Epidermal growth factor receptor gene numerical aberrations are frequent events in actinic keratoses and invasive cutaneous squamous cell carcinomas

Please cite this paper as: Epidermal growth factor receptor gene numerical aberrations are frequent events in actinic keratoses and invasive cutaneous squamous cell carcinomas. Experimental Dermatology 2010; 19: 151–153. Abstract: Epidermal growth factor receptor (EGFR) gene amplification and protein overexpression are common in several cancers. EGFR status has seldom been studied in cutaneous squamous carcinomas (SCCs), or their precursors, actinic keratoses (AKs). We evaluated the presence of EGFR genomic aberrations and EGFR protein overexpression in 25 AKs and 35 invasive SCCs by means of fluorescence in situ hybridization (FISH) and immunohistochemistry. EGFR numerical aberrations were detected in 52% of AKs and 77.1% of SCCs (P = 0.042). EGFR amplification was identified in 12% of AKs and 20% of SCCs. No differences regarding EGFR numerical aberrations were observed when AKs with high‐grade dysplasia were compared with SCCs. A good correlation was observed between EGFR numerical aberrations and EGFR overexpression. Our results suggest that EGFR numerical aberrations occur in the early stages of epithelial carcinogenesis in skin, not playing a role in the progression from low‐grade SCCs into more aggressive phenotypes.     

MYC gene numerical aberrations in actinic keratosis and cutaneous squamous cell carcinoma

Background  The genetic alterations that drive the transition from actinic keratoses (AKs) to cutaneous squamous cell carcinomas (SCCs) have not been defined precisely. Amplification and/or overexpression of the MYC proto-oncogene have been demonstrated in several human, malignant tumours including head and neck SCCs.
Objectives  To evaluate the presence of MYC genomic aberrations in both AKs and SCCs.
Methods  Skin biopsy specimens corresponding to AKs, SCCs and control samples were included in two paraffin-embedded tissue microarrays. MYC cytogenetic profile was evaluated by fluorescence in situ hybridization (FISH). The results obtained were compared with MYC immunohistochemical expression.
Results  Twenty-three AKs and 30 SCCs were evaluated. MYC numerical aberrations were observed in eight of 23 (35%) AKs and 19 of 30 (63%) SCCs ( P  = 0·05). MYC numerical aberrations were more frequent in moderately to poorly differentiated SCCs (77%) when compared with well-differentiated SCCs (25%; P  = 0·027). A significant association between copy number gains of MYC by FISH analysis and MYC protein expression was demonstrated.
Conclusions  MYC gains and amplifications are frequent cytogenetic abnormalities in SCCs and may play a relevant role in promoting SCC undifferentiation and tumoral progression.     

The Majority of Cutaneous Squamous Cell Carcinomas Arise in Actinic Keratoses

Background: Retrospective studies have given conflicting results with respect to how many cutaneous squamous cell carcinomas (SCCs) arise in actinic keratoses (AK). Objective: This study was conducted to determine what percentage of SCCs arise in AKs and to obtain more information about two histological features of SCCs, namely, thickness and ulceration. Methods: A prospective study was done of all SCCs treated by the authors during one calendar year. Results: Two hundred eight patients with SCC were entered into the study. An AK was contiguous with an SCC in 72% of the cases. This was taken as evidence that the SCC arose in the AK. Men presented with thicker and more ulcerated SCCs than women, but these were not statistically significant: p = 0.06 for thickness and p = 0.07 for ulceration. Ulcerated SCCs were more likely to arise on the head and neck (p = 0.02), on patients who had multiple skin cancers (p = 0.005), and on patients who had a family history of skin cancer (p = 0.03). Conclusion: Actinic keratoses need to be removed before they turn into SCCs. The prognostic significance of ulceration of cutaneous SCCs needs to be determined.     

