Expression of CD44 isoforms in basal cell carcinomas

The expression of CD44 isoforms (CD44std, CD44v6, CD44v10) was investigated by an immuno-histochemical technique in 42 basal cell carcinomas (BCC) of the superficial and nodular variety. All BCCs studied displayed very low amounts of CD44std, a receptor for hyaluronic acid. Except for single CD44std-positive cells located preferentially in the central parts of the BCC nests, the bulk of the tumour formations were CD44std-negative. CD44v6 showed a heterogeneous distribution pattern accentuated in the peripheral palisading tumour cells. In superficial BCCs, the labelling intensity for CD44v6) increased with the size of the tumour nests. CD44v10 was not detectable in BCCs Our findings support the notion that CD44v6 is not linked to the metastatic proclivity of tumours originating from keratinocytes. We suggest that the very low expression of the receptor for hyaluronic acid (CD44std) may be one of the factors which block the formation of metastases from BCCs.     

Hyaluronan,CD44 and versican in epidermal keratinocyte tumours

BACKGROUND: The high molecular weight polysaccharide hyaluronan is a major component of the extracellular matrix between the vital cells of human skin epidermis. The levels of hyaluronan, and those of the hyaluronan receptor CD44 and the hyaluronan binding proteoglycan versican, correlate with the aggressiveness of different human carcinomas of epithelial origin. OBJECTIVES: To study skin keratinocyte tumours for the expression of hyaluronan, the hyaluronan receptor CD44 and the hyaluronan binding proteoglycan versican. METHODS: Paraffin-embedded sections of 114 basal cell carcinomas (BCC), 31 in situ carcinomas (ISC) and 35 squamous cell carcinomas (SCC) were stained with a hyaluronan specific probe, biotinylated hyaluronan binding complex, and with monoclonal antibodies against CD44 and versican. RESULTS: Compared with normal epidermis, ISC and well differentiated SCCs showed an enhanced hyaluronan signal on carcinoma cells while CD44 expression level resembled that of normal skin. Less differentiated SCCs showed reduced and irregular expression of both hyaluronan and CD44 on carcinoma cells. In BCCs, hyaluronan and CD44 signals were absent or very low on the surface of carcinoma cells. However, hyaluronan was frequently present on BCC cell nuclei, a feature completely absent in ISC, SCC and normal epidermis. An accumulation of hyaluronan in the connective tissue stroma around the tumour was more frequent in SCCs than BCCs. Versican staining was positive around hair follicles and dermal blood vessels of normal skin. Peritumoral versican signal was present in a part of the BCCs but not in other tumours. CONCLUSIONS: The completely different hyaluronan and CD44 expression patterns in BCC and SCC probably reflect the different origins of the tumours, with BCC an undifferentiated keratinocyte and SCC a keratinocyte at an early stage in the differentiation pathway. The difference in hyaluronan and CD44 expression between these tumours may also contribute to the difference in their capacity to metastasize.     

Colocalization of basic fibroblast growth factor and CD44 isoforms containing the variably spliced exon v3 (CD44v3) in normal skin and in epidermal skin cancers

Previous in vitro studies have shown CD44 isoforms containing the alternatively spliced exon v3 (CD44v3) to be modified with heparan sulphate (HS) and to bind HS-binding basic fibroblast growth factor (bFGF). Here, we demonstrate that exogenously added bFGF is also bound in vivo by CD44v3-positive keratinocytes in normal skin and by tumour cells in basal cell carcinoma and squamous cell carcinoma (SCC), two skin cancers of keratinocyte origin. bFGF binding and CD44v3 expression were colocalized in cultured human normal keratinocytes (HNK) and on the SCC cell line A431. By contrast, benign or malignant tumours of melanocyte origin failed to express CD44v3 and bound no bFGF. The bFGF binding to normal or transformed keratinocytes in vivo and in vitro was dependent on HS modification, as it was completely eliminated by pretreatment with heparitinase or by blocking with free heparin, whereas chondroitinase had no effect. In addition, specific removal of CD44v3 by antibody-induced shedding also diminished bFGF binding to keratinocytes. Furthermore, bFGF stimulated the proliferation of CD44v3-positive HNK and A431 in a dose-dependent fashion. This bFGF effect was again completely abolished by heparitinase or free heparin, but not by chondroitinase. In aggregate, our results suggest that a function of HS-modified CD44 isoforms such as CD44v3 in skin is to present the HS-binding growth factor bFGF, thereby stimulating the proliferation of normal or transformed keratinocytes.     

