Chromosome 17 aneusomy detected by fluorescence in situ hybridization in vulvar squamous cell carcinomas and synchronous vulvar skin

Abstract

Vulvar squamous cell carcinoma (SCC) affects a spectrum of women with granulomatous vulvar diseases, human papillomavirus (HPV) infections, and chronic inflammatory vulvar dermatoses. To determine whether there is evidence of chromosomal instability occurring in synchronous skin surrounding vulvar SCCs, we investigated abnormalities in chromosome 17 copy number. Samples of SCC, vulvar intraepithelial neoplasia (VIN), and surrounding vulvar skin were obtained from all vulvar excisions performed for squamous neoplasia at Albany Medical College from 1996 to 1997. Histological categorization, fluorescent in situ hybridization (FISH) for the alpha satellite region of chromosome 17, DNA content by image analysis, and Ki-67 labeling were evaluated. Controls of normal vulvar skin not associated with cancer were used for comparison. One hundred ten specimens were obtained from 33 patients with either SCC or VIN 3 and consisted of 49 neoplastic, 52 nonneoplastic, and 9 histologically normal vulvar skin samples. The majority of SCCs (88%) and a minority (18%) of VIN 3 excisions were associated with lichen sclerosus. Normal vulvar skin controls did not exhibit chromosome 17 polysomy (cells with more than four FISH signals), whereas 56% of normal vulvar skin associated with cancer did. Moreover, the frequency of polysomy significantly increased as the histological classification progressed from normal to inflammatory to neoplastic lesions. The largest mean value and variance for chromosome 17 copy number was identified in SCCs (2.4 +/- 1.0) with intermediate values identified, in decreasing order, for SCC in situ (2.1 +/- 1.0), VIN 2 (2.1 +/- 0.8), lichen sclerosus (2.0 +/- 0.5), lichen simplex chronicus (1.9 +/- 0.4), and normal skin associated with SCC (1.8 +/- 0.4) compared with control vulvar skin (1.5 +/- 0. 05). Concordance of chromosome 17 aneusomy between cancers and synchronous skin lesions was found in 48% of patients. Loss of chromosome 17 was identified 5% of all samples and was significantly associated with women with SCC in situ (HPV-related). Both DNA content and Ki-67 labeling positively and significantly correlated with mean chromosome 17 copy number (r = 0.1, P: = 0.007). A high degree of genetic instability (aneuploidy) occurs in the skin surrounding vulvar carcinomas. As these events could be detected in histologically normal skin and inflammatory lesions (lichen sclerosus), chromosomal abnormalities may be a driving force in the early stages of carcinogenesis. Differences in chromosomal patterns (loss or gain) support the concept of at least two pathways in vulvar carcinogenesis.

Figure 1.

Normalized chromosome 17 indices for control vulvar skins and all histological classifications of cancer-associated vulvar skin. The controls exhibit little variance, in marked contrast to vulvar skin associated with squamous cell carcinoma. Note the apparent increase in both variance (copy number heterogeneity) and mean copy number index in the progression from cancer-associated normal skin to nonneoplastic inflammatory dermatitis to squamous neoplasia. Loss of chromosome 17 (monosomy) was identified in known non-LS, HPV-related lesions. The mean chromosome 17 copy numbers are normalized to those found in control vulvar skin. Box-and-whisker plots of chromosome 17 copy number index are listed by histological categorization: ls, lichen sclerosus; lsc, lichen simplex chronicus (a.k.a. squamous hyperplasia); scca, squamous cell carcinoma; sccis, squamous cell carcinoma in situ; n, normal adjacent vulvar skin; vin, vulvar intraepithelial neoplasia. The line in the middle of the box represents the median. The box extends from the 25th percentile to the 75th percentile (interquartile range). The whiskers mark upper and lower adjacent values. Circles represent outliers. The y axis represents the normalized chromosome 17 index (study group/control group).

Figure 2.

Aneusomy of chromosome 17 in LS and SCC is common. A: LS with keratinocytes exhibiting more than two signals per cell. B: Invasive SCCs demonstrated the highest number and greatest frequency of high chromosome 17 copy number. Note that many of the cells contain more than three signals per cell.

Figure 3.

Multistep histological model of carcinogenesis in the setting of vulvar LS. Normalized chromosome 17 index reveals a significant upward trend (r = 0.1, P = 0.001 linear regression) toward increasing polysomy from histologically normal skin to SCC arising from a background of LS. The data were derived from 73 samples from 17 women with vulvar LS that developed SCC or VIN 3, differentiated type. Box-and-whisker plots of normalized chromosome 17 copy number are listed by histological categorization: normal adjacent vulvar skin (1), lichen simplex chronicus (2), lichen sclerosus (3), VIN (4), and SCC (5). The line in the middle of the box represents the median. The box extends from the 25th percentile to the 75th percentile (interquartile range). The whiskers mark upper and lower adjacent values. Circles represent outliers. The y axis represents the normalized chromosome 17 index (study group/control group).

Figure 4.

Multistep histological model of carcinogenesis in the setting of HPV-related lesions (VIN 3 undifferentiated type/squamous cell carcinoma in situ (SCCIS)). Normalized chromosome 17 index reveals a downward trend, loss of chromosome 17 (r = −0.03, P = 0.9 linear regression), from SCCIS to squamous cell carcinoma arising in association with SCCIS. The data were derived from 33 samples from 15 women with SCCIS, one of which developed invasive SCC (two samples). Box-and-whisker plots of normalized chromosome 17 copy number are listed by histological categorization: normal adjacent vulvar skin (1), lichen simplex chronicus (2), condyloma and VIN 2 (3), SCCIS (4), and SCC (5). The line in the middle of the box represents the median. The box extends from the 25th percentile to the 75th percentile (interquartile range). The whiskers mark upper and lower adjacent values. Circles represent outliers. The y axis represents the normalized chromosome 17 index (study group/control group).