Actinic keratosis and field cancerization

While actinic keratoses (AKs) have been considered precancerous until recently for being able to turn into squamous cell carcinomas (SCCs), it is now agreed that it would be more appropriate to call them cancerous. Although not all AKs turn into SCC and some of them may even have a spontaneous regression, there is an obvious association between SCC and AK. Approximately 90% of SCs have been reported to develop from AKs and AKs are the preinvasive form of SCCs. The presence of two or more AKs on a photodamaged skin is an indicator of field cancerization and represents an increased risk of invasive SCC. All lesions should be treated since it cannot be foreseen which of the lesions will regress and which will progress to SCC. AK can be a single lesion or it can involve multiple lesions in a field of cancerization; thus, AK treatment is grouped under two headings: (1) Lesion-specific treatment; and (2) Field-targeted treatment. Lesion-specific treatments are practicable in patients with a small number of clinically visible and isolated lesions. These treatments including cryotherapy, surgical excision, shave excision, curettage and laser are based on physical destruction of the visible lesions. Field-targeted treatments are effective in the treatment of visible lesions, subclinical lesions and keratinocyte changes in the areas surrounding the visible lesions. Field targeted treatment options are topical imiquimod cream, 5% 5-fluorouracil cream, ingenol mebutate, diclofenac gel, resimiquimod and photodynamic therapy.     

Retinoblastoma gene expression in human non-melanoma skin cancer

BACKGROUND: The aberrant expression of both the retinoblastoma and p53 tumor suppressor genes has been associated with more aggressive tumors, metastasis and lower survival. METHODS: We have evaluated immunohistochemically the expression of pRB in a panel of non-melanoma skin cancers containing p53 somatic mutations. RESULTS: Nuclear anti-p53 staining was detected in 18 (72%) differentiated squamous cell carcinomas, six (100%) undifferentiated squamous cell carcinomas and seven (28%) basal cell carcinomas. A correlation was observed between p53 expression and the proliferative activity of differentiated squamous cell carcinomas (P < 0.066), undifferentiated squamous cell carcinomas (P < 0.05) and basal cell carcinomas (P < 0.01). Tumors were selected for mutant p53 expression by PCR-directed DNA sequencing and pRB expression measured immunohistochemically. Anti-pRB reactivity was detected in the nuclei of basal and suprabasal layer cells of normal epidermis, and in the proliferative compartment of all the differentiated squamous cell carcinomas, and basal cell carcinomas. A correlation was observed between pRB expression and the proliferative activity of the differentiated squamous cell carcinomas (P < 0.01) and basal cell carcinomas (P < 0.025). However, anti-pRB reactivity was not detected in the six anti-p53 reactive undifferentiated squamous cell carcinomas.     

Mutant p53 expression in solar keratosis: an immunohistochemical study

Thirty-eight solar keratoses from 32 patients were studied for expression of mutant p53 protein by an immunohistochemical technique. Twenty-eight of the 38 solar keratoses (73.7%) showed positive and variable nuclear labelling, whereas 10 specimens were immunonegative. The nuclear immunopositivity which was seen in all variants was mostly diffuse in distribution. The adjacent "normal" epidermis of 8 keratoses showed positive mutant p53 labelling. Eight of the keratoses were associated with invasive squamous cell carcinoma of which only two were immunopositive. Cytoplasmic labelling was never a feature. The study demonstrates that mutant p53 protein is commonly expressed in all variants of solar keratosis and that its expression correlates with atypical keratinocyte proliferation. It is proposed that the demonstration of mutant p53 in the adjacent normal epidermis may be a potential marker of early neoplastic transformation.     

Suberythemal ultraviolet B radiation alters the expression of cell cycle-related proteins in the epidermis of human subjects without leading to photoprotection

Background  Deregulation of the cell cycle proteins is one of the critical factors leading to cutaneous carcinogenesis.
Objectives  To monitor the expression of cell cycle proteins in the epidermis of subjects after repeated exposure to ultraviolet (UV) B radiation, and to test for the development of photoprotection by subsequent irradiation with a single erythemal UVB dose.
Methods  A total of 26 healthy volunteers were divided into four groups: group 1 ( n  =   9) were given whole-body UVB irradiation for 10 consecutive days with 0·7 minimal erythema dose (MED), group 2 ( n  =   9) were irradiated as in group 1 followed 24 h later by a single UVB dose of 3 MED on buttock skin, group 3 ( n  =   4) were irradiated with a UVB dose of 3 MED on buttock skin, and group 4 ( n  =   4) were not irradiated. Skin biopsies were collected 24 h after the final irradiation and stained for cyclins A, B1, D1, and p16, p18, p21, p27, p53, pRB, Bax and Bcl-2.
Results  The expression of cyclin D1, p18 and p21 was significantly higher in groups 1 and 2 compared with the nonirradiated group 4 controls and, in group 2, the expression of pRB, p53 and Bax was also increased. In group 3, only p53 and Bax proteins were significantly elevated compared with group 4. The expression of cyclin D1, p16, p18, p27, pRB and Bcl-2 was higher in group 2 compared with group 3.
Conclusions  Suberythemal UVB radiation was sufficient to cause changes in the expression of several epidermal cell cycle proteins. When tested by irradiation with a single erythemal UVB dose following the repeated exposures, no photoprotection against the UV-induced alteration in cell cycle protein expression was apparent.     

