lead to misclassification of some patients, with substantial impact on risk for progression or regression of disease. A fourth confounder is differences in interpretation of lesion grade among pathologists. Finally, patient factors such as age may modify the risk of progression within each grade of CIN. Local immune factors may predispose to regression, and women with impaired immunity, such as those infected with human immunodeficiency virus (HIV) and those with renal allografts, are at increased risk for progression.^{103, 104} When determining the appropriateness of various strategies balancing cervical cancer prevention with the morbidity of treatment, clinicians should keep in mind these limitations and modify general recommendations to fit the needs of individual patients.

The understanding of the invasive potential of CIN began with studies of patients with CIS. McIndoe and colleagues describe a group of 131 women with persistent abnormal cytologic findings after diagnosis of CIS; over time and despite therapy, more than a fifth developed invasive cancer, and the 20-year likelihood of progression to invasion was estimated at almost 40%.¹⁰⁵ The lifetime risk of developing invasive cancer among women with untreated CIS is likely to be higher still.

Although most clinicians recommend treatment immediately after diagnosis for women with CIN II, studies of the natural history of this lesion have been performed without therapy. Using cytologic study with microcolposcopy but not biopsy, Richart and Barron estimate the risk of progression of untreated CIN II to CIS at more than 90% after 14 years of follow-up.⁵⁰ Conversely, a meta-analysis of 15 studies spanning almost 35 years yielded an estimate of progression for CIN II of 20%, with 5% progressing to invasive cancer, 40% regressing, and 40% persisting unchanged.¹⁰⁶

Because of its low risk for progression, CIN I has been studied most intensively. Ostor estimates the likelihood of regression at 60%, with only 10% progressing to CIN III and just 1% advancing to invasive cancer.¹⁰⁶ Women infected with high-risk HPV subtypes may have a greater risk of progression,¹⁰² but prospective studies using HPV typing as triage for women with colposcopically demonstrated low-grade lesions have not yet been reported. Epidemiologic data from the large registry in British Columbia suggest that age also determines risk for progression: among women younger than 34 years of age with new lesions, 84% regressed, whereas only 40% of older women did so.¹⁰⁷

A useful screening test should identify asymptomatic patients at high risk for a disease of significant morbidity and mortality at a point in the disease course where intervention can alter outcome. Cervical cytologic study does this well. At the same time, an optimal screening test meets these objectives at low cost and with acceptable sensitivity and specificity. Precise definition of the sensitivity and specificity of cervical cytologic study is impossible because the gold standard against which it must be compared is histologic evaluation of the entire cervical transformation zone; the performance of random cone biopsies on cytologically normal women solely to determine these rates cannot be justified ethically. Still, the false-positive rate of cervical cytologic study has been determined using colposcopy and cone biopsy or cervical loop excision. This rate appears to vary substantially with the grade of CIN identified cytologically, with up to 30% of women with low-grade smears lacking identifiable CIN.^{108, 109} The false-negative rate for cytologic study among patients with cancer can range as high as 50%; because blood and inflammatory cells may obscure neoplastic cells in such patients, this rate appears to be lower for women with CIN.¹¹⁰

The accuracy of cytologic study depends on the prevalence of CIN in the population being screened. Epidemiologic risk factors are described in another chapter; populations with a high proportion of women with high-risk factors merit more intensive screening. The incidence of CIN exceeds that of invasive cancer of the cervix. This probably is a result both of the natural history of CIN, as described earlier, and of the identification and eradication of CIN in screened populations before invasion develops. Because CIN is not a reportable disease, its incidence is speculative, based on extrapolation of findings at a few centers; however, if, as noted earlier, 5% of smear results are abnormal, the US incidence must be several hundred thousand.

The false-negative rate falls significantly when serial smears are taken. Using published data and statistical analysis, Eddy estimates that the risk of cervical cancer fell 64% when screening was performed at 10-year intervals, a result that improved to 91% with 3-year screening intervals.¹¹¹ Increasing the frequency of sampling to annually results in only a small further decrease in risk. Epidemiologic risk factors that favor annual screening include early initiation of sexual activity, a history of multiple lifetime sexual partners, tobacco use, and immunosuppression. Unfortunately, high-risk women may lack sufficient sophistication to respond correctly to queries about screening.¹¹² Across socioeconomic classes, the reliability of women's recollection of the interval since the last smear and of their history of abnormal findings may be unreliable.¹¹³ Without documentation, a lifetime history of normal smear results cannot be assumed.

