Berek and Novak's Gynecology 15th Ed.

19 Intraepithelial Disease of the Cervix, Vagina, and Vulva[JL1]

Francisco Garcia

Kenneth D. Hatch

Jonathan S. Berek

• Cervical intraepithelial neoplasia (CIN) most often arises in an area of metaplasia in the transformation zone at the advancing squamocolumnar junction (SCJ). Metaplasia advances from the original SCJ inward, toward the external os and over the columnar villi, to establish the transformation zone. CIN is most likely to begin either during menarche or after pregnancy, when metaplasia is most active; after menopause, metaplasia is less active and a woman has a lower risk of developing CIN.

• Untreated, most CIN 1 and some CIN 2 lesions spontaneously regress; nevertheless, CIN refers to a lesion that may progress to invasive carcinoma. This term is equivalent to the term dysplasia, which means abnormal maturation; consequently, proliferating metaplasia without atypical mitotic activity should not be called dysplasia. Squamous metaplasia should not be diagnosed as dysplasia (or CIN) because it does not progress to invasive cancer.

• More than 90% of CIN is attributed to human papillomavirus (HPV) infection. Only certain types of HPV cause high-grade intraepithelial lesions and cancer (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68). Type 16 is the most common form of HPV found in invasive cancer and in CIN 2 and CIN 3; it is found in 47% of women with cancer.

• Potentially premalignant squamous lesions fall into three categories: (i) atypical squamous cells (ASC), (ii) low-grade squamous intraepithelial lesions (LSIL), and (iii) high-grade squamous intraepithelial lesions (HSIL).The ASC category is subdivided into two categories: those of unknown significance (ASC-US) and those in which high-grade lesions must be excluded (ASC-H).

• The LSIL category includes CIN 1 (mild dysplasia) and the changes of HPV, termed koilocytotic atypia. The HSIL category includes CIN 2 and CIN 3 (moderate dysplasia, severe dysplasia, and carcinoma in situ).

• The spontaneous regression rate of biopsy-proven CIN 1 is 60% to 85% in prospective studies. The regressions typically occur within a 2-year follow-up with cytology and colposcopy. For LSIL that persists longer than 2 years, the choice of treatment is optional. Expectant management is still appropriate in some patients, and ablative therapies, including cryotherapy and laser ablation, are acceptable treatment modalities.

• When a cytologic specimen suggests the presence of HSIL, colposcopy and directed biopsy should be performed. Although high-grade CIN can be treated with a variety of techniques, the preferred treatment for CIN 2 or 3 in nonadolescent patients is loop electrosurgical excision procedure (LEEP).

• Atypical endocervical cells pose a risk for adenocarcinoma in situ (AIS), which must be considered a precursor of adenocarcinoma.

• After sampling to rule out invasive disease, vaginal intraepithelial neoplasia (VAIN 3 lesions can be treated with laser or outpatient excisional therapy. Patients with vaginal intraepithelial neoplasia (VAIN 1 and most VAIN 2 and HPV infection do not require treatment. These lesions are multifocal and often regress, but may recur after ablative therapy.

• Vulvar intraepithelial neoplasia, grade 3 (VIN 3), is treated by simple excision, laser ablation, or superficial (partial) vulvectomy, with or without split-thickness skin grafting. Excision of small foci of disease produces excellent results, and although multifocal or extensive lesions may be difficult to treat by this approach, it offers the most cosmetic result. VIN 1 or 2 is generally associated with dystrophic changes or HPV and can be managed expectantly.

• Intraepithelial disease frequently occurs in the cervix, vagina, and vulva, and it may coexist in these areas. The cause and epidemiologic basis are common to all three locations, and treatment typically is ablative, excisional, and conservative. Early diagnosis and management are essential to prevent disease from progressing to invasive cancer.

Cervical Intraepithelial Neoplasia

The concept of preinvasive disease of the cervix was introduced in 1947, when it was recognized that epithelial changes could be identified that had the appearance of invasive cancer but were confined to the epithelium (1). Subsequent studies showed that these lesions, if left untreated, could progress to cervical cancer (2). Improvements in cytologic assessment led to the identification of early precursor lesions called dysplasia, a name that acknowledges the malignant potential of these lesions. Historically carcinoma in situ (CIS) was treated very aggressively (most often with hysterectomy), whereas dysplasias were believed to be less significant and were not treated or were treated by colposcopic biopsy and cryosurgery. The concept of cervical intraepithelial neoplasia (CIN) was introduced in 1968, when Richart suggested that dysplasias have the potential for progression (3). Most untreated CIN 1 and some CIN 2 lesions regress spontaneously; nevertheless, high-grade CIN refers to a lesion that may progress to invasive carcinoma when left untreated (4). Proliferating metaplasia without mitotic activity should not be called dysplasia or CIN because it does not progress to invasive cancer.

The criteria for the diagnosis of intraepithelial neoplasia may vary according to the pathologist, but the significant features are cellular immaturity, cellular disorganization, nuclear abnormality, and increased mitotic activity. The extent of the mitotic activity, immature cellular proliferation, and nuclear atypia identifies the degree of neoplasia. If the presence of mitoses and immature cells is limited to the lower third of the epithelium, the lesion usually is designated as CIN 1. Involvement of the middle and upper thirds is diagnosed as CIN 2 and CIN 3, respectively (Fig. 19.1).

Figure 19.1 Diagram of cervical intraepithelial neoplasia compared with normal epithelium.

Cervical Anatomy

The cervix is composed of columnar epithelium, which lines the endocervical canal, and squamous epithelium, which covers the exocervix (5). The point at which they meet is called the squamocolumnar junction (SCJ) (Figs. 19.2and 19.3).

Figure 19.2 The cervix and the transformation zone.

Figure 19.3 Diagram of the cervix and the endocervix.

The Squamocolumnar Junction

The SCJ rarely remains restricted to the external os. Instead, it is a dynamic point that changes in response to puberty, pregnancy, menopause, and hormonal stimulation (Fig. 19.4). In neonates, the SCJ is located on the exocervix. At menarche, the production of estrogen causes the vaginal epithelium to fill with glycogen. Lactobacilli act on the glycogen to lower the pH, stimulating the subcolumnar reserve cells to undergo metaplasia (5).

Figure 19.4 Different locations of the transformation zone and the squamocolumnar junction during a woman’s lifetime. The arrows mark the active transformation zone.

Metaplasia advances from the original SCJ inward, toward the external os and over the columnar villi. This process establishes an area called the transformation zone. The transformation zone extends from the original SCJ to the physiologically active SCJ, as demarcated by the squamocolumnar junction. As the metaplastic epithelium in the transformation zone matures, it begins to produce glycogen and eventually resembles the original squamous epithelium, both colposcopically and histologically (Fig. 19.5A and B).

Figure 19.5 A: Active metaplasia in the transformation zone. B: Maturing metaplasia in the transformation zone.

In most cases, CIN is believed to originate as a single focus in the transformation zone at the advancing SCJ. The anterior lip of the cervix is twice as likely to develop CIN as the posterior lip, and CIN rarely originates in the lateral angles. Once CIN occurs, it can progress horizontally to involve the entire transformation zone, but it usually does not replace the original squamous epithelium. This progression usually results in CIN with a sharp external border. Proximally, CIN involves the cervical clefts, and this area tends to have the most severe CIN lesions. The extent of involvement of these cervical glands has significant therapeutic implications because the entire gland must be destroyed to ensure elimination of the CIN (5). The only way to determine where the original SCJ was located is to look for nabothian cysts or cervical cleft openings, which indicate the presence of columnar epithelium. After the metaplastic epithelium matures and forms glycogen, it is called the healed transformation zone and is relatively resistant to oncogenic stimuli. The entire SCJ with early metaplastic cells is susceptible to oncogenic factors, which may cause these cells to transform into CIN. Therefore, CIN is most likely to begin either during menarche or after pregnancy, when metaplasia is most active. Conversely, after menopause a woman undergoes little metaplasia and is at a lower risk of developing CIN from de novohuman papillomavirus (HPV) infection. Oncogenic factors are introduced through sexual contact in general and intercourse in particular. Although several agents, including sperm, seminal fluid histones, trichomonas, chlamydia, and herpes simplex virus, were studied, it is established that persistent high-risk oncogenic HPV infection is the overwhelming risk factor for the development of CIN.

Normal Transformation Zone

The original squamous epithelium of the vagina and exocervix has four layers (5):

1. The basal layer is a single row of immature cells with large nuclei and a small amount of cytoplasm.

2. The parabasal layer includes two to four rows of immature cells that have normal mitotic figures and provide the replacement cells for the overlying epithelium.

3. The intermediate layer includes four to six rows of cells with larger amounts of cytoplasm in a polyhedral shape separated by an intercellular space. Intercellular bridges, where differentiation of glycogen production occurs, can be identified with light microscopy.

4. The superficial layer includes five to eight rows of flattened cells with small uniform nuclei and a cytoplasm filled with glycogen. The nucleus becomes pyknotic, and the cells detach from the surface (exfoliation). These cells form the basis for Papanicolaou (Pap) testing.

Columnar Epithelium

Columnar epithelium has a single layer of columnar cells with mucus at the top and a round nucleus at the base. The glandular epithelium is composed of numerous ridges, clefts, and infoldings and, when covered by squamous metaplasia, leads to the appearance of gland openings. Technically, the endocervix is not a gland, but often the term gland openings is used.

Metaplastic Epithelium

Metaplastic epithelium, found at the SCJ, begins in the subcolumnar reserve cells (Fig. 19.4). Under stimulation of lower vaginal acidity, the reserve cells proliferate, lifting the columnar epithelium. The immature metaplastic cells have large nuclei and a small amount of cytoplasm without glycogen. As the cells mature normally, they produce glycogen, eventually forming the four layers of epithelium. The metaplastic process begins at the tips of the columnar villi, which are exposed first to the acid vaginal environment. As the metaplasia replaces the columnar epithelium, the central capillary of the villus regresses, and the epithelium flattens out, leaving the epithelium with its typical vascular network. As metaplasia proceeds into the cervical clefts, it replaces columnar epithelium and similarly flattens the epithelium. The deeper clefts may not be completely replaced by the metaplastic epithelium, leaving mucus-secreting columnar epithelium trapped under the squamous epithelium. Some of these glands open onto the surface; others are completely encased, with mucus collecting in nabothian cysts. Gland openings and nabothian cysts mark the original SCJ and the outer edge of the original transformation zone (5) (Fig. 19.5A and B).

