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Keywords:

  • HPV ;
  • p16;
  • prognosis;
  • vulvar cancer

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

Vulvar squamous cell carcinoma (VSCC) accounts for >90% of the malignant tumours of the vulva. Most VSCCs originate in intraepithelial lesions, named vulvar intraepithelial neoplasia (VIN), that precede the development of VSCC by a variable period of time. Strong evidence has accumulated showing that there are two different aetiopathogenic pathways for the development of VSCC and VIN, one associated with infection by human papillomavirus (HPV), and a second independent of HPV infection. These two different types of VSCC have different epidemiological, pathological and clinical characteristics, and should therefore be considered as two separate entities. Histologically, HPV-associated VSCCs are of the basaloid or warty type, and arise from VIN of the usual type. Inactivation of p53 and the retinoblastoma tumour suppressor gene product by the viral gene products E6 and E7 is involved in the process of malignant transformation. HPV-independent VSCCs are histologically keratinizing, are associated with differentiated VIN and lichen sclerosus, and frequently show mutations of p53. p16INK4a and p53 immunostaining can be useful for classifying VSCC into HPV-associated or HPV-independent. Although large, multicentre studies are needed to definitively assess the involvement of HPV in the prognosis of VSCC, most studies have not found clear differences in survival between HPV-associated and HPV-independent tumours.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

Vulvar squamous cell carcinoma (VSCC) accounts for <5% of gynaecological malignancies but for >90% of malignant tumours of the vulva.[1, 2] Although this carcinoma was initially considered to be a single disease, strong evidence that has accumulated over the last two decades has shown that there are at least two different aetiopathogenic pathways for the development of VSCC, one associated with infection by high-risk human papillomavirus (HPV), and a second independent of HPV infection.[3-6] These two different types of VSCC have different epidemiological, clinical, pathological and molecular characteristics, and should therefore be considered as two separate entities. Nevertheless, this is still poorly reflected in the literature.

Precursors of VSCC

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

Epithelial disorders are found adjacent to VSCC in 50–70% of patients. Most of these intraepithelial changes appear to have some degree of dysplasia.[5-8] It is widely accepted that most VSCCs originate in these intraepithelial lesions, which precede the development of invasive cancer by a variable period of time. On the basis of the terminology of the uterine cervix, in which both precursor lesions and invasive carcinomas are much more frequent, the International Society for the Study of Vulvar Disease (ISSVD) introduced, in 1986, the term vulvar intraepithelial neoplasia (VIN) to designate the precursors of VSCC.[9] VIN has become an increasingly recognized clinical problem.[1] Similarly to VSCC, the precursor lesions of VIN develop along two separate pathogenic pathways: HPV-associated and HPV-independent. In 2004, the ISSVD proposed the current two-tier classification for VIN, reflecting these two different pathways of vulvar pre-cancer development: HPV-associated usual VIN (uVIN), often also referred to as classic or bowenoid VIN, and HPV-independent differentiated VIN (dVIN), also referred to as simplex VIN.[10]

Lichen sclerosus (LS) has also been suggested as a precursor of HPV-independent VSCC, but the mechanism of carcinogenic progression from LS has not been fully delineated.[5, 11-13] The association between the two entities has been established mainly because LS is a frequent finding in the skin adjacent to VSCC,[7, 14] and because some longitudinal cohort studies have shown that women with LS have a significantly higher risk of developing VSCC.[15, 16] Nevertheless, most of the women with LS do not develop VSCC,[15, 16] and the proportion of VSCC cases associated with LS is lower than the proportion of tumours associated with dVIN.[17] Interestingly, LS can be associated with dVIN, particularly in patients with VSCC,[14, 18] and atypical or ‘dysplastic’ changes in areas of LS have been documented by several investigators.[16, 19]

