Previous classification schemes considered papillomatous-exophytic condyloma acuminata and flat condyloma, commonly known as genital warts, as separate entities from intraepithelial neoplasia, but in the LAST classification both are included in the LSIL category. Condyloma acuminata are considered something of a “special case,” as they are such distinctive lesions, and the term has been so long in use, it is unlikely to be easily renamed. The LAST project advises maintaining the term in parenthesis for clarification. Flat lesions with similar cytologic features, while they are occasionally seen on the vulva [20] (Fig. 9.1), are far less common in this site than on the cervix and, when present, are usually closely associated with concurrent vulvar HSIL.

The vulva is the most common site for lower genital tract condylomata. The incidence of vulvar condylomata varies according to the population being studied but in Western countries is generally about 1 % of sexually active women, with a peak prevalence occurring between the ages of 16 and 29 years [21, 22]. Although usually asymptomatic, patients may experience itching, pain, or bleeding. Typical exophytic lesions are easily identified grossly, either by the patient herself, who brings it to the attention of her clinician, or by the examining physician. The flat condyloma lesion, including LSIL, is often macular or papular in appearance and may become more apparent on application of a 3 % solution of acetic acid (white vinegar), which will impart a white appearance (acetowhite) to the abnormal epithelium. Lesions are commonly multifocal and may involve the vestibule and perianal areas, as well as the vagina, cervix, urethra, and anal canal. When vulvar lesions are identified, close examination of these adjacent areas is indicated [23]. Lesions may become confluent and involve a significant portion of the vulvar skin or mucosa, particularly during pregnancy [24, 25] (Fig. 9.2).

Many risk factors have been identified for the development of LSIL of the vulva. The presence of SILs elsewhere in the lower anogenital tract is one; up to 50 % of women with vulvar exophytic condyloma acuminatum also have past, concurrent, or subsequent diagnoses of cervical or vaginal SIL [26, 27]. Other commonly associated conditions are vaginitis, pregnancy, diabetes, oral contraceptive use, and poor hygiene. Immunosuppression is an increasingly common predisposing factor, and women with human immunodeficiency virus (HIV), organ transplants, or autoimmune diseases often struggle with widespread lesions throughout the lower anogenital tract that can be very difficult to eradicate. As a sexually transmitted disease, the risk of SIL or condyloma acuminatum of the vulva is increased with increasing numbers of sexual partners. The presence of vulvar condyloma acuminatum in children should raise the question of sexual activity or abuse, although warts on the genitalia of children may be due to common cutaneous HPV subtypes [28].

All LSILs are HPV related, and the vast majority on the vulva are associated with types 6 and 11, although a significant minority may contain high-risk HPV subtypes [5, 10, 12, 21, 29–31]. Those associated with only low-risk virus are unequivocally benign and have no potential to progress to HSIL or malignancy. There is, however, disagreement as to the biologic behavior of those lesions harboring high-risk viral types [10], although it is clear that if any of these low-grade vulvar lesions do progress to higher-grade lesions, it is a very uncommon occurrence. Due to the as yet unclear evidence as to the biologic behavior of these lesions, as well as the expense of testing so many lesions to identify so few with high-risk virus, HPV typing by in situ hybridization or polymerase chain reaction (PCR) is not recommended for vulvar LSIL, with the exception of cases occurring in children [20].

Condylomata are highly infectious, and the time between the incident viral infection and the development of a clinically recognizable lesion is estimated at 2.9–5 months [21, 32]. Although spontaneous regression may occur in a minority of lesions [23, 33], most will require treatment. A variety of topical treatments are available, and most patients will require more than one course of treatment to achieve complete eradication of the lesion. The time to clearance varies depending on the treatment used, with a median of 5.9 months [32]. Treatment is often painful and may result in edema, erythema, and ulceration in addition to significant discomfort and associated sexual impairment which may persist following resolution of the lesion [23, 34]. Because treatment is aimed at destruction of the visible lesion rather than the underlying cause, even with no new exposure to HPV, successful treatment cannot preclude a possible recurrence.

