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Original article
Fascin and cyclin D1 immunoreactivity in non-neoplastic vulvar squamous epithelium, vulvar intraepithelial neoplasia and invasive squamous carcinoma: correlation with Ki67 and p16 protein expression C J R Stewart,1,2 M L Crook1 1
Department of Histopathology, King Edward Memorial Hospital, Perth, Western Australia, Australia 2 School for Women’s and Infants’ Health, University of Western Australia, Perth, Western Australia, Australia Correspondence to Dr Colin Stewart, Department of Histopathology, King Edward Memorial Hospital, Perth, Western Australia 6008, Australia; [email protected]. au Received 21 August 2013 Revised 27 September 2013 Accepted 10 October 2013 Published Online First 11 November 2013
ABSTRACT Aims To investigate cyclin D1 and fascin immunoreactivity in normal, reactive and neoplastic vulvar skin correlating the findings with p16 protein and Ki67 expression. Methods 66 vulvar biopsy or resection specimens demonstrating normal appearances, reactive epidermal changes, usual-type vulvar intraepithelial neoplasia (uVIN), differentiated-type VIN (dVIN), p16-positive squamous cell carcinoma (SCC) and p16-negative SCC were examined immunohistochemically for cyclin D1, fascin, Ki67 and p16 protein. Where applicable, expression patterns were compared in microanatomically distinct areas, particularly at the invasive front (deep tumour margin) of SCC. Results Normal epidermis showed parabasal Ki67 and cyclin D1 staining while fascin labelled cells in the lower one-third of the epithelium. Reactive and dVIN specimens demonstrated mildly increased Ki67 and cyclin D1 expression that maintained parabasal polarity, whereas uVIN and p16-positive SCC were characterised by loss of cyclin D1 staining. However, in 14 of 20 p16-positive SCC small infiltrative tumour groups and single infiltrating cells at the invasive front showed a cyclin D1-positive/ Ki67-negative phenotype. In contrast, p16-negative SCC generally showed diffuse and concordant cyclin D1 and Ki67 labelling, including at the invasive margin. Fascin expression was increased in all VIN and SCC lesions. Conclusions Variations in cyclin D1 and Ki67 expression between p16-positive and p16-negative vulvar SCCs suggest different mechanisms of invasion in these tumour subgroups. Fascin is upregulated in vulvar squamous neoplasia but immunostaining does not discriminate in situ from invasive lesions nor putative human papilloma virus (HPV)-associated and HPVindependent SCCs.
INTRODUCTION
To cite: Stewart CJR, Crook ML. J Clin Pathol 2014;67:319–325.
Squamous cell carcinoma (SCC) of the vulva is the fourth most common malignant tumour arising in the female genital tract. The annual incidence is 1.3–3.6/100 000 in developed countries and appears to be increasing.1 2 While most superficially invasive tumours are curable by local excision, 20% of cases have lymph node metastases at presentation requiring unilateral or bilateral groin node dissection. In patients with positive nodes, the 5-year survival drops to 43%.1
Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
It is now recognised that there are two main pathways to vulvar squamous neoplasia.3 4 First, there are human papilloma virus (HPV)-associated neoplasms which more often affect young women, are commonly associated with additional HPV-related lesions of the anogenital tract and typically arise from usual (basaloid, classical or warty)-type vulvar intraepithelial neoplasia (VIN). In common with high-grade squamous intraepithelial lesions of the cervix, vagina and anal mucosa, usual-type VIN (uVIN) shows immunophenotypic changes that reflect HPV integration and cell cycle dysregulation, including diffuse expression of p16 protein and markedly increased Ki67 expression.3–5 In contrast, non-HPV-related vulvar SCCs typically arise in older women and often are associated with a background chronic dermatosis, particularly lichen sclerosus. Many non-HPV-related SCCs are of keratinising type and they may be associated with differentiated-type VIN (dVIN). Although dVIN presents greater diagnostic difficulty to histopathologists and is probably under-diagnosed, it appears to represent a more aggressive and rapidly progressive precursor lesion than the more overtly atypical uVIN.4 6–8 Despite this progress in understanding the pathogenesis of vulvar SCCs, little is known about the mechanisms that drive invasion in these tumours and it is also unclear whether HPV-related and HPV-independent neoplasms progress through similar or divergent pathways. However, recent studies in other types of malignancy have suggested that two proteins, cyclin D1 and fascin-1 (hereafter fascin), may be involved in the induction or promotion of tumour invasion.9–12 Cyclin D1 has a wellestablished role in cell cycle regulation and diffusely increased cyclin D1 expression has been demonstrated in a variety of epithelial and non-epithelial tumours, often correlating with adverse clinicopathological features. However, in some tumours cyclin D1 immunoreactivity is more localised, typically being restricted to infiltrative cells at the tumour–stromal interface (‘invasive front’).13–17 In this context, cyclin D1 expression appears independent of, and indeed may be inversely related to, cell proliferation. The major activity of fascin involves the stabilisation of actin filaments, thereby promoting the formation of cytoplasmic protrusions, which are critical to cellular migration and invasion in both physiological and neoplastic 319
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Original article contexts. Increased fascin reactivity has been demonstrated in many tumours and as with cyclin D1 staining may be preferentially localised to malignant cells at the deep tumour margin.18–23 The purpose of the present study was to compare cyclin D1, fascin, p16 and Ki67 expression in a series of vulvar biopsy samples demonstrating normal and reactive squamous epithelium, VIN of usual and differentiated types and corresponding p16-positive and p16-negative SCCs.
