Levi S. Downs Jr. and Jori S. Carter
Over the past 3 decades, the work of population scientists, laboratory-based researchers, and clinicians has together promoted the understanding of the pathogenesis of preinvasive disease of the lower genital tract and its associated cancers. Elucidating the progression from preinvasive disease to invasive cancer was the first step in implementing the Pap test as one of the most successful cancer screening program in developed nations and has dramatically lessened the impact of cervical cancer in the United States and other developed countries. Furthermore, with the identification of the human papillomavirus (HPV) as the principal and necessary cause of cervical cancer, the development and application of HPV vaccines will potentially further reduce the burden of cervical cancer and other HPV-induced malignancies.
EPIDEMIOLOGY
Key Points
1. The HPV is the most common sexually transmitted infection and is the necessary cause for cervical dysplasia and cancer. HPV16 and 18 are the most common high-risk types implicated in carcinogenesis.
2. Additional risk factors for genital dysplasia including tobacco smoking, immunosuppression, early age at first intercourse, and multiple sexual partners.
3. The HPV proteins E6 and E7 are critical for malignant transformation; E6 binds and inactives the tumor suppressor gene p53, whereas E7 binds and inactivates the tumor supressor gene pRb.
Incidence of Cervical Dysplasia
In the United States, 2 to 3 million women are diagnosed with cervical cytologic abnormalities annually. The most common abnormality found by liquid-based cytology is atypical squamous cells of undetermined significance (ASC-US), accounting for 2% to 5% of all Pap test results. In contrast, low-grade squamous intraepithelial lesions (LSIL) account for 2% of Pap test results, and approximately 0.5% of Pap test results are high-grade squamous intraepithelial lesions (HSIL); less than 0.5% are suggestive of invasive cancer. Atypical glandular cells of undetermined significance (AGC) account for an additional 0.2% to 0.8% of Pap test results.
Every year, between 250,000 and 1 million women in the United States are diagnosed with cervical dysplasia. Histologic diagnosis of cervical dysplasia is based on a tissue biopsy and uses the Bethesda nomenclature; this differs from the nomenclature used for cytologic abnormalities diagnosed on Pap testing. Cervical intraepithelial neoplasia (CIN) is the formal histologic diagnosis of cervical dysplasia and is graded as 1, 2, or 3 based on the proportion of atypical cells in the cervical epithelium. CIN can occur at any age; the peak incidence is in women between the ages of 25 to 35 years (Figure 4-1).
FIGURE 4-1. Incidence of CIN2 and 3 and cervical cancer. Rates shown here are per 100,000 women undergoing routine cytologic screening for CIN2 and 3, and per 100,000 women for cervical cancer. The peak incidence of invasive cervical cancer is observed approximately 25 to 30 years later than for CIN2/3. (Sources: CIN2/3 incidence among screened women [Kaiser Permanente Northwest Health Plan, Portland, Oregon, 1998-2002], Cervical cancer incidence among unscreened women [Connecticut, 1940-1944].)
Human papillomavirus (HPV) is the necessary cause of cervical dysplasia and cervical cancer. HPV is the most common sexually transmitted infection, and is estimated that 6.2 million new infections occur annually in the United States. The overall HPV prevalence in women in the United States is estimated to be 27%. Genital HPV infection has the highest prevalence in young adults under the age of 25 years, with a 25% prevalence in girls aged 14 to 19 years and 45% in women aged 20 to 24 years.1 More than 100 HPV types have been identified, and more than 40 of these infect the anal/genital area. HPV is a double-stranded DNA virus that encodes 8 open reading frames. The corresponding proteins are described as early proteins, including 6 proteins that are involved with the HPV life cycle and replication. The 2 late proteins, L1 and L2, are the structural proteins of the virus and form the major and minor capsid, respectively (Figure 4-2).
FIGURE 4-2. The 8 open reading frames of the HPV virus. The 6 early proteins (E1-E6) are involved with life cycle and replication; the 2 late proteins (L1 and L2) form the major and minor capsid.
HPV type is determined by the degree of homology within the region of the DNA that codes for the L1 protein. A new HPV type is identified when the entire genome has been sequenced and there is more than 10% difference from a known L1 sequence. HPV is not only transmitted by sexual intercourse; it is important to recognize that nonpenetrative contact may also lead to new HPV infections. In a 2-year longitudinal study of HPV-negative women, 10% of those having sexual contact, but not sexual intercourse, were positive for HPV at the end of the observation period. It appears that most of these occur within the first few years of initiating sexual activity. Prospective studies suggest a cumulative incidence of 50% within 3 years of the onset of sexual activity. For most girls/women, genital HPV infections clear within 1 to 2 years of initial detection. The median time to clearance is 8 to 12 months, with more than 90% of infections having cleared within 2 years. It is difficult to determine whether HPV infections become dormant in basal cells and later become reactivated as “latent HPV.”2
Persistent infection with 1 of the approximately 15 carcinogenic HPV types places women at increased risk for high-grade cervical dysplasia and cervical cancer. There is no consensus on the exact duration of genital HPV infection that constitutes persistent infection; although this has been described differently in many studies, in general it appears to be between 18 and 24 months of detection of the same HPV type. It is believed that persistent infection of 10 to 20 years or more is required for cervical cancer to develop. Although young women under the age of 25 years can be diagnosed with cervical cancer, this is a rare occurrence, and there is little understanding of the circumstances that lead to a more rapid progression of the steps leading to epithelial transformation to malignancy in these rare cases.
HPVs are grouped according to their carcinogenicity. Fifteen high-risk HPV types are found in cervical cancers. These high-risk types may also be found in premalignant lesions of the cervix, but they are much more common in cancer cases than in controls; this provides the epidemiologic evidence of carcinogenicity that is used to classify high-risk and low-risk HPV types. HPV16 is recognized as the most carcinogenic of the high-risk HPV types. HPV type 16 has been identified in 46% of all high-grade premalignant lesions of the cervix and approximately 55% of all cervical cancers. High-risk HPV type 18 can be detected in 16% of all cervical cancers. Other high-risk HPV include types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82. In contrast, the low-risk HPV types 6 and 11 are the most common types detected in anogenital warts.
Incidence of Vaginal Dysplasia
Vaginal dysplasia is a relatively rare entity and comprises only 0.5% of all female genital tract lesions. The overall incidence in the United States is reported to be between 0.2 and 2 per 100,000 women. It is most commonly diagnosed in women older than 60 years of age who have current or prior diagnoses of cervical or vulvar preinvasive or invasive lesions, as they have many risk factors in common, such as HPV infection and immunosuppression. HPV DNA is present in approximately 80% of vaginal carcinoma in situ. Additional risk factors for vaginal dysplasia include prior pelvic or vaginal radiation and chronic inflammation from pessary use or prolapse. Vaginal dysplasia is usually asymptomatic with absence of a visible lesion. It is often detected by colposcopy during evaluation of an abnormal Pap test, commonly after hysterectomy for cervical dysplasia. When evident, lesions are generally white with sharp borders and are best evaluated after application of dilute acetic acid.
Vaginal intraepithelial neoplasia (VAIN) was first described in 1952 in a woman who had a total hysterectomy for cervical carcinoma in situ several years prior. VAIN was given its nomenclature in 1989 by the International Society for the Study of Vulvar Disease and includes grades 1 through 3 (mild dysplasia, moderate dysplasia, and severe dysplasia/carcinoma in situ) which are defined by the extent of microscopic cellular epithelial abnormalities.
Incidence of Vulvar Dysplasia
In the United States, the incidence of vulvar dysplasia has drastically increased by 411% between 1973 and 2000, with an incidence rate of 0.56 to 2.86 per 100,000 women, and it is becoming more common in younger women aged 20 to 35 years.3 Approximately 50% of women with vulvar dysplasia have dysplasia at other sites involving the genital tract, most commonly of the cervix.