Because exophytic condylomata are so distinctive in gross appearance, they are not always biopsied but may be presumptively diagnosed and treated based on appearance. There is lack of consensus among experts as to whether all lesions which appear as typical condylomata ought to be biopsied or not, but it is recommended to biopsy all those occurring in postmenopausal women and those which have failed to regress with topical therapy [35]. The low-grade vulvar lesions encountered in clinical practice of pathology are therefore likely to represent only a minority of cases, those in which the diagnosis was uncertain or which were too large to manage medically.

Typical exophytic condyloma acuminata (Fig. 9.3) are papillary lesions with a central fibrovascular stalk. The epithelial surface is always acanthotic and often hyperkeratotic, parakeratotic, or both. The fibrovascular stalk is lacking in flat lesions. Both papillary and flat lesions exhibit similar epithelial changes, characterized by proliferation of squamous cells with abnormal nuclear features and little cellular maturation in the lower third of the epithelium (Fig. 9.4). The nuclear abnormalities include increased nuclear size, irregularity of nuclear membranes, multinucleation, nuclear pleomorphism, hyperchromasia, and increased nuclear to cytoplasmic ratios (Fig. 9.5). Koilocytic change is defined by the presence of sharply delineated perinuclear halo encircling the atypical nucleus, the morphologic manifestation of production of viral particles in the nucleus of infected cells, and death of the cell with retraction of the cytoplasm from the nuclear membrane (Fig. 9.5). Koilocytosis is usually present but may be minimal to absent focally (Fig. 9.6) or occasionally throughout entire lesions [20]. Individual cell keratinization, dyskeratosis, and accentuation of intracellular bridges may also be seen (Fig. 9.7). The proliferative nature of the lesion is evidenced by basilar and parabasal stratification, increased mitotic figures confined to the lower third of the epithelium, and cytoplasmic maturation beginning to appear in the middle third and relatively normal maturation of the upper third. Extension of immature cells or mitotic figures into the upper two-thirds of the epithelium or the finding of abnormal mitotic figures in any level precludes the diagnosis of LSIL, and lesions with such features should be interpreted as HSIL. Some nuclear abnormalities are commonly present throughout the full thickness of the epithelium in LSIL, and this finding does not indicate a need to upgrade the diagnosis to HSIL. Severe nuclear atypia, however, is not a typical feature of LSIL, and its presence does warrant upgrading to a diagnosis of HSIL.

Immunohistochemistry for p16INK4a (p16), a cell cycle protein which is upregulated and overexpressed in oncogenic HPV infection, is valuable in the evaluation of HSIL but is of limited use in the evaluation of LSIL of the vulva. While it may be tempting to use p16 as a routine screening tool for dysplasia, it should be noted that this is not an advisable or recommended use for this stain, particularly when the diagnostic impression is of a low-grade lesion. There are, however, certain situations in which staining with p16 may be considered in such lesions, as summarized in Table 9.3. It is recommended [1] for use in cases where there is high clinical suspicion of a high-grade lesion but the initial impression is of a low-grade lesion, in which case it may help to identify occult areas of higher-grade disease [36]. Staining with p16 can also be useful in the triage of the occasional lesions with an intermediate morphology, such as were formerly designated VIN 2, to determine whether the lesion is best interpreted as high or low grade, as positive “en bloc” staining in SIL of uncertain grade correlates with the presence of high-risk HPV types when the morphology is also supportive [37, 38]. Staining for p16 is of no use in the much more common situation of distinguishing between low-grade lesions and reactive or reparative changes, as LSIL is usually p16 negative [39, 40] and non-HPV-related squamous abnormalities may be p16 positive. Ki-67, a marker of cell cycle activation and cellular proliferation, has a similarly limited role in evaluation of LSIL and is restricted to the more thorough evaluation of proliferation abnormalities in cases in which the cytologic features are equivocal [41]. Because HPV infection in LSIL activates the cell cycle in order to accomplish viral reproduction, and this process is occurring in the maturing squamous cells, Ki-67 will be expressed in some cells in the upper levels of the epithelium in LSIL (Fig. 9.8), while in normally proliferating epithelium, expression of Ki-67 is limited to the basal and parabasal cells.