Cervical cytologic study was adopted without rigorous testing as an important way to detect cervical cancer because of its manifest superiority to techniques of inspection, palpation, and iodine staining with blind biopsy available at its introduction. Despite the absence of clinical trials, cervical cytologic study remains the standard tool for cervical cancer screening. Several reports correlate significant declines in the incidence of cervical cancer with the institution of widespread screening programs.^{114, 115, 116, 117} It has been proposed that cervical cancer could be eradicated if all women complied strictly with screening guidelines.¹¹⁸ Nevertheless, despite widespread incorporation of cytologic screening into clinical practice, cervical cancer remains a significant public health problem.

The reasons for this are many and have been reviewed.¹¹⁸ Unfortunately, many women fail to comply with screening recommendations, and many patients with cervical cancer acknowledge that they have not been screened adequately.^{113, 119} The reasons for this are not clear, but studies suggest that significant reasons for inadequate screening include ignorance of guidelines, dislike of pelvic examination, lack of access to the medical care system, fear of cancer, fear of pain from diagnostic procedures, and mistrust of medical authorities.¹²⁰ Women who fail to receive adequate screening tend to be older and less educated and belong to minority ethnic groups.^{120, 121} Compliance with screening guidelines has been shown to be improved by education and physician-initiated reminders.^{122, 123}

Even when women comply with screening guidelines, inadequate cervical sampling may allow malignant and premalignant lesions to progress untreated. Badly lacerated cervices and endophytic neoplasms may contribute to this problem. Initially, Papanicolaou described vaginal pool aspirates as the source of cellular material for cytologic analysis. However, modern understanding of the location of CIN has led to the development of techniques for sampling the cervical transformation zone. Currently, sampling should be done from both the ectocervix and the endocervix. The Bethesda system originally classified smears that lacked an endocervical component as “satisfactory but limited” because of the risk that abnormalities at the squamocolumnar junction may have been missed.⁸² Endocervical cells are commonly absent in true-negative smear results from women with cervical cancer.¹²⁴

A variety of instruments exists to facilitate cytologic sampling of the cervix and endocervix, but none has been shown to be superior in the yield of cervical dysplasia diagnosed (Figs. 2 and 3). An endocervical brush increases the yield of endocervical cells sevenfold more than a moistened cotton-tipped applicator but may induce bleeding that can obscure smears.^{125, 126} Therefore, the ectocervix should be sampled first when an endocervical brush is used. Single-step instruments are available, but little literature is available to determine comparative efficacy.

Improper handling of smears also can result in missed lesions. Rapid fixation is essential to proper reading because air-drying introduces artifacts that make diagnosis of dysplasia difficult or impossible. Some suggest that submitting separate endocervical and ectocervical smears increases the sensitivity of cytologic screening, but this has not been shown definitively. Problems in cytologic analysis, including overworked cytotechnicians and inadequate quality control, likely contribute to the underdiagnosis of cervical cancer precursors.¹¹⁸

To minimize human error, computerized screening for cytologic abnormalities has been developed. Systems currently use neural network artificial intelligence technology with iterative improvement.^{127, 128} When used for automated rescreening, these devices review smears classified by cytotechnologists as normal, selecting smears with the most abnormal cells for review. In contrast, when used for primary screening, smears with the least abnormal cells are identified and reported as normal, whereas the remaining slides are re-screened by cytotechnologists. Re-screening improves the sensitivity of cytologic study, although most additional lesions identified are of low grade. Cost is greater than for conventional manual analysis.

An additional strategy for minimizing human error in cytologic study has been the development of liquid-based monolayer technology.^{129, 130} Cells sampled from the cervix are placed in fixative and applied mechanically to slides for review either by cytotechnologists or through an automated screening system. Obscuring of neoplastic cells by blood, leukocytes, and clumping is minimized. Again, the increased sensitivity of monolayer cytology is achieved primarily through the detection of low-grade lesions, whereas cost is increased above that of conventional smears. The liquid fixative can be used for HPV detection to enhance screening.¹³¹

A final source of missed opportunities for cervical cancer prevention can occur when communication between physician and cytopathologist is compromised. The Bethesda system for classification of abnormalities was developed to standardize nomenclature to minimize misunderstandings between cytologists and treating physicians, and was modified in 2001. This classification is summarized in Table 1. The initial classification notes whether the smear is satisfactory for evaluation or unsatisfactory. Reasons for unsatisfactory smears include obscuring blood or pus, broken slides, inadequate cellularity, and insufficient patient information. The intermediate category “satisfactory but limited” was eliminated in the 2001 Bethesda System; it was often confusing to clinicians and led to unnecessary repeat testing.

