Bowenoid papulosis. Classification as a low-grade in situ carcinoma of the epidermis on the basis of histomorphologic and DNA ploidy studies

The nature of bowenoid papulosis was investigated by a comparative investigation of 12 biopsy specimens of this lesion, 19 biopsy specimens of Bowen's disease, 14 biopsy specimens of squamous cell carcinoma and 10 biopsy specimens of seborrheic keratosis. In addition to conventional histomorphologic and cytomorphologic studies, nuclear DNA measurements on single cells isolated from tissue blocks were performed using a TV image analysis system combined with an automatic microscope. Two parameters, the "5c exceeding rate" (5cER) and the "2c deviation index" (2cDI), were computed from the single-cell DNA values to arrive at a "DNA diagnosis" and a "DNA malignancy grade" (DNA-MG). All specimens of bowenoid papulosis and Bowen's disease were morphologically diagnosed as in situ carcinomas of the epidermis; a DNA diagnosis of malignant was rendered in all of these specimens due to the detection of aneuploid nuclei (5cER greater than or equal to 1). DNA diagnoses of malignant were also rendered on all specimens of squamous cell carcinoma (100% sensitivity) while DNA diagnoses of benign were rendered in all specimens of seborrheic keratosis (100% specificity). The mean DNA-MG for bowenoid papulosis (0.69) was significantly lower than that for Bowen's disease (1.04) and squamous cell carcinoma (1.15). The mean morphologic (Broder's) grade of malignancy was also lower for bowenoid papulosis than for Bowen's disease and squamous cell carcinoma. HPV 16 DNA was detected in 10 of 12 specimens of bowenoid papulosis. Thus, the results of DNA image cytometry and morphologic investigation suggest that bowenoid papulosis is a low-grade carcinoma in situ of the epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The oligosaccharidic component of the glycoconjugates in lichen planus, granuloma annulare, seborrheic keratosis and plamoplantar keratoderma: lectin histochemical study.

It is well known that cell surface glycoconjugates play an important role in cell proliferation, adhesion and differentiation. The aim of this investigation was to define the changes of the glycoconjugate saccharidic moieties in the epidermis and derma of patients affected by several skin pathologies such as seborrheic keratosis, lichen planus, granuloma annulare and palmoplantaris keratoderma. Bioptical specimens from skin lesions as well as from normal skin were fixed in Carnoy's fluid and routinely processed. The sections were treated with HRP-lectins (PNA, DBA, SBA, WGA, ConA, LTA and UEAI). Cytochemical controls were performed for specificity of lectin-sugar reaction. Some sections were pre-treated with neuraminidase prior to staining with HRP lectins. In comparison with normal human skin, epidermal lectin binding pattern in the considered diseases showed considerable qualitative and quantitative variations. In general, in all the considered pathologies, a lack and/or a decrease in lectin binding at the epidermal layers was observed; among the various diseases, differences in cellular localisation of the sugar residues were also noted. In such respect, an exception was represented by seborrheic keratosis, where the cells of the basal layer showed PNA reactivity, which was absent in the basal layer of the normal skin. Although seborrheic keratosis and lichen planus have been studied by others authors, our findings are not in total accordance concerning lectin binding; this is probably due to the different fixatives employed. Our findings seem to reveal significant changes in keratinocyte glycoconjugate oligosaccharides in the previously mentioned diseases, providing clues to their pathogenesis.     

CyclinD1在鲍温病中的表达

目的:探讨cyclinD1(细胞周期蛋白D1)在鲍温病皮肤组织中的表达及其临床意义。方法:采用免疫组化S-P法观察16例鲍温病标本中cyclinD1的表达模式。结果:正常人皮肤组织中,cyclinD1阳性细胞主要分布在基底层及毛囊漏斗部的基底层细胞上。在16例鲍温病标本中14例细胞核内cyclinD1过度表达,阳性细胞分布于肿瘤组织。结论:cyclinD1的异常表达可能与鲍温病的发生相关。     

Quantitative analysis of viral load per haploid genome revealed the different biological features of Merkel cell polyomavirus infection in skin tumor