The differential diagnoses of vulvar LSIL and condyloma acuminatum include numerous lesions, especially fibroepithelial polyp, squamous papilloma, seborrheic keratosis, warty HSIL, verrucous carcinoma, and warty carcinoma. Polyps, while they do have a fibrovascular core, are typically lacking the acanthosis, hyperkeratosis, and parakeratosis so commonly seen in LSIL of the vulva. Immunohistochemistry for Ki-67 will distinguish LSIL and condyloma acuminatum from fibroepithelial polyps and squamous papilloma in problematic cases, as the staining is limited to the lower third of the epithelium in these lesions but extends into the upper layers in LSIL and condyloma acuminatum [41]. The papillary architecture and hyperkeratosis of seborrheic keratosis can be suggestive of condyloma acuminatum, particularly at low power. Further complicating the differential between these two entities is the fact that seborrheic keratosis, particularly of the vulvar skin, is frequently found to contain HPV DNA [42–44], and some authors have gone so far as to maintain that such lesions on the vulva are, in fact, simply a variant of condyloma [42] although this remains highly controversial. The absence of koilocytic atypia in seborrheic keratosis and of keratin horn cyst in condyloma will usually resolve the diagnosis. Distinction from warty HSIL rests on the identification of increased mitotic activity, abnormal mitotic figures, mitotic figures in the upper two-thirds of the epithelium, and more severe nuclear atypia, in addition to positive staining with p16, all of which are features which point to a high-grade diagnosis. It should be remembered that, although uncommon, lesions with the typical morphologic features of condyloma can contain areas of associated HSIL, in which case it is appropriate to classify it as condyloma acuminatum with HSIL (Fig. 9.9). Although the superficial portion of verrucous and warty types of squamous carcinoma may resemble LSIL, identification of invasion at the base of the lesion will indicate the correct diagnosis.

The risk factors for HPV-related HSIL are essentially the same as those for LSIL, and the patient population is similar. Patients with HPV-related HSIL are usually in their 30s or 40s [9, 26, 45], are frequently smokers, and often have a history of or concurrent multifocal vulvar lesions and/or multicentric HPV-related disease or other sexually transmitted diseases [9, 14, 16, 17, 26, 27, 45–49]. Pruritis is the most common symptom, reported in approximately 60 % of patients [14, 23, 41]. Other symptoms may include pain, ulceration, or dysuria, and approximately 20 % of patients have no symptoms at all but may have observed an abnormal area on self-examination [12, 14, 45, 49].

The gross appearance of the lesion is variable and does not predict the histological appearance. Lesions may appear red, white, or pigmented and may be raised, flat, or ulcerated (Fig. 9.10a, b). Most lesions are well demarcated and asymmetrical. They are usually multiple but may be unifocal as well, and the incidence of multicentricity decreases with increasing age [49–51]. The most common sites are on the labia majora, labia minora, and fourchette [14], but the lesions may be found anywhere on the vulva and the perineum. Like LSIL, lesions may involve the perianal skin and extend into the anal mucosa.

“Bowenoid papulosis” is a clinical term which has been applied in circumstances in which multifocal, pigmented, or violaceous papular lesions are identified on the vulva and perineum of a young woman, often during pregnancy or postpartum [9, 19]. Histologically, such lesions are indistinguishable from HSIL, but they are thought to have less potential to progress, and indeed many cases have been found to regress spontaneously [19, 49, 52]. Because the diagnosis rests on specific clinical features, bowenoid papulosis is not acceptable as a histopathologic diagnosis, but close communication between the pathologist and clinician in cases which meet the clinical criteria can allow for optimal management decisions.

Most HSILs of the vulva have been found to contain high-risk HPV types [9, 10, 12, 17, 20, 26, 29–31, 53], most commonly HPV type 16. In this HPV-dependent pathway, the estimated time of progression from incident infection to the development of clinical disease has been estimated at 18.5 months [30].