Table 1. The 2001 Bethesda System¹³² (modified from the original publication)

The Bethesda system also describes whether a smear is normal, shows benign abnormalities, or contains neoplastic or potentially neoplastic cells. Benign abnormalities included in the Bethesda classification system include changes consistent with such infections as candidiasis, trichomoniasis, bacterial vaginosis, and infection with herpes simplex or Actinomyces, as well as reactive changes such as metaplasia and reparative changes that follow radiation, atrophy, inflammation, or intrauterine contraceptive devices.

Epithelial abnormalities are classified under the Bethesda system as glandular or squamous. In addition to adenocarcinoma and the finding of endometrial cells out of cycle or after menopause, atypical glandular cells are categorized as to their likely origin and whether or not they favor neoplasia. Squamous abnormalities include atypical squamous cells of uncertain significance (ASC-US), as well as atypical squamous cells for which HSIL cannot be ruled out (ASC-H). The ASC-US category should represent no more than 5% of smears from patients of average risk; clinics with many high-risk patients may have a higher proportion of abnormal smears. Some 30% of women with ASCUS smears have dysplasia; the incidence of invasive cancer in this population is low. Cells that show changes consistent with mild dysplasia or HPV infection are classified as consistent with low-grade squamous intraepithelial lesions (LSIL). Most patients with such smears have condylomata or CIN I, fewer than 1–2% of women with LSIL smears have invasive cancer, about 5≥10% may have high-grade dysplasia (CIN II–III), and about 30% have no lesions. Cells that show changes consistent with CIN II–II are classified as high-grade squamous intraepithelial lesions (HSIL). Finally, some smears may be classified as showing frank invasive cancer; this diagnosis is based on the finding of a “tumor diathesis,” blood and inflammatory cells admixed with neoplastic cells. Such a diagnosis requires histologic confirmation before treatment.

Because the sensitivity of cytologic study as a screen for cervical cancer and its precursors is imperfect, alternative techniques for the identification of these abnormalities have been proposed. However, none has yet supplanted cytologic study, and all are currently experimental or limited to certain populations. Most have been tested as adjuncts to cytologic study, improving the sensitivity of Pap smear screening rather than replacing it.

The most widely studied alternative technique for cervical cancer screening is the use of HPV typing to identify women with papillomavirus infections. Women with positive screen results for HPV are at increased risk for CIN, and women infected with HPV types 16, 18, 31, 33, and 35 are more likely to have high-grade CIN⁷⁴. However, although the sensitivity of HPV typing appears to be excellent, its specificity is limited.^{77, 79} HPV typing is not indicated for women with smears read as HSIL or cancer, for whom the prevalence of high-grade CIN justifies immediate colposcopy. The high rate of positivity in women with LSIL smears negates its use.¹³³ The added expense of HPV testing does not appear to be a cost-effective improvement over serial cytologic study for women with atypical smears,¹³⁴ although it may have a role in clinical settings where access to colposcopy is restricted.⁷⁸ The utility of HPV testing may change as new assays are tested, and more definitive recommendations are likely to follow completion of a multicenter trial of HPV testing and other strategies for the management of ASC-US and LSIL smears.¹³³ The use of HPV testing as a primary screen has shown promise in developing countries but remains experimental.^{135, 136}

Colpophotography is a second alternative to cytologic screening, again one most often used as an adjunct rather than a replacement for Pap smears.^{77, 78} It involves the use of special photographic equipment to capture an image of the cervix after staining with acetic acid. Slides then are sent to a diagnostic center, where they are projected for magnified viewing by expert colposcopists who assess the transformation zone for abnormalities. Patients with abnormal or equivocal results can be referred for formal colposcopy.