Human Papillomavirus

The cytologic changes of HPV were first recognized by Koss and Durfee in 1956 and given the term koilocytosis (6). Their significance was not recognized until 20 years later, when Meisels and colleagues reported these changes in mild dysplasia (7) (Fig. 19.6). Molecular biologic studies showed high levels of HPV DNA and capsid antigen, indicating productive viral infection in these koilocytic cells (8). The HPV genome is found in all grades of cervical neoplasia (9). Infection with HPV is the primary cause of cervical cancer (10). As the CIN lesions become more severe (Fig. 19.7), the koilocytes disappear, the HPV copy numbers decrease, and the capsid antigen disappears, indicating that the virus is not capable of reproducing in less differentiated cells (11). Instead, portions of the HPV DNA become integrated into the host cell. Integration of the transcriptionally active DNA into the host cell appears to be essential for malignant transformation (12). Malignant transformation requires the expression of E6 and E7 HPV oncoproteins (13). Because HPV will not grow in cell culture, there is no direct evidence of the carcinogenesis of HPV. However, a cell culture system for growing keratinocytes was described that allows for stratification and differentiation of specific keratinase types (14). When normal cells are transfected with the plasmid-containing HPV-16, the transfected cells produce cytologic abnormalities identical to those seen in intraepithelial neoplasia. The E6 and E7 oncoproteins are identifiable in the transfected cell lines, providing strong laboratory evidence of a cause-and-effect relationship (15). Cervical cancer cell lines that contain active copies of HPV-16 or -18 (SiHa, HeLa, C 4–11, Ca Ski) express HPV-16 E6 and E7 oncoproteins (16).

Figure 19.6 Cervical intraepithelial neoplasia grade 1 (CIN 1) with koilocytosis. The normal maturation process and differentiation from the basal and parabasal layers to the intermediate and superficial layers are maintained. In the upper layers, koilocytes are characterized by perinuclear halos, well-defined cell borders, and nuclear hyperchromasia, irregularity, and enlargement.

Figure 19.7 Cervical biopsy showing normal cells, cervical intraepithelial neoplasia 2 and 3 (CIN 2, CIN 3). In CIN 3, the normal maturation is lost.

HPV DNA can be detected in most women with cervical neoplasia (17,18). There are more than 120 types of HPV identified, with 30 of these HPV types primarily infecting the squamous epithelium of the lower anogenital tract of men and women (19,20). Detection of HPV is associated with a 250-fold increase risk of high-grade CIN (21). The percentage of intraepithelial neoplasia attributed to HPV infection approaches 90% (18).

Only certain types of HPV account for about 90% of high-grade intraepithelial lesions and cancer (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59 and -68) (18). Type 16 is the most common HPV found in invasive cancer and in CIN 2 and CIN 3, and it is found in 47% of women with cancer in all stages (22). It is the most common HPV type found in women with normal cytology.

HPV-16 infection is not very specific; it can be found in 16% of women with low-grade lesions and in up to 14% of women with normal cytology. Human papillomavirus type-18 is found in 23% of women with invasive cancers, 5% of women with CIN 2 and CIN 3, 5% of women with HPV and CIN 1, and fewer than 2% of patients with negative findings (18). Therefore, HPV-18 is more specific than HPV-16 for invasive tumors.

Usually, HPV infections do not persist. Those that do persist may remain latent for many years. Most women who are exposed have no clinical evidence of disease, and the infection is eventually suppressed or eliminated (17). Other women exhibit low-grade cervical lesions that mostly regress spontaneously. In the vast majority of cases, the infection will clear in 9 to 15 months (23). A small minority of women exposed to HPV develops persistent infection that may progress to CIN (17,24). Persistent high-risk HPV infection increases the risk of high-grade disease 300-fold and is required for the development and maintenance of CIN 3 (25,26). Factors that may have a role in persistence and progression include smoking, contraceptive use, infection with other sexually transmitted diseases, or nutrition (17,22). Any factor that influences the integration of HPV DNA into the human genome may cause progression to invasive disease (27).

Human Papillomavirus Vaccine

Because HPV infection is a necessary factor in the development of cervical neoplasia, an important step in primary prevention was the development of a prophylactic vaccine to protect against HPV infection. The development of prophylactic vaccination became possible through the development of protein mimics that simulate the outermost protein capsid of the virus and are termed viruslike particles (VLPs) (28). Gardasil (Merck, NJ, USA), a quadrivalent vaccine containing the VLPs for HPV-6, -11, -16, and -18, was approved by the U.S. Food and Drug Administration (FDA) in 2006; and Cervarix(GlaxoSmithKline, Middlesex, U.K.), a bivalent vaccine, was approved in 2009, and contains the VLPs to types 16 and 18. Clinical trials demonstrated that both the bivalent and quadrivalent vaccines are highly efficient in preventing CIN 2, CIN 3, or adenocarcinoma in situ caused by HPV-16 and -18 in women from 15 to 26 years of age (29,30). For the population who are seronegative and HPV DNA negative for HPV-16 and -18 at vaccination and received all three vaccinations, the efficacy is 100% (31,32). This protection is documented to last as long as 6.4 years after vaccination(31).

In a small subset of women who were seropositive for HPV-16 or -18, but DNA negative, the vaccine efficacy remained at 100% (29,30). This suggests that past exposure and clearance of the virus does not reduce the efficacy of the vaccine. On the other hand, the vaccines do not show efficacy in women who are HPV-16 and -18 DNA positive at the time of entry onto the study. This indicates that the vaccines are not able clear an active infection and cannot be used to treat CIN.

Since HPV-16 and -18 are contained in 52% of CIN and 70% of invasive cancers, the data concerning cross-protection from the other high-risk types are important (33). The bivalent vaccine demonstrates protection from persistent infection with HPV types 31, 33, and 45 and protection from CIN-related HPV-31 (29). Quadrivalent vaccine data showing cross-protection against CIN 2 or greater and against HPV 31 exist, but this indication is not included in the FDA-approved labeling (34).

The 3-year follow-up studies of both vaccine products exhibit a reduction in referrals to colposcopy by 26% and 20%, respectively (29,35). This is accompanied by a reduction in excisional procedures of 69% and 42% for the bivalent and quadrivalent products, respectively (29,35). The additional reduction may come from cross-protection against nonvaccine specific HPV types.

The quadrivalent vaccine prevents HPV-6 and -11 infections that cause genital warts. The clinical trials for women show a 99% efficacy in protection from HPV-6, -11, -16, and -18 caused external warts in women who received all three of the vaccine shots and 80% of those who did not (36). In the vaccine trials in men and boys the protection was 89% at 29 months, leading to approval of this quadrivalent vaccine in males (37).

The quadrivalent vaccine trials evaluated the protection from vulva intraepithelial neoplasia (VIN) and vaginal intraepithelial neoplasia (VAIN). The women who were HPV negative at vaccination and followed the protocol had a 100% reduction in HPV-16, -18 caused VIN 2 or 3, and VAIN 2 or 3 lesions compared to placebo (38). When the entire vaccinated population was evaluated, the reduction in VIN 2 or 3 and VAIN 2 or 3 was 50% (38). These data demonstrate that high-risk HPV types other than 16 and 18 are responsible for a significant proportion of VIN and VAIN. It reveals that women who were already positive for HPV-16 and -18 were not protected from VIN and VAIN. The bivalent trials were not designed to assess vulvar or vaginal end points.

Duration of vaccine efficacy is important because we know that HPV infection peaks in the early 20s and cervical cancer occurs in the 40s. The vaccines are approved for women up to 26 years of age and to be protective they should be effective beyond 10 years. In order to induce a significant antibody response to the antigen, it is combined with an adjuvant. The quadrivalent adjuvant is aluminum hydroxyphosphate sulfate, which proved to be superior to a simple aluminum adjuvant in binding to HPV-16 in mice. By contrast, the bivalent adjuvant is an aluminum hydroxide combined with a monophosphoryl lipid A. This is theorized to function as a link between the HPV and the activation of the innate immune system. This adjuvant produced higher antibody titers than aluminum-induced titers at the 4-year follow-up visit (39). It remains unclear whether a booster shot will be needed as the adolescent cohort reaches its mid-20s where the exposure to HPV peaks. Because the first vaccinations were given less than 10 years ago, the studies to determine the advisability of a booster are ongoing.

The American Committee on Immunization Practices developed recommendations for the utilization of both the quadrivalent and bivalent vaccines in young girls and women. In 2007 the American Cancer Society issued a set of clinical guidelines that remain relevant to the use of these agents (40). Under these guidelines, routine HPV vaccination is recommended for girls at 11 to 12 years of age, but may be provided as early as 9 years and as late as 18 years. For young women between the ages of 19 to 26, there are insufficient data to determine the value of universal vaccination. A decision to vaccinate a woman in that age group should be based on an informed discussion with her health care provider regarding her risk of previous HPV exposure and potential benefit and harm from the vaccination (41–44). Screening practices for cervical intraepithelial neoplasia and cancer should remain unchanged in both vaccinated and unvaccinated women.

Screening

Pap Test Classification: The Bethesda System

In 1988, the first National Cancer Institute (NCI) workshop held in Bethesda, Maryland, resulted in the development of the Bethesda System for cytologic reporting (45). A standardized method of reporting cytology findings facilitated peer review and quality assurance. The terminology was refined in the Bethesda III System (2001). According to this system, potentially premalignant squamous lesions fall into three categories: (i) atypical squamous cells (ASC), (ii) low-grade squamous intraepithelial lesions (LSIL), and (iii) high-grade squamous intraepithelial lesions (HSIL) (46). The ASC category is subdivided into two categories: those of unknown significance (ASC-US), and those in which high-grade lesions must be excluded (ASC-H). Low-grade squamous intraepithelial lesions include CIN 1 (mild dysplasia) and the changes of HPV, termed koilocytotic atypia.The HSIL category includes CIN 2 and CIN 3 (moderate dysplasia, severe dysplasia, and carcinoma in situ). A comparison of the various terms is shown in Table 19.1.