HSIL may recur or progress to invasive carcinoma. The frequency of recurrence on the vulva is higher than for HSIL in other sites in the lower anogenital tract [54], and reported rates vary from 30 % to over 50 % [55–57]. Recurrence is more likely in patients who continue to smoke after initial diagnosis [46, 58], in patients with multifocal disease [45, 59], and in patients with positive margins on the initial resection [35, 58, 59], although the latter association has recently been refuted [14].

Up to 25.7 % of patients with HSIL of the vulva have been found to have prior, concurrent, or subsequent vulvar squamous cell carcinoma [12, 55, 60]. Subsequent carcinoma may occur at the original site of the HSIL or in a new location. Several clinical risk factors for the progression to carcinoma have been identified. In addition to continued smoking, larger size of the initial lesion and basaloid morphology increase the likelihood of progression [26, 58]. Patient age, multicentricity and multifocality of disease, and immunosuppression have all been reported as affecting the risk of progression, but to date the data remain conflicting on all counts [9, 16, 19]. The reported rates of progression for treated vulvar HSIL have ranged from 2.0 % to 10 % [16, 19, 26, 58, 61], and the median times to a cancer diagnosis have been reported as 2.4–9 years [19, 58]. Untreated lesions have been found to progress to carcinoma with a median of 3.9 years, with all these cases resulting in carcinoma within 8 years [19]. In general, the long-term risk for developing carcinoma in women previously treated for HSIL is 2.5–7 % [62–64]. Early diagnosis and treatment can prevent this occurrence, and data showing that while the diagnosis of HSIL has been increasing in frequency, the diagnosis of invasive vulvar carcinoma has not [15], suggest that this has indeed been the case.

There has been a trend towards more conservative management of HSIL over the last 20 years. While total vulvectomy was once the standard treatment for these patients, most disease is now managed with wide local superficial partial excision or laser ablation [14, 26, 49]. Topical treatments are available and currently under investigation, but to date none have been shown to be as effective as surgical treatment [26]. Close clinical follow-up is very important, as patients with HPV-related HSIL remain at risk for recurrent or new vulvar lesions, as well as lesions elsewhere in the lower anogenital tract for the rest of their lives. The American College of Obstetrics and Gynecology advises follow-up at 6 and 12 months following the initial diagnosis, and if no lesions are discovered in that time, yearly thereafter [35].

HPV-related HSIL is characterized by the presence of nuclear abnormalities similar to those seen in LSIL but with more pronounced proliferation and decreased maturation. Mitotic figures may be found in all layers of the epithelium and abnormal forms may be seen (Figs. 9.11 and 9.12). Little to no maturation is seen in the middle to upper thirds. Marked nuclear pleomorphism and nuclear hyperchromasia are present, especially in cases with warty histological features.

Two distinct histological patterns may be recognized, alone or mixed. The warty (condylomatous) type of HSIL is characterized by a spiky or undulating surface, giving the lesions a warty or condylomatous gross appearance. The epithelium is markedly thickened, with wide, deep rete pegs separated by thin dermal papillae that often closely approach the surface (Fig. 9.13). The epithelium shows disorganization, but there is evidence of maturation in the upper layers. Hyperkeratosis is prominent, often with accompanying hypergranulosis, parakeratosis, and koilocytic change which may be present towards the surface (Fig. 9.13). Multinucleated cells may be present, as well as dyskeratotic cells (Fig. 9.14), and nuclear atypia may be quite severe (Fig. 9.15). Nuclei are enlarged and nuclear chromatin hyperchromatic, with irregular nuclear membrane contours and prominent pleomorphism. An appreciable amount of eosinophilic cytoplasm is present, and the cell borders are easily delineated.

In the basaloid pattern of HSIL, the surface is relatively flat, and consequently these lesions do not typically appear papillary or exophytic but rather macular to papular. Hyperkeratosis, parakeratosis, and koilocytosis may be present but to a lesser degree than is seen in the warty type. The epithelium is thickened by a relatively uniform population of immature cells with scant cytoplasm, poorly defined cytoplasmic membranes, and enlarged nuclei with chromatin hyperchromasia (Fig. 9.16).