Table 1 Summary of antibody sources and immunohistochemical methods Antibody
Source
Clone
Dilution
Cyclin D1 Fascin p16 Ki67
Dako, Carpinteria, California, USA Dako, Carpinteria, California, USA Santa Cruz, Dallas, Texas, USA Dako, Glostrup, Denmark
SP4 55K-2 JC8 MIB-1
1:100 1:200 1:200 1:50
MATERIALS AND METHODS Sixty-six vulvar punch biopsies or resection specimens were selected from the histopathology files of King Edward Memorial Hospital Perth. All specimens had been formalin fixed and processed routinely to paraffin wax. The diagnostic categories included normal skin ( present at the margin of 38 specimens), reactive epidermal changes (n=16), high-grade uVIN without invasion (n=12), p16-positive SCCs (n=20) eight of which also demonstrated uVIN and p16-negative SCCs (n=18) seven of which were associated with dVIN and nine with lichen sclerosus. The reactive cases included six lichen sclerosus, five spongiotic dermatitis, four candida infection and one lichen planus. In this study, all uVIN and p16-positive SCCs showed confluent (‘block’) nuclear and cytoplasmic p16 immunoreactivity which has been shown to be a reliable surrogate marker of high-risk HPV infection in vulvar squamous neoplasia as in other anogenital sites (figure 1).4 5
Immunohistochemistry Serial 4-micron sections from one representative block of each case were stained immunohistochemically using the panel of antibodies summarised in table 1. Overall staining patterns in squamous cells were assessed as being negative or minimal (50% of cells positive), but where applicable differences in staining in microanatomically distinct areas were noted. Cytokeratin staining (clone AE1/AE3) was performed as previously described in selected SCC to highlight the distribution of the invasive tumour cells.24
RESULTS The immunohistochemical findings are summarised in table 2. Ki67 demonstrated only nuclear reactivity, while p16 showed both nuclear and cytoplasmic staining. Cyclin D1 expression was predominantly nuclear, but occasional cells also showed cytoplasmic staining. Fascin staining mainly showed cytoplasmic distribution, but concurrent nuclear staining was noted in a minority of keratinocytes in both reactive and neoplastic lesions.
Table 2 Summary of immunohistochemical staining patterns in normal, reactive and neoplastic vulvar squamous epithelium p16
Ki67
Cyclin D1
Fascin
Normal (n=38)
Negative
Parabasal cells
Parabasal cells
Reactive (n=16)
Negative (occasional single cells positive) Diffusely positive
Increased parabasal staining
Increased parabasal staining
Increased in all cell layers
p16-positive SCC (n=20)
Usually negative (random single cells positive) Diffusely positive
p16-negative SCC (n=18)
Usually negative
Mildly increased (basal cells focally positive) Mainly periphery of larger nests, reduced at the IF Diffusely positive or mainly at periphery of larger nests
Usually negative (random single cells positive) Increased parabasal staining
Lower one-third of epidermis As normal (diffuse staining in two cases) Diffusely positive
uVIN (n=12)
dVIN (n=7)
Figure 1 (A) Usual-type VIN (left) with invasive squamous carcinoma (right) showing ‘block’ p16 protein expression (B). Note that the immunohistochemistry highlights small clusters of infiltrating cells at the invasive front of the tumour. 320
Diffusely positive
Usually negative/ focal except at the IF
Diffusely positive
Diffusely positive or periphery of larger nests
Diffusely positive
dVIN, differentiated-type vulvar intraepithelial neoplasia; IF, invasive front; SCC, squamous cell carcinoma; uVIN, usual-type vulvar intraepithelial neoplasia.
Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
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Original article Normal and reactive squamous epithelium Parabasal cells in the normal epidermis showed Ki67 and cyclin D expression and this was mildly increased in the reactive specimens, typically with staining of 2–3 cell layers. Fascin labelled cells in the lower one-third of the normal epidermis, including basal cells. Non-neoplastic superficial keratinocytes did not express fascin except in two dermatitis specimens which showed almost fullthickness staining. Cyclin D1 and fascin focally labelled endothelial and inflammatory cells together with the ductal epithelium of
normal follicles and eccrine glands (figures 2 and 3), while p16 staining was restricted to sparse inflammatory and stromal cells.