Vulvar dysplasia was initially described by Bowen in 1912, in which a lesion of the thigh and buttocks was termed precancerous dermatosis. These lesions became known as precancerous lesions of the vulva and have adopted several different terminologies over the years, including Bowen disease, erythroplasia of Queyrat, atypical hyperplasia, lichen sclerosis et atrophicus, leukoplakia, leukokeratosis, leukoplakic vulvitis, hyperplastic vulvitis, kraurosis vulvae, neurodermatitis, atypical hyperplasia, dysplasia, atypia, carcinoma in situ, and carcinoma simplex. The original consensus for terminology was described in 1976 by the International Society for the Study of Vulvovaginal Disease, which recommended the adoption of the terms vulvar dystrophies, vulvar atypia, and squamous cell carcinoma in situ. The terminology was further revised in 1989 to include the use of the term non-neoplastic epithelial disorder when describing dysplastic conditions such as lichen sclerosis and squamous cell hyperplasia. The 1989 revisions also included the current use of the term vulvar intraepithelial neoplasia (VIN), with a grade of 1 to 3 (mild, moderate, and severe/in situ) based on the extent of microscopic cellular epithelial abnormalities, which replaced the terms atypia and carcinoma in situ. In 2004, the terminology was again revised to include the nomenclature that is currently in use to describe vulvar lesions.
Vulvar lesions are subdivided into 2 major categories: usual type VIN (including warty, basaloid, and mixed warty/basaloid types) which is associated with HPV infection and is generally seen in younger women, and differentiated type VIN, which is not associated with HPV and is generally seen in older women. A third category, unclassified type VIN, was included for rare cases that do not fit either usual or differentiated by histologic criteria.4
Risk Factors
Several risk factors for persistent HPV infection, cervical dysplasia, and cervical cancer have been identified. Because these 3 outcomes exist on a continuum, in general, the risk factors are similar for each diagnosis, with duration of exposure being the main differentiating factor. As previously described, persistent genital infection with high-risk HPV types is the most important risk factor for cervical dysplasia, and a history of high-grade cervical dysplasia is the most important risk factor for cervical cancer. When considering the specific high-risk HPV types, it appears that HPV16 infection is more likely to persist beyond 24 months and is more oncogenic than other high-risk HPV types. This is confirmed by epidemiologic data showing that HPV16 is detected in more cervical cancers than any other HPV type. In general, for all HPV infections, the longer that a type-specific infection has persisted, the more likely it is to persist. Additionally, older women with detectable HPV are more likely to have a persistent HPV infection.
Studies have found that tobacco smoking is associated with an increased risk of genital dysplasia.5 There also appears to be a close association with long-term oral contraceptive use.6 Coinfection with other sexually transmitted diseases has also been investigated as risk factors for cervical dysplasia. Chlamydia trachomatis infection is associated with cervical dysplasia. Studies investigating the role of herpes simplex virus and Trichomonas vaginalis have provided inconsistent results. There are not consistent data to support an association with nutritional intake, nutritional supplements, or alcohol use as cofactors for genital dysplasia.
Recent studies have sought to identify the role of host immunity and genetic factors such as human leukocyte antigen class I and II genes and viral factors such as HPV variants or multiple-type HPV infections, viral load, or HPV genome integration sites. More work is needed in this area, and no consistent trends have been identified.
Acquired immunosuppression, specifically human immunodeficiency virus (HIV) infection, and immunosuppressive therapy, specifically in organ transplant recipients, are closely associated with increased risk of genital dysplasia. The prevalence of all cervical cytologic abnormalities is increased in HIV-infected women, with a 3-fold increase in the prevalence of high-grade squamous intraepithelial lesions and cancer. Women receiving lifelong intense immunosuppressive therapy as a strategy to reduce the risk of organ rejection have up to a 2- to 6-fold increased risk of cervical dysplasia, 3-fold increased risk for cervical cancer, and a 50-fold increased risk for vulvar cancer.7
Other behavioral factors that are associated with and increased risk of preinvasive disease of the genital tract include early age at first intercourse, multiple sexual partners, and male partners with multiple partners.
HPV and Pathogenesis
Epidemiologic and molecular research has fully defined the relationship between persistent HPV infection and premalignant disease of the lower genital tract. High-risk HPV types 16 and 18 are the types most frequently found in cervical cancer worldwide and therefore are the most well studied. High-risk HPV infection has also been associated with the development of premalignancies and cancers of other sites, such as anal cancer, penile cancer, and malignancies of the head and neck. In contrast to cervical cancer, these cancers are preferentially associated with HPV16. Because the most is known about the molecular biology and pathogenesis at the cervix, this section focuses on specific steps that lead to premalignancy in this organ. It should be recognized, however, that there is increasing evidence that a similar pathogenesis appears to occur in other epithelial tissues where HPV has been implicated as a cause of premalignancy and cancer.
Genital HPV types preferentially infect the cervical transformation zone. Although the majority of HPV infections clear within 1 to 2 years, persistence of infection promotes the development of low-grade and high-grade cytologic and histologic abnormalities. Even after premalignant lesions are identified, some may regress, whereas others progress to an invasive malignancy after what appears to be a period of latency. In HPV-positive cancers, all malignant cells contain at least 1 copy of the viral genome that is actively transcribed. This leads to the overexpression of the viral oncoproteins E6 and E7; the long-term, continuous expression of these oncoproteins in epithelial cells leads to high-grade dysplasia and potentially malignancy.
When host epithelial cells are infected with HPV, it has been shown that the viral genome integrates into the host cell’s DNA. This occurs more frequently with high-risk HPV types, and frequency of integration increases with increasing degree of dysplasia/cancer. When integration occurs, the continuous expression of the E6 and E7 genes leads to the transformation of epithelial cells to the malignant phenotype. Both E6 and E7 have specific transforming properties. E6 induces degradation of the tumor suppressor protein p53 via the ubiquitin pathway. p53 is a cellular transcription factor that can trigger cell cycle arrest of apoptosis in response to cellular stress such as hypoxia or DNA damage. The role of p53 is to ensure the integrity of the cellular genome, preventing cell division after DNA damage or delaying it until damage has been repaired. By blocking the function of p53, E6 allows for the accumulation of chromosomal abnormalities, greatly increasing the chance of progression from normal epithelium to high-grade dysplasia and cancer. In a similar manner, E7 binds to the tumor suppressor protein retinoblastoma (pRb1) and its related pocket proteins, p107 and p130. The 3 tumor suppression proteins are critically involved in cell cycle regulation. When these proteins bind to E7, this activates the transcription of a group of genes that encode proteins essential for cell cycle progression. This allows cells to enter into S phase, when, in the absence of E7, they would otherwise undergo cell cycle arrest in G1 phase.8
Pathogenesis of Non-HPV Vulvar Dysplasia
Vulvar intraepithelial neoplasia may arise through different mechanisms. HPV-associated vulvar dysplasia is most commonly associated with the HPV16 subtype and is often multifocal and strongly associated with cigarette smoking and has a pathogenesis similar to that of cervical dysplasia. This type of VIN includes the basaloid or warty variants. In contrast, the differentiated VIN variant is not generally associated with HPV infection and is morphologically similar to invasive squamous cell carcinoma in appearance.9 Non-neoplastic epithelial disorders including lichen sclerosis, lichen simplex chronicus, and squamous cell hyperplasia are often associated with the differentiated type VIN. Although not considered premalignant lesions independently, a well-accepted hypothesis in the pathogenesis of differentiated VIN is that chronic pruritus caused by these disorders, and the resultant inflammation from scratching, plays a role in the progression from lichen sclerosis to lichen simplex chronicus and results in squamous cell hyperplasia, which then progresses to differentiated VIN, and ultimately to invasive carcinoma. Mixed dystrophy refers to lichen sclerosis which is associated with variable degrees of squamous cell hyperplasia and illustrates the progression and spectrum of changes seen in lichen sclerosis.