Colposcopy is used in parts of Europe as a component of the standard gynecologic examination and as part of a screen for cervical cancer and its precursors. However, costly equipment and the need for intensive training make colposcopy prohibitively expensive as an initial screen. It may have a role in populations with a high incidence of CIN, such as women infected with the HIV. HIV-infected women often have multiple risk factors for the development of cervical cancer, and their immunosuppression appears to allow CIN and cancer to develop at an accelerated rate.^{137, 138} In some studies, the prevalence of CIN HIV-infected women exceeds 30% – sufficient frequency to consider screening colposcopy.¹³⁹ However, the Centers for Disease Control and Prevention recommend that women with HIV be screened cytologically; if initial screening results are normal or if initial smear shows reactive, inflammatory findings, screening should be repeated after 6 months.¹⁴⁰ The Bethesda guidelines for management do not apply to this high-risk group, and colposcopy appears to be indicated for all HIV-infected women with other abnormal smears, including ASCUS.^{140, 141}

In 2001 the American Society for Colposcopy and Cervical Pathology (ASCCP) and members of other national and international organizations joined to create consensus guidelines for the management of women with abnormal cervical screening results. Since that time a number of key studies looking at low-grade lesions, as well as the addition of HPV DNA testing to screening practices, have once again altered the way in which we interpret such results. In 2006 a consensus conference – again comprised of members of the ASCCP and other professional societies – convened at the National Institutes of Health to develop revised guidelines for the management of women with abnormal cervical cancer screening tests; their comprehensive recommendations are summarized below.¹⁴² It is critical to remember that each patient is individual, and that management decisions should be based on these guidelines, on the local prevalence of CIN, and on individual risk factors. Also, it should be noted that the guidelines below focus on the evaluation of abnormal cervical cytology and the detection of CIN; the management of CIN is discussed in the chapter 'Treatment of Cervical Intraepithelial Neoplasia'.

Atypical squamous cells – undetermined significance (ASC-US)

According to the 2006 ASCCP guidelines, for women over 20 years of age there are three acceptable ways to manage ASC-US on initial diagnosis. These include repeat PAP smears at 6-month intervals, HPV DNA testing, and referral for colposcopy. Women who are ASC-US and HPV negative can be followed with repeat cytology at 1 year, while women who have ASC-US and are HPV positive should undergo colposcopy. 

If colposcopy reveals a lesion at the transformation zone, biopsies should be taken and it is acceptable to obtain endocervical curettings. Endocervical sampling should always be performed if the colposcopy is unsatisfactory and/or no lesions are identified. Post-colposcopy, patients without CIN identified should be followed with either repeat cytology at 6 and 12 months, or with repeat HPV DNA testing at 12 months. If repeat cytology is chosen, PAP tests should be obtained every 6 months until two consecutive results are negative (“negative for intraepithelial lesion or malignancy”). If testing for HPV DNA is chosen, the ASCCP recommends not testing more frequently than every 12 months. 

According to the ASCCP guidelines there are special populations that need to be taken into account when interpreting cervical cytology results, including adolescents, immunosuppressed, postmenopausal, and pregnant women. Adolescents include women 20 years of age and younger; it is recommended that these women NOT undergo HPV DNA analysis. If ASC-US is identified in this population, annual cytology is recommended for follow-up. At 1 year from initial diagnosis, only a diagnosis of HSIL or greater warrants colposcopy referral. At 2 years from initial diagnosis, adolescents with ASC-US or greater should be referred for colposcopy. These guidelines stem from an accumulation of data showing that adolescents have a high prevalence of HPV and low-grade lesions which often regress spontaneously, and only rarely progress to invasive cancers. 

ASCCP guidelines regarding immunosuppressed and postmenopausal women with ASC-US indicate that these populations should be treated identically to the general population. Pregnant women over age 20 differ only in that colposcopy can be deferred until at least 6 weeks' postpartum, and that endocervical sampling is unacceptable in this population given the risk of rupturing fetal membranes.

Atypical squamous cells – cannot exclude HSIL (ASC-H)

Colposcopy is recommended for women with ASC-H according to the 2006 ASCCP guidelines. Management of CIN 2,3 – if identified – is discussed in the chapter 'Treatment of Cervical Intraepithelial Neoplasia'. However, in patients without CIN 2,3 at the time of colposcopy, acceptable follow-up includes repeat cytology at 6 and 12 months, or HPV DNA testing at 12 months. If repeat cytology returns consistent with ASC-US or greater, or if HPV is identified, repeat colposcopy is indicated. Patients may return to routine screening once the PAP smear is negative at two consecutive intervals, or if repeat HPV testing is negative.