Table 19.1 Comparison of Cytology Classification Systems

Bethesda System	Dysplasia/CIN System	Papanicolaou System
Within normal limits	Normal	I
Infection (organism should be specified)	Inflammatory atypia (organism)	II
Reactive and reparative changes
Squamous cell abnormalities
Atypical squamous cells	Squamous atypia
(1) of undetermined significance (ASC-US)	HPV atypia, exclude LSIL	IIR
(2) exclude high-grade lesions (ASC-H)	Exclude HSIL
	HPV atypia
Low-grade squamous intraepithelial lesion (LSIL)	Mild dysplasia CIN 1
High-grade squamous intraepithelial lesion (HSIL)	Moderate dysplasia CIN 2	III
	Severe dysplasia CIN 3	IV
	Carcinoma in situ
Squamous cell carcinoma	Squamous cell carcinoma	V
CIN, cervical intraepithelial neoplasia, HPV, human papillomavirus.

Cellular changes associated with HPV (i.e., koilocytosis and CIN 1) are incorporated within the category of LSIL because the natural history, distribution of various HPV types, and cytologic features of both of these lesions are the same (27). Long-term follow-up studies showed that lesions properly classified as koilocytosis progress to high-grade intraepithelial neoplasia in 14% of cases and that lesions classified as mild dysplasia progress to severe dysplasia or CIS in 16% of cases (4,46). It was initially thought that lesions classified as koilocytosis would contain only low-risk HPV types, such as HPV-6 and -11, whereas high-risk HPV types, such as HPV-16 and -18, would be limited to true neoplasms, including CIN 1, thus justifying the distinction. Histopathologic and molecular virologic correlation showed a similar heterogeneous distribution of low- and high-risk HPV types in both koilocytosis and CIN 1 (47). Studies evaluating the dysplasia, CIS, and CIN terminology consistently demonstrated problems with interobserver and intraobserver reproducibility (48). The greatest lack of reproducibility is between koilocytosis and CIN 1 (49). On the basis of clinical behavior, molecular biologic findings, and morphologic features, HPV changes and CIN 1 appear to be the same disease. The rationale for combining CIN 2 and CIN 3 into the category of HSIL is similar. The biologic studies reveal a comparable mix of high-risk HPV types in the two lesions, and the separation of the lesions is not reproducible (48,49). The management of CIN 2 and CIN 3 is similar.

Pap Test Accuracy

Screening for cervical cancer precursors using exfoliative cervico-vaginal cytology, the Pap test was successful in reducing the incidence of cervical cancer by 79% and the mortality by 70% since 1950 (50). However, 20% of women in the United States do not undergo regular screening and have not had a Pap test in the previous 3 years. The annual incidence rate dropped from 8 to 5 cases per 100,000 women, so approximately 8,200 women per year are diagnosed with cervical cancer (50–52). Some cases of cervical cancer continue to occur in patients who have regular Pap tests. A literature review of cervical cytology testing techniques was conducted by the Agency for Healthcare Research and Quality (53). The conclusion was that the sensitivity of conventional cytologic testing in detecting cervical cancer precursor lesions was 51%, with an estimated false-negative rate of 49%. In three reviews of the accuracy of cervical cytology assessment, the sensitivity of the Pap test in detecting CIN 2 or 3 ranged from 47% to 62% and the specificity ranged from 60% to 95% (54–56). Nearly 30% of new cancer cases each year occur among women who underwent Pap testing. Errors of sampling, fixation, interpretation, or follow-up may be responsible for the missed cases (57). Prior overestimates of Pap test sensitivity of approximately 80% led to erroneous recommendations of screening frequency (58).

The conventional Pap test technique needs to be improved in order to reduce false-negative errors. Sampling errors occur because a lesion is too small to exfoliate cells or the device did not pick up the cells and transfer them to the fixation media. Historically, preparation errors occurred because of poor fixation on the glass slide, leading to air drying and its consequences for interpretation. The slide preparations could be too thick and obscured by vaginal discharge, blood, or mucus. These problems were obviated with the widespread utilization of liquid-based media. Interpretive errors may still occur when the slide contains diagnostic cells that the screening technician or automated detection device fails to identify.

The ubiquitous use of liquid-based medium to collect the cytologic sample and preserve the collected cervical cells significantly decreased specimen sampling and preparation errors. With this technique, liquid samples are processed to provide a uniform, thin layer of cervical cells without debris on a glass slide. The Agency for Healthcare Research and Quality assessment of liquid-based cytology improved the sensitivity of the Pap test to the stated goal of 80%. The cell sample is collected with an endocervical brush used in combination with a plastic spatula or with a plastic broom. The sample is rinsed in a vial containing liquid alcohol-based preservative. With this technique, 80% to 90% of the cells are transferred to the liquid media, as compared with the 10% to 20% transferred to the glass slide with conventional cytologic testing. Using liquid-based media eliminates air drying. The cells are retrieved from the vial by passing the liquid through a filter, which traps the larger epithelial cells, separating them from the small blood and inflammatory cells. This process yields a thin layer of diagnostic cells properly preserved and more easily interpreted by the cytologist. This technique reduces by 70% to 90% the rate of unsatisfactory samples encountered with conventional cytologic testing (59). Liquid-based cytology is commonly performed by most of the laboratories in the United States.

Another technology approved by the FDA for primary screening and rescreening samples of cervical cytology initially interpreted as normal is the automated image-guided slide screening system. This technique uses an automated microscope coupled to a special digital camera. The system scans the slide and uses computer imaging techniques to analyze each field of view on the slide. Computer algorithms rank each slide on the probability that the sample may contain an abnormality. The selected slides are reviewed by a cytotechnologist or a cytopathologist. This technique reduced the false-negative rate by 32% (60).

Bethesda System Modifications

The Bethesda System for reporting the results of cervical cytology developed as a uniform system of cytology reporting that would provide clear guidance for clinical management (45). It creates a standardized framework for laboratory reports that includes a descriptive diagnosis and an evaluation of specimen adequacy. The Bethesda System was modified to reflect the development of new technologies and research findings.

In the Bethesda System, specimen adequacy is categorized as satisfactory or unsatisfactory for evaluation. If a specimen is found to be unsatisfactory, cervical cytology is repeated promptly. Otherwise the specimen is classified as satisfactory. If sampling of the transformation zone is inadequate or obscuring factors are present, cervical cytology can be repeated in 6 to 12 months. The general categorizations are (i) negative for intraepithelial lesion or malignancy, (ii) epithelial cell abnormality, and (iii) other. In the category of negative for intraepithelial lesions or malignancy,evidence for the presence of trichomonas vaginalis, candida, bacterial vaginosis, actinomyces, and herpes simplex virus may be noted. Included in this category are reactive cellular changes, glandular cells status after hysterectomy, and atrophy. The category epithelial cell abnormality includes squamous cell and glandular cell abnormalities. The rest of the categories of ASC, LSIL and HSIL remain unchanged from the classification noted above (Table 19.2).

Table 19.2 Bethesda System 2001

Specimen Type: Indicate conventional smear (Pap smear) vs. liquid based vs. other

Specimen Adequacy

• Satisfactory for evaluation (describe presence or absence of endocervical/transformation zone component and any other quality indicators, e.g., partially obscuring blood, inflammation, etc.) • Unsatisfactory for evaluation… (specify reason)   • Specimen rejected/not processed (specify reason)   • Specimen processed and examined, but unsatisfactory for evaluation of epithelial abnormality because of (specify reason)

General Categorization (optional)

• Negative for intraepithelial lesion or malignancy • Epithelial cell abnormality: See Interpretation/Result (specify “squamous” or “glandular” as appropriate) • Other: See Interpretation/Result (e.g., endometrial cells in a woman 40 years of age)

Automated Review

If case examined by automated device, specify device and result.

Ancillary Testing

Provide a brief description of the test methods and report the result so that it is easily understood by the clinician.

Interpretation/Result

Negative for Intraepithelial Lesion or Malignancy (when there is no cellular evidence of neoplasia, state this in the General Categorization above and/or in the Interpretation/Result section of the report, whether or not there are organisms or other nonneoplastic findings)

Organisms

• Trichomonas vaginalis • Fungal organisms morphologically consistent with Candida spp. • Shift in flora suggestive of bacterial vaginosis • Bacteria morphologically consistent with Actinomyces spp. • Cellular changes consistent with herpes simplex virus

Other Nonneoplastic Findings (optional to report; list not inclusive):

• Reactive cellular changes associated with:   • inflammation (includes typical repair)   • radiation   • intrauterine contraceptive device (IUD) • Glandular cells status posthysterectomy • Atrophy

Other

• Endometrial cells (in a woman 40 years of age) (specify if “negative for squamous intraepithelial lesion”)

Epithelial Cell Abnormalities

Squamous Cell

• Atypical squamous cells   • of undetermined significance (ASC-US)   • cannot exclude HSIL (ASC-H) • Low-grade squamous intraepithelial lesion (LSIL) encompassing: HPV/mild dysplasia/CIN 1 • High-grade squamous intraepithelial lesion (HSIL) encompassing: moderate and severe dysplasia, CIS/CIN 2 and CIN 3   • with features suspicious for invasion (if invasion is suspected) • Squamous cell carcinoma

Glandular Cell

• Atypical   • endocervical cells (not otherwise specified [NOS] or specify in comments)   • endometrial cells (NOS or specify in comments)   • glandular cells (NOS or specify in comments) • Atypical   • endocervical cells, favor neoplastic   • glandular cells, favor neoplastic • Endocervical adenocarcinoma in situ • Adenocarcinoma   • endocervical   • endometrial   • extrauterine • NOS

Other Malignant Neoplasms (specify)

Educational Notes and Suggestions (optional)

Suggestions should be concise and consistent with clinical follow-up guidelines published by professional organizations (references to relevant publications may be included).

From Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002;287:2114–2119. Available online at: www.bethesda2001.cancer.govwww.cancer.gov/newscenter/pressreleases/2002/bethesda2001

The group of glandular cell abnormalities includes atypical glandular cells (AGC), which are further described by the terms endocervical, endometrial, or glandular cells not otherwise specified, depending on the presumptive origin, or modified to atypical glandular cells favor neoplasia in the presence of evidence of their origin (Table 19.2).

Cervical Cancer Precursors

Guidelines based on the literature were developed to guide cervical cancer screening, follow-up, and treatment. The evolving state of the science and our improved understanding of HPV and cervical carcinogenesis, and clinical practice and liability considerations, occasionally lead to subtle differences in the interpretation of these guidelines. Ultimately guidelines cannot substitute for an informed discussion of risks and benefits between a patient and health care provider in order to make decisions about treatment.

