High frequency of loss of heterozygosity in vulval intraepithelial neoplasia (VIN) is associated with invasive vulval squamous cell carcinoma (VSCC)

Abstract

Vulval intraepithelial neoplasia (VIN) is thought to be the premalignant phase of human papillomavirus (HPV)-associated vulval squamous cell carcinoma (VSCC).Various molecular events have been suggested as markers for progression from VIN to VSCC, but loss of heterozygosity (LOH) in vulval neoplasia has rarely been studied in this context. We performed LOH analysis by polymerase chain reaction (PCR) amplification of polymorphic microsatellite markers at 6 chromosomal loci (17p13-p53, 9p21-p16, 3p25, 4q21, 5p14 and 11p15). The presence of HPV was assessed using consensus PCR primers and DNA sequencing. Key words: VIN; vulval; cancer; LOH; HPVVulval intraepithelial neoplasia (VIN) is thought to be the premalignant phase of invasive vulval squamous cell carcinoma (VSCC). This hypothesis is based on the observation that VIN frequently occurs adjacent to VSCC, 1 that VIN and a subgroup of VSCC are associated with similar risk factors [smoking, 2 immunosuppression 3 and human papillomavirus (HPV) infection 2,4 ] and that VIN is a monoclonal neoplastic condition. 5 The risk of progression of VIN to VSCC is unclear. 6,7 Limited evidence is available about molecular events in vulval carcinogenesis. Loss of heterozygosity (LOH) is a common molecular event in malignancy, but has been studied only in relatively small numbers of VIN and VSCC. 8 -11 We set out to document the LOH rates in VIN and VSCC in a larger series, to examine the relationship between the 2 conditions. Events common to both conditions could be early events in vulval carcinogenesis. Events occurring in VIN associated with VSCC but not in lone VIN could be markers for risk of progression to VSCC.Because human papillomavirus (HPV) is thought to be involved in the development of VIN-associated VSCC, but is not found so often in VSCC occurring in the absence of VIN, 2 we also performed HPV analysis, to compare LOH in HPV-positive and HPV-negative VSCC. MATERIAL AND METHODS SamplesPatients with VIN and VSCC diagnosed between 1989 and 1997 were identified using the computerized database of the pathology departments of St. Bartholomew's and the Royal London Hospitals. Samples containing both normal and neoplastic tissue were as follows: 43 cases of VIN III alone, 42 cases of VSCC alone and 21 cases of VIN associated with concurrent VSCC (18 of which had the concurrent VSCC still remaining on the specimen blocks after serial sectioning). Of the 60 VSCC cases, 24 were stage I, 11 were stage II, 9 were stage III, 3 were stage IV and in 13 information for accurate staging was not available. Of the 21 cases of VIN associated with VSCC, 18 were VIN III, 2 were VIN II and 1 was VIN

High-resolution genomic profiling of human papillomavirus-associated vulval neoplasia

“…Indeed, few studies have previously examined the genetic changes associated with highgrade VIN using lower resolution techniques. One investigation (Rosenthal et al, 2001) reported that, of 6 loci (3p, 4q, 5p, 9p, 11p and 17p) examined for LOH using microsatellite marker analysis, 3p was the most frequently affected with LOH in 8 of 28 (29%) informative HPV-positive VIN3 samples, comparable to this study. Elsewhere, metaphase CGH was used to analyse the genetic events Reanalysed data from a previous study on cervical carcinomas (Scotto et al, 2008b).…”

“…Limited data are available on the genetic alterations associated with VSCC and VIN precursor lesions; previous studies have used microsatellite marker analysis and metaphase comparative genomic hybridisation (CGH) rather than higher resolution arraybased techniques (Pinto et al, 1999;Jee et al, 2001;Rosenthal et al, 2001;Allen et al, 2002;Micci et al, 2003;Bryndorf et al, 2004;Huang et al, 2005). In contrast, the genetic changes associated with CIN and CxSCC have been extensively studied, most recently using array-based methods (Hidalgo et al, 2005;Wilting et al, 2006Wilting et al, , 2009Choi et al, 2007;Kloth et al, 2007;Scotto et al, 2008a, b).…”

Molecular evidence of a common clonal origin and subsequent divergent clonal evolution in vulval intraepithelial neoplasia, vulval squamous cell carcinoma and lymph node metastases

“…LOH autoradiographs from these samples were shown in our previous report. 7 Similarly, samples exhibiting Ͼ50% reduction in the intensity ratio of the VIN-or VSCC-modified androgen receptor alleles compared to unmodified alleles were classed as having skewed X-chromosome inactivation, indicative of a monoclonal origin. Examples are shown in Figure 1.…”

“…These LOH frequencies were known from our previous study. 7 Where identical losses occurred at more than 1 locus in a sample pair, the odds of this occurring were calculated as the product of the probabilities for loss at each locus. The total probability of observing common losses in the different sample groups was calculated as the product of the probabilities of each sample in that group.…”

“…We then wanted to document the patterns of LOH events and X-chromosome inactivation in VSCC samples and contiguous and noncontiguous VIN, to examine whether VIN and VSCC could have arisen from the same clone. Because we had already studied the frequency of LOH at 6 chromosomal loci in lone VIN and VSCC, 7 it was possible to calculate the odds of identical losses occurring by chance in both VSCC and associated VIN. Finally, as primary cancers and their metastases are thought to have the same clonal origin, we compared the number of identical losses occurring in primaries and their metastases with the number of identical losses occurring in VSCC and associated VIN.…”