HPV in VSCC and VIN

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

The proportion of VSCCs associated with HPV infection varies widely in different reports, ranging from 15% to 79%.[18, 20-28] The prevalence of HPV DNA in VSCC observed in the different published series is shown in Table 1. The presence of HPV DNA in VIN has been detected in 52–100% of the lesions[18, 20-26, 29-31] (Table 2). HPV sequences are detected in most cases of uVIN, and in few, if any, cases of dVIN.[12, 32-34] Not surprisingly, HPV is also present in most of the VSCCs that develop from uVIN, but not in tumours that develop from dVIN.[20, 21, 26, 30] These differences in the relative percentages of VSCC and VIN that are positive and negative for HPV between series probably reflect some geographical variability in the incidence. However, technical issues, such as the sensitivity of the molecular test used for HPV detection or the possibility of contamination, which is a frequent problem in HPV testing using techniques based on polymerase chain reaction (PCR), may result in an overestimation or underestimation of HPV positivity and be responsible, at least in part, for these differences. In summary, between one-fifth and one-half of VSCCs and over four-fifths of VINs are associated with HPV infection. It is thus remarkable that, whereas most of the VINs are associated with HPV, most of the VSCCs arise independently of the viral infection.

Table 1. Percentages of invasive vulvar squamous cell carcinomas (VSCCs) positive and negative for human papillomavirus (HPV), and the prevalence of HPV16 in cases positive for HPV
ReferenceYear n HPV typing testPrevalence of HPV (%)HPV16 in HPV-positive cases (%)
  1. n, Number of cases included in the study; PCR, polymerase chain reaction.

Monk[26]199555PCR L1 consensus primers6049
Kim[27]199618PCR L1 consensus primers3971
Pinto[22]199916PCR L1 consensus primers50
Carter[23]200138PGMY9/117955
Riethdorf[21]200471GP5+/GP6+ and p16INK4a3576
van der Nieuwenhof[18]2009130Short PCR fragment L13544
Kowalewska[28]201046Linear array HPV test1571
Alonso[20]201198SPF10 and p16INK4a1974
Gargano[24]2012121PGMY9/116981
Tsimplaki[25]20126PapilloCheck HPV50100
Table 2. Percentages of vulvar intraepithelial neoplasias (VINs) positive and negative for human papillomavirus (HPV), and the prevalence of HPV16 in cases positive for HPV
ReferenceYear n HPV typing testType of VINPrevalence of HPV (%)HPV16 in HPV-positive cases (%)
  1. n, Number of cases included in the study; PCR, polymerase chain reaction; uVIN, usual VIN.

Trimble[30]199654Omniprobe Assay (Digene)Not specified89
Pinto[22]199916PCR L1 consensus primersuVIN67
Carter[23]200118PGMY9/11Not specified9174.6
Riethdorf[21]200467GP5+/GP6+ and p16INK4aNot specified5288
van der Avoort[34]200637SPF10uVIN6665
van der Nieuwenhof[18]200913Short PCR fragment L1uVIN10044
Garland[29]200962PCRuVIN8442
Smith[31]200965PGMY09/11Not specified9850
Alonso[20]201148SPF10uVIN83
Gargano[24]201166PGMY9/11Not specified9448
Tsimplaki[25]201228PapilloCheck HPVuVIN7165

Interestingly, HPV16 is by far the most common type identified in both VSCCs and VINs (Tables 1 and 2), although other HPV types, such as 18, 31, 33, and 45, have also been reported.[7, 32] Prophylactic HPV vaccines, which cover HPV16 and HPV18, have been associated with a significant reduction in the incidence of VIN in young women.[29] Thus, a decrease in the incidence of vulvar lesions associated with HPV infection is expected to occur in the future because of the development of these prophylactic vaccines, which have become a promising new tool for the prevention of HPV-associated premalignant and malignant lesions. It has also been shown that vaccination with synthetic long peptides from the HPV16 oncoproteins E6 and E7 seems to have a therapeutic effect on HPV16-positive VIN.[35]

Low-risk HPV types, specially HPV6 and HPV11, have been found in a small percentage of VINs and VSCCs,[3, 36-38] but their role in the development of vulvar lesions other than exophytic condyloma acuminata is not clear.