The 2002 American Cancer Society (ACS) recommended that screening with conventional Pap testing should occur every year. If liquid-based cytology is being used, screening can be extended to every 2 years. Beginning at the age of 30, women may be screened every 3 years using a combination of cytology and a high-risk HPV DNA test. Screening should begin at the age of 21 or within 3 years of the onset of sexual activity, and screening can stop at age 70 if there were no abnormal Pap test result in the previous 10 years. Under these recommendations, screening after hysterectomy for benign (non-HPV related) disease is not necessary (61).

The American College of Obstetricians and Gynecologists (ACOG) updated its guidelines (62). The ACOG recommends that women not initiate cervical cancer screening until they are 21, regardless of the onset of sexual activity. This acknowledges the very low prevalence of invasive cancer in very young women, the long multiyear process of cervical carcinogenesis, and the very low but real risks for preterm birth associated with outpatient excisional procedures. Likewise screening frequency was revised to every 2 years from age 21 to 29 (with either conventional slide or liquid-based cytology), and every 3 years for women after age 30 years if three consecutive negative, i.e., negative for intraepithelial lesion or malignancy (NILM) Pap tests can be documented. More frequent screening continues to be recommended for HIV-positive women (twice first year and annually after), those who are immune-suppressed, diethylstilbestrol (DES) daughters, and for those with a history of CIN 2 or greater (screen annually for 20 years). Discontinuation of screening is reasonable between 65 to 70 years, with reassessment of risk factors annually to determine if reinitiating screening is appropriate. Likewise in the setting of posthysterectomy for benign indications it is reasonable to discontinue screening in the absence of a history of high-grade CIN or cancer (Table 19.3).

Table 19.3 Comparison of Screening Guidelines from the American Cancer Society and the American College of Obstetricians and Gynecologists

Comparison of Cervical Cytology Screening
Guideline	American Cancer Society	American College of Obstetricians and Gynecologists
Initial screening	• Age 21 or 3 y after vaginal sex	• Age 21 or 3 y after vaginal sex
Interval	• Every year for conventional Pap • Every 2 years for liquid-based Pap • Every 2–3 y after age 30 with 3 consecutive normals	• Every year for either liquid-based Pap or conventional • Every 2–3 y after age 30 with 3 consecutive normals
Discontinue			• Age 70 if 3 consecutive normals in 10 y	• No upper limit of age

In 2003, the FDA approved HPV DNA testing combined with cervical cytology as a screening technique for women older than age 30. When the results of both tests are negative, the woman does not have to be retested for 3 years. The negative predictive value of a double negative test exceeds 99% (19). Because most HPV infections are transient, clear spontaneously, and do not lead to real cancer precursors (especially in young women), it should not be used for screening in women younger than 30 (63). Women who have negative test results for both cytology and HPV have a 1 in 1,000 chance of having CIN 2 or worse detected in the following 6 months (64). Prospective studies report less than 2 per 1,000 women will develop CIN 2 or greater in the following 3 years (64–66).

Atypical Squamous Cells

The ASC category is restricted to those test results disclosing abnormal cells that are truly of unknown significance. The ASC category does not include benign, reactive, and reparative changes, which are classified as normal in the Bethesda system. Because of the subjective diagnostic criteria and the fear of medical–legal action, the diagnosis is relatively common, ranging from 3% to 25% in some centers (67). When standardized diagnostic criteria are used, the rate of ASC results should be 3% to 5% (68). The ASC category is subdivided into ASC-US and ASC-H.

The cytologic diagnosis of ASC-US is associated with a 10% to 20% incidence of CIN 1 and a 3% to 5% risk for CIN 2 or 3 (69–72). It is apparent that CIN 1 is most often a benign HPV infection and will regress spontaneously in more than 60% of cases; therefore, the goal of triage of an ASC-US Pap test result is to identify more advanced CIN 2 and 3 lesions (73).

The option of repeat Pap testing is suboptimal because of a false-negative rate of 20% to 50% for identifying CIN lesions and practical issues with patient compliance. About 50% of patients will undergo colposcopy because of subsequent abnormal Pap test results, making this option nearly as costly as immediate colposcopy (69). Immediate colposcopy is assumed to be the most sensitive method of detecting CIN 2 or 3 (69,72). Because 80% of patients will not have significant lesions, it is important to avoid overinterpretation of the colposcopic findings and to be conservative in performing biopsies. There is the risk that pathologists will overinterpret the biopsy results and the patient will be diagnosed with CIN when metaplasia is the only finding.

Several studies documented the usefulness of HPV testing in the assessment of ASC-US Pap test results (74–76). These studies demonstrate that HPV testing can identify 90% of the patients with CIN 2 or 3 lesions. To compare the aforementioned triage method in a prospective, randomized fashion, the NCI funded an ASC-US/LSIL Triage Study (ALTS) (77). Patients with ASC-US or LSIL were randomized to three triage arms: (i) immediate colposcopy, (ii) HPV test, and (iii) conservative management by repeat Pap test. There were 1,163 women in the immediate colposcopy group, and 14 refused the examination. The results of colposcopy are assumed to reflect the prevalent disease rates, which were as follows: CIN 1, 14.3%; CIN 2, 16.1%; and CIN 3, 5%. Thus, 75% of the women with ASC-US had negative colposcopy results and either did not have a biopsy (25%) or had a biopsy with negative results. The HPV test results were positive in 56.1% of the patients, and 6.1% of the patients did not return for colposcopy. Of the 494 who underwent colposcopy, the results were as follows: CIN 1, 22.5%; CIN 2, 11.9%; and CIN 3, 15.6%. The sensitivity of HPV test was 95.9% for the detection of CIN 2 and 96.3% for the detection of CIN 3.

In the conservatively managed group, only one follow-up Pap test was reported. To be effective, Pap testing must be done every 6 months. Despite this, the results of the single follow-up Pap test were included. Using a cutoff that includes any positive finding of ASC-US or greater, the sensitivity is 85% for CIN 2 and 85.3% for CIN 3, with 58.6% of patients being referred for colposcopy. If LSIL is used as a cutoff, 26.2% of the patients are referred, with sensitivity of 64.0% for both CIN 2 and 3. Using HSIL as the cutoff, 6.9% are referred, and the sensitivity falls to 44%. The conclusion of the ALTS trial is that HPV triage is highly sensitive in identifying CIN 2 and 3 lesions and that it cuts the rate of referral for colposcopy by approximately one-half (78). When mathematical models are used to simulate the natural history of HPV and cervical cancer in a cohort of U.S. women, a 2- to 3-year screening strategy that uses cytologic assessment in combination with either HPV DNA testing or reflex HPV testing appears more effective and less costly in reducing the rate of cancer than annual conventional cytology (79).

Low-Grade Squamous Intraepithelial Lesions

The cytologic diagnosis of LSIL is reproducible and accounts for 1.6% of cytologic diagnoses (68). About 75% of the patients have CIN, with 20% being CIN 2 or 3 (69–71). These patients require additional evaluation. The ALTS trial closed the HPV test arm early because the HPV positivity rate was 82% and was not a valid discriminator in determining the presence of disease. The ALTS trial found that a cytology interpretation of LSIL is associated with a 25% risk of histologic CIN 2 or 3 within 2 years. No effective triage strategy was identified to spare many women from colposcopic referral without increasing their risk of CIN 3 and invasive carcinoma (80). Guidelines confirm the validity of the current practice of performing colposcopy to evaluate a single LSIL result (75).

High-Grade Squamous Intraepithelial Lesions

Any woman with a cytologic specimen suggesting the presence of HSIL should undergo colposcopy and directed biopsy (75). This is because two-thirds of patients with this cytologic finding will have CIN 2 or greater. After colposcopically directed biopsy and determination of the distribution of the lesion, excisional or ablative therapy that addresses the entire transformation zone should be performed.

Diagnosis

Colposcopy Findings

Acetowhite Epithelium

Epithelium that turns white after application of acetic acid (3%–5%) is called acetowhite epithelium (53). The application of acetic acid coagulates the proteins of the nucleus and cytoplasm and makes the proteins opaque and white (5).

The acetic acid does not affect mature, glycogen-producing epithelium because the acid does not penetrate below the outer one-third of the epithelium. The cells in this region have very small nuclei and a large amount of glycogen (not protein). These areas appear pink during colposcopy. Dysplastic cells are those most affected. They contain large nuclei with abnormally large amounts of chromatin (protein). The columnar villi become “plumper” after acetic acid is applied, making these cells easier to see. They appear slightly white, particularly in the presence of the beginning signs of metaplasia. The immature metaplastic cells have larger nuclei and show some effects of the acetic acid. Because the metaplastic epithelium is very thin, it is not as white or opaque as CIN but instead appears gray and filmy (5).

Leukoplakia

Translated literally, leukoplakia is white plaque (5). In colposcopic terminology, this plaque is white epithelium, visible before application of acetic acid. Leukoplakia is caused by a layer of keratin on the surface of the epithelium. Immature squamous epithelial cells have the potential to develop into keratin-producing cells or glycogen-producing cells. In the vagina and on the cervix, the normal differentiation is toward glycogen. Keratin production is abnormal in the cervicovaginal mucosa. Leukoplakia can be caused by HPV; keratinizing CIN; keratinizing carcinoma; chronic trauma from diaphragm, pessary, or tampon use; and radiotherapy.

Leukoplakia should not be confused with the white plaque of a monilial infection, which can be completely wiped off with a cotton-tipped applicator. The most common reason for leukoplakia is HPV infection (Fig. 19.8). Because it is not possible to see through the thick keratin layer to the underlying vasculature during colposcopy, such areas should undergo biopsy to rule out keratinizing carcinoma.

Figure 19.8 Colposcopy of cervical intraepithelial neoplasia 2 (CIN 2) associated with human papillomavirus (HPV) infection of the cervix.

Punctation

Dilated capillaries terminating on the surface appear from the ends as a collection of dots and are referred to as punctation (Fig. 19.9). When these vessels occur in a well-demarcated area of acetowhite epithelium, they indicate an abnormal epithelium—most often CIN (5) (Fig. 19.10). The punctate vessels are formed as the metaplastic epithelium migrates over the columnar villi. Normally, the capillary regresses; however, when CIN occurs, the capillary persists and appears more prominent.