uVIN In most cases, there was no difference in staining between the pure uVIN lesions and those associated with SCC, although occasionally the latter showed increased cyclin D1 expression adjacent to foci of invasion. There was increased Ki67 and fascin immunoreactivity involving most of the epidermis other than surface parakeratotic cells. In contrast, cyclin D1 was negative or reduced, and when present showed loss of parabasal polarity with staining of randomly distributed cells within the epidermis (figure 3).
dVIN There was a mild increase in Ki67 and cyclin D1 expression similar to that seen in reactive epithelium and generally maintaining parabasal distribution (figure 4), although focal Ki67 staining of basal cells was also observed. Fascin was usually diffusely positive, but p16 was negative or labelled only scattered single keratinocytes.
p16-positive SCC The majority of tumours showed focal Ki67 expression that was most consistent at the periphery of the larger invasive cell groups. Keratinised cells and ‘squamous pearls’ were negative.
Figure 2 (A) Deep margin of squamous carcinoma showing diffuse expression of p16 protein. (B) The tumour is cyclin D1 negative, but endothelial cells and normal eccrine ducts (right) are focally positive. (C) Fascin labels tumour cells, eccrine ducts and benign stromal elements. Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
Figure 3 (A) Usual-type VIN showing diffuse p16 protein expression. Note that a normal dermal follicle (right) is not stained. (B) Cyclin D1 shows an inverse staining pattern. 321
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Original article
Figure 4 (A) Differentiated-type VIN at the margin of a squamous carcinoma (not shown) with dermal changes of lichen sclerosus. (B) There is increased cyclin D1 expression in parabasal keratinocytes.
In 13 cases, discontinuous infiltrating cells at the invasive tumour front showed reduced Ki67 labelling compared with the adjacent larger tumour aggregates (figure 5). In contrast, cyclin D1 was typically negative or minimally positive in the larger tumour nests but highlighted small invasive tumour groups and single cells along the deep margin in 14 cases (figure 6). Fascin consistently labelled tumour cells and reactive stromal elements (figure 2).
Figure 5 (A) Deep margin of p16-positive squamous carcinoma. Note desmoplastic and inflammatory stroma with small infiltrative tumour nests showing cytological alteration at the invasive front (right). (B) Immunostaining shows focal Ki67 expression within the larger tumour groups, but the small infiltrative nests are negative.
DISCUSSION We have investigated the distribution of cyclin D1 and fascin in a series of normal, reactive and neoplastic vulvar specimens correlating the findings with Ki67 and p16 expression. The VIN
p16-negative SCC Similar staining patterns were observed in the p16-negative tumours associated with dVIN and those in which no clear in situ component was identified. The SCC typically showed diffuse (>50%) Ki67 labelling and usually this was concordant with cyclin D1 expression, particularly within the higher-grade elements. Better differentiated tumour areas showed predominant Ki67 and cyclin D1 staining at the periphery of the larger tumour aggregates, while keratinised cells were negative with both markers. Tumour cells at the invasive front usually showed coexpression of Ki67 and cyclin D1 (figure 7) and the latter also highlighted discontinuous malignant cells such as foci of neural spread (figure 8). Fascin was diffusely positive in the malignant cells but also labelled reactive cellular elements such that it could be difficult to distinguish neoplastic and inflammatory cells on the immunostained slides (figure 9). 322
Figure 6 Deep margin of p16-positive squamous carcinoma. Cyclin D1 expression is largely restricted to the small infiltrative tumour groups at the invasive front (right). Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
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Original article
Figure 8 (A) A p16-negative squamous carcinoma showing focal intraneural spread (arrow). (B) The tumour cells are highlighted by cyclin D1 staining.