Epidemiology

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

Not surprisingly, uVIN and HPV-associated VSCC share many epidemiological characteristics with the much more frequent cervical intraepithelial neoplasia (CIN) and carcinoma of the uterine cervix. They mainly occur in relatively young women, and have the same risk factors for development and progression as cervical lesions, showing a strong association with the number of sexual partners, smoking habits, and impaired immunological status.[5, 34, 39-41] In contrast, dVIN and HPV-negative VSCCs are commonly found in elderly patients,[40] and frequently occur in women with chronic dermatological diseases, such as LS and lichen simplex chronicus.[40, 42]

The incidence of both types of VSCC increases with age,[42, 43] with the average age at diagnosis being in the 7th to 8th decades of life. Over the years, an increase has been observed in the incidence of VIN, and in some countries the number of women with uVIN has almost doubled during the last 10 years.[40, 42] In contrast, only a marginal increase in the frequency of VSCC has been reported during the same period, basically in young women and probably also because of HPV infection.[42, 44] It is therefore not clear whether the increasing number of VIN cases indicates a genuine increase in its incidence or an increased awareness and better recognition of these lesions.

Women with human immunodeficiency virus (HIV) infection are at increased risk of developing VIN and, to a lesser extent VSCC, than HIV-negative women, and the risks increase with increasing immunosuppression.[45-47]

Histological features of VIN

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

uVIN is histologically similar to CIN and to other HPV-associated intraepithelial lesions, such as vaginal intraepithelial neoplasia, anal intraepithelial neoplasia, and penile intraepithelial neoplasia.[48] It is the prototypical type of VIN, and is recognizable because of its obvious architectural and cytological abnormalities, which make the lesion easily identifiable as an intraepithelial neoplasm, often even at low magnification. The epidermis is thickened, and frequently shows hyperkeratosis and/or parakeratosis. There is a loss of cell maturation, a high nuclear-to-cytoplasmic ratio, nuclear hyperchromasia, pleomorphism, and numerous mitotic cells in all epidermal layers.[8] Apoptotic cells with dense eosinophilic cytoplasm and pyknotic nuclei are common. Involvement of the follicular epithelium by dysplastic cells and abundant melanophages in the papillary dermis are frequent findings in uVIN.[8]

uVIN has been divided into basaloid (undifferentiated) and warty (condylomatous) subtypes.[8] In basaloid uVIN, undifferentiated cells resembling basal cells replace the whole thickness of the epidermis, which is usually flat and non-papillomatous (Figure 1A). The epidermis of warty uVIN has wide and deep rete ridges that result in a characteristic condylomatous appearance. Cytological signs of viral infection (koilocytotic changes, multinucleation, and coarse granules) and acanthosis are common (Figure 1B). There is, however, frequent overlap of these two patterns, and mixtures of basaloid and warty patterns are commonly found in the same lesion. Thus, both subtypes are considered to be morphological variants of a single disease rather than separate entities.[6, 8] On rare occasions, the atypical cells show pale cytoplasm and are either isolated or arranged in clusters, simulating extramammary Paget's disease.[8] This variant has been referred to as pagetoid VIN.

image

Figure 1. Vulvar intraepithelial neoplasia of the usual (classic, bowenoid) type (uVIN). A, Basaloid subtype with undifferentiated cells with a basaloid appearance replacing the whole thickness of the flat, non-papillomatous epidermis. B, Warty subtype of uVIN showing wide and deep rete ridges giving the lesion a characteristic condylomatous appearance. C, uVIN with intermediate basaloid/warty features. D, The same field as shown in (C) with strong, basal and continuous immunostaining with p16INK4a.

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In the grading system currently used by the World Health Organization (WHO), uVIN (classic VIN in the WHO terminology) is divided into three grades (VIN1–VIN3), similarly to CIN lesions in the uterine cervix, depending on the proportion of epithelium containing undifferentiated cells. However, flat, low-grade lesions showing superficial maturation and koilocytotic atypia (VIN1), which are frequent in the uterine cervix, are uncommon in the vulva. In fact, most lesions in the vulva showing clear-cut changes of HPV infection with low-grade dysplasia are typical exophytic condyloma acuminata and should be diagnosed as such, and not as VIN1, because they represent infections by low-risk HPV types (HPV6 and HPV11) which very infrequently, if ever, progress to VSCC.[49] Consequently, the ISSVD has proposed abandonment of the VIN1 category.[10] On the other hand, high subjectivity in the differentiation between VIN2 and VIN3 has led to these two categories being combined, according to the ISSVD, into a single category termed uVIN. So-called bowenoid papulosis is considered to be an old term designating a lesion that is histologically undistinguishable from uVIN, and is consequently classified as such.