Figure 19.9 Diagram of punctation. The central capillaries of the columnar villi are preserved and produce the punctate vessels on the surface.

Figure 19.10 Human papillomavirus (HPV)/cervical intraepithelial neoplasia 2 (CIN 2) presents as a white lesion with surface spicules.

Mosaic

Terminal capillaries surrounding roughly circular or polygonal-shaped blocks of acetowhite epithelium crowded together are called mosaic because their appearance is similar to mosaic tile (Fig. 19.11). These vessels form a “basket” around the blocks of abnormal epithelium. They may arise from a coalescence of many terminal punctate vessels or from the vessels that surround the cervical gland openings (5). Mosaicism tends to be associated with higher-grade lesions and CIN 2 (Fig. 19.12) and CIN 3 (Fig. 19.13).

Figure 19.11 A: Mosaic pattern and punctation. This pattern develops as islands of dysplastic epithelium proliferate and push the ends of the superficial blood vessels away, creating a pattern that looks like mosaic tiles. B: Diagram of mosaic pattern.

Figure 19.12 Human papillomavirus (HPV)/cervical intraepithelial neoplasia 3 (CIN 3). Cribriform pattern of HPV at periphery with mosaicism and punctation near the squamocolumnar junction.

Figure 19.13 Cervical intraepithelial neoplasia grade 3 (CIN 3).

Atypical Vascular Pattern

Atypical vascular patterns are characteristic of invasive cervical cancer and include looped vessels, branching vessels, and reticular vessels.

Endocervical Curettage

ASCCP guidelines do not require endocervical curettage. In cases when an endocervical sample is needed, a cytobrush is sufficient for sampling the endocervical canal.

Cervical Biopsy

The cervical biopsy is performed at the area most likely to have dysplasia. If the lesion is large or multifocal, multiple biopsies may be necessary to ensure a complete sample of the affected tissue.

Correlation of Findings

Ideally, both the pathologist and colposcopist should review the colposcopic findings and the results of cytologic assessment, cervical biopsy, and endocervical sample before deciding therapy. This is particularly valuable when operators are learning the technique of colposcopy. Cytology results should not be sent to one laboratory and the histology results to another. When the cytology and biopsy results correlate, the colposcopist can be reasonably certain that the worst lesion was identified. If the cytology indicates a more significant lesion than the histology, the patient may require further evaluation, including repeat colposcopy, additional biopsies, and excisional diagnostic procedures under certain circumstances. An algorithm for the evaluation, treatment, and follow-up of abnormal Pap test results is presented in Figure 19.14.

Figure 19.14 An algorithm for the evaluation, treatment, and follow-up of an abnormal Pap test.

Management

ASCCP 2006 Consensus Guidelines

The American Society for Colposcopy and Cervical Pathology (ASCCP) Consensus Guidelines were developed in 2001 for women with cytologic abnormalities and cervical cancer precursors and to incorporate the 2001 Bethesda System (46,81,82). Improved understanding of the pathogenesis and natural history of cervical HPV infection and cervical cancer precursors, combined with an appreciation of the impact of treatment of CIN on future pregnancy among young women, and the management of adenocarcinoma in situ led to a critical review of the Guidelines (75,83).

In 2006, the American Society for Colposcopy and Cervical Pathology (ASCCP) and the NCI sponsored a consensus conference to update evidence-based guidelines for abnormal cervical cancer screening. Comprehensive and specific guidelines for the management of cervical cytologic and histologic entities are available on the Web (84). The ASCCP 2006 Consensus Guidelines for management based on cytologic findings are presented in Figure 19.15 and those based on histologic findings are in Figure 19.16.

Figure 19.15 A–J Algorithms from the 2006 Consensus Guidelines for the Management of Women with Cervical Cytologic Abnormalities. Reprinted from the Journal of Lower Tract Disease, vol. 11, issue 4, with the permission of the ASCCP (© American Society for Colposcopy and Cervical Pathology 2007. No copies of the algorithms may be made without the prior consent of the ASCCP.)

Figure 19.16 A–F Algorithms from the 2006 Consensus Guidelines for the Management of Women with Cervical Histologic Abnormalities. Reprinted from the Journal of Lower Tract Disease, vol. 11, issue 4, with the permission of the ASCCP (© American Society for Colposcopy and Cervical Pathology 2007. No copies of the algorithms may be made without the prior consent of the ASCCP.)

Atypical Squamous Cells

Based on these recommendations, women with ASC-US should be managed initially with either (i) two repeat Pap tests with referral for colposcopy for any significant abnormality, (ii) immediate colposcopy, or (iii) testing for high-risk type HPV (Fig. 19.15A).

Testing for HPV DNA is the preferred method when liquid-based cytology is used or when co-collection is feasible. Women who have positive test results should be referred for colposcopy, and those with negative results should receive yearly cytology assessment (75). Adolescent women with ASC-US or LSIL should have a repeat cytology in 12 months (Fig. 19.15B). Women with ASC-H should be referred to colposcopy, they do not benefit from triage with high-risk HPV testing (Fig 19.15C). The management of cytologic abnormalities with findings of LSIL and HSIL are presented in Figure 19.15 D-F, and AGC in Figures 19.15H and 19.15I, and for adolescent women with HSIL in Figure 19.15H.

CIN 1

The spontaneous regression rate of biopsy-proven CIN 1 is 60% to 85% in prospective studies. The regressions typically occur within a 2-year follow-up with cytology and colposcopy (4,75,85–88). Patients who have biopsy proven CIN 1 (after a cytologic finding of ASC, ASC-H, LSIL) with satisfactory colposcopy and who agree to the evaluation every 6 months may be followed with Pap testing performed at 6 and 12 months or HPV DNA testing at 12 months. After two negative test results or a single negative HPV DNA test, routine screening may be resumed (75). Colposcopy and repeat cytology at 12 months or a diagnostic excisional procedure may be necessary if the CIN 1 biopsy was preceded by an HSIL or AGC cytology (75). Regression of CIN 1 decreases after 24 months, with the regression rate becoming the same as for CIN 2 by 5 years (89).

For patients with persistent CIN 1 after 24 months, the choice of treatment is optional. Expectant management is acceptable, as long as the patient is cooperative with follow-up. Patients with chronic systemic disease associated with immunosuppression, such as those requiring steroids, chemotherapy, or antirejection drugs, may have chronically persistent low-grade abnormalities. Ablative therapies, including cryotherapy or laser ablation, seem preferable to excisional procedures including loop electrosurgical excision procedure (LEEP) (75). A randomized prospective trial comparing cryosurgery with laser and LEEP showed no difference in persistent disease rate (4%) or recurrent disease rate (17%). Cryosurgery has the advantage of low cost and ease of use. The disadvantages are lack of tissue specimen, inability to adapt to lesion size, and posttreatment vaginal discharge. If colposcopy findings are unsatisfactory, ablative therapy should be avoided.

CIN 2 and 3

All CIN 2 and 3 lesions require treatment in women 21 years of age and older (75). This recommendation is based on a meta-analysis showing that CIN 2 progresses to CIS in 20% of cases and to invasion in 5%. Progression of CIS to invasion is 5% (90).

In recognition of the small but real risk for preterm birth, young women less than 20 years may be offered a program of surveillance with cytology and colposcopy at 6-month intervals with treatment only for persistence at 24 months (75). The heterogeneity of CIN 2 lesions is significant and regression rates are higher than for CIN 3. The histological distinction between CIN 2 and CIN 3 remains subjective and these diagnoses are combined in the 2006 Consensus Guidelines.

Although CIN can be treated with a variety of techniques, the preferred treatment for CIN 2 and 3 is LEEP. This allows a specimen to be sent for evaluation and enables the pathologist to identify occult microinvasive cancer or adenomatous lesions to ensure these lesions were treated adequately. The persistent and recurrent disease rate posttreatment is estimated at 4% to 10% (91,92).

Most of the excisional and ablative techniques used to treat CIN can be performed in an outpatient setting, which is one of the main objectives in the management of this disease.

Because all therapeutic modalities carry an inherent recurrence rate of up to 10%, surveillance at 6-month intervals with cytology and colposcopy or alternatively with high-risk HPV testing at 6 to 12 months is required (75). Surveillance is continued until two consecutive negative results occur. Referral to colposcopy for any abnormal results is mandatory.

Ablative therapy is appropriate when the following conditions exist:

1. There is no evidence of microinvasive or invasive cancer on cytology, colposcopy, endocervical curettage, or biopsy.

2. The lesion is located on the ectocervix and can be seen entirely.

3. There is no involvement of the endocervix with high-grade dysplasia as determined by colposcopy and endocervical curettage.

Management Summary

This summary of the management of these histologic lesions is based on ASCCP 2006 Consensus Guidelines (adapted from MJ Campion [93]) (Fig 19.16):

CIN 1 Preceded by ASC-US, ASC-H or LSIL Cytology

1. Women with a histologic diagnosis of CIN 1 preceded by ASC-US, ASC-H or LSIL cytology should be followed by yearly HPV-DNA testing or Pap tests every 6–12 months (Fig. 19.16A). If HPV DNA testing remains positive or if repeat cytology is reported as ASC-US or greater, repeat colposcopy is recommended.

2. If the HPV DNA test is negative or two consecutive Pap smears are reported as negative, return to routine screening is recommended.

3. If CIN 1 persists for 2 years or more, continued follow-up or treatment is appropriate. Treatment can be ablative or excisional.

4. If colposcopy is unsatisfactory or the endocervical sample is positive for a high-grade CIN, a diagnostic excisional procedure is recommended.

5. Two consecutive negative Pap tests in follow-up of low-grade lesions is not absolute indication that the disease regressed.

CIN 1 Preceded by HSIL or AGC-NOS Cytology

Women with a histologic diagnosis of CIN 1 diagnosed during the assessment of pap tests showing HSIL (CIN 2 or 3) or atypical glandular cells-not otherwise specified (AGC-NOS) can be managed by either an excisional diagnostic procedure or 6-monthly colposcopy and cytology for 1 year (Fig 19.16B). Those with a cytological finding of AGC-NOS must have a satisfactory colposcopic examination and negative endocervical sampling.

If the patient is followed expectantly, a diagnostic excisional procedure is recommended if the repeat cytology at either 6 or 12 months is HSIL or AGC-NOS. If the histology of a colposcopically directed biopsy confirms high-grade CIN, the management is according to guidelines regardless of the cytology result.