Figure 7 Deep margin of p16-negative squamous carcinoma associated with a prominent inflammatory infiltrate. (A) Cytokeratin highlights the distribution of the tumour cells. Note that most tumour cells coexpress Ki67 (B) and cyclin D1 (C) but that the keratinised tumour cell nest (left) is Ki67/cyclin D1 negative. and SCC lesions examined herein showed uniformly positive or negative p16 staining, but it is acknowledged that not all vulval squamous neoplasms demonstrate such clear-cut immunophenotypic findings and that cases with mixed or overlapping features do occur.25 26 We found that parabasal cells in the normal epidermis coexpressed cyclin D1 and Ki67, while reactive epidermis typically showed increased labelling in 2–3 cell layers but with preservation of parabasal polarity. Similar changes were present in dVIN Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
indicating that staining with these markers is not likely to have diagnostic value in histologically challenging biopsies. In contrast, uVIN showed consistently reduced cyclin D1 reactivity despite the increase in Ki67 labelling thus indicating discordant expression of these proliferation-associated proteins. Similar findings have been reported in cervical squamous and glandular intraepithelial lesions and in HPV-associated neoplasms of the head and neck,13 14 27–29 and this most likely reflects HPV-related disruption of intracellular feedback loops involving the retinoblastoma protein. Our findings suggest that the reduced expression of cyclin D1 could also be used diagnostically in conjunction with ‘positive’ staining markers such as p16 protein and Ki67 to support a diagnosis of uVIN, and to distinguish the basaloid and differentiated subtypes of VIN. Previous studies have recorded increased or abnormal expression of cyclin D1 (variably defined) in 21–51% of biopsy specimens,30–33 as well as in vulvar SCC cell lines in vitro.34 However, these have not commented upon specific localisation of protein expression and most have not distinguished between HPV-associated and HPV-independent neoplasms.35 We found a difference in cyclin D1 immunoreactivity between the p16-negative and p16-positive SCCs, the former usually showing diffuse cyclin D1 expression and concordance with Ki67 staining similar to dVIN. In contrast, p16-positive SCC typically showed low cyclin D1 expression except at the invasive front where immunostaining often highlighted small groups of malignant cells at the tumour–stromal interface. These results 323
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Original article p16-positive SCCs suggest that these tumours not only have different aetiologies but also different mechanisms of invasion. However, it should be noted that the staining patterns of p16-positive and p16-negative SCCs were not completely uniform suggesting that different invasive pathways may also occur within each tumour subgroup. Previously, we have suggested that the localised upregulation of proteins such as cyclin D1 and fascin occurs during epithelial–mesenchymal transition (EMT),14 18 21 24 a process that is considered important in the process of invasion in many common cancers. Interestingly, Rodrigues et al recently reported decreased β-catenin and increased vimentin expression at the invasive front of vulvar SCCs consistent with EMT.42 However, EMT-related alterations were less common in HPV-associated tumours and this would appear contradictory to the present findings. At present there are insufficient data to determine the role of EMT in vulvar SCC and clearly further studies are required in this regard.43 To our knowledge, there is only one earlier report of vulvar fascin expression in the English language literature and that study focused upon the immune/inflammatory components of lichen sclerosus and SCC with no specific comment on epithelial staining.44 In the current study, VIN lesions of both usual and differentiated types showed increased fascin expression similar to squamous dysplasia of the oral mucosa and oesophagus.45 46 There was also diffuse fascin expression in both p16-negative and p16-positive SCCs, sparing only foci of keratinisation. Thus, increased fascin expression characterises in situ and invasive SCC at various anatomical sites but does not appear related to HPV. Although fascin expression has been localised preferentially to the infiltrating tumour margin in some malignancies, supporting a role in the promotion of tumour invasion, this was not the case in vulvar SCC and, as noted above, VIN lesions were also consistently fascin positive. Therefore, while fascin upregulation may have a role in the development and progression of vulvar squamous neoplasia it would not appear to be useful in distinguishing in situ and invasive neoplasms or the subtypes of vulvar SCC.
Take-home messages
Figure 9 (A) Deep margin of p16-negative squamous carcinoma. (B) Cytokeratin staining highlights small clusters of infiltrative tumour cells (right). (C) Fascin labels the tumour but also many reactive stromal cells and inflammatory cells, including a multinucleate histiocyte (arrow). are consistent with earlier studies in which HPV/p16 proteinpositive vulvar SCC showed lower cyclin D1 expression.31 32 We also found an inverse correlation between cyclin D1 and Ki67 expression at the invasive front of many p16-positive SCCs. These findings suggest that cellular proliferation is inhibited during active invasion in a subset of vulvar SCC and, as in other types of malignancy,15 36–41 cyclin D1 may have a more significant role in potentiating invasion than proliferation in some settings. Moreover, the variations in the extent and distribution of cyclin D1 staining between p16-negative and 324
▸ Vulvar squamous carcinoma develops via HPV-associated and HPV-independent pathways, but little is known about the mechanisms of invasion in these tumours. ▸ This study showed differences in cyclin D1 expression in VIN lesions of usual and differentiated types and in corresponding p16-positive and p16-negative squamous carcinomas. In p16-positive tumours, infiltrative tumour cells at the deep tumour margin (invasive front) often showed a discordant cyclin D1-positive/Ki67-negative phenotype. Fascin expression was increased in all cases of VIN and squamous carcinoma. ▸ Different molecular pathways may be involved during the progression of HPV-positive and HPV-negative vulvar squamous carcinomas. Fascin upregulation may contribute towards the development and progression of vulvar squamous neoplasia, but it does not distinguish in situ and invasive lesions or aetiological association with HPV infection.
Contributors CJRS reviewed the histology and immunohistochemical findings and prepared the manuscript. MLC performed the immunohistochemical staining. Both authors approved the manuscript. Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
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Original article Competing interests None.
23
Ethics approval KEMH Perth, Ethics Reference 2013066 EW. Provenance and peer review Not commissioned; externally peer reviewed.