The differentiation between uVIN and early invasive VSCC may be difficult. Features suggesting invasion are irregularity in the size and shape of the nests, cytoplasmic eosinophilia of the invading cells, and desmoplastic stroma.[50] Oblique sections of uVIN may show islands of atypical cells that seem to be detached within the dermis, simulating invasion. In contrast to invasive nests, these dermal islands are bulbous and smooth, and lack a desmoplastic stromal reaction.

Typically, uVIN lesions show strong immunostaining for p16INK4a and are negative for p53 (Figure 1C,D). These markers can be useful for confirmation of the diagnosis.[7, 51] It should be noted that, as established in the uterine cervix,[52] only lesions showing diffuse basal staining should be considered to be positive for p16INK4a. It has recently been shown that ProEx C, an immunochemical reagent recognising a combination of two markers (MCM2 and TOP2A), is almost always positive in uVIN, as it is in high-grade CIN,[53] and may be used as a marker for uVIN. However, although no comparative studies on the vulva have been published, experience concerning the uterine cervix suggests that it probably does not improve the results obtained with p16INK4a.[54]

The characteristics of dVIN are much more subtle than the well-defined characteristics of uVIN. The hallmark of dVIN is abnormal atypical keratinocytes in the context of a fully differentiated vulvar epithelium. The epidermis is thickened, and has a parakeratotic surface reaction. The rete ridges are often elongated, and are frequently branched. Most of the epidermis is composed of abnormal, enlarged squamous cells with large vesicular nuclei and macronucleoli. The cytoplasm is abundant and brightly eosinophilic, and the intercellular bridges are typically very prominent (Figure 2A). LS and other inflammatory dermatological conditions, such as lichen simplex chronicus, are found in a significant number of patients, either immediately adjacent to dVIN or elsewhere in the same specimen.[8, 55] Owing to this high degree of cellular differentiation and the absence of widespread architectural disarray, it is frequently confused with a benign dermatosis,[8, 56] and significant interobserver variability in the diagnosis of these lesions is likely to occur. In fact, many gynaecopathologists diagnose dVIN mainly in women with VSCC, either synchronously in the skin adjacent to the invasive cancer or metachronously in patients with a diagnosis of VSCC,[8, 55, 57] and, in contrast to uVIN, dVIN is not a frequent diagnosis in an isolated lesion. It should be noted that dVIN is considered to be a form of high-grade VIN by all classification systems, and is consequently not graded. Thus, ‘differentiated’ in the context of VIN refers to the overall mature appearance of the epithelium, and not to the grade of the lesion.

image

Figure 2. Vulvar intraepithelial neoplasia of the differentiated (simplex) type (dVIN). A, Abnormal atypical keratinocytes in the context of a fully differentiated vulvar epithelium. The epidermis is thickened and has a parakeratotic surface reaction. B, Immunostaining for p53 with abundant positive cells extending above the basal layer into higher levels of the epidermis. C,D, dVIN with basaloid morphology. Atypical undifferentiated cells occupy the whole thickness of the epithelium, and mimic usual vulvar intraepithelial neoplasia.