Merkel cell polyomavirus (MCPyV) has recently been identified in Merkel cell carcinoma (MCC), an aggressive cancer that occurs in sun-exposed skin. Conventional technologies, such as polymerase chain reaction (PCR) and immunohistochemistry, have produced conflicting results for MCPyV infections in non-MCC tumors. Therefore, we performed quantitative analyses of the MCPyV copy number in various skin tumor tissues, including MCC (n?=?9) and other sun exposure-related skin tumors (basal cell carcinoma [BCC, n?=?45], actinic keratosis [AK, n?=?52], Bowen's disease [n?=?34], seborrheic keratosis [n?=?5], primary cutaneous anaplastic large-cell lymphoma [n?=?5], malignant melanoma [n?=?5], and melanocytic nevus [n?=?6]). In a conventional PCR analysis, MCPyV DNA was detected in MCC (9 cases; 100%), BCC (1 case; 2%), and AK (3 cases; 6%). We then used digital PCR technology to estimate the absolute viral copy number per haploid human genome in these tissues. The viral copy number per haploid genome was estimated to be around 1 in most MCC tissues, and there were marked differences between the MCC (0.119-42.8) and AK (0.02-0.07) groups. PCR-positive BCC tissue showed a similar viral load as MCC tissue (0.662). Immunohistochemistry with a monoclonal antibody against the MCPyV T antigen (CM2B4) demonstrated positive nuclear localization in most of the high-viral-load tumor groups (8 of 9 MCC and 1 BCC), but not in the low-viral-load or PCR-negative tumor groups. These results demonstrated that MCPyV infection is possibly involved in a minority of sun-exposed skin tumors, including BCC and AK, and that these tumors display different modes of infection.     

A quantitative histochemical study of three oxidative enzymes in solar keratoses and Bowen's disease

Synopsis Succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH) activities have been studied using quantitative enzyme histochemical techniques in the epidermis of five patients with solar keratoses and Bowen's disease. Non sun-exposed buttock skin was compared with the skin from the actual lesion and adjacent, clinically normal paralesional skin. SDH activity was significantly increased in the basal layer and decreased in the granular layer in the epidermis of both lesion and paralesional skin, although the total epidermal activities were unchanged when compared to non-exposed buttock skin. G6PDH activity was increased in the granular layer of paralesional epidermis and of lesions. No change in LDH activity was detected. Inclusion of phenazine methosulphate in the reaction mixtures resulted in a three-fold increase in formazan deposition without altering the localization.It is concluded that the quantitative changes and alteration in localization of SDH and G6PDH reported in solar keratoses are accompanied by similar changes in adjacent, clinically normal sun-exposed skin and differ from normal non-exposed skin. It is suggested that these changes may precede the development of the solar keratoses and that these findings may indicate a significant metabolic alteration in the events that lead to neoplasia.     

Detection of non-papillary, non-invasive transitional cell G1 carcinoma as revealed by increased DNA instability and other cancer markers

The method to reveal DNA-instability as demonstrated by immunohistochemical staining with anti-cytidine antibody after acid hydrolysis (DNA-instability test) was used as a marker of malignancy. The test was applied to paraffin-embedded sections taken from 15 urinary bladders, renal pelvic cavities, and ureters bearing multiple carcinoma in situ (CIS) and totally 31 papillary urothelial cancers. The serial sections of the same tissues were also subjected to immunohistochemical staining for PCNA, p53, DFF45, and VEGF. The DNA-instability test was positive in 100% cancer lesions irrespective of the grades, and apparently normal urothelium, and hyperplastic and dysplastic urothelial lesions also showed the areas with clones positively stained with DNA-instability testing, and the percent numbers of positive areas in them were 28.3%, 37.7%, and 61.5%, respectively. These clones, which were present in apparently normal urothelium and in hyperplastic and dysplastic urothelial lesions, showed higher percent values of PCNA-positive-cells, in comparison to the values estimated in the areas with negatively stained DNA-instability testing, and the former values were statistically not different from those in carcinoma lesions. Furthermore, the percent numbers of areas positive for p53, DFF45, and VEGF, with positive DNA-instability testing were also much higher than those with negative DNA-instability testing in apparently normal urothelium, and hyperplastic and dysplastic urothelial lesions, and the former values were again comparable to those in cancer lesions with no statistical differences. These clones were regarded as already being malignant and should be the direct precursors of progressed cancer lesions. They will make progression through two different pathways, one to papillary non-invasive G1 cancers by neovascularization induced by paracrine secretion of VEGF, and another to flat CIS G2 without secretion of VEGF; thus the clones should be regarded as non-papillary, non-invasive Gl TCC, or CIS G1. It should be always taken into account that the probability for apparently normal urothelium, and hyperplastic and dysplastic urothelial lesions to contain cancer clones, will be high already, especially in tumor-bearing bladders.     