24
25
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Fascin and cyclin D1 immunoreactivity in non-neoplastic vulvar squamous epithelium, vulvar intraepithelial neoplasia and invasive squamous carcinoma: correlation with Ki67 and p16 protein expression C J R Stewart and M L Crook J Clin Pathol 2014 67: 319-325 originally published online November 11, 2013
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Original article
Fascin and cyclin D1 immunoreactivity in non-neoplastic vulvar squamous epithelium, vulvar intraepithelial neoplasia and invasive squamous carcinoma: correlation with Ki67 and p16 protein expression C J R Stewart,1,2 M L Crook1 1
Department of Histopathology, King Edward Memorial Hospital, Perth, Western Australia, Australia 2 School for Women’s and Infants’ Health, University of Western Australia, Perth, Western Australia, Australia Correspondence to Dr Colin Stewart, Department of Histopathology, King Edward Memorial Hospital, Perth, Western Australia 6008, Australia; [email protected]. au Received 21 August 2013 Revised 27 September 2013 Accepted 10 October 2013 Published Online First 11 November 2013
ABSTRACT Aims To investigate cyclin D1 and fascin immunoreactivity in normal, reactive and neoplastic vulvar skin correlating the findings with p16 protein and Ki67 expression. Methods 66 vulvar biopsy or resection specimens demonstrating normal appearances, reactive epidermal changes, usual-type vulvar intraepithelial neoplasia (uVIN), differentiated-type VIN (dVIN), p16-positive squamous cell carcinoma (SCC) and p16-negative SCC were examined immunohistochemically for cyclin D1, fascin, Ki67 and p16 protein. Where applicable, expression patterns were compared in microanatomically distinct areas, particularly at the invasive front (deep tumour margin) of SCC. Results Normal epidermis showed parabasal Ki67 and cyclin D1 staining while fascin labelled cells in the lower one-third of the epithelium. Reactive and dVIN specimens demonstrated mildly increased Ki67 and cyclin D1 expression that maintained parabasal polarity, whereas uVIN and p16-positive SCC were characterised by loss of cyclin D1 staining. However, in 14 of 20 p16-positive SCC small infiltrative tumour groups and single infiltrating cells at the invasive front showed a cyclin D1-positive/ Ki67-negative phenotype. In contrast, p16-negative SCC generally showed diffuse and concordant cyclin D1 and Ki67 labelling, including at the invasive margin. Fascin expression was increased in all VIN and SCC lesions. Conclusions Variations in cyclin D1 and Ki67 expression between p16-positive and p16-negative vulvar SCCs suggest different mechanisms of invasion in these tumour subgroups. Fascin is upregulated in vulvar squamous neoplasia but immunostaining does not discriminate in situ from invasive lesions nor putative human papilloma virus (HPV)-associated and HPVindependent SCCs.
INTRODUCTION
To cite: Stewart CJR, Crook ML. J Clin Pathol 2014;67:319–325.
Squamous cell carcinoma (SCC) of the vulva is the fourth most common malignant tumour arising in the female genital tract. The annual incidence is 1.3–3.6/100 000 in developed countries and appears to be increasing.1 2 While most superficially invasive tumours are curable by local excision, 20% of cases have lymph node metastases at presentation requiring unilateral or bilateral groin node dissection. In patients with positive nodes, the 5-year survival drops to 43%.1
Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
It is now recognised that there are two main pathways to vulvar squamous neoplasia.3 4 First, there are human papilloma virus (HPV)-associated neoplasms which more often affect young women, are commonly associated with additional HPV-related lesions of the anogenital tract and typically arise from usual (basaloid, classical or warty)-type vulvar intraepithelial neoplasia (VIN). In common with high-grade squamous intraepithelial lesions of the cervix, vagina and anal mucosa, usual-type VIN (uVIN) shows immunophenotypic changes that reflect HPV integration and cell cycle dysregulation, including diffuse expression of p16 protein and markedly increased Ki67 expression.3–5 In contrast, non-HPV-related vulvar SCCs typically arise in older women and often are associated with a background chronic dermatosis, particularly lichen sclerosus. Many non-HPV-related SCCs are of keratinising type and they may be associated with differentiated-type VIN (dVIN). Although dVIN presents greater diagnostic difficulty to histopathologists and is probably under-diagnosed, it appears to represent a more aggressive and rapidly progressive precursor lesion than the more overtly atypical uVIN.4 6–8 Despite this progress in understanding the pathogenesis of vulvar SCCs, little is known about the mechanisms that drive invasion in these tumours and it is also unclear whether HPV-related and HPV-independent neoplasms progress through similar or divergent pathways. However, recent studies in other types of malignancy have suggested that two proteins, cyclin D1 and fascin-1 (hereafter fascin), may be involved in the induction or promotion of tumour invasion.9–12 Cyclin D1 has a wellestablished role in cell cycle regulation and diffusely increased cyclin D1 expression has been demonstrated in a variety of epithelial and non-epithelial tumours, often correlating with adverse clinicopathological features. However, in some tumours cyclin D1 immunoreactivity is more localised, typically being restricted to infiltrative cells at the tumour–stromal interface (‘invasive front’).13–17 In this context, cyclin D1 expression appears independent of, and indeed may be inversely related to, cell proliferation. The major activity of fascin involves the stabilisation of actin filaments, thereby promoting the formation of cytoplasmic protrusions, which are critical to cellular migration and invasion in both physiological and neoplastic 319
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Original article contexts. Increased fascin reactivity has been demonstrated in many tumours and as with cyclin D1 staining may be preferentially localised to malignant cells at the deep tumour margin.18–23 The purpose of the present study was to compare cyclin D1, fascin, p16 and Ki67 expression in a series of vulvar biopsy samples demonstrating normal and reactive squamous epithelium, VIN of usual and differentiated types and corresponding p16-positive and p16-negative SCCs.