CD44 and hyaluronate in the differential diagnosis of dermatofibroma and dermatofibrosarcoma protuberans

The histological distinction between dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP) may be extremely difficult. CD34 and Factor XIIIa have been used to differentiate DF from DFSP. However, there is an overlap and relative lack of specificity of their expressions. CD44 is a widely distributed integral membrane glycoprotein, which is expressed as a multitude of isoforms generated by alternative splicing of at least 10 different variant exons and post-translational modifications. CD44 is currently thought to be the principal cell surface receptor for hyaluronate (HA), the major component of the extracellular matrix. In this study we aimed to assess the expression of standard CD44 (CD44s) and its isoforms (CD44v3, CD44v4, CD44v5, CD44v6, CD44v7, CD44v7v8, and CD44v10), and HA in DF and DFSP. Immunohistochemical staining was performed on the biopsy specimens of 15 cases of DF and four cases of DFSP, using antibodies that recognize the CD44s, different CD44 isoforms and the hyaluronate binding protein (HABP). Tumor cells displayed a strong CD44s immunoreactivity in all cases of DF whereas a faint HA positivity was observed in the tumor stroma. The DF cells were negative for CD44v3, CD44v4, CD44v6, CD44v7 and CD44v7v8 but showed a strong reactivity for CD44v5 and CD44v10. In contrast, CD44s' expression was significantly reduced or absent in all DFSP lesions and the tumor stroma displayed strong staining for HA. Our results indicate that CD44 and HA can be used as additional diagnostic markers to distinguish DF from DFSP.     

Overexpression of protein kinase C-α and -β isozymes by stromal dendritic cells in basal and squamous cell carcinoma

Protein kinase C (PKC) isozymes transduce signals from cell surface receptors and thereby regulate important cellular functions in skin including keratinocyte growth and differentiation. Overexpression of individual PKC isozymes results in aberrant cell growth and in certain instances tumorigenicity. PKC is implicated in tumour promotion in mouse skin. Abnormal expression of PKC has been reported in several human cancers. We have, therefore, investigated expression of PKC-α and -β in basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) by immunohistochemistry. Sections were stained with specific antibodies to PKC-α, PKC-β, CDla, T cells, B cells and dermal dendritic cells (factor XIIIa), using an immunoperoxidase technique, PKC-α and PKC-β were not detected in tumour cells in BCCs or SCCs. In SCCs, PKC-β immunostaining revealed positively stained inflammatory and dendritic cells scattered through the stroma: PKC-α immunostaining was essentially negative. In contrast, in BCCs, PKC-α+ and PKC-β+ dendritic and spindle-shaped cells were observed in the stroma, immediately adjacent to the tumour islands. Double-labelling experiments showed that a proportion (approximately 20%) of PKC-β+ dendritic cells also expressed factor XIIIa. BCCs depend on stroma for growth: PKC regulates expression of type IV collagenase and stromelysis III and interactions between tumour and stroma may be important in determining tumour invasion and metastasis, Therefore, overexpression of PKC-α and -β by stromal dendritic cells in BCCs suggests that PKC activation may be involved in stromal/tumour interactions in these tumours.     

Atypical Response of Xeroderma Pigmentosum to 5‐Fluorouracil: A Histopathological Image Analysis Study Reveals New Insight into Etiopathogenesis

The histological distinction between dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP) may be difficult. CD34 and Factor XIIIa have been used to differentiate DF from DFSP, but there is a lack of specificity. CD44 is a membrane glycoprotein which has multiple isoforms generated by alternative splicing of variant exons. CD44 is the principal cell surface receptor for hyaluronate (HA). In this study we explored the expression of standard CD44 (CD44s) and its isoforms (CD44v3, CD44v4, CD44v5, CD44v6, CD44v7, CD44v7v8, and CD44v10), and HA in DF and DFSP. Immunohistochemistry was performed on the biopsy specimens of 15 cases of DF and 4 cases of DFSP, using antibodies for the CD44s and its isoforms, and hyaluronate binding protein (HABP). Tumor cells displayed a strong CD44s immunoreactivity in all cases of DF whereas a faint HA positivity was observed in the tumor stroma. DF cells were negative for CD44v3, CD44v4, CD44v6, CD44v7 and CD44v7v8 but they showed a strong reactivity for CD44v5 and CD44v10. CD44s expression was significantly reduced or absent in all DFSP lesions and the tumor stroma displayed a strong staining for HA. Our results indicate that CD44 and HA can be used as additional diagnostic markers to distinguish DF from DFSP.     