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Immunostaining for p53 has been proposed as being of value in identifying dVIN. p53 has been reported to label dVIN in 90% of cases. Characteristically, p53-positive cells extend above the basal layer into higher levels of the epidermis (Figure 2B).[8, 45, 55] However, a number of dVINs do not show mutations of TP53 or are associated with deletions of TP53, and these cases have been shown to be immunonegative for p53.[58] On the other hand, p53 has been shown to be overexpressed in a significant number of cases of LS.[59-61] Thus, the value of p53 immunostaining in confirming the diagnosis of dVIN seems to be limited. Ki67 has also been reported to be helpful in distinguishing dVIN from normal vulvar epithelium.[51] p16INK4a is characteristically negative in dVIN.[7, 62]

We have recently shown that HPV-negative dVIN may occasionally have basaloid morphology, with atypical undifferentiated cells occupying the whole thickness of the epithelium, and mimic uVIN (Figure 2C,D). Negative immunostaining for p16INK4a and positive immunostaining for p53 with suprabasal extension of positive cells is helpful for the appropriate classification of this lesion and its differential diagnosis from true HPV-positive basaloid uVIN.[63]

When dVIN invades the dermis, nests of eosinophilic keratinocytes appear to explode from the basilar epidermis or from elongated rete ridges. Early invasion in dVIN lesions is not uncommon. In superficial biopsies, the distinction between dVIN and early invasive VSCC may be extremely difficult, because of the cytological similarities between dVIN and VSCC.[8, 55]

Malignant potential of VIN

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

Although the risk of progression of uVIN cannot be neglected, it seems to be low. Progression to VSCC in untreated patients has been reported to vary from 9% to 16%,[64, 65] and to be ~3% in treated patients.[65] It seems to be slightly higher for the basaloid subtype.[66] Spontaneous regression has been reported in 1.2% of patients with uVIN.[65, 67] Women younger than 35 years with multifocal lesions have a higher rate of regression, and this probability decreases with age.[64, 65, 67, 68] Pregnancy has been related to both spontaneous regression[64, 65, 67] and progression[69] of the premalignant lesion.

The risk of progression of dVIN seems to be higher than that of uVIN, showing a high oncogenic potential, despite its differentiated appearance.[40, 70] Moreover, the time taken for progression to VSCC development seems to be significantly shorter for dVIN than for uVIN.[42] However, it should be noted that, as previously stated, although most VSCCs arise independently from HPV infection, dVIN is an uncommon lesion.[5, 18] The main hypotheses proposed to explain this paradoxical situation are a high rate of underdiagnosis or underreporting of dVIN, owing to its subtle histological features,[17] and the possibility that dVIN represents a transient lesion that rapidly progresses to VSCC.[65, 71, 72]

Clinical features of VIN

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

Although the clinical presentation of uVIN is diverse, its diagnosis is, in general, easy. It is almost invariably identified as a distinct lesion showing sharp margins. The lesions can be pigmented or white, or appear as chronic erythema. They can be flat, raised, or ulcerated.[8, 73] Multicentricity and multifocality, with distinct lesions simultaneously involving different organs of the lower female anogenital tract, are often observed.[74-78]

The frequency of multicentric lesions is particularly high in young women, and decreases in women >50 years of age.[8, 40] Thus, thorough examination of the vulva, cervix, vagina and anus after a diagnosis of uVIN is strongly recommended, especially in younger women. A decreased immune response to HPV infection has been suggested as a possible cause of these multicentric HPV infections.[37] Interestingly, patients with multiple lesions involving different areas of the female genital tract show, in general, the same HPV type in all of the lesions.[39]

In contrast, the diagnosis of dVIN represents a major clinical challenge. These lesions almost always develop in areas of chronic vulvar dermatosis. Red hyperkeratotic lesions with a rough irregular surface might raise suspicion of dVIN in patients with a history of chronic vulvar skin alterations. Pruritus and/or pain are observed in ~60% of the women,[79] and a long history of itching and burning is often reported. However, many dVIN lesions are asymptomatic.