p53 immunoreactivity in non-melanoma skin cancer from immunosuppressed and immunocompetent individuals: a comparative study of 246 tumours

p53 immunoreactivity was examined in 132 cutaneous non-melanoma tumours from renal transplant recipients and in 114 histologically matched specimens from immunocompetent individuals. Skin lesions examined included 52 viral warts, 50 clysplastic keratoses, 51 intraepidermal carcinomas (IEC), 50 invasive squamous cell carcinomas (SCC) and 43 basal cell carcinomas (BCC). Overall, 51% (51/101) pre-malignant skin lesions and 45% (42/93) non-melanoma skin cancers (NMSC) showed p53 immunoreactivity, with extensive (> 50% cells positive) p53 staining in 27% (27/101) of pre-malignant and 20% (19/93) of malignant lesions. 17% (9/52) viral warts showed p53 immunoreactivity, but this was limited to focal or basal p53 staining. p53 immunoreactivity in all tumours was less in transplant than in non-transplant patients and this reached statistical significance for SCCs (p = 0.03).     

Activation of Src-family tyrosine kinases in hyperproliferative epidermal disorders

Background:  Src-family tyrosine kinases (SFKs) are important regulators of keratinocyte growth and differentiation. In a broad range of cell types, persistent activation of SFKs correlates with increased cell proliferation. In this study, we determined if SFK activity is increased in cutaneous neoplasia and psoriasis, common hyperproliferative epidermal disorders.
Methods:  Formalin-fixed tissue sections of unremarkable epidermis, psoriasis, actinic keratoses (AKs), squamous cell carcinoma in situ (SCIS) and squamous cell carcinoma (SCC) were subjected to immunohistochemical staining for activated SFKs.
Results:  All psoriasis specimens displayed significantly greater staining for activated SFKs than sections of unremarkable skin. In the psoriasis biopsies, the degree of epidermal hyperplasia was proportional to the level of activated SFK staining. All AKs, SCISs and SCCs exhibited more prominent staining than sections of unremarkable epidermis. No discernable difference in activated SFK staining was seen between AKs, SCIS and SCC specimens.
Conclusions:  This study shows increased staining of activated SFKs in human biopsy specimens of psoriasis and cutaneous neoplasia. These data provide direct evidence for increased activation of SFKs in the pathogenesis of hyperproliferative epidermal disorders.     

DeltaNp63alpha promotes apoptosis of human epidermal keratinocytes

In this study we show that deltaNp63alpha overexpression in primary human epidermal keratinocytes causes decreased cell proliferation and increased apoptosis. These changes are associated with increased levels of p21 and p27, decreased cyclin D1 and cyclin E levels, reduced mitochondrial membrane potential, and enhanced procaspase and poly(ADP-ribose) polymerase cleavage. Bcl-xS and Bax levels are increased and Bcl-xL level is reduced. p53 levels are increased in the deltaNp63alpha-expressing cells and p53 overexpression reproduces features of the deltaNp63alpha phenotype. Increased p53 expression results in reduced deltaNp63alpha, suggesting that p53 may negatively regulate deltaNp63alpha level. DeltaNp63alpha also induces apoptosis in HaCaT and SCC-13 cells, which encode inactive p53 genes, suggesting that the response is p53 independent in these cell lines. Both deltaNp63alpha and TAp63alpha reduce SCC-13 cell survival. These studies indicate that both deltaNp63alpha and TAp63alpha can negatively regulate keratinocyte survival.     