Early progression stage of malignancy of uterine cervical dysplasia as revealed by immunohistochemical demonstration of increased DNA-instability

The degree of DNA-instability as revealed by the immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (DNA-instability test) was used as a marker of malignancy. This was applied to mild dysplasia (42 cases), moderate dysplasia (43 cases), severe dysplasia (27 cases), squamous cell carcinoma in situ (CIS) (21 cases), invasive squamous cell carcinoma (SCC) (31 cases) and normal (7 cases) human uterine cervix. The expression of tumour suppressor gene p53 and oncogene bcl-2 was detected immunohistochemically. Proliferative activity was evaluated by PCNA immumohistochemistry and the quantitative analysis of the number, mean area, the largest area and maximum shape irregularities of AgNOR in a nucleus were performed for all these cases. The distribution of numeric chromosomal aberrations of chromosome 17 was also investigated in some of these cases. The results showed that 31 SCC (100%), 21 CIS (100%), 21 severe dysplasia (77.77%), 28 moderate dysplasia (65.11%), and 14 mild dysplasia (33.33%) were positively stained by the DNA-instability test diffusely or sporadically, indicating their malignancy. Reflecting the malignant character, these cases showed a remarkable increase in the PCNA-index with the loss of polarity of PCNA positive cell distribution and also an increase in number, mean and largest sizes and maximum shape irregularity of AgNOR dots. The mean chromosome index for chromosome 17, p53 and bcl-2 immunostaining positivity were also found to be significantly increased in moderate and severe dysplasia and in cancerous cases in comparison to normal and mild dysplasia cases. Moreover, the DNA-instability-test positive dysplasia cases showed statistically significant increased values of PCNA-index, AgNOR parameters, mean chromosome index, p53 and bcl-2 expression in comparison to those of DNA-instability-test negative dysplasia cases. In conclusion, some mild dysplasia (33.33%) and most of the moderate (65.11%) and severe dysplasia (77.77%) were regarded as malignant in nature, existing at an early stage of progression of malignancy.     

Corticotropin Expression by Human Melanocytes in the Skin

Highly dendritic melanocytes have been observed in rapidly proliferating seborrheic keratosis, epidermis overlying melanomas, and in melanomas. On staining for the presence of POMC with monoclonal antibody against human ACTH, the melanocytes show cytoplasmic positivity. Short term organ cultures of whole skin from the marginal zone of vitiligo patients show that 22.7% of controls and 45.5% on dark incubation in adriamycin and 87.5% exposed to a pulse of UV on adriamycin treatment show melanocytes positive for ACTH. The surrounding keratinocytes in the epidermis and in the seborrheic keratosis are negative, whereas in melanomas, isolated groups of melanocytes are positive for ACTH. These findings indicate that ACTH is expressed by the melanocytes in the G₂-phase, the activity being enhanced on UV exposure. Thus UV dependent pigmentation is associated with POMC production in human skin. From this work it is evident that the melanocyte network varies the MSH/ACTH levels in correlation with repigmentation and depigmentation in the marginal zone in vitiligo by expressing POMC locally and is related to the UV-sensitivity of the melanocytes.     

Are there differences in the cell cycle of normal and malignant cells? An approach to the analysis of the clonal expansion of cells in malignant epithelial proliferations in situ

The Williams-Bjerknes model for clonal expansion of transformed cells in epithelia was used to examine a series of related questions about the growth of in situ proliferations in epidermis. Bowens disease, actinic keratosis and lentigo maligna lesions were reconstructed on a three-dimensional basis, and all were found to behave differently: the evidence supported the proposal that the 'carcinogenic advantage' enjoyed by transformed cells was proliferative in nature, and indeed varied between the three lesions. We conclude that the clonal expansion of a single cell, with different proliferative carcinogenic advantage, can explain the three-dimensional appearances of these in situ proliferations; furthermore, the well-known 'multifocality' of in situ epidermal proliferations can also be explained by the clonal expansion of a single transformed cell.     