Histological features of invasive squamous cell carcinomas

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

The WHO classification includes three main subtypes of vulval squamous carcinoma, namely basaloid, warty, and keratinizing.[80] Basaloid tumours are composed of cells with scanty cytoplasm and little keratinization, resembling the cells of the basal layer of the epidermis (Figure 3A), whereas the warty type exhibits bulbous or irregular nests, often with keratinization, and prominent koilocytotic-like changes (Figure 3B). The most common type of VSCC, the keratinizing type, is usually formed by differentiated cells with an absence of koilocytosis, and frequently shows abundant keratin pearls (Figure 4A). Although most tumours are easily classified, some overlapping features between these histological types exist, and result in difficulties in classification.

image

Figure 3. Human papillomavirus (HPV)-positive vulvar squamous cell carcinomas. A, Basaloid tumour composed of cells with a basaloid appearance and scanty cytoplasm with little keratinization. B, Warty type exhibiting irregular nest with central keratinization and prominent koilocytotic-like changes characterized by pleomorphic and often multiple irregular nuclei that are either hyperchromatic and shrunken or have clumped or smudged chromatin. C, Keratinizing-type HPV-positive tumour. DF, p16INK4 immunohistochemistry demonstrating diffuse basal staining in all three tumours shown in (AC). Keratin pearls do not stain for p16INK4.

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image

Figure 4. Human papillomavirus (HPV)-negative vulvar squamous cell carcinomas. A, Keratinizing tumour composed of nests of well-differentiated cells showing central keratinization. B,C, HPV-negative tumour showing basaloid features. D, Immunostaining for p16INK4 showing a completely negative result. E, p53 staining of a conventional keratinizing tumour. F, p53 staining of an HPV-negative tumour showing basaloid features.

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Analogously to the situation described for VIN lesions, basaloid and warty types have been associated with HPV infection, whereas keratinizing tumours are typically HPV-negative.[3, 4] However, it has been shown that there is some overlap between the histological types and the association with HPV, with some HPV-positive VSCCs being of the keratinizing type (Figure 3C), and a small number of HPV-negative VSCCs showing basaloid (Figure 4B,C) or warty features.[7, 20] Thus, the proposed histological separation of VSCCs into basaloid and warty types, both of which are considered to be associated with HPV infection, and the keratinizing type, which is considered not to be associated with HPV, is of limited reliability. The presence of dVIN and/or LS in the skin adjacent to a tumour is strong evidence of its HPV-independent nature, whereas the presence of uVIN strongly supports an HPV-associated neoplasm.[7]

It should be noted that non-keratinizing squamous cell carcinoma, the most frequent histological variant in the uterine cervix,[80] and the most frequent histological variant of HPV-associated tumour in other areas, such as the head and neck, is rarely diagnosed in the vulva, whereas basaloid or warty tumours, the prototypical HPV-associated tumours in the vulva, are rarely diagnosed in the uterine cervix. These differences are not related to true differences in histological type, but are probably a consequence of historical terminological conventions. A unified terminology for HPV-associated tumours, adequate for all areas harbouring these neoplasms, should be proposed.

Other rare types of VSCC have been reported, such as acantholytic (adenoid or pseudoglandular)[81, 82] and spindle cell (pseudosarcomatous) variants, and carcinomas with a prominent fibromyxoid stromal pseudovascular reaction.[83] In our experience, all of these unusual variants are usually HPV-negative. Similarly, verrucous carcinoma, initially thought to be associated with low-risk HPV types, has recently been shown to be not related to HPV infection.[84]

As observed with the intraepithelial lesions, p16INK4a immunostaining can be a useful tool with which to properly classify VSCC into HPV-associated or HPV-independent lesions. In our experience, the sensitivity and specificity of p16INK4a immunostaining for detecting HPV-associated carcinomas are close to 100%, and are higher than the sensitivity and specificity of histological classification[7, 20] (Figures 3D–F and 4D). p16INK4a immunostaining can be considered to be almost fully equivalent to HPV detection, and may thus represent a much more reliable tool for classifying VSCCs according to their relationship with HPV infection. This immunohistochemical technique, which is available in most pathology laboratories and is highly reproducible, may be used as an acceptable alternative to the much more complex techniques of HPV detection based on PCR.[7, 52] Nevertheless, HPV-positive tumours may exceptionally show negative staining for p16INK4a, and we have observed isolated examples in the vagina[85] and the uterine cervix. A significant percentage (50–70%) of HPV-independent VSCCs show p53 overexpression (Figure 4E,F) which contrasts with the almost constant negative p53 staining of HPV-associated tumours.[7]