Low-grade squamous intraepithelial lesion (LSIL)

Based on data from the ALTS study,^{143, 144} the 2006 ASCCP guidelines recommend treating LSIL and ASCUS-HPV positive women similarly, with the exception of some special populations. In the general population, a diagnosis of LSIL is cause for colposcopic evaluation. Women with identifiable lesions should have biopsies taken; women without visible lesions or with an unsatisfactory colposcopy exam should have endocervical curettings obtained. If CIN 2,3 is identified, a separate algorithm for management has been established by the ASCCP, and is discussed in the chapter 'Treatment of Cervical Intraepithelial Neoplasia'. If the colposcopy evaluation is negative for findings of CIN 2,3, patients may be followed up with HPV testing at 1 year, or with repeat cervical cytology at 6 and 12 months. Positive testing at follow-up (ASC-US or greater cytology, or high-risk HPV positive) warrants repeat colposcopy examination. Patients may return to standard screening guidelines once cytology is negative on two consecutive visits or HPV is negative at 1-year follow-up.

Special populations are again treated uniquely with respect to a diagnosis of LSIL on PAP testing. Adolescents (women under 20 years of age) should not be tested for HPV; the ASCCP goes as far to say that if HPV testing is undertaken inadvertently, that the results should not be used to influence management. Adolescents with LSIL should be evaluated with yearly PAP tests. Referral to colposcopy should be made only for HSIL or greater at 12 months out, and ASC-US or greater at 24 months out. 

At the other end of the spectrum, postmenopausal women with a diagnosis of LSIL may be managed in one of three ways according to the 2006 guidelines, including reflex HPV testing, repeat cytology at 6 and 12 months, or colposcopic evaluation. If HPV DNA testing is negative, or if colposcopy is negative for CIN, then repeat PAP testing at 12 months is considered appropriate management. If repeat cytology reveals ASC-US or greater, or if HPV is positive, then repeat colposcopy is warranted. Patients may return to routine cervical screening once two consecutive PAP tests are negative.

Finally, pregnant women over the age of 20 with LSIL cytology warrant colposcopic evaluation as per the ASCCP guidelines, though it is acceptable to defer that colposcopy until at least 6 weeks' postpartum. If colposcopy is undertaken during pregnancy, endocervical sampling is not recommended. As long as there is no evidence of CIN 2,3 or cancer, the most recent guidelines recommend postpartum follow-up without further cytology or colposcopy during the pregnancy.

High-grade squamous intraepithelial lesion (HSIL)

According to the 2006 conclusions and guidelines outlined by the ASCCP, the finding of HSIL on cytologic screening carries a reasonably high risk for cervical disease, and should be managed accordingly. In the general population (excluding adolescents and pregnant women), women with HSIL should be managed either with colposcopy including endocervical sampling, or with a loop electrosurgical excision procedure (LEEP). If CIN 2,3 is identified, the histology algorithm set forth by the ASCCP for management should be utilized. If CIN 2,3 is not identified, however, the patient and provider may elect for close observation or a diagnostic excisional procedure.  Observation entails repeat cytology and colposcopy at 6 and 12 months; it is important to note that in this case, the colposcopy must be satisfactory and the endocervical curettings must be negative. If HSIL is again encountered at 6 or 12 months on cytology, then a diagnostic excisional procedure is recommended. Similarly, a diagnostic excisional procedure should be performed if the colposcopy is unsatisfactory.

In adolescent populations, the ASCCP recommends colposcopy, and NOT immediate loop electrosurgical excision, for a diagnosis of HSIL on screening cytology. If CIN 2,3 is not encountered on the initial colposcopy, then the patient may be followed with repeat cytology and colposcopy every 6 months for up to 24 months. This is provided that the colposcopy is satisfactory and the endocervical evaluation is negative. Biopsy is only recommended if a high-grade lesion is identified on colposcopy or if HSIL persists for 1 year. If HSIL is persistent on cervical cytology for 24 months without a diagnosis of CIN 2,3, at that point a diagnostic excisional procedure is recommended. Similarly, a diagnostic excisional procedure is recommended if colposcopy examination is not satisfactory. 

Pregnant women should undergo colposcopy for a result of HSIL on screening cytology. However, the ASCCP recommends that only lesions consistent with CIN 2,3 or invasive cancer are biopsied. Biopsy of lesions appearing low-grade can be deferred until follow-up colposcopy no sooner than 6 weeks' postpartum.  Endocervical sampling and diagnostic excisional procedures are considered unacceptable unless invasive cancer is suspected.  

HPV DNA testing

As an evolving aide in the screening process for cervical dysplasia, the ASCCP guidelines comment only briefly on HPV DNA testing as an adjunct to routine cytology. Only women 30 years of age and older should undergo routine HPV testing with PAP smears; most laboratories perform reflex testing for ASC-US diagnoses in younger women. Women 30 years or older with negative cytology and negative HPV may space out their cervical screening to be performed every 3 years. Those with negative cytology but who are HPV positive are recommended to have repeat cytology and HPV DNA testing in 1 year. Finally, women with other epithelial abnormalities (ASC-H, LSIL, HSIL) should be managed as above, regardless of HPV diagnosis. 