Table 1 Summary of antibody sources and immunohistochemical methods Antibody
Source
Clone
Dilution
Cyclin D1 Fascin p16 Ki67
Dako, Carpinteria, California, USA Dako, Carpinteria, California, USA Santa Cruz, Dallas, Texas, USA Dako, Glostrup, Denmark
SP4 55K-2 JC8 MIB-1
1:100 1:200 1:200 1:50
MATERIALS AND METHODS Sixty-six vulvar punch biopsies or resection specimens were selected from the histopathology files of King Edward Memorial Hospital Perth. All specimens had been formalin fixed and processed routinely to paraffin wax. The diagnostic categories included normal skin ( present at the margin of 38 specimens), reactive epidermal changes (n=16), high-grade uVIN without invasion (n=12), p16-positive SCCs (n=20) eight of which also demonstrated uVIN and p16-negative SCCs (n=18) seven of which were associated with dVIN and nine with lichen sclerosus. The reactive cases included six lichen sclerosus, five spongiotic dermatitis, four candida infection and one lichen planus. In this study, all uVIN and p16-positive SCCs showed confluent (‘block’) nuclear and cytoplasmic p16 immunoreactivity which has been shown to be a reliable surrogate marker of high-risk HPV infection in vulvar squamous neoplasia as in other anogenital sites (figure 1).4 5
Immunohistochemistry Serial 4-micron sections from one representative block of each case were stained immunohistochemically using the panel of antibodies summarised in table 1. Overall staining patterns in squamous cells were assessed as being negative or minimal (50% of cells positive), but where applicable differences in staining in microanatomically distinct areas were noted. Cytokeratin staining (clone AE1/AE3) was performed as previously described in selected SCC to highlight the distribution of the invasive tumour cells.24
RESULTS The immunohistochemical findings are summarised in table 2. Ki67 demonstrated only nuclear reactivity, while p16 showed both nuclear and cytoplasmic staining. Cyclin D1 expression was predominantly nuclear, but occasional cells also showed cytoplasmic staining. Fascin staining mainly showed cytoplasmic distribution, but concurrent nuclear staining was noted in a minority of keratinocytes in both reactive and neoplastic lesions.
Table 2 Summary of immunohistochemical staining patterns in normal, reactive and neoplastic vulvar squamous epithelium p16
Ki67
Cyclin D1
Fascin
Normal (n=38)
Negative
Parabasal cells
Parabasal cells
Reactive (n=16)
Negative (occasional single cells positive) Diffusely positive
Increased parabasal staining
Increased parabasal staining
Increased in all cell layers
p16-positive SCC (n=20)
Usually negative (random single cells positive) Diffusely positive
p16-negative SCC (n=18)
Usually negative
Mildly increased (basal cells focally positive) Mainly periphery of larger nests, reduced at the IF Diffusely positive or mainly at periphery of larger nests
Usually negative (random single cells positive) Increased parabasal staining
Lower one-third of epidermis As normal (diffuse staining in two cases) Diffusely positive
uVIN (n=12)
dVIN (n=7)
Figure 1 (A) Usual-type VIN (left) with invasive squamous carcinoma (right) showing ‘block’ p16 protein expression (B). Note that the immunohistochemistry highlights small clusters of infiltrating cells at the invasive front of the tumour. 320
Diffusely positive
Usually negative/ focal except at the IF
Diffusely positive
Diffusely positive or periphery of larger nests
Diffusely positive
dVIN, differentiated-type vulvar intraepithelial neoplasia; IF, invasive front; SCC, squamous cell carcinoma; uVIN, usual-type vulvar intraepithelial neoplasia.
Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
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Original article Normal and reactive squamous epithelium Parabasal cells in the normal epidermis showed Ki67 and cyclin D expression and this was mildly increased in the reactive specimens, typically with staining of 2–3 cell layers. Fascin labelled cells in the lower one-third of the normal epidermis, including basal cells. Non-neoplastic superficial keratinocytes did not express fascin except in two dermatitis specimens which showed almost fullthickness staining. Cyclin D1 and fascin focally labelled endothelial and inflammatory cells together with the ductal epithelium of
normal follicles and eccrine glands (figures 2 and 3), while p16 staining was restricted to sparse inflammatory and stromal cells.
uVIN In most cases, there was no difference in staining between the pure uVIN lesions and those associated with SCC, although occasionally the latter showed increased cyclin D1 expression adjacent to foci of invasion. There was increased Ki67 and fascin immunoreactivity involving most of the epidermis other than surface parakeratotic cells. In contrast, cyclin D1 was negative or reduced, and when present showed loss of parabasal polarity with staining of randomly distributed cells within the epidermis (figure 3).
dVIN There was a mild increase in Ki67 and cyclin D1 expression similar to that seen in reactive epithelium and generally maintaining parabasal distribution (figure 4), although focal Ki67 staining of basal cells was also observed. Fascin was usually diffusely positive, but p16 was negative or labelled only scattered single keratinocytes.
p16-positive SCC The majority of tumours showed focal Ki67 expression that was most consistent at the periphery of the larger invasive cell groups. Keratinised cells and ‘squamous pearls’ were negative.