Depletion of cell surface CD44 in nonmelanoma skin tumours is associated with increased expression of matrix metalloproteinase 7

Background  Expression of matrix metalloproteinase (MMP)-7 and MMP-9 is low in the normal epidermis and is induced by physiological processes such as wound healing, but also malignant transformation of epidermal cells. The activity of both MMPs has been associated with the hyaluronan (HA) receptor CD44. We previously reported that the levels of CD44 and HA differ between the two types of epidermal tumours, basal (BCC) and squamous cell carcinoma (SCC), as well as between different grades of SCC.
Objectives  To investigate if the immunostaining patterns of MMP-7 and MMP-9 correlate to those of CD44 and HA in BCC and SCC.
Methods  Paraffin sections from 71 BCCs, 21 in situ SCCs and 27 SCCs were immunostained for MMP-7 and -9.
Results  Positive immunostaining for MMP-7 and MMP-9 was found in tumour cells of both BCC and SCC, while the staining intensity tended to be stronger in SCC. The staining intensity of MMP-7 was inversely correlated with that of CD44 in both tumour types. In well-differentiated SCC, the intensity of MMP-7 was generally weak, while CD44 staining was strong and homogeneously distributed. In poorly differentiated SCC, an increase in MMP-7 was seen, and the staining intensity of CD44 became weak and was locally absent. No correlation was seen between MMP-9 and CD44 or either of the two MMPs and HA.
Conclusions  Our results show that in nonmelanoma skin tumours MMP-7 and -9 are present in the tumour cells, and suggest a link between MMP-7 activity and the depletion of cell surface CD44.     

Expression of stem cell factor in basal cell carcinoma

Summary Stem cell factor (SCF) distribution in basal cell carcinomas (BCCs) was examined by immunohisto-chemistry. Eighteen BCCs (11 nodular, three superficial, two cystic, one adenoid and one morphoeic type) showed positive expression of SCF in the tumour islands. The centre of the tumour island was strongly positive in nodular, superficial and morphoeic types. In cystic BCCs. SCF-positive tumour cells were also located in the peripheral lesion around the cystic space. SCF was also detected on fibroblast-like cells and mast cells in the stroma. SCF was positively stained within the upper keratinocytes in the overlying epidermis, more strongly as compared with normal skin. The mast cell number (mean ± SD)) was significantly increased in the peritumoral stroma (85.7 ± 28. 3/mm²) compared with normal skin (32.1 ±4.2/mm²) ( P <0.005). SCF was also positive in the tumour nests of four cases of trichoepithelioma, in which fibrosis of the surrounding stroma was found histologically. This study demonstrates that abundant SCF produced by the tumour cells may account for the increased number of stromal mast cells, which induce fibroplasia of the surrounding stroma.     

Detection of CD44 splice variants in formalin-fixed, paraffin-embedded specimens of human skin cancer

The standard form of CD44 (CD44s) and CD44 isoforms, containing sequences encoded by one or several of 10 different variant CD44 exons (v1-v10), are thought to play a crucial role in the growth and metastasis of certain human tumors. Recently, monoclonal antibodies (mAbs) directed against all CD44 isoforms (panCD44), or against epitopes encoded by specific variant exons (CD44v) have been developed, which unfortunately only stain cryopreserved tissues. We wished to develop a technique to unmask chemically CD44s and CD44v epitopes in paraffin-embedded specimens of human skin cancers, so that they would be accessible for these mAbs. To address this issue, CD44s and CD44v expression was compared in cryopreserved and in formalin-fixed, paraffin-embedded biopsies obtained from the same basal cell carcinomas (BCC), squamous cell carcinomas (SCC), primary malignant melanomas (PMM) and metastatic malignant melanomas (MMM). Formalin-fixed tumors were de-paraffinized and treated briefly with an antigen retrieval fluid (TUF™) at 95°C or left untreated. In untreated paraffin-embedded tissues, no CD44s or CD44v staining was detected. In contrast, in antigen retrieval fluid-treated biopsies CD44s and CD44v expression was identical to that in cryopreserved specimens of the same tumor with the exception of mAbs detecting v7/8 and v10. We conclude that antigen retrieval unmasks certain epitopes in formalin-fixed, paraffin-embedded tissues, thus facilitating future research on the relevance of CD44s and CD44v expression for human skin carcinogenesis.     