Differential expression of proliferation- and apoptosis-related markers in lentigo maligna and solar keratosis keratinocytes

Keratinocytes influence the number, morphology, and proliferation of melanocytes. An interference in the melanocyte-keratinocyte relationship may contribute to melanoma development. This study examined the expression of apoptotic and proliferative markers in keratinocytes in lentigo maligna to characterize the epidermis permissive to these lesions. Formalin-fixed and paraffin-embedded tissues from 25 samples of lentigo maligna, 20 samples of solar keratoses, and 5 samples each of normal sun-exposed and non-sun-exposed skin (controls) were immunostained with antibodies directed against the proapoptotic markers bax and p53, the antiapoptotic marker bcl-2, and the proliferation marker ki-67. Eight percent of the lentigo maligna samples were positive for keratinocyte expression of bcl-2, 24% were positive for p53, and 76% were positive for bax; respective findings for solar keratoses were 35%, 85%, and 90%. Comparison with normal sun-exposed skin yielded lower rates of keratinocyte proliferation in 56% of the lentigo maligna samples, similar rates in 36%, and higher rates in 8%; for solar keratoses, proliferation was higher than controls in 60% of samples, similar in 35%, and lower in 5%. All these differences were statistically significant. These findings indicate that there are variable patterns of epidermal reaction to chronic sun exposure. The epidermis in lentigo maligna shows overall low proliferation and an apparently low apoptotic tendency. The dysfunctional epidermis may be permissive to aberrant melanocyte proliferation in the early stages of melanoma development.     

Validation of a diagnostic algorithm for the discrimination of actinic keratosis from normal skin and squamous cell carcinoma by means of high‐definition optical coherence tomography

Actinic keratoses (AKs) commonly arise on sun‐damaged skin. Visible lesions are often associated with subclinical lesions on surrounding skin, giving rise to field cancerization. To avoid multiple biopsies to diagnose subclinical/early invasive lesions, there is an increasing interest in non‐invasive diagnostic tools, such as high‐definition optical coherence tomography (HD‐OCT). We previously developed a HD‐OCT‐based diagnostic algorithm for the discrimination of AK from squamous cell carcinoma (SCC) and normal skin. The aim of this study was to test the applicability of HD‐OCT for non‐invasive discrimination of AK from SCC and normal skin using this algorithm. Three‐dimensional (3D) HD‐OCT images of histopathologically proven AKs and SCCs and images of normal skin were collected. All images were shown in a random sequence to three independent observers with different experience in HD‐OCT, blinded to the clinical and histopathological data and with different experience with HD‐OCT. Observers classified each image as AK, SCC or normal skin based on the diagnostic algorithm. A total of 106 (38 AKs, 16 SCCs and 52 normal skin sites) HD‐OCT images from 71 patients were included. Sensitivity and specificity for the most experienced observer were 81.6% and 92.6% for AK diagnosis and 93.8% and 98.9% for SCC diagnosis. A moderate interobserver agreement was demonstrated. HD‐OCT represents a promising technology for the non‐invasive diagnosis of AKs. Thanks to its high potential in discriminating SCC from AK, HD‐OCT could be used as a relevant tool for second‐level examination, increasing diagnostic confidence and sparing patients unnecessary excisions.     

Fluorescence diagnosis in actinic keratosis and squamous cell carcinoma

Background: As different tissue types have distinct capabilities to accumulate protoporphyrin IX, fluorescence diagnosis with aminolevulinic acid‐induced porphyrins (FDAP) could be used to discriminate between different types of tissue. Previous results demonstrated higher fluorescence ratios in squamous cell carcinoma (SCC) compared with actinic keratoses (AKs). Objectives: The lesional : non‐lesional fluorescence ratio of AKs was compared with the ratio of SCC. Other factors influencing macroscopic fluorescence were also assessed, including stratum corneum thickness, which has been demonstrated to account for heterogeneous fluorescence in psoriasis and in AKs. Methods: After 1 week of keratolytic pretreatment, FDAP was performed in 13 patients with 36 lesions suspected for AK or SCC. Biopsies were taken for histopathological diagnosis and measurement of stratum corneum thickness. Results: No significant differences were found in the fluorescence ratio (lesional : non‐lesional skin) between AKs and SCCs, although macroscopic fluorescence was significantly higher in Bowen's disease and micro‐invasive SCCs. Conclusions: There could be a potential applicability of FDAP to differentiate premalignant lesions with a tendency to progress into SCC and squamous cutaneous lesions already progressing into early invasive cancer from other squamous cutaneous (pre)malignancies. The amount of hyperkeratosis, invasiveness and degree of differentiation seem to be responsible for variations in fluorescence intensity.     