Figure 2 (A) Deep margin of squamous carcinoma showing diffuse expression of p16 protein. (B) The tumour is cyclin D1 negative, but endothelial cells and normal eccrine ducts (right) are focally positive. (C) Fascin labels tumour cells, eccrine ducts and benign stromal elements. Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
Figure 3 (A) Usual-type VIN showing diffuse p16 protein expression. Note that a normal dermal follicle (right) is not stained. (B) Cyclin D1 shows an inverse staining pattern. 321
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Original article
Figure 4 (A) Differentiated-type VIN at the margin of a squamous carcinoma (not shown) with dermal changes of lichen sclerosus. (B) There is increased cyclin D1 expression in parabasal keratinocytes.
In 13 cases, discontinuous infiltrating cells at the invasive tumour front showed reduced Ki67 labelling compared with the adjacent larger tumour aggregates (figure 5). In contrast, cyclin D1 was typically negative or minimally positive in the larger tumour nests but highlighted small invasive tumour groups and single cells along the deep margin in 14 cases (figure 6). Fascin consistently labelled tumour cells and reactive stromal elements (figure 2).
Figure 5 (A) Deep margin of p16-positive squamous carcinoma. Note desmoplastic and inflammatory stroma with small infiltrative tumour nests showing cytological alteration at the invasive front (right). (B) Immunostaining shows focal Ki67 expression within the larger tumour groups, but the small infiltrative nests are negative.
DISCUSSION We have investigated the distribution of cyclin D1 and fascin in a series of normal, reactive and neoplastic vulvar specimens correlating the findings with Ki67 and p16 expression. The VIN
p16-negative SCC Similar staining patterns were observed in the p16-negative tumours associated with dVIN and those in which no clear in situ component was identified. The SCC typically showed diffuse (>50%) Ki67 labelling and usually this was concordant with cyclin D1 expression, particularly within the higher-grade elements. Better differentiated tumour areas showed predominant Ki67 and cyclin D1 staining at the periphery of the larger tumour aggregates, while keratinised cells were negative with both markers. Tumour cells at the invasive front usually showed coexpression of Ki67 and cyclin D1 (figure 7) and the latter also highlighted discontinuous malignant cells such as foci of neural spread (figure 8). Fascin was diffusely positive in the malignant cells but also labelled reactive cellular elements such that it could be difficult to distinguish neoplastic and inflammatory cells on the immunostained slides (figure 9). 322
Figure 6 Deep margin of p16-positive squamous carcinoma. Cyclin D1 expression is largely restricted to the small infiltrative tumour groups at the invasive front (right). Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
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Original article
Figure 8 (A) A p16-negative squamous carcinoma showing focal intraneural spread (arrow). (B) The tumour cells are highlighted by cyclin D1 staining.
Figure 7 Deep margin of p16-negative squamous carcinoma associated with a prominent inflammatory infiltrate. (A) Cytokeratin highlights the distribution of the tumour cells. Note that most tumour cells coexpress Ki67 (B) and cyclin D1 (C) but that the keratinised tumour cell nest (left) is Ki67/cyclin D1 negative. and SCC lesions examined herein showed uniformly positive or negative p16 staining, but it is acknowledged that not all vulval squamous neoplasms demonstrate such clear-cut immunophenotypic findings and that cases with mixed or overlapping features do occur.25 26 We found that parabasal cells in the normal epidermis coexpressed cyclin D1 and Ki67, while reactive epidermis typically showed increased labelling in 2–3 cell layers but with preservation of parabasal polarity. Similar changes were present in dVIN Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
indicating that staining with these markers is not likely to have diagnostic value in histologically challenging biopsies. In contrast, uVIN showed consistently reduced cyclin D1 reactivity despite the increase in Ki67 labelling thus indicating discordant expression of these proliferation-associated proteins. Similar findings have been reported in cervical squamous and glandular intraepithelial lesions and in HPV-associated neoplasms of the head and neck,13 14 27–29 and this most likely reflects HPV-related disruption of intracellular feedback loops involving the retinoblastoma protein. Our findings suggest that the reduced expression of cyclin D1 could also be used diagnostically in conjunction with ‘positive’ staining markers such as p16 protein and Ki67 to support a diagnosis of uVIN, and to distinguish the basaloid and differentiated subtypes of VIN. Previous studies have recorded increased or abnormal expression of cyclin D1 (variably defined) in 21–51% of biopsy specimens,30–33 as well as in vulvar SCC cell lines in vitro.34 However, these have not commented upon specific localisation of protein expression and most have not distinguished between HPV-associated and HPV-independent neoplasms.35 We found a difference in cyclin D1 immunoreactivity between the p16-negative and p16-positive SCCs, the former usually showing diffuse cyclin D1 expression and concordance with Ki67 staining similar to dVIN. In contrast, p16-positive SCC typically showed low cyclin D1 expression except at the invasive front where immunostaining often highlighted small groups of malignant cells at the tumour–stromal interface. These results 323