Lichen sclerosis is a non-neoplastic epithelial disorder of unclear etiology and is most commonly seen in postmenopausal white women. Lesions appear as pale white, flat, plaque-like areas which can resemble thinned parchment paper in advanced cases (Figure 4-3). Histologically, lichen sclerosis shows a thinned epidermis with blunting or loss of rete ridges, a middle layer of homogenous collagenized subepithelial edema, and a lower band of lymphocytic infiltration. When lichen sclerosis is associated with lichen simplex chronicus, it shows epidermal thickening instead of thinning associated with superficial dermal chronic inflammatory infiltrate with vertical collagen streaks in the papillary dermis. Lichen sclerosis with squamous cell hyperplasia has the presence of epidermal hyperplasia without inflammation, atypia, or evidence of a specific dermatosis.
FIGURE 4-3. Lichen sclerosis. White, plaque-like areas can resemble parchment paper.
Lichen planus is a dermatosis most commonly seen in women older than 40 years, although it may be present across a wide age range. When symptomatic, women present with burning and pruritus. It is often associated with similar lace-like plaques in the oral or vaginal mucosa. There is a variable appearance histologically, but it is diagnosed by the presence of a bandlike chronic lymphocytic inflammatory infiltrate and the presence of colloid bodies formed as a result of degenerated keratinocytes. Lichen planus can evolve into erosive vulvar disease, which has been associated with invasive vulvar squamous cell carcinoma.10
DIAGNOSIS
Key Points
1. The American Cancer Society recommends Pap test screening 3 years after the initiation of sexual intercourse or by 21 years of age. In contrast, the American Congress of Obstetricians/Gynecologists recommends that screening should not begin until 21 years of age.
2. Abnormal Pap tests should trigger subsequent colposcopic examination to obtain directed tissue specimens for histologic evaluation of dysplasia or cancer.
3. The most common presenting symptom of vulvar dysplasia is a pruritic lesion.
Cervical Dysplasia
As early as 1932, it was recognized that carcinoma in situ was a precursor to cervical cancer. Papanicolaou and Traut subsequently demonstrated that the exfoliated cells from the ectocervix could be used to detect carcinoma in situ and invasive cancer. In 1969, Richart hypothesized that cervical cancer develops from noninvasive stages, thereby introducing the terminology cervical intraepithelial neoplasia (CIN).
Since the 1950s, the Pap smear has been the central component of population-based cervical cancer screening in the United States. Pap-based screening has led to a more than 70% reduction in cervical cancer mortality by making it possible to identify and treat premalignant lesions of the cervix before these lesions develop into cancer. Similarly, the goal of treating vulvar and vaginal dysplasia is primarily to decrease or eliminate the risk of any individual developing cancer of these organs. The Pap smear was originally performed using a wooden or plastic spatula to scrape the ectocervix in a 360-degree circumference. The exfoliated cells were then smeared onto a glass slide, preserved with a spray fixative, processed, and examined by cytopathologists. The degree of cellular atypia and abnormal morphology lead to classifications based on the risk of a histologic abnormality.
The conventional Pap smear is arguably suboptimal due to the frequency of both false-positive and false-negative results, which likely result from the poor quality of sampling and the preparation method. The frequent presence of blood cells or inflammatory cells, poor cell fixation, and inhomogeneous distribution of cells contributes to greater difficulty in detecting epithelial cell abnormalities and impairs the reproducibility of diagnosis. In an attempt to improve test sensitivity, liquid-based cytology may demonstrate superiority to conventional Pap techniques. Exfoliated cells are collected from the ectocervix with a brush designed to match the contour of the cervix, and the cells are rinsed into a vial with a preservative solution. The solution is processed to remove inflammatory and blood cells and debris, and a single-layer slide of epithelial cells is created to facilitate detection of cellular abnormalities. Because only a portion of the preservative is used, the remaining liquid can be used for other purposes, such as HPV detection or other testing. Many prospective studies have suggested that this process improves the sensitivity and positive predictive value for the detection of moderate- or high-grade dysplasia. However, recent studies have challenged these original reports. A large cohort of almost 90,000 women has shown that liquid-based cytology has similar sensitivity and positive predictive value for preinvasive disease of the cervix as that of conventional Pap smears.11 Despite these recent reports showing similar sensitivities between the 2 methods, the additional benefit of using liquid-based cytology for HPV detection or other testing, faster reading times, and decreased processing costs through automation makes this approach superior to conventional Pap smears. Pap testing with liquid-based cytology has become the primary sampling method used in the United States.
Population-based screening programs have been successful in reducing cervical cancer mortality in developed nations. The optimal screening program has not been defined, and the approach varies throughout the world based on heath system financing and resources allocated to screening programs. For example, screening frequency varies from 5-year intervals in the Netherlands and parts of France to yearly intervals in Germany.
The American Cancer Society (ACS) and the American College of Obstetrics and Gynecology (ACOG) both publish guidelines on cervical cancer screening (Table 4-1). In general, the consensus guidelines of both organizations are supported by the United States Preventative Services Task Force. The 2003 ACS guidelines and the 2009 updated recommendations from ACOG are similar and are presented next. The main area of difference between the 2 organizations is age at which to initiate screening. The ACS recommendations are to begin screening 3 years after the initiation of sexual intercourse or by 21 years of age. In contrast, the updated 2009 ACOG guidelines recommend that screening should not begin until 21 years of age.12 The recommendation to begin screening at age 21 is based on the very low incidence rate of cervical cancer before age 21 years and the anxiety and harm that diagnosis and treatment of premalignant lesions in this age group may cause. Because the majority of abnormal cytology and premalignant lesions diagnosed in this age group will regress without the need for treatment, the risks of Pap testing may outweigh any benefits. These risks are particularly relevant with the incidence of cervical cancer in women under 21 years of age as low as 1 to 2 per 1,000,000, or only 0.1% of all cervical cancers.
Table 4-1 Comparison of ACS and ACOG Guidelines for Screening for Cervical Cancer12
Once cervical cancer screening begins, both ACS and ACOG recommend that screening occurs at a frequency of once every 2 years until age 29 years. Women ≥ 30 years of age with a history of 3 consecutive negative Pap smears may elect to have combination testing with cytology and high-risk HPV detection. With a normal Pap test and absence of high-risk HPV, further screening is deferred for 3 years, at which time the combination screen should be repeated. If HPV testing is not performed along with cytology after age 30 years of age, then testing should be performed every 2 to 3 years. ACS recommendations specify that women who choose to have high-risk HPV testing in this setting should be informed that (1) HPV infection usually is not detectable or harmful; (2) almost everyone who has had sexual intercourse has been exposed to HPV, and infection is very common; (3) a positive HPV test result does not reflect the presence of a sexually transmitted disease, but rather a sexually acquired infection; and (4) a positive HPV test result does not indicate the presence of cancer, and the large majority of women who test positive for an HPV infection will not develop advanced cancer.
Women who have an intact cervix should continue screening at the 2- to 3-year frequency until age 65 years (per ACOG guidelines) or 70 years (per ACS guidelines). Women may then choose to discontinue routine screening if they have no abnormal cytology or histologic diagnoses of the cervix for the prior 10 years and there is documentation of 3 consecutive normal Pap tests. Screening is recommended to continue, without a maximum age, for women in good health who have not previously undergone screening or if information regarding prior screening is not available. Women who are immunocom-promised by organ transplantation, chemotherapy, or chronic corticosteroid treatment or who are HIV positive should be tested twice during the first year after their immunosuppression-related diagnosis and then annually thereafter. There is no recommended age to stop screening in these women. Immunocompromised women should continue screening for as long as they are in good health and are likely to benefit from early detection and treatment of preinvasive disease.
Cytology screening is not recommended for women who have had a hysterectomy for reasons other than gynecologic cancer or cervical dysplasia. If a women has a history of CIN 2 or 3, or if it is not possible to document the absence of CIN 2 or 3 from pathologic reports, cytologic screening should take place for 10 years, and there should be documentation of 3 consecutive normal cytologic screenings at the end of this period.