Modes of action of diclofenac 3%/hyaluronic acid 2.5% in the treatment of actinic keratosis

Background: The efficacy of topically applied diclofenac 3 % in combination with hyaluronic acid 2.5 % in the treatment of actinic keratoses (AKs) has been demonstrated in several clinical studies, but the exact mode of action is still unclear. This study evaluates the potential molecular and cellular main modes of action of topically applied diclofenac in the treatment of AKs. Patients and methods: In this prospective study 20 male patients with AKs were treated for 90 days with topically applied diclofenac 3 %/hyaluronic acid 2.5 %. Before and after treatment, skin biopsies were taken from the treatment area and were investigated histologically and immunohistochemically as well as compared to healthy skin. For this purpose, markers for inflammation (COX‐2, CD3, CD8), apoptosis (p53), cell cycle arrest (p53, p21), proliferation (Ki67), and angiogenesis (CD31) were examined. Results: The immunohistochemical analysis demonstrated a significant decrease in expression of COX‐2, CD3 and CD8. Furthermore, there was a clear reduction of CD31 expression as a marker for angiogenetic processes. Additionally, there was a tendency toward a reduction in markers for proliferation and apoptosis. Conclusions: The efficacy of diclofenac 3 %/hyaluronic acid 2.5 % in the treatment of AKs is probably due to anti‐inflammatory and anti‐angiogenic effects, potentially associated with anti‐proliferative and apoptosis‐inducing underlying mechanisms.     

Current perspective on actinic keratosis: a review

Actinic keratoses (AKs) are common, with prevalence in the U.S.A. estimated at almost 40 million in 2004 and annual costs of > $1 billion (U.S.D.). However, there is no universally accepted definition of AK and thus it is difficult to identify reliably. AKs are lesions of epidermal keratinocytic dysplasia that result from chronic sun exposure and have the ability to progress to invasive squamous cell carcinoma (SCC), but clinicians disagree about whether AKs are premalignant lesions, superficial SCCin situ or epiphenomena of chronically sun‐damaged skin. Yearly AK to SCC progression rates of 0·6% were reported in an elderly population with multiple prior keratinocyte carcinomas (KCs); and rates of spontaneous AK regression have been reported to be > 50%, but regressed lesions often reappear. As AKs have both cosmetic consequences and potential for malignant transformation, there are multiple reasons for treatment. There is no current agreement on the most efficacious treatment, but 5‐fluorouracil has been shown to both prevent and treat AKs, and imiquimod and photodynamic therapy may have the best cosmetic outcomes. AKs may be treated to improve appearance and relieve symptoms, but the keratinocytic dysplasia that gives rise to malignancy, and sometimes appears as an AK, may be what actually threatens patient health. Thus, treatments should aim to decrease the risk of KC or facilitate KC diagnosis by reducing the potential for misidentification created when a KC appears in a field of AKs. Improved agreement among clinicians on AK definition may improve management.     

Epigenetic abnormalities in cutaneous squamous cell carcinomas: frequent inactivation of the RB1/p16 and p53 pathways

BACKGROUND: Aberrant methylation of CpG islands in the promoter regions of cancer-related genes has been demonstrated in many human tumours. However, the methylation profile of these regions in cutaneous squamous cell carcinomas (SCCs) has not been well studied. OBJECTIVES: To examine epigenetic abnormalities of a wide range of cancer-related genes in SCCs. METHODS: We investigated the methylation status of 11 candidate cancer-related genes (CDH1, p16(INK4a), p14(ARF), DAPK1, MGMT, RB1, RASSF1, p15(INK4b), PTEN, PRDM2 and p53) in 20 cases of SCC by methylation-specific polymerase chain reaction, and comparatively examined the protein production of E-cadherin (CDH1), p16, RB1, p14, BMI1 and cyclin A by immunohistochemical analysis. RESULTS: The frequency of cancer-related gene methylation in SCCs was: CDH1 (95%), p16 (20%), p14 (15%), DAPK1 (15%), MGMT (15%), RB1 (5%), RASSF1 (5%), p15 (0%), PTEN (0%), PRDM2 (0%) and p53 (0%). Almost all cases with hypermethylation of CDH1, p16, RB1 and p14 showed no obvious production of each protein, suggesting that promoter hypermethylation of these genes contributes to the loss of protein production. The results of methylation analysis, in combination with the results of our previous mutation analysis of CDKN2A locus and p53, revealed that 70% of SCCs have alterations in the RB1/p16 or p53 pathway. CONCLUSIONS: Our findings indicate that the promoter hypermethylation of cancer-related genes, especially CDH1, is frequently shown in SCCs, and dysregulation of the RB1/p16 and/or p53 pathway through either genetic or epigenetic mechanisms, except for epigenetic abnormalities of p53 itself, should contribute to the carcinogenesis of SCCs.     