As noted earlier, screening tests for CIN are imperfect, with limited sensitivity and specificity. The definitive diagnosis of CIN requires colposcopy and biopsy, with treatment based on histologic findings.

The colposcopic exam begins with careful visual inspection of the vagina and cervix. A warm speculum moistened with water, not lubricant, is inserted into the vagina so that the entire cervix is visualized. After the cervix is wiped clean of excess mucus and debris, if present, repeat cytologic samples may be obtained, an especially important procedure when a significant delay has occurred since the PAP smear that prompted the colposcopic evaluation.

The cervix next is inspected with the colposcope, a stereoscopic binocular microscope capable of at least 16× magnification, a center light with green filter, and an adjustable stand. Colposcopes are available with a variety of accessories, including adjustable magnification, real-time video, still cameras, ocular arms that allow simultaneous examination by a student or preceptor, and CO₂ lasers (Fig. 5).

Fig. 5. The colposcope. (Courtesy of Carl Zeiss, Inc.)

The cervix is examined through the colposcope in a clockwise fashion, concentrating on the squamocolumnar junction, the border between squamous epithelium of the exocervix, and the columnar epithelium of the endocervix, where most dysplasia appears to originate. If no obvious malignant lesions are noted, 3–5% acetic acid then is liberally applied to the cervix. This not only cleans the cervix, it also alters the reflectivity of superficial cells, accentuating atypia and highlighting vasculature. The cervix again is inspected, concentrating on the transformation zone, the area of metaplastic transition between the native squamous epithelium, which is red and smooth, and the columnar epithelium, which has a white, grape-like appearance after the application of acetic acid. Once inspection with white light has been completed, inspection is completed with a green filtered light, which enhances vascular patterns.

Colposcopic abnormalities include leukoplakia, acetowhite epithelium, punctation, mosaicism, and atypical vessels. White epithelium occurs from an accumulation of cells with an increased nuclear-cytoplasmic ratio; leukoplakia is white in its native state, whereas acetowhite epithelium appears only after the application of acetic acid. Dull white lesions with rolled peeling edges that are quick to stain may represent higher grade lesions than more transparent, slow-staining lesions with indefinite margins.¹⁴⁵ Punctation results from visualization of capillaries that lie perpendicular to the surface epithelium, with coarse punctate patterns associated with higher grade lesions than fine ones. Mosaicism represents capillaries running parallel to and underneath the surface epithelium, with low-grade lesions also having a finer pattern. Atypical vessels are associated with high-grade lesions or invasive cancers and usually show a corkscrew or hairpin configuration, as opposed to the pronounced but normally branching vasculature associated with inflammation or nabothian cysts. In performing colposcopy, keep in mind the association of high-grade lesions with larger transformation zones (63 mm_² or more)¹⁴⁶ and take time to evaluate them thoroughly.

Colposcopy can only exclude the presence of an invasive cancer when the cervical transformation zone is adequately seen. An adequate colposcopy is one in which the entire transformation zone is visualized, along with the entire extent of any lesion beginning at the transformation zone; colposcopy is termed inadequate when complete visualization cannot be achieved. During a woman's lifetime, this zone migrates into the endocervix by a process of squamous metaplasia of the columnar epithelium. The three periods of most active metaplasia, which are under the influence of estrogen, progesterone, and vaginal pH, are in the fetus, at adolescence, and at the time of the first pregnancy. The position of the squamocolumnar junction is directly related to age, gravidity, and parity. It is located in the endocervical canal in only 26% of women aged 16–20 years, but in 100% of women older than 60 years and in 67% of grand multiparas.¹⁴⁷

If the entire transformation zone cannot be visualized because of migration into the endocervical canal, an endocervical speculum may be useful. If this is done, an estimate of the depth of the squamocolumnar junction should be made so that therapy, if required, can be carried to an appropriate level. When the location of the squamocolumnar junction is obscured by mucus or debris, visualization can be improved by manipulation with a cotton-tipped applicator. In postmenopausal women with inadequate colposcopy, 4–6 weeks of vaginal estrogen therapy before repeat colposcopy may improve visualization of the squamocolumnar junction. When colposcopy is inadequate after dysplastic cytology, cone biopsy is indicated to exclude invasive cancer.