Spatial, temporal, and hormonal regulation of epidermal keratin expression during development of the frog, Xenopus laevis.

To study the mechanism of hormone-induced keratin expression in the epidermis during Xenopus metamorphosis, a monospecific antibody was raised against a unique carboxy-terminal peptide of the 63-kDa keratin. Immunohistological analysis demonstrated that the onset of 63-kDa keratin expression showed distinct regional and temporal differences. The expression started at stage 54 in the hindlimb epidermis, at stage 57 in the head, and over 1 month later at stage 63 in the tail. The amount of 63-kDa keratin was further regulated during epidermal stratification and differentiation. The 63-kDa keratin was expressed first in basal epidermal cells before stratification began. The outer layer of the larval epidermis (periderm) did not express the 63-kDa keratin. As the cells moved out of basal layer, they stained more intensely with the anti-keratin antibody indicating that 63-kDa keratin synthesis is up-regulated during differentiation. Similar results were obtained with cultures of purified epidermal cells grown in high calcium conditions. Since we have shown that thyroid hormone (T3) induces 63-kDa keratin gene expression and hydrocortisone (HC) modulates T3 action we examined the effects of T3 and HC at the single cell level with the anti-keratin antibody. Immunostaining demonstrated that T3 alone and T3 plus HC increased the number of 63-kDa keratin-positive cells as well as the amount of 63-kDa keratin per cell. Unexpectedly these hormones had the same effects on head and tail epidermal cells even though the latter cells degenerate during metamorphosis. The major difference between tail and head cells was that the percentage 63-kDa keratin-producing cells was much greater in the head than in the tail.     

Clonal evolution and progression of 20-methylcholanthrene-induced squamous cell carcinoma of mouse epidermis as revealed by DNA instability and other malignancy markers

We examined the clonal evolution of skin malignant lesions by repeated topical applications of 20-methylcholanthrene (20-MC) to the skin, which induces hyperplastic epidermis, papillomatous lesion and invasive carcinoma in mice. The lesions were examined histologically and immunohistochemically with anti-single-stranded DNA after acid hydrolysis (DNA-instability test), p53, VEGF, DFF45, PCNA and AgNORs parameters analyses. Multiple clones with increased DNA instability comparable to that of invasive carcinoma were noted in early-stage (2-6 weeks) hyperplastic epidermis, and their number increased in middle (7-11 weeks), and late-stages (12-25 weeks) of hyperplastic epidermis, indicating that they belong to the malignancy category. All papillomatous lesions and invasive carcinomas showed a positive DNA-instability test. Positive immunostaining for various biomarkers and AgNORs parameters appeared in clones with a positive DNA-instability test in early-or middle-stage hyperplastic epidermis, and markedly increased in late-stage hyperplastic epidermis, papillomatous lesions and invasive carcinomas. The percentage of PCNA-positive vascular endothelial cells was significantly higher in VEGF-positive lesions with a positive DNA-instability test and became higher toward the late-stage of progression. Cut-woundings were made to papillomatous and invasive carcinoma lesions, and the regeneration activity of vascular endothelial cells was determined by using flash labeling with tritiated thymidine (3H-TdR). In small papillomatous lesions, vascular endothelial cells showed regenerative response, but the response was weak in large lesions. No such response was noted in invasive carcinomas; rather, cut-wounding induced collapse of blood vessels, which in turn induced massive coagulative necrosis of cancer cells. These responses can be interpreted to reflect exhausted vascular growth activity due to excessive stimulation by VEGF-overexpression, which was persistently seen from hyperplastic epidermis to invasive carcinoma.     

Langerhans cells in cutaneous tumours: immunohistochemistry study using a computer image analysis system

Immunohistochemistry, based on antibody anti-S100 protein, was used to evaluate the Langerhans cells (LC) in benign and malign skin neoplasias. These cells were quantitatively estimated using a computer image analysis (OPTIMAS software system, Version 6.1) in skin biopsies diagnosed as basal cell carcinoma (BCC), epidermoid carcinoma (EpC), trichoepithelioma (TE), keratoacanthoma (KA), seborreic keratosis (SK) and actinic keratosis (AK). The antibody anti-S100 protein recognized them. No significant variations were observed in the number of LC among malignant tumour (BCC = 23.25 ± 5.81 and EpC = 20.88 ± 4.24). Benign lesions (AK = 33.04 ± 7.11; TE = 55.74 ± 9.35; SK = 42.38 ± 9.92, and KA = 47.62 ± 10.4) presented a higher number of LC when they were compared among them and to malignant and normal tissues. No significant differences were observed in LC area and volume between benign and malign neoplasias. These results indicate possibly differences in the immunogenicity between benign and malign epidermic tumours. In conclusion, the experimental computer assessment method was reliable and consistent to morphometric analysis of tumoural tissues.     