After two consecutive negative for intraepithelial neoplasia or malignancy results in follow-up, routine cytologic screening can be resumed.

If CIN 1 is preceded by HSIL or AGC-NOS cytology and colposcopy is unsatisfactory, a diagnostic excisional procedure is recommended, except in special circumstances (e.g., pregnancy).

CIN 1 in Adolescents and Pregnancy

For adolescents with CIN 1, follow-up with annual cytology is recommended (Fig 19.16C). Only those with HSIL or greater at 12 months should be referred for colposcopy.

At 24-months, those with ASC-US or greater should be referred.

Prospective follow-up by HPV DNA testing in this age group is not useful because of the high rate of positive results.

CIN 2 or 3

Both excisional and ablative procedures are acceptable treatment modalities for women with histologically proven CIN 2 or 3 with satisfactory colposcopy (Fig. 19.16D). An excisional procedure is recommended for residual/recurrent CIN 2 or 3.

Ablation is unacceptable for women with a histologic diagnosis of CIN 2 or 3 and unsatisfactory colposcopy.

Cytologic and colposcopic follow-up of CIN 2 or 3 is necessary only in specific circumstances. Acceptable posttreatment follow-up options include cytology alone, every 6 months, combined cytology and colposcopy every 6 months, and HPV DNA testing at 6 to 12 months.

If HPV DNA testing is positive or if the repeat cytology is ASC-US or greater, referral for colposcopy and endocervical sampling is recommended.

If HPV DNA testing is negative or if two consecutive posttreatment cytology results are negative for intraepithelial lesion or malignancy, routine screening for at least 20 years is recommended, and should be annual for at least 5 years. A negative HPV DNA test is an excellent predictor of a normal posttreatment for CIN 2 or 3.

If CIN 2 or 3 is histologically found at the margins of an excised specimen or in an endocervical sample obtained immediately after the procedure, cytologic follow-up with endocervical sampling every 4 to 6 months is preferred. The role of colposcopy in this follow-up option is not clearly defined in the Guidelines, although in practice, cytology and colposcopy will be performed in most clinical settings.

A repeat diagnostic excisional procedure is acceptable. The Guidelines allow for hysterectomy if a repeat diagnostic excisional procedure is not feasible. An undisclosed invasive cancer within the endocervical canal must be excluded prior to hysterectomy.

For women with histologically proven residual/recurrent CIN 2 or 3, the Guidelines permit a repeat excisional procedure or hysterectomy.

CIN 2 or 3 in Adolescence and Pregnancy

For adolescents with a histological diagnosis of CIN 2 or 3 not otherwise specified, the Guidelines state that either treatment or observation by cytology and colposcopy every 6 months for up to 24 months is acceptable provided colposcopy is satisfactory (Fig 19.16E). Allowing for the subjectivity in this histological distinction, observation is preferred for a diagnosis of CIN 2 alone, but treatment is acceptable.

Treatment is recommended for a histologic diagnosis of CIN 3 or if colposcopy is unsatisfactory. Although invasive cervical cancer is very rare in this age group, prospective follow-up of a histological diagnosis of CIN 2 or 3, not otherwise specified, in young women should be limited to those women likely to be compliant with the recommendations.

After 2 consecutive negative for intraepithelial lesion or malignancy results, implying negative cytology and colposcopy with satisfactory colposcopic examinations, adolescents and young women can return to routine cytologic screening. An annual screening interval should be recommended.

Treatment is recommended if CIN 3 is diagnosed histologically or if CIN 2 or 3 persists for 24 months.

Cervical Adenocarcinoma in situ (AIS)

Hysterectomy remains the preferred management recommendation for women with a histological diagnosis of AIS on a specimen from a diagnostic excisional procedure (Fig 19.16F).

A histologic diagnosis of AIS from a punch biopsy or a cytological diagnosis of AIS is not sufficient to justify hysterectomy without a diagnostic excisional procedure. The difficulty in defining colposcopic limits of AIS lesions, the frequent extension of disease within the endocervical canal and the presence of multifocal, “skip-lesions” (i.e., lesions that are not contiguous) compromise conservative excisional procedures.

Negative margins in an excisional specimen do not mean the lesion is completely excised.

If future fertility is desired, conservative excisional management is acceptable. The overall failure rate of excision is less than 10%. Margin status is a useful clinical predictor of residual disease as is endocervical sampling at the time of excision.

If a conservative excisional procedure is performed and margins are involved or the endocervical sample at the time of excision shows AIS or CIN, reexcision is recommended.

A reassessment at 6 months using a combination of cytology, colposcopy, HPV DNA testing, and endocervical sampling is acceptable. Long-term follow-up is recommended for women who do not undergo hysterectomy for AIS.

Treatment Modalities

Cryotherapy

Cryotherapy destroys the surface epithelium of the cervix by crystallizing the intracellular water, resulting in the eventual destruction of the cell. The temperature needed for effective destruction must be in the range of (–20° to –30°C). Nitrous oxide (–89°C) and carbon dioxide (–65°C) produce temperatures below this range and, therefore, are the most commonly used gases for this procedure.

The technique believed to be most effective is a freeze-thaw-freeze method in which an ice ball is achieved 5 mm beyond the edge of the probe. The time required for this process is related to the pressure of the gas; the higher the pressure, the faster the ice ball is achieved. Cryotherapy is an effective treatment for CIN with very acceptable failure rates under certain conditions (94–97). It is a relatively safe procedure with few complications. Cervical stenosis is rare but can occur. Posttreatment bleeding is uncommon and is usually related to infection.

Cure rates are related to the grade of the lesion; CIN 3 has a greater chance of treatment failure (Table 19.4). Townsend showed that cures are related to the size of the lesion; those covering most of the ectocervix have failure rates as high as 42%, compared with a 7% failure rate for lesions less than 1 cm in diameter (99). Positive findings on endocervical curettage can reduce the cure rate significantly. Endocervical gland involvement is important because the failure rate in women with gland involvement was 27%, compared with 9% in those who did not have such involvement (98).

Table 19.4 Results of Cryotherapy for Cervical Intraepithelial Neoplasia (CIN) Compared with Grade of CIN

Cryotherapy should be considered acceptable therapy when the following criteria are met:

1. CIN 1 that has persisted for 24 months, or CIN 2

2. Small lesion

3. Ectocervical location only

4. Negative endocervical sample

5. No endocervical gland involvement on biopsy

Laser Ablation

Although rarely used in practice, laser ablation was used effectively for the treatment of CIN (Table 19.5). The expense of the equipment combined with the necessity for special training limited the use of laser ablation. Most early CIN is now managed expectantly, so the need for ablation is decreasing.

Table 19.5 Success Rate for Laser Vaporization

Loop Electrosurgical Excision

Loop electrosurgical excision is a valuable tool for the diagnosis and treatment of CIN (106–116). It offers the advantage of performing an operation that is simultaneously diagnostic and therapeutic during one outpatient visit (100–105,117–122).

The tissue effect of electricity depends on the concentration of electrons (size of the wire), the power (watts), and the water content of the tissue. If low power or a large-diameter wire is used, the effect will be electrocautery, and the thermal damage to tissue will be extensive. If the power is high (35–55 watts) and the wire loop is small (0.5 mm), the effect will be electrosurgical, and the tissue will have little thermal damage. The actual cutting is a result of a steam envelope developing at the interface between the wire loop and the water-laden tissue. This envelope is pushed through the tissue, and the combination of electron flow and acoustical events separates the tissue. After the excision, a 5-mm diameter ball electrode is used, and the power is set at 50 watts. The ball is placed near the surface so that a spark occurs between the ball and the tissue. This process is called electrofulguration, and it results in some thermal damage that leads to hemostasis. If too much fulguration occurs, the patient will develop an eschar with more discharge, and the risk for infection and late bleeding will be higher.

Research shows that LEEP is associated with an increased risk of overall preterm delivery, preterm delivery after premature rupture of membranes, and low-birth-weight infants in subsequent pregnancies at greater than 20 weeks gestation (123). Loop excision should not be used before an intraepithelial lesion is identified with histopathology. Although “see and treat” may be appropriate in certain settings and among populations for whom follow-up is not possible, the potential excision of the entire transformation zone along with varying amounts of the cervical canal, may compromise birth outcomes (114,116). This is particularly true for young women, who may have large, immature transformation zones with extensive acetowhite areas. Complications following loop electrosurgical excision are minimal and compare favorably with those following laser ablation and conization. Intraoperative hemorrhage, postoperative hemorrhage, and cervical stenosis can occur but at low rates, as noted in Table 19.6. The SCJ is visible in more than 90% of patients after this procedure. Effectiveness of LEEP and comparison of LEEP to other excision procedures are shown in Tables 19.7 through 19.9.

Table 19.6 Therapeutic Efficiency of Cervical Conization: Comparison between Laser and Knife Techniques

Table 19.7 Perioperative and Postoperative Bleeding from Cervical Conization: Comparison between Laser and Knife Techniques

Table 19.8 Complications of Electrosurgical Excision

Table 19.9 Unsuspected Invasion in Electrosurgical Excision Specimens

Conization

Conization of the cervix plays an important role in the management of CIN. Before the availability of colposcopy, conization was the standard method of evaluating an abnormal Pap test result. Conization is both a diagnostic and therapeutic procedure and has the advantage over ablative therapies of providing tissue for further evaluation to rule out invasive cancer (117,118,120,124).

Conization is indicated for diagnosis in women with HSIL or AGC-adenocarcinoma in situ and may be considered under the following conditions:

1. Limits of the lesion cannot be visualized with colposcopy.

2. The SCJ is not seen at colposcopy.

3. Endocervical curettage (ECC) histologic findings are positive for CIN 2 or CIN 3.

4. Substantial lack of correlation between cytology, biopsy, and colposcopy results.

5. Microinvasion is suspected based on biopsy, colposcopy, or cytology results.

6. The colposcopist is unable to rule out invasive cancer.

Lesions with positive margins are more likely to recur after conization (117,118,120) (Table 19.10). Endocervical gland involvement is predictive of recurrence (23.6% with gland involvement compared with 11.3% without gland involvement) (125). When compared with conization, LEEP is the simpler technique, and short-term results are similar to those obtained with conization or laser excision (91,126). In a prospective study examining the long-term effects of LEEP, conization, and laser excision, no difference in recurrence of dysplasia or in pregnancy outcomes was found (127) (Tables 19.11 and 19.12).