It is important to recognize that Pap testing is a screening test and indicates women who are at risk for having a histologic diagnosis of cervical dysplasia. When sufficient risk exists that a patient may have cervical dysplasia or cancer, then most often the next step in diagnosis is to perform a colposcopic examination to obtain directed tissue specimens for histologic determination of the presence (or absence) of cervical dysplasia or cancer. The colposcope is a lighted binocular microscope which magnifies the surface of the tissue being examined. It is used to visualize the cervix, vagina, and vulva. Tissue can be magnified between 2 × and 25 × power. Colposcopes are equipped with various light filters to help better identify vascular patterns that may be associated with varying degrees of dysplasia or cancer. A dilute solution of acetic acid is applied to the cervix during colposcopy. The acetic acid dehydrates cells, thus exaggerating the increased nuclear-to-cytoplasmic ratio found in the cells of dysplastic tissue and cancer. Due to this increased density of DNA, dysplastic cells will reflect the light from the colposcope, and the observer sees a so-called acetowhite lesion. In addition to these acetowhite changes, other colposcopic features suggestive of dysplasia include the margin of the lesion, the presence of vascular patterns referred to punctations or mosaicisms, and size of the lesion relative to the overall size of the cervix. Furthermore, the colposcopist may apply an iodine paint (or Lugol solution) to the cervix to aid in the detection of dysplastic lesions. The iodine solution is thought to stain intra-cellular glycogen. Normal cells will absorb the iodine and appear brown when viewed through the colposcope. Dysplastic cells, however, absorb less iodine due to their increased nuclear to cytoplasmic ratio and appear yellow or variegated brown/yellow. These features of cervical dysplasia help the colposcopist determine where to biopsy and how many biopsies need to be performed.
The second objective of colposcopy is to exclude a diagnosis of invasive cancer. It is important that the colposcopist be attuned to the visual changes that are predictive of invasive cancer. Fungating lesions or areas of hyperemia and friable areas may be evidence of dysplasia or invasive cancer. In addition, large, complex acetowhite lesions obliterating the cervical os or lesions with irregular and exophytic contour are very concerning for cancer. Lesions that appear to be thick, chalky-white with raised or rolled out margins, or lesions bleeding on touch should be biopsied to evaluate for possible preclinical invasive cancer. An important feature of invasive cancer is the appearance of atypical blood vessels. Atypical vessels occur with blood vessels breaking out from mosaic formations. The atypical vessel patterns are varied and may take the form of hairpins, corkscrews, commas, or have irregular branching patterns with irregular caliber.
Vaginal Dysplasia
As with cervical dysplasia, there are minimal clinical features associated with vaginal dysplasia. The diagnosis of vaginal dysplasia follows the steps described previously for cervical dysplasia. The screening Pap test may result in a diagnosis of a cytologic abnormality; when this occurs after the cervix has been surgically removed, then colposcopy is specifically performed to detect vaginal dysplasia. A detailed examination of the complete vagina is warranted, and it may be necessary to reposition the speculum in this setting so that the lateral and anterior and posterior walls may be fully examined with acetic acid solution. It is often helpful to use Lugol solution during complete colposcopy of the vagina. Abnormal findings should be biopsied and evaluated in a manner similar to that of cervical lesions seen during colposcopic examination.
Vulvar Dysplasia
Most women with vulvar dysplasia are asymptomatic, and the diagnosis is made with a high index of suspicion, colposcopy, and biopsy. When clinically evident, the most common symptom is pruritus. Other symptoms include burning, dyspareunia, erythema, edema, and pain. Lesions have a raised surface and are pigmented in 25% of cases and can also be grey or red (Figure 4-4). Multifocality is a common feature. Lesions are frequently located at the posterior vulva or periclitoral regions and can extend to adjacent structures. Many women have concomitant or previous dysplasia in another location in the genital tract and should be evaluated with colposcopy and liberal biopsies. Half of lesions involved with VIN become acetowhite after the application of 3% to 5% acetic acid, which should be applied for at least 5 minutes before examination. Thorough examination with the colposcope should follow application of acetic acid, and punch biopsies should be taken at each site of a suspicious lesion. These small punch biopsies can be easily performed in the clinic setting after the superficial injection of lidocaine and incorporate the full thickness of the skin for diagnosis. If needed, hemostasis can be achieved with silver nitrate or a single suture across the area of the biopsy.
FIGURE 4-4. Vulvar dysplasia. Lesions have a raised surface and may be pigmented.
PATHOLOGY
Key Points
1. CIN and VIN are reported via a 3-level system, which is based on the proportion of epithelium occupied by abnormal cells measured from the basement membrane.
2. Adenocarcinoma in situ describes cellular atypia involving the glandular epithelium, which may be multicentric (with “skip lesions”) and extend to multiple quadrants.
Cervical Cytology
The most frequently used terminology for reporting Pap test results are the Bethesda System criteria, which was last updated in 200113 (Table 4-2). Normal squamous and endocervical cells without evidence of HPV infection are considered as “negative for intraepithelial lesion of malignancy.” Pap tests with normal endometrial cells may also be placed into this diagnostic category. It is not uncommon that Pap tests of the vagina performed after a hysterectomy show evidence of normal glandular cells. Reasons for this include exfoliated cells from vaginal endometriosis, Bartholin glands, periurethral and perivaginal glands, vaginal adenosis, remnants after surgical or ablative therapies, or prolapsed fallopian tubes. When the glandular cells appear completely benign, they can be considered negative and are included in this diagnostic group.
Table 4-2 The 2001 Bethesda System (Abridged)
The category of atypical squamous cells of undetermined significance (ASC-US) is used to describe a finding of equivocal cells. These cells may be atypical secondary to HPV infection, but other reasons include atrophic or reactive processes. Regardless of etiology, the nuclear size of these cells are 2 to 3 times increased over normal cells, with a slight increase in nuclear-to-cytoplasmic ratio. There is mild nuclear hyperchromasia and chromatin irregularity with mild variations in nuclear shape. Atypical parakeratosis may also be seen.
Specimens that are interpreted as atypical squamous cells, favor high grade (ASC-H), contain cells that are suspicious for the presence of high-grade dysplasia but are too limited in volume to permit classification as a high-grade squamous intraepithelial lesion. These specimens typically contain immature cells with atypia and nuclei that are 1.5 to 2.5 times increased over normal. There is an increased nuclear-to-cytoplasmic ratio, similar to high-grade squamous intraepithelial lesion (HSIL), and variations in nuclear size and shape, with nuclear membrane irregularity.
Specimens that are described as low-grade squamous intraepithelial lesions (LSIL) show atypia in mature cells with nuclei that are 3 times normal size. There is a slight increase in nuclear-to-cytoplasmic ratio, with hyperchromasia and coarsely granular chromatin. There are variations is nuclear size and shape, with binucleation and multinucleation. There is irregularity to nuclear membranes, which is variable, and perinuclear cavitation or koilocytosis, sometimes described as a cytoplasmic halo (Figure 4-5).
FIGURE 4-5. Low-grade squamous intraepithelial lesions. Cytology shows nuclear atypia, increased nuclear to cytoplasmic ratios, and perinuclear cavitation (koilocytosis).
In contrast to LSIL, Pap tests with HSIL show atypia in immature cells, with variability in cell size, including small cells. There is marked increase in nuclear-to-cytoplasmic ratio, with hyperchromasia with fine to coarse chromatin. There is variation in nuclear size and shape, with marked nuclear membrane irregularity (Figure 4-6).
FIGURE 4-6. High-grade squamous intraepithelial lesions. Cytology shows atypia in immature cells, with marked increase in the nuclear to cytoplasmic ratio.
Cervical Histology
The histologic diagnosis of cervical dysplasia and pre-cancer is referred to as CIN. The standard reporting for histologic diagnoses of CIN involves a 3-level system: CIN1, 2, and 3. The traditional grading of dysplasia is based on the proportion of epithelium occupied by basaloid, undifferentiated cells, with higher grade assigned with the progressive loss of epithelial maturation.