Keratinization in the epidermis of amphibians and the lungfish: comparison with amniote keratinization

Keratinization in the epidermis of amphibians and the lungfish has been studied by electron microscopy, autoradiography and immunocytochemistry to determine whether histidine-rich proteins, filaggrin and loricrin are present. In the lungfish and amphibian tadpoles, anti-keratin antibodies (AE1 and AE3) stain the whole epidermis but not the AE2 antibody, a marker for keratinization. In adult epidermis, the AE2 antibody mainly stains keratinized layers, AE1 mainly stained basal cells, less suprabasal cells and no pre-keratinized and keratinized layers, and AE3 stains all epidermal layers. This staining pattern resembles that of amniote epidermis. Little tritiated histidine is taken up in toad epidermis at 4-6 h post-injection but 24 h after injection the radioactivity is most concentrated in the replacement layer beneath the corneus. This indicates that protein synthesis takes place in the epidermis but, due to the metabolic conversion that takes place in 24 h, it is unlikely that histidine-rich proteins are formed. Neither filaggrin-like nor loricrine-like immunoreactivities are present in amphibian and lungfish epidermis. This indicates absence of histidine-rich matrix proteins and corneous cell envelope proteins and only mucus is present among keratin filaments. Filaggrine-like and loricrin-like proteins are characteristic of amniotes epidermis and might have originated in basic amniotes (cotylosaurs).     

FB1, a monoclonal antibody reacting with a keratin 14 epitope, stains only a small subset of psoriatic basal keratinocytes

Abstract
A murine monoclonal antibody, FB1, reacted with the basal keratinocytes of human stratified epithelia. One-dimensional and two-dimensional immunoblotting assays, performed on keratins extracted from HaCat cells and normal human keratinocytes, showed that FBI recognizes K14. When LL002, another K14 monoclonal antibody is added, the FB1 stained area in the 2D-immunoblot seems to cover a fraction of the LL002 spot. Immunohistochemical data obtained from studies on normal human tissues supported the K14 specificity of FB1, but when compared with two other monoclonal antibodies, LL002 and RCK107 reacting with K14, some differences appeared. These differences were mainly seen in sweat glands, hair follicles, psoriatic epidermis and salivary glands. In psoriatic epidermis, FB1 showed a heterogeneous pattern of staining of the basal cell compartment. Intense reactivity was only observed at the bottom of the rete ridges. Staining diminished and finally disappeared in the basal cells above the dermal papillae. This observation supports the view that an increased germinative cell population in psoriasis involves a partially differentiated amplifying compartment in which the number of cell divisions is increased.     

Monoclonal Antibody MAT-1 Against Human Tyrosinase Can Detect Melanogenic Cells on Formalin-Fixed Paraffin-Embedded Sections

Immunohistochemical localization of tyrosinase was examined with a monoclonal antibody (MoAb MAT-1) against human tyrosinase on routine formalin-fixed paraffin-embedded sections of 3 normal skin specimens, 15 melanocytic tumors (6 pigmented nevi, 3 juvenile melanomas and 6 malignant melanomas) and 3 non-melanocytic tumors. In the melanotic melanomas, almost all tumor cells were clearly stained with the antibody. In the nevocytic nevi, the nevus cells in lower epidermis and upper dermis were positive for MoAb MAT-1, but negative in middle and lower dermis. All three juvenile melanomas, one amelanotic melanoma, and three non-melanocytic tumors were entirely negative for MoAb MAT-1. Thus, MoAb MAT-1 could recognize the cells with melanogenic activity on routine formalin-fixed paraffin-embedded sections. However, the staining quality was not adequate for normal epidermal melanocytes, indicating that small technical innovations in the immunostaining process such as formalin fixation after PBS washing are required. Nevertheless, MoAb MAT-1 can be expected to be very useful for identifying melanogenic cells on paraffin-embedded sections, because we have to date no other antibody available for it.     