Table 19.10 Grade of Discomfort of Large-Loop Excision versus Laser Conization

Side Effect	Loop Excision (n = 98)	Laser (n = 101)
Not unpleasant	80 (92%)	32 (32%)
Moderately unpleasant	16 (16%)	50 (50%)
Very unpleasant	2 (2%)	19 (18%)
Operative time	20–50 sec	4–15 min
	(mean, 16 sec)	(mean, 6.5 min)
From Gunasekera PC, Phipps JH, Lewis BV. Large loop excision of the transformation zone (LLETZ) compared to carbon dioxide laser in the treatment of CIN: a superior mode of treatment. Br J Obstet Gynaecol1990;97:995–998, with permission.

Table 19.11 Results of Loop Electrosurgical Excision

Author (ref. no.)	Patients Treated	Patients Recurred
Prendiville et al. (106)	102	2
Whiteley et al. (107)	80	4
Bigrigg et al. (109)	1,000	41
Gunasekera et al. (110)	98	7
Luesley et al. (114)	616	27
Murdoch et al. (115)	600	16
Total	2,496	97 (3.9%)

Table 19.12 Recurrence of Cervical Intraepithelial Neoplasia After Cone Biopsy

Hysterectomy

Hysterectomy is considered a treatment of last resort for recurrent high-grade CIN. In a study of 38 cases of invasive cancer occurring after hysterectomy among 8,998 women (0.4%), the incidence of significant bleeding, infection, and other complications, including death, is higher with hysterectomy than with other means of treating CIN (128). There are some situations in which hysterectomy remains a valid and appropriate (although not mandatory) method of treatment for CIN:

1. Microinvasion

2. CIN 3 at the endocervical limits of conization specimen in selected patients

3. Poor compliance with follow-up

4. Other gynecologic problems requiring hysterectomy, such as fibroids, prolapse, endometriosis, and pelvic inflammatory disease

5. Histologically confirmed recurrent high-grade CIN

Glandular Cell Abnormalities

Atypical Glandular Cells

The Bethesda System created the term atypical glandular cells to describe the spectrum of glandular cell abnormalities. This classification is subdivided with the qualifier into the categories favor neoplasiaand not otherwise specified (NOS). The latter qualified with the cell of origin when identifiable as either endocervical or endometrial origin. Included in the glandular cell category is endocervical carcinoma in situ and adenocarcinoma (129).

Atypical glandular cells are important because of their risk for significant disease. In a series of 63 patients from whom subsequent cervical biopsy or hysterectomy specimens were evaluated, 17 women had CIN 2 or 3, five women had adenocarcinoma in situ, and two women had invasive adenocarcinoma (125). An additional eight patients had CIN 1, and two women had endometrial hyperplasia. Overall, 32 patients (50.8%) had significant cervical lesions. This is a much higher positive rate than that for ASC-US Pap test results.

Adenocarcinoma

In adenocarcinoma in situ (AIS), the endocervical glandular cells are replaced by tall columnar cells with nuclear stratification, hyperchromasia, irregularity, and increased mitotic activity (130). Cellular proliferation results in crowded, cribriform glands. The normal branching pattern of the endocervical glands is maintained. Most neoplastic cells resemble those of the endocervical mucinous epithelium. Endometrioid and intestinal cell types occur less often. About 50% of women with cervical AIS have squamous CIN. Some of the AIS lesions represent incidental findings in specimens removed for treatment of squamous neoplasia. Because AIS is located near or above the transformation zone, conventional cervical specimens may not be effective for detecting glandular disease. Obtaining good endocervical specimens by cytobrush may improve detection of AIS. If the focus of AIS is small, cervical biopsy and endocervical curettage may have negative findings. In such cases, a more comprehensive survey of the cervix by diagnostic conization may be necessary. This type of specimen allows exclusion of coexisting invasive adenocarcinoma. The term microinvasion should not be used to describe adenocarcinomas. After the gland is invaded, it is methodologically problematic to estimate a true “depth of invasion” because the invasion may have originated from the mucosal surface or the periphery of the underlying glands. The “breakthrough” of the basement membrane cannot be described; therefore, the tumor is either AIS or invasive adenocarcinoma.

With the recent apparent increase in invasive adenocarcinoma of the endocervix, more attention is directed toward AIS. There is evidence that AIS may progress to invasive cancer (108). In a series of 52 cases of adenocarcinoma of the uterine cervix, the results of 18 endocervical biopsies were interpreted as negative 3 to 7 years before the presentation with cancer (130). In five of these cases, AIS was found. In a study of the anatomic distribution of AIS in 23 women, all patients had AIS involving both the surface and the glandular endocervical epithelium, often with the deepest glandular cleft involved (131). The entire endocervical canal was at risk; nearly one-half of the patients had lesions 1.5 to 3 cm from the external os. Overall, 15 patients had unifocal disease, 3 had multifocal disease, and 5 had AIS of undermined type; 11 of the 23 patients had squamous intraepithelial lesions and AIS. In a study of 40 patients with AIS who had cervical conization, 23 of 40 patients (58%) had coexisting squamous intraepithelial lesions and 2 had invasive squamous cell carcinoma (128). Of the 22 patients who underwent hysterectomy, the margins on the cone specimen were positive in 10 patients, and 70% had residual AIS, including 2 patients with foci of invasive adenocarcinoma. One of the 12 patients with negative margins had focal residual adenocarcinoma in the hysterectomy specimen, and 18 women had conization only with negative margins and no relapse of disease after a medium interval of 3 years. Thus, positive margins on the conization specimen are significant findings in these patients (132).

In a more alarming study of 28 patients with AIS, of the 8 patients with positive margins who underwent repeat conization or hysterectomy, 3 had residual AIS and 1 patient had invasive adenocarcinoma (133). Of 10 patients with negative margins who underwent hysterectomy or repeat conization, 4 had residual AIS. One patient in whom the cone margin could not be evaluated had invasive adenocarcinoma. Of the 15 patients treated conservatively with repeat conization of the cervix and close follow-up, 7 (47%) had a recurrent glandular lesion detected after the conization, including invasive adenocarcinoma in 2 women. A glandular lesion was not suspected in 48% of the patients, based on Pap test and endocervical curettage results obtained before conization of the cervix.

AIS must be considered a serious cancer precursor of adenocarcinoma. The entire endocervical canal is at risk, and detection of the lesion with cytologic assessment or endocervical curettage may not be reliable. Any patient with a positive cone margin should undergo repeat conization. If fertility is not desired, a hysterectomy should be performed because of the risk of recurrence, even in the presence of negative margins.

Vaginal Intraepithelial Neoplasia

VAIN often accompanies CIN and is believed to share a common etiology (134). Such lesions may be extensions onto the vagina from the CIN, or they may be satellite lesions occurring mainly in the upper vagina. Because the vagina does not have a transformation zone with immature epithelial cells to be infected by HPV, the mechanism of entry for HPV is by way of microabrasions resulting primarily from insertional sexual activity. As these abrasions heal with metaplastic squamous cells, the HPV may begin its growth in a manner similar to that in the cervical transformation zone. VAIN lesions are asymptomatic. Because they often accompany active HPV infection, the patient may report vulvar warts or an odoriferous vaginal discharge from vaginal warts.

Screening

Women with an intact cervix should undergo routine cytologic screening. Because VAIN is nearly always accompanied by CIN, the Pap test result is likely to be positive when VAIN is present. The vagina should be carefully inspected by colposcopic examination at the time of colposcopy for any CIN lesion. Particular attention should be paid to the upper vagina. Women who have persistent abnormal Pap tests without evident cervical pathology and those with abnormal cytology after treatment of CIN should be examined carefully for VAIN. For women in whom the cervix was removed for high-grade cervical neoplasia, Pap testing should be performed at regular intervals (e.g., yearly), depending on the diagnosis and severity of lesion.

Diagnosis

Colposcopic examination and directed biopsy are the mainstays of diagnosis of VAIN. Typically, the lesions are located along the vaginal ridges, are ovoid in shape and slightly raised, and often have surface spicules. VAIN 1 lesions usually are accompanied by a significant amount of koilocytosis, indicating their HPV origin (Fig. 19.17). VAIN 2 exhibits a thicker acetowhite epithelium, a more raised external border, and less iodine uptake (Fig. 19.18A). When VAIN 3 occurs, the surface may become papillary, and the vascular patterns of punctation and mosaic may occur (Fig. 19.18B). Early invasion is typified by vascular patterns similar to those of the cervix.

Figure 19.17 Human papillomavirus (HPV)/vaginal intraepithelial neoplasia grade 1 (VAIN 1). Note the surface spicules with partial uptake of Lugol’s stain.

Figure 19.18 A: Vaginal intraepithelial neoplasia grade 2 (VAIN 2). B: Vaginal intraepithelial neoplasia grade 3 (VAIN 3).

Treatment

Patients with VAIN 1 and HPV infection do not require treatment. These lesions often regress, are multifocal, and recur quickly when treated with ablative therapy. VAIN 2 lesions can be managed expectantly or treated by ablation. VAIN 3 lesions are more likely to harbor an early invasive lesion. In a study of 32 patients who underwent upper vaginectomy for VAIN 3, occult invasive carcinoma was found in 9 patients (28%) (135). It is recommended in older patients that VAIN 3 lesions located in the dimples of the vaginal cuff be excised to rule out occult invasive cancer. VAIN 3 lesions that are adequately sampled to rule out invasive disease can be treated with laser therapy. The major advantage of laser vaporization therapy is the ability to control the depth and width of destruction by direct vision through the colposcope. The other advantage of laser therapy is the rapid posttreatment healing phase. This process takes about 3 to 4 weeks, after which time a new epithelium has formed completely and, in most cases, has a mature glycogen-containing epithelium.

Tissue Interaction

When the laser beam contacts tissue, its energy is absorbed by the water in the cells, causing it to boil instantly. The cells explode into a puff of vapor (thus the term laser vaporization). The protein and mineral content is incinerated by the heat and leaves a charred appearance at the base of the exposed area. The depth of laser destruction is a function of the power of the beam (in watts), the area of the beam (in millimeters squared), and the length of time the laser remains in the tissue. The beam must be moved uniformly across the tissue surface to prevent deep destruction. The laser beam vaporizes a central area and leaves a narrow zone of heat necrosis surrounding the laser crater. The goal of laser vaporization is to minimize the area of tissue necrosis. This goal is accomplished by using high wattage (20 watts) with medium beam size (1.5 mm) and moving the beam uniformly but quickly over the surface. The zone of thermal necrosis will be 0.1 mm when the laser is used in this manner. Some lasers have a function called super pulse, in which the laser beam is electronically switched off and on thousands of times per second, thereby allowing the tissue to cool between pulses to create less thermal necrosis.