Prognosis of VSCC

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

In recent years, HPV detection has been shown to provide relevant information in several settings where, as in the vulva, two different carcinoma pathways, HPV-associated and HPV-independent, exist. In the head and neck region, HPV-associated tumours have consistently shown a better prognosis than HPV-independent neoplasms. This better survival rate of patients with HPV-associated tumours has been reported particularly for oropharyngeal and sinonasal tumours,[86-89] and has been related to a better response to chemotherapy and radiotherapy.[87, 88] Thus, current diagnostic protocols for head and neck squamous cell carcinomas include HPV detection and/or p16INK4a immunohistochemical staining as prognostic biomarkers that allow the identification of a subset of tumours with a better prognosis, in which radiotherapy and chemotherapy have a relevant role in the control of the disease.[87]

In a recent study conducted at our institution,[20] which included a series of HPV-positive and HPV-negative groups that were similar in terms of FIGO stage distribution, percentage of involvement of surgical margins, ulceration, and tumour size or invasion depth at diagnosis, no significant differences were observed in survival between patients with HPV-positive and HPV-negative VSCC. Moreover, radiotherapy did not provide any significant increase in survival in HPV-positive women. In keeping with our study, a number of different series have shown that HPV is not an independent prognostic factor in VSCC.[22, 33, 81, 90] In contrast, several studies have suggested that patients with HPV-positive VSCC might have better survival than those with HPV-negative tumours.[4, 26, 34, 91-93] Nevertheless, the evidence provided by these studies is mainly indirect, as many of them do not give data on HPV detection.[91, 94] Van de Nieuwenhof et al.[18] included 130 cases of VSCC, and found better survival in patients with VSCC showing adjacent uVIN than in patients with VSCC showing dVIN. However, the differences were not significant in the analysis of VSCC stratified according to HPV status. One report has shown impaired prognosis for HPV-positive tumours,[95] but these results have not been confirmed in any other study, with the exception of a recent report showing similar data but not reaching statistical significance.[96] The overall survival at 5 years for patients with HPV-associated and HPV-independent VSCC reported in the studies that provide this data are summarized in Table 3.

Table 3. Overall survival at 5 years for patients with vulvar squamous cell carcinoma associated with and independent of human papillomavirus (HPV) infection
ReferenceYear n Overall survival at 5 years (%) P
HPV-associatedHPV-independent
  1. n, Number of cases included in the study.

Monk[26]19955572440.010
Pinto[22]20021663710.447
van de Nieuwenhof[18]200913080780.646
Lindell[92]20107585400.030
Alonso[20]20119867710.789
Choschzick[96]2011394075>0.05

In conclusion, although large, multicentre studies are needed to definitively assess the prognostic implication of HPV for the prognosis of VSCC, there seems to be no clear difference in survival between patients with HPV-positive and HPV-negative tumours. Two main hypotheses have been proposed to explain this situation. First, the HPV-independent pathway for VSCC in the vulva may be different from the HPV-negative pathway in head and neck tumours, in which smoking and alcohol are important aetiological factors, and result in less aggressive tumours. However, this improved prognosis for HPV-associated tumours has recently also been observed by our group in squamous cell carcinomas of the vagina.[97] Alternatively, the frequent possibility of adequate surgical excision in VSCC, with the consequent lesser relevance of radiotherapy and chemotherapy for control of the disease, may result in a reduction in the differences in survival between the two aetiological groups.

The application of other ancillary techniques to the assessment of prognosis in VSCC has also shown inconclusive results. DNA ploidy has been found to be of prognostic relevance in some studies,[98] but not in others.[99] Some studies have found that tumours showing immunohistochemical p53 expression, a frequent finding in HPV-independent tumours, have a poorer prognosis,[100] whereas others have failed to show any prognostic usefulness for p53 staining.[96, 101] A diffuse staining pattern for the cell proliferation marker Ki67 has been reported to be associated with reduced survival, but the results seem to be inconsistent.[102]