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Original article p16-positive SCCs suggest that these tumours not only have different aetiologies but also different mechanisms of invasion. However, it should be noted that the staining patterns of p16-positive and p16-negative SCCs were not completely uniform suggesting that different invasive pathways may also occur within each tumour subgroup. Previously, we have suggested that the localised upregulation of proteins such as cyclin D1 and fascin occurs during epithelial–mesenchymal transition (EMT),14 18 21 24 a process that is considered important in the process of invasion in many common cancers. Interestingly, Rodrigues et al recently reported decreased β-catenin and increased vimentin expression at the invasive front of vulvar SCCs consistent with EMT.42 However, EMT-related alterations were less common in HPV-associated tumours and this would appear contradictory to the present findings. At present there are insufficient data to determine the role of EMT in vulvar SCC and clearly further studies are required in this regard.43 To our knowledge, there is only one earlier report of vulvar fascin expression in the English language literature and that study focused upon the immune/inflammatory components of lichen sclerosus and SCC with no specific comment on epithelial staining.44 In the current study, VIN lesions of both usual and differentiated types showed increased fascin expression similar to squamous dysplasia of the oral mucosa and oesophagus.45 46 There was also diffuse fascin expression in both p16-negative and p16-positive SCCs, sparing only foci of keratinisation. Thus, increased fascin expression characterises in situ and invasive SCC at various anatomical sites but does not appear related to HPV. Although fascin expression has been localised preferentially to the infiltrating tumour margin in some malignancies, supporting a role in the promotion of tumour invasion, this was not the case in vulvar SCC and, as noted above, VIN lesions were also consistently fascin positive. Therefore, while fascin upregulation may have a role in the development and progression of vulvar squamous neoplasia it would not appear to be useful in distinguishing in situ and invasive neoplasms or the subtypes of vulvar SCC.
Take-home messages
Figure 9 (A) Deep margin of p16-negative squamous carcinoma. (B) Cytokeratin staining highlights small clusters of infiltrative tumour cells (right). (C) Fascin labels the tumour but also many reactive stromal cells and inflammatory cells, including a multinucleate histiocyte (arrow). are consistent with earlier studies in which HPV/p16 proteinpositive vulvar SCC showed lower cyclin D1 expression.31 32 We also found an inverse correlation between cyclin D1 and Ki67 expression at the invasive front of many p16-positive SCCs. These findings suggest that cellular proliferation is inhibited during active invasion in a subset of vulvar SCC and, as in other types of malignancy,15 36–41 cyclin D1 may have a more significant role in potentiating invasion than proliferation in some settings. Moreover, the variations in the extent and distribution of cyclin D1 staining between p16-negative and 324
▸ Vulvar squamous carcinoma develops via HPV-associated and HPV-independent pathways, but little is known about the mechanisms of invasion in these tumours. ▸ This study showed differences in cyclin D1 expression in VIN lesions of usual and differentiated types and in corresponding p16-positive and p16-negative squamous carcinomas. In p16-positive tumours, infiltrative tumour cells at the deep tumour margin (invasive front) often showed a discordant cyclin D1-positive/Ki67-negative phenotype. Fascin expression was increased in all cases of VIN and squamous carcinoma. ▸ Different molecular pathways may be involved during the progression of HPV-positive and HPV-negative vulvar squamous carcinomas. Fascin upregulation may contribute towards the development and progression of vulvar squamous neoplasia, but it does not distinguish in situ and invasive lesions or aetiological association with HPV infection.
Contributors CJRS reviewed the histology and immunohistochemical findings and prepared the manuscript. MLC performed the immunohistochemical staining. Both authors approved the manuscript. Stewart CJR, et al. J Clin Pathol 2014;67:319–325. doi:10.1136/jclinpath-2013-201920
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Original article Competing interests None.
23
Ethics approval KEMH Perth, Ethics Reference 2013066 EW. Provenance and peer review Not commissioned; externally peer reviewed.
24
25
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Fascin and cyclin D1 immunoreactivity in non-neoplastic vulvar squamous epithelium, vulvar intraepithelial neoplasia and invasive squamous carcinoma: correlation with Ki67 and p16 protein expression C J R Stewart and M L Crook J Clin Pathol 2014 67: 319-325 originally published online November 11, 2013
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