Although various grading systems for colposcopy have been proposed, colposcopy alone is inadequate to determine the grade of dysplasia, when present, or to reliably exclude small invasive cancers. Rather, colposcopy is used to direct biopsies to the most abnormal areas of the cervix. Greatest accuracy results when biopsy specimens are taken liberally from all areas of colposcopic abnormality. Of 195 cases reported from a public hospital colposcopy clinic, when the colposcopic impression was only a condyloma, 24% of the biopsies revealed high-grade lesions.¹⁴⁸

Colposcopically directed biopsies are performed with a Kevorkian-Younge instrument, which can remove a sample 2–3 mm wide and several millimeters deep; larger instruments, such as the cup or Tischler forceps, are more appropriate for suspected cancers. The specimen, placed on a paper towel to aid the pathologist in orientation and thus avoid tangential sectioning, then is fixed in formalin or Bouin's solution. Each specimen should be placed in a separate container labeled with the location of the biopsy site. Locations on the cervix are described using the 12 hours of a clock face as reference. The colposcopic appearance of the cervix should be sketched or photographed for comparison with findings at follow-up examination.

ECC (endocervical curettage) has been advocated as an important part of the diagnostic evaluation of women with abnormal results on screening cytologic study. When ECC is performed, a Kevorkian curette is used to scrape twice around the circumference of the endocervix from the internal os to the squamocolumnar junction (Fig. 6). Tissue, mucus, blood, and debris all should be collected into fixative. The purpose of ECC is to identify whether neoplastic cells are present in the endocervical canal beyond the limits of colposcopic visualization. When ECC shows dysplasia, cone biopsy is indicated to exclude invasive cancer in the endocervical canal.

Fig. 6. Instruments used for colposcopy. From the top: Graves speculum, Kevorkian-Younge biopsy forceps, Kevorkian curet, and endocervical speculum.

The need for ECC is controversial. The sensitivity of ECC in identifying endocervical dysplasia is less than that of brush cytologic study (55% versus 92%), although it increases to 83% if abundant tissue is retrieved. However, specificity is higher, at 75% as opposed to 38% for the cytobrush;¹⁴⁹ because suspicion of endocervical disease requires cone biopsy for confirmation, with attendant morbidity and cost, we prefer the more specific test. Some argue that ECC need not be done in most premenopausal women because of the rarity of endocervical lesions in these patients.¹⁵⁰ Further, a study of 28 nonpregnant adolescents younger than 17 years of age evaluated for abnormal cytologic findings in our dysplasia clinic revealed no positive ECC results. However, others conclude that ECC should be done in selected cases,¹⁵¹ and still others argue that it should always be done.¹⁵² In one study of 1500 women, nine of the 11 women with abnormal cytologic findings, adequate colposcopy results, negative biopsy results, and positive ECC findings had dysplasia that would have been missed without ECC.¹⁵³ A review of 540 patients demonstrated that ECC led to procedures that resulted in diagnosis of a lesion more severe than that identified by colposcopy in 3.8% of women with adequate colposcopy and 21.1% in those with inadequate colposcopy.¹⁵² Most persuasively, a study of cervical cancer developing after cryotherapy suggested that omission of ECC was commonly associated with failure.¹⁵⁴ Therefore, an ECC is part of the colposcopic evaluation of every nonpregnant women examined in the dysplasia clinic at Cook County Hospital.

Colposcopic biopsies involve moderate discomfort and pain. A variety of methods have been used to decrease pain and anxiety related to the procedure. Topical anesthetic applied to the cervix immediately before biopsy has led to mixed results, leading to a significant reduction of pain in one study using 20% benzocaine¹⁵⁵ but not in another using lidocaine.¹⁵⁶

Colposcopy alone is insufficient for the specific diagnosis of cervical neoplasia when unsatisfactory in visualizing the squamocolumnar junction, when accompanied by a positive ECC result, when it has failed to identify a lesion consistent with that suggested by cytologic study, or when microinvasion is identified. In these cases, cone biopsy should be performed, and 5–20% of all women evaluated for abnormal cytologic findings will need such a procedure.¹⁵⁷ Cone biopsy can be done using either a scalpel or a diathermy loop. Comparative trials suggest that in expert hands, loop excision requires less anesthetic, can be performed in clinic, results in less blood loss, and yields an equivalent specimen.^{158, 159} However, when conization is performed using the diathermy loop, care must be taken to excise the entire lesion; in cases of inadequate colposcopy, this may require use of loops up to 2 cm in depth or multiple endocervical excisions under colposcopic guidance. Loop excision does result in coagulation artifact at the margin of excision. Therefore, we prefer cold-knife conization when adenocarcinoma in situ is suspected or when microinvasion is present. In addition, knife conization may be preferable for the multiparous, lacerated cervix because it allows the excision to be tailored to the abnormal anatomy.