Molecular features

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References

There is currently strong evidence that uVIN and VSCC are different steps in the spectrum of a single entity. The clonal relationship and evolution from uVIN to VSCC have been shown with X-chromosome inactivation and loss of heterozygosity analysis.[10, 103] Ueda et al.[104] were the first to show, by the demonstration of monoclonal expansion, that both VSCC and the adjacent uVIN originate from a single cell. Since then, different authors have reported that premalignant lesions and the malignancies that develop from their progression are monoclonal proliferations sharing different molecular events.[11, 22, 103]

It is likely that the mechanisms involved in the development of uVIN and the corresponding VSCC are similar to those involved in cervical carcinogenesis. The viral oncoproteins E6 and E7 have a main role in cellular transformation. E6 degrades the tumour suppressor p53, abrogating its function, and consequently leading to the absence of cell cycle arrest.[105] The HPV oncoprotein E7 inactivates the retinoblastoma tumour suppressor gene product, resulting in hyperproliferation of host cells and overexpression of the cell cycle-related biomarkers p16INK4a and p14arf.[34] Therefore, as previously stressed, HPV-associated premalignant lesions and carcinomas show diffuse immunostaining for p16INK4a and p14arf, and are negative for p53.[34, 62] In this regard, it has been suggested that the identification of HPV DNA sequences may not be sufficient to accurately differentiate between the HPV-associated and HPV-independent types of vulvar lesions. Indeed, the presence of HPV DNA sequences as such does not indicate a causal role in carcinogenesis, and may represent a non-significant or transient infection.[42, 106, 107] As p16INK4A overexpression is strongly associated with the activity of the viral oncoprotein E7 and with cell cycle disruption, a key event in HPV-associated carcinogenesis, recent studies have proposed the use of p16INK4A immunostaining as the first step in an algorithm to identify clinically relevant HPV infections.[108] Moreover, p16INK4A immunostaining strongly supports the aetiological involvement of HPV in neoplasm development, and may help in the confident attribution to a tumour of HPV infection.

In contrast to the relatively large amount of data on HPV-associated tumours, the HPV-independent pathway of VSCC has been much less well studied, and the molecular mechanisms involved in its development have not yet been fully elucidated. Genetic mutations in TP53[55, 96] or PTEN[109] have been detected not only in a high percentage of HPV-negative VSCCs, but also in dVIN, suggesting that these are early changes in HPV-independent vulvar carcinogenesis. These mutations of TP53 frequently correlate with immunohistochemical overexpression of the p53 protein, a frequent finding in HPV-independent VSCC and dVIN.[55, 96] A strong correlation between high p53 expression and DNA aneuploidy has been observed.[110] However, not all HPV-independent vulvar cancers follow the p53 pathway, and the mechanisms for tumour initiation and progression in these cancers without TP53 mutation are unknown.

Allelic imbalance and microsatellite instability in some chromosomes have also been suggested to play a role in the development of HPV-independent VSCC.[22] Comparative genomic hybridization has shown various chromosomal alterations that might differentiate between HPV-associated and HPV-independent VSCC, with HPV-positive tumours frequently showing gains of 3q and HPV-negative tumours gains of 8q.[111] It has recently been shown that a gain of 3q26 is present in most dVINs, and in fewer than half of uVINs.[13] The authors suggest that the detection of a 3q26 imbalance could be of additional diagnostic value in the diagnosis of VIN lesions in combination with other molecular markers.[13] Nevertheless, other studies have reported different patterns of chromosomal alterations.[22, 112] Thus, the identification of chromosomal aberrations is not currently useful for the diagnosis of VIN.

Recently, epigenetic alterations, such as hypermethylation of the RASSF2A, MGMT and TSP1 gene promoters, have frequently been seen in VSCC associated with dVIN or LS, suggesting a possible role of the silencing of these genes in the HPV-independent pathway of vulvar carcinogenesis.[113]

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Precursors of VSCC
  5. HPV in VSCC and VIN
  6. Epidemiology
  7. Histological features of VIN
  8. Malignant potential of VIN
  9. Clinical features of VIN
  10. Histological features of invasive squamous cell carcinomas
  11. Prognosis of VSCC
  12. Molecular features
  13. References
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