Cryosurgery should not be used in the vagina because the depth of injury cannot be controlled and inadvertent injury to the bladder or rectum may occur. Superficial fulguration with electrosurgical ball cautery may be used under colposcopic control to observe the depth of destruction by wiping away the epithelial tissue as it is ablated. Excision is an excellent method for treatment of upper vaginal lesions in a small area. Occasionally, total vaginectomy will be required for a VAIN 3 lesion occupying the entire vagina. It should be accompanied by a split-thickness skin graft. This aggressive treatment for widespread vaginal lesions should not be used for VAIN 2.

The malignant potential of VAIN appears to be less than that of CIN. In a review of 136 cases of CIS of the vagina over a 30-year period, 4 cases (3%) progressed to invasive vaginal cancer despite the use of various treatment methods (134).

Vulvar Intraepithelial Disease

Vulvar Dystrophies

In the past, terms such as leukoplakia, lichen sclerosis et atrophicus, primary atrophy, sclerotic dermatosis, atrophic and hyperplastic vulvitis, and kraurosis vulvae were used to denote disorders of epithelial growth and differentiation (136). In 1966, Jeffcoate suggested that these terms did not refer to separate disease entities because their macroscopic and microscopic appearances were variable and interchangeable (137). He assigned the generic term chronic vulvar dystrophy to the entire group of lesions.

The International Society for the Study of Vulvar Disease (ISSVD) recommended that the old dystrophy terminology be replaced by a new classification under the pathologic heading nonneoplastic epithelial disorders of skin and mucosa. This classification is shown in Table 19.13. In all cases, diagnosis requires biopsy of suspicious-looking lesions, which are best detected by careful inspection of the vulva in a bright light aided, if necessary, by a magnifying glass (138).

Table 19.13 Classification of Epithelial Vulvar Diseases

Nonneoplastic epithelial disorders of skin and mucosa

Lichen sclerosis (lichen sclerosis et atrophicus)   Squamous hyperplasia (formerly hyperplastic dystrophy)   Other dermatoses

Mixed nonneoplastic and neoplastic epithelial disorders

Intraepithelial neoplasia

Squamous intraepithelial neoplasia   VIN 1   VIN 2   VIN 3 (severe dysplasia or carcinoma in situ)   Nonsquamous intraepithelial neoplasia   Paget’s disease   Tumors of melanocytes, noninvasive

Invasive tumors

VIN, vulvar intraepithelial neoplasia.

From Committee on Terminology, International Society for the Study of Vulvar Disease. New nomenclature for vulvar disease. Int J Gynecol Pathol 1989;8:83, with permission.

The malignant potential of these nonneoplastic epithelial disorders is low, particularly now that the lesions with atypia are classified as VIN. Patients with lichen sclerosis and concomitant hyperplasia may be at particular risk (139).

Vulvar Intraepithelial Neoplasia

As with the vulvar dystrophies, there is confusion regarding the nomenclature for VIN. Four major terms are used: erythroplasia of Queyrat, Bowen’s disease, carcinoma in situ simplex, and Paget’s disease. In 1976, the ISSVD decreed that the first three lesions were merely gross variants of the same disease process and that all of these entities should be included under the umbrella term squamous cell carcinoma in situ (stage 0) (114). In 1986, the ISSVD recommended the term vulvar intraepithelial neoplasia (Table 19.13).

VIN is graded as 1 (mild dysplasia), 2 (moderate dysplasia), or 3 (severe dysplasia or CIS) on the basis of cellular immaturity, nuclear abnormalities, maturation disturbance, and mitotic activity. In VIN 1, immature cells, cellular disorganization, and mitotic activity occur predominantly in the lower one-third of the epithelium, whereas in VIN 3, immature cells with scanty cytoplasm and severe chromatinic alterations occupy most of the epithelium (Fig. 19.19). Dyskeratotic cells and mitotic figures occur in the superficial layer. The appearance of VIN 2 is intermediate between VIN 1 and VIN 3. Additional cytopathic changes of HPV infection, such as perinuclear halos with displacement of the nuclei by the intracytoplasmic viral protein, thickened cell borders, binucleation, and multinucleation, are common in the superficial layers of VIN, especially in VIN 1 and VIN 2. These viral changes are not definitive evidence of neoplasia but are indicative of viral exposure (140). Most vulvar condylomas are associated with HPV-6 and -11, whereas HPV-16 is detected in more than 80% of VIN cases by molecular techniques.

Figure 19.19 Carcinoma in situ of the vulva (vulvar intraepithelial neoplasia grade 3, VIN 3).

VIN 3 can be unifocal or multifocal. Typically, multifocal VIN 3 presents with small hyperpigmented lesions on the labia majora (Fig. 19.20). Some cases of VIN 3 are more confluent, extending to the posterior fourchette and involving the perineal tissues. The term bowenoid papulosis (bowenoid dysplasia) was used to describe multifocal VIN lesions ranging from grade 1 to 3. Clinically, patients with bowenoid papulosis present with multiple small pigmented papules (40% of cases) that are usually less than 5 mm in diameter. Most women with these lesions are in their 20s, and some are pregnant. After childbirth, the lesions may regress spontaneously. The term bowenoid papulosis is no longer recommended by the ISSVD.

Figure 19.20 Vulvar carcinoma in situ: carcinoma in situ (VIN 3) extending into the hair follicle.

Paget’s Disease of the Vulva

Extramammary Paget’s disease of the vulva (AIS) was described 27 years after the description by Sir James Paget of the mammary lesion that now bears his name (141). Some patients with vulvar Paget’s disease have an underlying adenocarcinoma, although the precise frequency is difficult to ascertain.

Histology

Most cases of vulvar Paget’s disease are intraepithelial. Because these lesions demonstrate apocrine differentiation, the malignant cells are believed to arise from undifferentiated basal cells, which convert into an appendage type of cell during carcinogenesis (Fig. 19.21). The “transformed cells” spread intraepithelially throughout the squamous epithelium and may extend into the appendages. In most patients with an underlying invasive carcinoma of the apocrine sweat gland, Bartholin gland, or anorectum, the malignant cells are believed to migrate through the dermal ductal structures and reach the epidermis. In such cases, metastasis to the regional lymph nodes and other sites can occur.

Figure 19.21 Paget’s disease of vulva. The epidermis is permeated by abnormal cells with vacuolated cytoplasm and atypical nuclei. This heavy concentration of abnormal cells in the parabasal layers is typical of Paget’s disease.

Paget’s disease must be distinguished from superficial spreading melanoma. All sections should be studied thoroughly using differential staining, particularly periodic acid–Schiff (PAS) and mucicarmine stains. Mucicarmine has routinely positive results in the cells of Paget’s disease and negative results in melanotic lesion.

Clinical Features

Paget’s disease of the vulva predominantly affects postmenopausal white women, and the presenting symptoms are usually pruritus and vulvar soreness. The lesion has an eczematoid appearance macroscopically and usually begins on the hair-bearing portions of the vulva (Fig. 19.22). It may extend to involve the mons pubis, thighs, and buttocks. Extension to the mucosa of the rectum, vagina, or urinary tract is described (142). The more extensive lesions are usually raised and velvety in appearance.

Figure 19.22 Paget’s disease of the labium majus.

A second synchronous or metachronous primary neoplasm is associated with extramammary Paget’s disease in about 4% of patients, which is much less common than previously believed (143). Associated carcinomas were reported in the cervix, colon, bladder, gallbladder, and breast. When the anal mucosa is involved, there usually is an underlying rectal adenocarcinoma (139).

Treatment

VIN The treatment of VIN 3 varies from wide excision to the performance of a superficial or “skinning” vulvectomy (144–147). Although the treatment originally recommended for CIS of the vulva was wide excision, fears that the disease is preinvasive led to the widespread use of superficial vulvectomy (146). Because progression is relatively uncommon, typically occurring in 5% to 10% of cases, extensive surgery is not warranted (144). This is particularly important because many VIN 3 lesions are found in premenopausal women.

The therapeutic alternatives for VIN 3 are simple excision, laser ablation, and superficial vulvectomy with or without split-thickness skin grafting.

Excision of small foci of disease produces excellent results and has the advantage of providing a histopathologic specimen. Although multifocal or extensive lesions may be difficult to treat by this approach, it offers the potential for the most cosmetic result. Repeat excision is often necessary but can usually be accomplished without vulvectomy (145,147).

The carbon dioxide laser can be used for multifocal lesions but is unnecessary for unifocal disease. The disadvantages are that it can be painful and costly and does not provide a histopathologic specimen (148).

Superficial vulvectomy is appropriate to treat extensive and recurrent VIN 3 (147). The goal of the surgery is to extirpate all of the disease while preserving as much of the normal vulvar anatomy as possible. The anterior vulva and the clitoris should be preserved if possible. In some patients, the disease extends up the anus, which must be resected. An effort should be made to close the vulvar defect primarily, reserving the use of skin grafts for instances in which the vulvar defect cannot be closed because the resection is so extensive. Split-thickness skin grafts can be harvested from the thighs or buttocks, but the latter is more easily concealed (149).

Paget’s Disease

Unlike squamous cell VIN 3, in which the histologic extent of disease correlates closely with the macroscopic lesion, Paget’s disease usually extends well beyond the gross lesion (150). This extension results in positive surgical margins and frequent local recurrence unless a wide local excision is performed (151). Underlying adenocarcinomas are apparent clinically, but this finding does not occur invariably; therefore, the underlying dermis should be removed for adequate histologic evaluation. For this reason, laser therapy is unsatisfactory in treating primary Paget’s disease. If underlying invasive carcinoma is present, it should be treated in the same manner as a squamous vulvar cancer. This treatment usually requires radical vulvectomy and at least an ipsilateral inguinal-femoral lymphadenectomy.

Recurrent lesions are almost always in situ, although there was at least one report of an underlying adenocarcinoma in recurrent Paget’s disease (143). It is reasonable to treat recurrent lesions with surgical excision.

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