Major complications of cone biopsy include the immediate problems of hemorrhage, uterine perforation, and anesthetic risk. Delayed bleeding also may occur. Other late complications may include loss of cervical mucus, infertility, cervical stenosis, and incompetent cervix. Such complications are uncommon. Discrepant results have been reported from studies of the effect of conization on cervical competence and associated premature delivery.^{160, 161}

To ensure adequate excision of all lesional tissue and the entire cervical transformation zone, colposcopy or painting with Lugol's solution should be done immediately before cervical conization. The placement of absorbable hemostatic sutures at 3-o'clock and 9-o'clock positions and injection of dilute phenylephrine or vasopressin may facilitate manipulation and minimize blood loss. Some advocate the injection of sterile saline to compress small vessels and facilitate hemostasis.¹⁶² Results of ECC performed immediately after excision correlates with prognosis for recurrence.¹⁶³ To orient the pathologist, the specimen should be marked without injuring the epithelium.

Although the goal of colposcopy in nonpregnant women is the identification of preinvasive neoplasms before ablation, the goal of colposcopy in pregnant patients is the identification or exclusion of invasive cancer. Preinvasive lesions have no effect on pregnancy and should not prompt treatment, but a diagnosis of invasive cancer may require pregnancy termination to save the mother's life. The technique of colposcopy is identical in pregnant and nonpregnant patients, but metaplastic acetowhite epithelium may be more prominent in pregnancy, and the engorged pregnant cervix may present unusually prominent vessels or glands. ECC should never be undertaken during pregnancy because of the risks of rupture of the fetal membranes and perforation of the softened cervix. The safety of a cytobrush in pregnant women has been evaluated without apparent complication but is not advocated by the manufacturer.¹⁶⁴

Lurain and Gallup followed 131 women in the second and third trimesters of pregnancy until after delivery who had abnormal cytologic findings but no colposcopic evidence of invasive cancer without biopsy; no cancers were missed.¹⁶⁵ Among 401 pregnant women who had colposcopy with biopsy, colposcopic impression was accurate within one degree in 87%; only 3% had a more advanced histologic type. Women older than 30 years of age had a 6% incidence of cancer, whereas among younger women, the incidence was just 1%, suggesting that biopsy is most appropriate for older women or those with findings consistent with CIS.¹⁶⁶ Profuse bleeding may follow biopsy during pregnancy, but usually it can be controlled by rapid application of direct pressure and thickened Monsel's solution. If undertaken, biopsy should be limited to a single site. Dysplasia detected during pregnancy has a greater rate of regression than when detected in a nongravid woman.¹⁶⁷ Once evaluation and treatment no longer pose hazards to the fetus, colposcopy with ECC is repeated, usually about 6 weeks after delivery.

Cone biopsy rarely is required during pregnancy. When indicated, it should be performed cautiously after placement of a circumferential hemostatic suture. The biopsy should be as shallow as possible, and some authorities advocate a “wedge” biopsy instead.¹⁶⁸ One analysis of 448 cone biopsies performed during pregnancy revealed no increase in pregnancy loss and an estimated blood loss in excess of 500 mL in 9%.¹⁶⁹ Anecdotal reports of loop excision to exclude malignancy exist, but no series large enough to allow estimation of efficacy or morbidity has been reported.

Finally, hysterectomy may rarely be required for the diagnosis of patients with abnormal screening results. Usually, these patients are postmenopausal with cervices that are flush with the vaginal fornices. Often, prior cone biopsy has left insufficient tissue for repeat conization without risk of bladder injury. At times, prior surgery has resulted in agglutination of the cervix, leaving the clinician unable to rely on serial cytologic study to exclude persistent dysplasia or progressive carcinoma high in the canal. Once clinical examination has excluded parametrial involvement by cancer, hysterectomy can be undertaken. The cervix should be serially sectioned, as for a cone biopsy. If deeply invasive carcinoma is found, cure rates equivalent to those after radical hysterectomy can be achieved with radiation or radical reoperation.^{170, 171}