The architectural abnormalities associated with CIN1 are the morphologic manifestations of an active and productive HPV infection. These morphologic changes include pronuclear cytoplasm cavitation with thickening of the cytoplasmic membrane, nuclear atypia, and anisocytosis. Nuclear atypia is present, with nuclear enlargement, hyperchromasia, and irregularity and wrinkling of the nuclear membrane. The loss of maturation is demonstrated by the presence of cells with nuclei near the surface epithelium; these nuclei are somewhat smaller than basal cells. The term koilocytosis refers to the combination of the cytoplasmic cavitation (vacuoles) and nuclear atypia. Further, these cells may be binucleated or multinucleated. CIN1 is defined by the limitation of these abnormal, atypical cells to the bottom one-third of the epidermal layer.
An important feature of HPV-induced lesions anywhere in the genital tract is the presence of cellular atypia. In the absence of atypia, these changes are nonspecific, which may be a reflection of atrophy-related vacuolar degeneration or non-HPV infections such as Gardnerella vaginalis or candidiasis or squamous epithelium containing abundant amounts of glycogen. These are the most important abnormalities to consider in the differential diagnosis of an LSIL Pap test and represent the diagnostic challenges in correctly identifying CIN1. Specific testing for HPV DNA may help to differentiate these lesions (Figure 4-7).
FIGURE 4-7. CIN1. Abnormal, atypical cells are limited to the bottom one-third of the epidermal layer.
In CIN2 and CIN3, immature basaloid cells occupy more than the lower one-third of the epithelium. CIN2 is defined as atypical cells confined to the lower two-thirds of the epidermal layer; CIN3 is considered the presence of atypical cells extending more than two-thirds from the basement membrane. Carcinoma in situ is defined as the entire epidermal layer comprising atypical cells, without penetration through the basement membrane. In general, there is nuclear crowding, pleomorphism, and loss of the normal cell polarity. Nuclear enlargement that is more pronounced in the lower portion of the epithelium is present, but it occurs throughout the epithelium. This lesion is differentiated from CIN1 in that the nuclear chromatin is more coarsely granular, normal or abnormal mitotic figures are present, and cytoplasm is usually scant. In the superficial layers of the epithelium, individual dyskeratotic cells may be seen (keratinization occurring below the most superficial cells of the epithelium). In addition, there may be marked variability in nuclear size (anisonucleosis) (Figure 4-8).
FIGURE 4-8. CIN2 and 3. Abnormal, atypical cells are limited to the lower two-thirds of the epidermal layer for CIN2; for CIN3, atypical cells extend more than two-thirds from the basement membrane.
Included in the differential diagnosis of CIN2, CIN3 is immature metaplasia and atrophy, the 2 most common lesions mistaken for dysplasia. Although there is a lack of maturity in the epithelium of immature metaplasia and there is a high nuclear-to-cytoplasmic ratio, the absence of nuclear pleomorphism helps to differentiate this lesion from high-grade dysplasia. Immature metaplasia does not contain abnormal mitoses. Additionally there may be mucinous epithelium on the surface of immature metaplastic squamous epithelium, this is rarely seen in high-grade dysplasia. Atrophic epithelium may be mistaken for high-grade dysplasia. Although atrophic epithelium has high nuclear-to-cytoplasmic ratio, atrophic epithelium is thin and shows no nuclear pleomorphism, mitotic activity, atypia, or lack of polarity. Often a repeat biopsy after a trial of vaginal estrogen therapy may help clarify this difference; vaginal estrogen will not alter the histologic appearance of CIN2 or 3.
Adenocarcinoma in situ (ACIS) demonstrates histologic features including preservation of normal glandular architecture and involvement of part or all of the epithelium by enlarged, hyperchromatic, stratified nuclei with coarse chromatin, small nucleoli, mitoses, and apoptosis. The cytoplasm can be depleted or abundant, vacuolated, granular and basophilic, or eosinophilic (Figure 4-9). This histology may frequently coincide with a squamous intraepithelial lesion. It is often difficult to identify these lesions because they are located high in the endocervical canal and the cytologic criteria for identifying neoplastic glandular lesions is less clear. An important feature of these glandular lesions is that they may be multicentric. Several foci of ACIS may coexist and be interspersed with normal epithelium within the glandular architecture, leading to histologic findings of “skip lesions.”
FIGURE 4-9. Adenocarcinoma in situ. Normal glandular architecture is preserved, but atypical cells involve the epithelium. Lesions may be multicentric and interspersed with normal glandular epithelium.
Vulvar Dysplasia
The histologic diagnosis of premalignant lesions of the vulva is referred to as VIN with a 3-level system: VIN1, 2, and 3. As seen in CIN, VIN is graded into 1 of these 3 levels depending on the level of involvement of the affected epidermis by cellular disarray, atypia, and mitotic activity. In VIN1 (mild dysplasia), the lowest third of the epidermis is involved. In VIN2 (moderate dysplasia), the lower two-thirds are involved, and in VIN3 (severe dysplasia/carcinoma in situ), more than two-thirds of the epithelium are involved. The affected epidermis is apparent when epithelial cells have a high nuclear-to-cytoplasmic ratio and lack cytoplasmic maturation above the basal and parabasal layers. Mitotic figures are present above the basal layer, and multinucleation and dyskeratosis may be seen, often including formation of intraepithelial squamous pearls. The presence of abnormal mitoses is almost always seen in VIN2 and 3 (Figure 4-10).
FIGURE 4-10. VIN2 and 3. Abnormal mitoses are characteristic for high-grade vulvar dysplasia.
The grading system for VIN parallels that of cervical dysplasia. However, whereas CIN1 lesions of the cervix are relatively common, VIN1 lesions are rare, as most lesions are diagnosed as VIN2 or 3. Another difference between CIN and VIN is that the origin of atypical cells in CIN develops from the endocervical glandular mucosa and the metaplastic squamous epithelium of the transformation zone. VIN originates from mature stratified squamous epithelium of vulvar epidermis or squamous mucosa. In this way there are often more pathologic similarities of VIN to premalignant squamous lesions of the oral cavity and vocal cords.
There are 2 variants of the usual type of VIN, basaloid and warty. The basaloid variant is similar to CIN in its morphology of epithelial changes. There is very little maturation of the keratinocytes, except some keratinization or parakeratosis, which may be seen at the surface. It is associated with uniform, small cells that have hyperchromatic and coarse nuclear chromatin.
The warty variant has larger cells, pleomorphic nuclei, and abnormal mitotic figures. There is often the presence of a prominent granular layer. The nuclear chromatin is clumped and coarse without evidence of nucleoli, despite the presence of mitotic figures. The surface has features similar to condyloma acuminatum with koilocytosis and multinucleation.
The differentiated type of VIN has a thickened epithelium with the presence of parakeratosis. The keratinocytes tend to be large and pleomorphic, with eosinophilic cytoplasm in the cells within the basal and parabasal layer in the base of rete ridges. Within the rete, there is sometimes the presence of keratin pearl formation. Nuclear chromatin is often vesicular with prominent nucleoli, most prominent in the basal and parabasal layers.
Included in the differential diagnosis of VIN are basal cell carcinoma, superficial spreading malignant melanoma, Paget disease, pagetoid urothelial intraepithelial neoplasia, and multinucleated atypia of the vulva. Specifically, differentiated VIN can be mistaken for atypical squamous cell hyperplasia, and the discrimination can be made by evidence of eosinophilic cytoplasm at the base of the rete ridges in differentiated VIN. Immunoperoxidase staining can differentiate the presence of Paget disease or melanoma: Paget disease will stain positive for mucin with mucicarmine stain and stain positive for carcinoembryonic antigen and cytokeratin; melanoma in situ will stain positive for S-100 protein, HMB-45, and Melan-A. An important issue to consider in the evaluation of VIN is that the application of podophyllin on condyloma acuminata on the vulva will cause mitotic arrest, which can easily be misinterpreted as VIN. Therefore, biopsy should be obtained at least after 1 to 2 weeks from the last application of podophyllin.