It should be recognized that lesions are not always easily categorized as warty or basaloid. Mixed forms, containing morphologic features of both types, are not uncommon and may be designated as such. Because there is no clinical or outcome differences between warty and basaloid types, it is also acceptable not to specify any subtype, so long as the distinction from differentiated vulvar intraepithelial neoplasia (d-VIN) is made clear.

Immunohistochemical study for Ki-67 in HSIL of the vulva shows widespread reactivity in the basal third and extending upwards into the upper two thirds (Fig. 9.17). Of greater utility in the diagnosis of vulvar HSIL is p16, which has been shown to be a reliable indicator of infection with high-risk HPV in the vulva [47, 53, 65–68]. As such, it is strongly and diffusely positive throughout the epithelium in the oncogenic HPV-associated warty/basaloid HSILs [40, 53, 68, 69]. The pattern of reactivity may be nuclear or nuclear and cytoplasmic and should be reactive in a continuous “block” pattern throughout at least the lower one third of the epithelium (Fig. 9.18). Focal, patchy staining is nonspecific. Rare cases of HPV-negative basaloid HSIL have been identified in association with HPV-negative cancers and have been found to be HPV negative and p16 negative as well [70]. Unlike in LSIL, p16 immunostaining is useful in, and recommended for, distinguishing between HSIL from potential mimics (Table 9.3) [1] and its use is reported to increase interobserver agreement and accuracy of diagnosis in cases of HSIL [37, 67, 68, 71–73].

The differential diagnosis of HSIL varies according to the morphology of the lesion. Basaloid lesions are generally easy to recognize as lesions of malignant potential, with their pronounced lack of maturation, but may be mistaken for basal cell carcinoma which shows a similar lack of maturation. The lack of identifiable stromal invasion in HSIL should distinguish it from basal cell carcinoma. It should also be kept in mind that while basaloid HSIL is a relatively common diagnosis on the vulva, basal cell carcinoma in this location is distinctly uncommon. Lesions of warty or mixed morphologies may show overlapping features with numerous other conditions, both benign and malignant. Although both condyloma and warty HSILs may have prominent koilocytic changes, HSIL can be distinguished from condyloma acuminatum by the presence of atypical, pleomorphic cells in the deeper levels of the epithelium and by the presence of increased mitotic activity, including abnormal mitotic figures in the upper layers. Additional features which may be of use in distinguishing low-grade from high-grade lesions are enumerated in Table 9.4. As discussed previously, it should be kept in mind that the two are not mutually exclusive; HSIL may be found within or adjacent to condyloma and in such cases both entities may be diagnosed in the same biopsy. Other lesions that may be confused with warty HSIL include seborrheic keratosis and lichen simplex chronicus, which can show acanthosis and hyperkeratosis, but these lesions lack the nuclear atypia of HSIL. The large atypical cells present in the epithelium in cases of superficial spreading melanoma can sometimes be confused with all types of HSIL. Immunohistochemical studies for S-100, HMB-45, and/or Melan-A will resolve this dilemma, as HSIL will be negative for these melanoma markers but will be reactive for cytokeratin, which are negative in melanoma.

Several rare variants of HSIL have been described, usually in association with concurrent invasive carcinoma. Occasional cases have been recognized with a pagetoid morphology, in which clusters and nests of atypical squamous cells are found scattered within an otherwise normal epithelium [74, 75]. Synchronous non-pagetoid SIL was also present in two of three reported cases, providing for easy comparison of the neoplastic pagetoid cells with those in the more typical lesion. When this alone will not resolve the matter, true Paget cells will stain for mucin, but the VIN cells will be negative. More recently, a variant of HSIL has been described containing cells with mucinous cytoplasm admixed throughout the epithelium of an otherwise typical HSIL, which has been termed “VIN with mucinous differentiation” [76]. Both pagetoid VIN and VIN with mucous differentiation are oncogenic HPV related and reported lesions were also positive diffusely reactive for p16INK4a. Distinction from Paget disease may be made on morphologic grounds, but awareness of these Paget-like entities should allow for the proper interpretation.