Cell cycle regulators in the keratinocyte (cyclin-cdk)

Abstract It has recently become clear that cyclin-dependent kinase (cdk) complex regulates the cell cycle by phosphorylating Rb protein, a tumor suppressor protein. It is likely that this complex is a target of various growth factors and anti-growth factors (UV TGF-β etc.) in keratinocyte (KC). It has also been suggested that abnormalities in the cell cycle regulating mechanism such as increased activity of cyclin-cdk due to mutation of p53, a tumor suppressor gene, and overexpression of cyclin D may be concerned with carcinogenesis of KC. Thus, recent studies indicate that the cyclin-cdk complex is a common target of proliferation and carcinogenesis in KC.     

p63 expression in normal skin and usual cutaneous carcinomas

BACKGROUND: p63 is a p53 homologue that is mapped to chromosome 3q27. This gene encodes six different isoforms, which have either transactivating or dominant negative effects on p53-reporter genes. It has been described that in contrast to p53, p63 seems not to be associated with tumor predisposition, as neither p63 knockout mouse models nor germline p63 mutations are related to an increased risk of tumorigenesis. It has been demonstrated that p63 is a reliable keratinocyte stem cell marker and that it is involved in the maintenance of the stem cell population. Scant data on p63 expression in normal skin, basal cell carcinomas (BCCs), keratoacanthomas and squamous cell carcinomas (SCCs) have been reported. We herein evaluated p63 expression in 16 BCCs, one keratoacanthoma and 13 SCCs. METHODS: Immunohistochemistry according to the streptavidin-biotin-peroxidase technique, using the antibody 4A4 raised against all p63 isoforms, was performed. p63 expression was evaluated in epidermal cells and skin appendages. Semi-quantitative evaluation (-, +, ++, +++) of p63 expression in BCCs, keratoacanthoma and SCCs was carried out. Only nuclear expression was considered as specific. RESULTS: p63 was expressed in the nuclei of epidermal basal and suprabasal cells, in the cells of the germinative hair matrix and the external root sheath of hair follicles, in the basal cells of the sebaceous gland and in the myoepithelial/basal cells of the sweat glands. All terminally differentiated cells were negative for p63. All BCCs showed ++ to +++ immunoreactivity. At variance, keratoacanthomas and grade I and II SCCs showed variable p63 reactivity in a basal layer-like distribution, whereas undifferentiated cells of grade III SCCs showed ++ to +++ positivity. A grade IV spindle SCC showed + immunoreactivity. The SCCs in situ showed remarkable expression of p63 in all cell layers. Terminally differentiated squamous cells were either negative or showed only focal immunoreactivity in the carcinomas. CONCLUSIONS: p63 is consistently expressed in the basal cells of epidermis and cutaneous appendages, including the basal/myoepithelial cells of sweat glands. Based on our findings, the balance of probabilities favors that p63 might play a role in the pattern of differentiation and in the oncogenesis of usual carcinomas of the skin.     

Less expression of cyclin E in cutaneous squamous cell carcinomas than in benign and premalignant keratinocytic lesions

In a previous study, we showed that overexpression of cyclin D, a G1 cyclin, is frequently associated with keratinocyte carcinogenesis as an early event. Another G1 cyclin, cyclin E, was recently suggested to be a prognostic marker for breast cancer. In order to evaluate the role of cyclin E in human keratinocyte carcinogenesis, we analysed the expression of cyclin E by immunohistochemistry in normal skin, seborrheic kaeratosis (SK), keratoacanthoma (KA), actinic keratosis (AK), Bowen's disease (BD), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Positive cells were seen rarely in normal epidermis, in 9 of 20 cases of SK, in 5 of 6 cases of KA, in 9 of 13 cases of AK and in all 27 cases of BD. Some of the cases of AK and BD had positive cells in the superficial epidermis, where atypicality is less obvious. In contrast, positive cells were seen in 4 of 25 cases of SCC and none of 15 cases of BCC. These results suggest that expression of cyclin E plays a role in the formation of benign and premalignant keratinocytic tumors, whereas down-regulation of cyclin E expression may be involved in carcinogenesis in human keratinocytes.