Squamous histology is the most common histology of premalignant disorders of the vulva. However, glandular histologies are present in vulvar extramammary Paget disease and Bartholin gland tumors (as well as other tumors that arise from other skin appendages, the urethra, and the Skene gland). Seen primarily in postmenopausal women, Paget disease presents often as multifocal, well-demarcated, scaly, velvet-like, moist, eczematous plaques dotted with small, pale islands. Vulvar Paget disease is primarily an intraepithelial lesion, but has the potential for dermal invasion (4%-26%) and is sometimes associated with an underlying adenocarcinoma (in up to 25% of cases). Frequently, lesions extend beyond the clinically apparent margins and may require extensive surgical resection. Because of this, positive margins are frequent and may account for a high rate of recurrence.
Histologically, Paget cells are large intraepidermal cells with a large nucleus with prominent nucleolus and abundant clear, pale cytoplasm, which stain positive for mucin. These cells may appear singly, in small clusters, or in large nests and may extend into the surrounding hair shafts and skin appendages (Figure 4-11).
FIGURE 4-11. Paget disease of the vulva. Paget cells are intraepidermal with abundant pale cytoplasm; they may appear singly, in small clusters, or in large nests.
TREATMENT
Key Points
1. The mainstay of treatment of high-grade CIN, VIN, and VAIN is surgical excision, although size and number of lesions in VIN and VAIN may influence decisions to treat with topical therapies or CO2 laser ablation.
2. CIN2 in compliant adolescent and young women may be managed with close surveillance instead of excisional biopsy.
3. ACIS should be managed with conization, and surveillance after excision appears to be safe if margins are negative for disease.
Management of Cervical Cytologic Abnormalities
The American Society for Colposcopy and Cervical Pathology (ASCCP), together with partner organizations and collaborators from the National Institutes of Health, have developed consensus guidelines for the treatment of women with abnormal cervical cancer screening tests, as well as guidelines for the management of CIN or ACIS.14 The following sections provide a summary of the important concepts included in the most recent full sets of guidelines.
Management of Atypical Squamous Cells
In women older than 20 years of age, the cytologic findings of ASC-US should be managed by DNA testing for high-risk types of HPV. As reported in the ASCUS-LSIL Triage Study (ALTS), women with ASC-US who are high-risk HPV negative can be followed up with repeat cytologic testing in 12 months. In a setting where specific high-risk types are reported, it is recommended to triage women who are positive for types 16 and 18 to colposcopy, whereas women positive for other, less carcinogenic high-risk types, can be followed up with cytologic testing in 12 months (similar to women who are negative for any high-risk type).15
Women with ASC-US and who are high-risk type positive (where specific typing is not available) or are specifically HPV16 or 18 positive, as well as women with ASC-H Pap results, should be referred for colposcopic evaluation. Endocervical curettage is preferred for women in whom no lesions are identified or for whom colposcopy is unsatisfactory. When CIN is not identified in these women, acceptable follow-up includes repeat HPV DNA testing at 12 months or cytologic testing at 6 and 12 months, depending on the situation.
An important population in which to discuss the management of Pap test results is adolescent women. Because of the high prevalence of high-risk HPV in this population and the high likelihood for moderate and severe dysplasia to regress, it is recommend that ASC-US be followed with repeat cytology in 12 months. This is the same for ASC-H and LSIL in this population. The guidelines recommend that for adolescents (girls age 20 years and younger), only those with HSIL Pap results be referred for colposcopy.
Management of Low- and High-Grade Squamous Intraepithelial Lesions
In general, women with LSIL and HSIL lesions should be referred for colposcopy. For women with LSIL, if no dysplasia is identified, then surveillance can be performed with testing for high-risk HPV DNA at 12 months or repeat cervical cytology at 6 and 12 months. If the DNA test is negative or if 2 consecutive repeat cytologic tests are negative, then the patient can return to routine cytologic screening. Adolescents with LSIL should not be referred to colposcopy and should be re-screened in 12 months.16 Postmenopausal women with LSIL can be managed with testing for high-risk HPV with those positive referred for colposcopy and repeat Pap testing at 6 and 12 months. If no abnormalities are found on either of these screens, then return to routine annual screening is safe.
HSIL Pap results are associated with a greater than 50% to 60% risk for high-grade cervical dysplasia. In young women, many of these lesions will spontaneously regress. Nonetheless, in nonadolescents, advanced “see and treat” management options based on cytology results may be appropriate. Options include immediate loop electrosurgical excision (LEEP) or colposcopy with endocervical assessment and directed biopsies. When CIN2 or CIN3 is not identified histologically, then a diagnostic excision procedure or observation with colposcopy and cytology at 6-month intervals for 1 year is acceptable. A diagnostic excisional procedure is recommended in women with HSIL Pap results who also have unsatisfactory colposcopy.
In adolescents with HSIL, immediate referral to colposcopy is the appropriate management. It is not acceptable to perform immediate LEEP on adolescents. When CIN2 or 3 is not identified histologically, then observation for up to 24 months using colpos-copy and cytology at 6-month intervals is preferred. If HSIL persists for 24 months, without identification of CIN, then a diagnostic excisional procedure is recommended. After 2 negative cytologic screens, adolescents can return to routine annual screening.
Management of Atypical Glandular Cells
Atypical glandular cells (AGC) is a rare Pap test diagnosis, but it may be associated with an invasive cancer between 3% and 17% of the time.6 Because this diagnosis can be associated with many different neoplastic entities, the initial evaluation includes multiple tests. Colposcopy with endocervical curettage should be performed for all women with AGC. In women older than 35 years of age, endometrial biopsy should also be performed. In women younger than 35 years with findings that suggest a risk for endometrial cancer, such as irregular vaginal bleeding or chronic anovulation, endometrial biopsy should be performed. HPV testing should be performed at the time of colposcopy in women with atypical endocervical, endometrial, or glandular cells not otherwise specified.
If colposcopy and biopsies are negative after AGC Pap diagnosis, then the recommended follow-up is repeat cytology and HPV DNA testing at 6 months for women who were positive for high-risk HPV and at 12 months for women who were HPV DNA negative. If on subsequent testing they are found to be high-risk HPV positive or have ASC-US or greater abnormality on cytology, then they should be referred for colpos-copy. If both tests are negative, then women can be returned to annual cytologic screening.
Management of Cervical Histologic Abnormalities
There are many treatment modalities that are used once a histologic diagnosis of dysplasia has been established. These can be divided into 2 broad categories: ablative techniques and excisional techniques. Ablative techniques destroy the abnormal cells without removing tissue, and such procedures include cryotherapy, laser ablation, and electrofulguration. Excisional techniques include cold-knife conization, LEEP, and laser conization. A recent Cochrane review of these various treatment modalities found similar effectiveness and side effects with ablative procedures of cryotherapy and laser ablation. Risk of residual dysplasia and recurrence appear to be similar. In similar fashion, the excisional procedures are equally effective. The immediate side effects of the procedure—hemorrhage, infection, and vaginal discharge—are similar for each modality.17
The most important long-term side effect of excisional procedures is related to pregnancy. These include an increased risk of preterm labor, low-birth-weight infants, and cesarean section. Pregnancy complications have been recognized as a potential outcome of cold-knife cone biopsies for some time. Recent studies have demonstrated a greater frequency of these outcomes than once suspected and that after just 1 procedure, women are at an increased risk for pregnancy complications. There is increasing evidence that LEEP procedures are also associated with an increased risk of pregnancy complications, including an up to 3-fold increase in the risk of preterm delivery.18
CIN1
As described earlier, most CIN1 lesions will spontaneously regress without treatment. Progression of CIN1 lesions to CIN2/3 is uncommon. In the ALTS trial, the risk of subsequent diagnosis of CIN2/3 within 2 years of a CIN1 diagnosis was 13%, almost identical the risk of CIN2/3 within 2 years from a normal cervical biopsy (12%).15 Based on this understanding, the recommendations for management of CIN1 are related to the abnormal Pap test that first led to the colposcopy referral. In cases in which the CIN1 diagnosis was preceded by ASC-US, ASC-H, or LSIL, the recommended management is surveillance with HPV DNA testing every 12 months or repeat cervical cytology every 6 to 12 months. If a patient has persistent CIN1 for 2 years, surveillance or treatment are acceptable options. In this setting either ablation or excisional techniques may be performed. However, if the preceding colposcopic examination is unsatisfactory, the endocervical sample contains CIN, or the patient has been previously treated, then an excisional procedure should be performed.