Differential expression of keratin 19 in normal human epithelial tissues revealed by monospecific monoclonal antibodies

Summary Three monospecific monoclonal antibodies (BA16, BA17 and A53—B/A2) recognizing different epitopes of the human keratin 19 were used to determine tissue distribution of this 40 kDa keratin polypeptide. Immunohistochemical methods revealed four different staining patterns among normal human epithelial tissues: firstly, complete negativity of the epidermis, sebaceous glands, hepatocytes and other tissues; secondly, homogeneous positivity as seen for example in the gall bladder and urinary bladder epithelium, endometrium and many other epithelia; thirdly, a mosaic of positive and negative cells among mammary gland luminal cells, prostate epithelia and some other epithelia and fourthly, a more complex heterogeneous pattern found in non-keratinizing squamous epithelia and hair follicles with generally the basal layer being the most strongly or sometimes exclusively stained. The pattern seen in non-keratinizing squamous epithelia varied considerably according to the fixation method and the antibody used as well as among different donors and in different areas of the same organ. The other three staining patterns were on the other hand nearly identical with all three antibodies on both frozen sections and sections of methacarn-fixed paraffinembedded tissues. Our results provide evidence for differential expression of the human keratin 19 at the single cell level, an observation which could be exploited in the study of epithelial differentiation and pathology.     

Monoclonal antibodies to various acidic (type I) cytokeratins of stratified epithelia

We determined the reactivity of two monoclonal antibodies to cytokeratins that are typically expressed in certain stratified epithelia and several human squamous cell carcinomas using immunoblotting techniques and immunofluorescence microscopy. Antibody KS 8.12 reacted specifically with cytokeratin polypeptides nos. 13 and 16, and stained noncornified squamous epithelia in a rather uniform way. The examination of diverse human carcinomas showed all squamous cell carcinomas to be positively stained with this antibody, whereas all adenocarcinomas were negative. Another antibody, KK 8.60, reacted with polypeptides nos. 10 and 11, and uniformly stained the suprabasal layers of the epidermis. In several noncornified squamous epithelia (e.g., tongue, exocervix), in thymus reticulum epithelial cells, and in moderately and well differentiated squamous cell carcinomas this antibody exhibited a nonuniform labeling pattern that allowed the detection of individual cytokeratin-10/11-positive cells scattered throughout the tissue. It is concluded that antibodies KS 8.12 and KK 8.60 represent specific molecular probes for the definition of certain stages of squamous differentiation in normal development as well as in pathological processes such as squamous metaplasia and carcinogenesis. We propose the use of these antibodies in the differential diagnosis of carcinomas and their metastases.     

Immunolocalization of the Tumor-Sensitive Calmodulin-Like Protein CALML3 in Normal Human Skin and Hyperproliferative Skin Disorders

Background and Objective

Calmodulin-like protein CALML3 is an epithelial-specific protein regulated during keratinocyte differentiation in vitro. CALML3 expression is downregulated in breast cancers and transformed cell lines making it an attractive marker for tumor formation. The objective of this study was to survey CALML3 localization in normal epidermis and in hyperproliferative skin diseases including actinic keratosis, squamous and basal cell carcinoma as well as verruca and psoriasis and to compare CALML3 immunoreactivity with the proliferation marker Ki-67.

Methods

Paraffin-embedded tissue sections from normal human skin and hyperproliferative skin disorders were examined by immunohistochemistry and analyzed for localization and expression of CALML3 and Ki-67.

Results

CALML3 was strongly expressed in differentiating layers of normal skin, staining the periphery in suprabasal cells and exhibiting nuclear localization in the stratum granulosum. CALML3 nuclear localization was inversely correlated to Ki-67 staining in each disease, indicating that CALML3 nuclear presence is related to terminal cell differentiation and postmitotic state.

Conclusions

Increased CALML3 expression in suprabasal layers is characteristic for differentiating keratinocytes in normal epidermis, and nuclear expression of CALML3 inversely correlates with expression of the proliferation marker Ki-67. This suggests that CALML3 is a useful marker for normal and benign hyperplastic epidermal development, whereas the loss of nuclear CALML3 indicates progression to a proliferative and potentially malignant phenotype.