When a CIN1 diagnosis is preceded by an HSIL or ACG Pap test, either an excisional procedure or observation with colposcopy and cytology at 6-month intervals for 1 year are acceptable options. If colposcopy is unsatisfactory or endocervical curettage is positive for dysplasia, then an excisional procedure is recommended. If during the observation period a repeat Pap test shows HSIL or AGC, then an excisional procedure should be performed. If during the year of observation there are 2 sequential Pap tests that are negative, then the patient can return to routine screening. Hysterectomy should not be considered as the initial treatment for CIN1.
CIN2 or 3
In general, lesions categorized as CIN2 are more likely to regress spontaneously than CIN 3 lesions. However, there is considerable variability in the categorization of the lesions between various pathologists. Based on this, treatment recommendations consider CIN2 as the threshold for recommending treatment, as there is risk for progression to CIN3 and invasive cancer.
Excision or surveillance may be considered in the treatment of CIN2 or 3, depending on the age of the patient. Young women have more robust immune systems, and thus surveillance of CIN2 is acceptable due to the higher rates of regression and risks of pregnancy complications with excisional procedures. These recommendations are supported by a prospective study of young women ages 13 to 24 years with CIN2. In 95 patients, 38% were observed to demonstrate resolution at 1 year, 63% by year 2, and 68% by year 3.19 Thus, in compliant adolescent patients, CIN2 may be managed with 6-month cytology and colpos-copy.16 For noncompliant or immunosuppressed patients, or in those for whom fertility is not desired, excisional biopsy is recommended.
CIN3 should be managed with excisional biopsy. In these situations, LEEP or conization is both therapeutic and diagnostic. The rationale for excision of CIN3 is underscored by the risks of progression to invasive disease, which is classically described as 12%.20
Surveillance after the treatment of CIN2 or 3 can be performed by testing for the presence of high-risk HPV types at 6- to 12-month intervals or cytology alone or cytology with colposcopy at 6-month intervals. If patients are found to have high-risk HPV, or abnormal cytology of ASC-US or greater, colposcopy is then recommended. After 2 consecutive negative screens, patients can return to annual screening and should continue to have screening for the subsequent 20 years. In women found to have positive margins on their excisional biopsy specimen, repeat testing at 4 to 6 months is the preferred management. Immediate re-excision is not necessary, as 50% to 60% of women with positive margins after LEEP or conization will not demonstrate recurrence on subsequent surveillance Pap testing.21,22
ACIS
Women with ACIS diagnosed on cervical biopsy should undergo conization to exclude the possibility of invasive adenocarcinoma. In the past, the management of ACIS was somewhat controversial due to the “skip lesions” that can be characteristic of this disease, and subsequent extrafascial hysterectomy was traditionally recommended due to the possibility of residual ACIS, even with negative surgical cone margins. These recommendations were based on studies suggesting up to a 19% incidence of identifying ACIS on hysterectomy specimens after a cone biopsy with negative margins.23
More recent data suggest that conservative management is a safe option in women who desire uterine preservation. A large meta-analysis of 671 women followed with surveillance only after a diagnosis of ACIS on cone biopsy identified only a 2.6% rate of recurrence with negative margins and a 19.4% rate of recurrence with positive margins.24 In this same study, invasive adenocarcinoma was more commonly associated with positive margins (5.2%) compared with negative margins (0.1%). These data support surveillance of women with ACIS and negative margins and consideration of hysterectomy versus re-excision if positive margins are identified at cone biopsy.
Management of Vulvar and Vaginal Dysplasia
Various treatment modalities are used in the management of VIN and VAIN and include topical agents, laser ablation, radiotherapy, and surgical excision. Prospective trials demonstrating the most effective treatment modality for these disease sites are limited, and treatment typically is recommended based on disease and patient factors such as size and number of lesions, as well as performance status of the patient.
Topical agents are noninvasive treatments that can be applied by the patient directly to the dysplastic lesion or to the entire vulvar and/or vaginal mucosal surface. In general, these agents are the recommended therapy for persistent low-grade lesions and multifocal disease, as well as for women who are poor surgical candidates. Five percent imiquimod cream is currently the most commonly used topical agent for the treatment of VIN. It is a topical immune-response modifier that affects local cytokine production and cell-mediated immunity. Two randomized controlled trials have shown that imiquimod is more effective than placebo. Mathiesen and colleagues25 randomized 32 women with VIN2 or 3 to imiquimod versus placebo and found an 81% complete response rate and 10% partial response rate 2 months after the completion of 16 weeks of treatment. Similarly, van Seters and colleagues26 demonstrated at least partial response in 81% and a complete response in 35% of 26 women treated with imiquimod. Imiquimod is usually applied on the affected area 2 to 3 times per week, for a total of 16 weeks. Up to two-thirds of patients reduce the frequency or length of treatment due to local side effects of erythema or erosions. Imiquimod has also been examined as a potential topical therapy in the vagina. Use of 1 sachet of 5% cream (0.25 g) once or twice weekly has been shown to induce complete resolution of low-grade VAIN lesions after 1 to 3 treatment cycles. Importantly, vulvar or vestibular excoriation was only reported in 2 of the 56 patients, and none demonstrated vaginal ulceration.27For high-grade lesions, a smaller cohort identified an 86% chance of resolution to normal or VAIN1 after use of imiquimod.28
Five-fluorouracil (5-FU) cream is another topical agent that induces a chemical degradation of the dysplastic lesion. For VIN, 5-FU is associated with response rates up to 75%; however, local side effects are significantly more severe as compared with imiquimod, with severe inflammation lasting for up to 2 weeks after the completion of a 6- to 10-week course. In the vagina, suppositories of 5% 5-FU may be used once to twice daily for 5 to 14 days or once weekly for 10 weeks. Small cohort studies suggest recurrence rates ranging from 9% to 54%, although completion of therapy has also been limited by the potentially severe side effect of mucosal excoriation.29,30
CO2 laser ablation has shown excellent success for the treatment of both VIN and VAIN. It is well tolerated and results in minimal sexual dysfunction. For VIN, success rates after a single laser treatment are approximately 75%. VIN lesions should be ablated to a depth of 1 mm for non-hairy lesions and 3 mm for hairy lesions. For high-grade VAIN, laser ablation is associated with a curate rate of 69%.29 Because tissue is not evaluated histologically with laser ablation, biopsies of vulvar and vaginal lesions should be performed preoperatively to exclude invasive disease.
Intracavitary radiation therapy is a modality shown to be effective for the treatment of VAIN. However, radiation is associated with relatively greater morbidity, including vaginal atrophy, stenosis, and shortening. This modality is generally reserved for patients who have failed other therapies, are poor surgical candidates, or have extensive multifocal disease. Small series have examined low-dose-rate vaginal brachytherapy for VAIN3 using personalized vaginal molds with delivery of 60 Gy to 5 mm below the vaginal mucosa. In 28 patients, only 1 recurrence was observed, corresponding to a 5-year local control rate of 93%.31
Surgical resection remains the mainstay of the treatment of VIN and VAIN. This modality has several advantages, including removal of diseased tissue while obtaining a histologic diagnosis of the entire lesion of interest. Depending on the extent of disease, surgical excision can be as minimal as a wide local excision or as extensive as a total vulvectomy or vaginectomy. Wide local excision of dysplastic lesions should be performed with a 5-mm margin and is ideal for localized lesions. Primary end-to-end reapproximation of the defect can usually be performed with interrupted dissolvable sutures. Skinning vulvectomy is reserved for large, extensive, or multifocal lesions. This procedure involves the removal of the vulvar skin along the avascular plane beneath the epidermis while preserving the subcutaneous tissue. The defect may require closure with a split-thickness skin graft. For high-grade VAIN lesions, wide local excisions should similarly be performed with a 5-mm margin; curative success with excision of VAIN3 has been reported as 69%.29
PREVENTION
In June 2006, the US Food and Drug Administration approved the first vaccine for the prevention of infection from HPV. Today there are 2 commercially available prophylactic HPV vaccines. Both consist of L1 capsid proteins made from recombinant DNA. Each also contains its unique adjuvant to assist in the immune response necessary to induce long-term protection from HPV infection. Gardasil (Merck Corporation) is a quadrivalent vaccine with antigens that mimic the L1 capsid of HPV types 6 and 11, included to decrease the risk of external genital warts, and HPV types 16 and 18, included to decrease the risk of high-grade dysplasia and ultimately cancer. Cervarix (GlaxoSmithKline) is a bivalent vaccine that is made up of the L1 capsid proteins from HPV types 16 and 18. Both vaccines are effective in preventing HPV16 and 18 infections and the associated high-grade CIN in women ages 15 to 26 years who do not have HPV infection at the time of vaccination.32,33 Neither shows therapeutic efficacy against current infection. In addition, the quadrivalent vaccine has been shown to prevent VIN and VAIN, as well as genital warts. Although 70% of cervical cancers and 50% of CIN2 and 3 lesions are associated with HPV types 16 and 18, only 35% of LSIL are associated with HPV types 16 and 18. For this reason, it is not anticipated that HPV vaccination will have a major impact on the number of abnormal Pap test results. The full duration of immunity after HPV vaccination is unknown. Phase 2 data provide the longest follow-up data and suggest that immunity persists for at least 5 years for the quadrivalent vaccine and 8.4 years for the bivalent vaccine. In general, HPV vaccination is safe. The only adverse events that were seen in increasing frequency in subjects participating in randomized trials of the vaccines were related to injection site reactions that included erythema, pain, and irritation. An important component of assessing vaccine safety are the post-treatment surveillance registries that specifically monitor any adverse events that occur after vaccination. Uncomplicated syncope has been the most frequently observed adverse event after vaccination. These registries will play a role in identifying any long-term side effects of vaccination. Finally, more recent studies have addressed the efficacy of vaccination in boys and older adult women and will assist in shaping public policy on HPV vaccination recommendations.
PROGNOSIS AND MANAGEMENT OF RECURRENCE
Key Points
1. Surveillance after treatment for CIN, VIN, or VAIN should include routine screening after the post-treatment period.
2. Risk factors for recurrence include positive margins of the excisional biopsy, smoking, immunosuppression, and multifocal disease.
After treatment for cervical, vulvar, or vaginal dysplasia, patients should be monitored closely for evidence of recurrence. There are consensus guidelines for post-treatment surveillance of cervical dysplasia. A recent study of more than 37,000 women treated for CIN1, 2, or 3 has demonstrated that these patients remain at risk for recurrent dysplasia and cervical cancer after treatment. The risk of recurrence is greatest in women treated for CIN2 or 3. The risk of developing recurrent CIN2/3 within the first 6 years after treatment was 9% in women previously treated for CIN3 and 6% in women treated for CIN2. The risk of recurrence was associated with older age at diagnosis and was associated with treatment type, with highest risk in women treated for CIN3 with cryotherapy. The overall incidence of cancer in the cohort of women treated for any dysplasia was 37 cancers per 100,000 woman-years, compared with 6 cancers in a comparison group of women with no history of cervical dysplasia. It is important to strongly encourage women treated for CIN2 and 3 to continue to participate in routine screening programs even after the immediate post-treatment surveillance period.34
The clinical course of VIN is varied, and evidence has shown that it may progress, resolve, or become persistent. Differentiated type VIN is more than 5 times more aggressive than usual type VIN, with a rate of 32.8% progression to invasive squamous cell carcinoma in differentiated VIN, as compared with 5.7% progression in usual type after 14 years of follow-up.35 A meta-analysis showed that untreated VIN3 overall has a 9% rate of progression to invasive vulvar carcinoma in women followed up over 12 to 96 months. In patients who were treated for VIN3, the rate of progression was 3.3%.36
At least one-third of patients with VIN will recur regardless of treatment modality, and women should receive long-term follow-up with surveillance of the entire lower genital tract every 6 months for 5 years, and then annually. Risk factors for recurrent disease include cigarette smoking, immunosuppression, multifocal disease, and positive margins on surgical excision. If excisional biopsy has been performed with positive margins, there is 3 times the risk of recurrence as compared with negative margins. For recurrent disease, the recommended treatment takes into consideration the same factors as for primary disease. Surgical excision is preferred, but if this is not feasible because of multiple prior excisions, or if the patient is not an optimal surgical candidate, then topical and laser treatment should be considered after biopsies have excluded invasive carcinoma.
FUTURE DIRECTIONS: HPV AND BIOMARKER DETECTION AS PRIMARY SCREENING
Despite the enormous impact that the Pap test and current management algorithms have had in reducing the burden of cervical cancer in developed countries, the application of this potentially costly and inefficient program is not applicable in poor and underdeveloped parts of the world. A potential approach to addressing this challenge is the development of a cost-effective cervical cancer screening approach that replaces or augments Pap testing. In general, these efforts have focused on HPV detection as a primary tool for cervical cancer screening. HPV testing is presently incorporated into algorithms for triage of women with equivocal cytologic abnormalities, follow-up of women with abnormal screening results, and in determining cervical cancer risk to individualize frequency of Pap testing. Studies also support the use of HPV detection to predict the therapeutic outcome after treatment for CIN. Meta-analyses demonstrate that primary screening for cervical cancer by HPV detection, using various methods of detection, identifies more than 90% of all CIN2, CIN3, or cancer and is 25% more sensitive than cytology alone. However, this approach is also associated with a 6% reduction in specificity, which leads to increased triage to additional testing and affects the cost-effectiveness gained by the improved sensitivity.37 Recent large studies have attempted to address the reduction in specificity (or positive predictive value) by altering the cutoff for HPV positivity or altering the sequence and components of screening tests and algorithms. In women ages 35 to 60 years, it has been demonstrated that HPV detection (with a cutoff for positive HPV DNA of 2 pg/mL) is 80% more sensitive than conventional Pap testing, and the positive predictive value is essentially unchanged.38 Another strategy that has improved sensitivity without compromising positive predictive value is primary screening with HPV DNA testing followed by cytologic triage and repeat HPV DNA testing of HPV-positive women whose subsequent cytologic examination was normal. With this algorithm, HPV testing was shown to be 30% more sensitive than conventional Pap testing, with a relatively small decrease in positive predictive value. This approach, however, still resulted in a 12% increase in the number of cytologic tests.39 Other approaches to molecular testing that are currently being evaluated deal with the decreased sensitivity of HPV DNA detection and include HPV typing for HPV types 16, 18, and 45; testing for markers of proliferative lesions by detection of p16 (a protein that is upregulated downstream of E7 inhibition of pRB activity); and mRNA coding for the viral oncogenes E6 and E7. Several studies have outlined the principles of these screening tests; however, the details and cost-efficacy are still under investigation, and the exact role of these new tests is yet to be defined.
With these advances in HPV testing, the incidence of lower genital tract dysplasias and malignancies may be significantly reduced. Much work is still necessary to target women who do not have access to medical care, both in the United States and in developing nations. Until widespread screening can be implemented, cervical, vulvar, and vaginal dysplasia will still be a significant clinical burden to women worldwide.
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