CD44V6在表皮肿瘤及恶性黑素瘤的表达

目的　研究CD44V6在鳞状细胞癌、基底细胞癌和恶性黑素瘤的表达。方法　免疫组织化学对石蜡包埋组织标本进行检测。结果　 1 0例鳞状细胞癌CD44V6膜表达阳性 ,且随癌细胞分化程度降低CD44V6表达下调。 1 0例基底细胞癌和 8例恶性黑素瘤不表达CD44V6。结论　CD44V6的表达与皮肤肿瘤的类型有关     

Expression of beta-catenin in basal cell carcinoma

Background beta-Catenin is a crucial member of the E-cadherin/catenin complex, which plays a major role in cell-cell adhesion. beta-Catenin is also known to be involved in signal transduction pathways. Many studies have demonstrated changes in the expression of beta-catenin in colorectal carcinomas, suggesting a role for beta-catenin in neoplastic development. Objectives Basal cell carcinoma (BCC) is a locally invasive tumour. The various subtypes show differences in biological behaviour. This study aimed to investigate the presence of differences in the immunoprofile of beta-catenin among histological variants of BCC. Methods Eighty BCCs were studied (32 nodular, 7 micronodular, 24 superficial and 17 infiltrative and morphoeic). Formalin-fixed, paraffin-embedded tissue sections were stained for beta-catenin using the avidin/biotin immunodetection technique. Results All the nodular BCCs showed membranous and weak cytoplasmic staining. Nuclear staining was seen in 15 of 32 (47%) cases, being stronger at the periphery of the nodules in 11 of 15 (73%) of these cases. In superficial BCCs the membranous staining was variable and cytoplasmic staining was increased. Nuclear staining was seen in 16 of 24 (67%) cases, being more notable at the periphery in 8 of 16 (50%) of these cases. All micronodular BCCs showed strong membranous staining, weak cytoplasmic and no nuclear staining. In the infiltrative and morphoeic BCCs membranous staining was completely lost at the advancing margins of the invading cell strands, with a marked increase in cytoplasmic staining; nuclear staining was observed in all these tumours. Conclusions The expression of beta-catenin varied between different types of BCC. Nuclear localization was most notable in the infiltrative and morphoeic variants, followed by the superficial variant, and seen least in nodular BCC. Its prominence at tumour margins suggests that this may be associated with more aggressive types of invasion.     

Regression in basal cell carcinoma: an immunohistochemical analysis

Summary Spontaneous regression of some cutaneous tumours is well recognized, and is thought to result from an immunological response to the tumour, Regression has previously been noted in basal cell carcinomas, but no studies defining the role of the immune response in the regression of this malignancy have been performed. We have examined 45 primary basal cell carcinomas (BCCs) (20 nodular, 25 superficial) and identified the cellular phenotypes and activation states of the cells infiltrating primary regressing and non-regressing BCCs, by immunocytochemistry. We have found a significantly increased number of CD3⁺ and CD4⁺ T cells infiltrating regressing compared with non-regressing tumours, and the expression of interleukin-2 receptor (an early activation marker for T cells) was also increased. There were no significant differences in class II major histocompatibility complex (MHC), CD1, or macrophage antigen expression in these groups. These findings suggest that activated CD4⁺ cytokine-secreting cells are important in the regression of BCCs.     

Distribution of CD44 variant isoforms in human skin: differential expression in components of benign and malignant epithelia

Expression of cell adhesion molecules regulates epithelial cell differentiation and organization of complex tissues such as skin. The CD44 family of adhesion molecules is generated by alternative splicing of up to 10 variant exons encoding inserts into the extracellular domain. Expression of CD44 variant exons has been correlated with metastatic potential of some epithelial malignancies. We studied the distribution of total and variant CD44 isoforms containing exons v4, v6, and v9 in normal skin, basal cell carcinoma, and in control tissues using immunohistologic assays. While normal epidermis and other stratified squamous epithelia reacted strongly with antibodies specific for standard CD44 (CD44S) and CD44 isoforms containing exons v4, v6, and v9, the epithelium of eccrine glands was reactive, often in a polarized distribution, only with antibodies specific for CD44S and isoforms containing exon v9. These studies suggest that differential expression of CD44 variant exons may be important in development and organization of epithelial structures within skin. Malignant cells in basal carcinoma tissues were found to have low reactivity with antibodies specific for CD44S or variant CD44 molecules. The low expression of CD44 molecules in basal cell carcinomas may play a role in the relatively low probability of metastasis of these neoplasms.     

Immunophenotypic analysis of the p53 gene in non-melanoma skin cancer and correlation with apoptosis and cell proliferation

BACKGROUND: Sunlight precipitates a series of genetic events that lead to the development of skin cancers such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). The p53 tumour suppressor gene, which plays a pivotal role in cell division and apoptosis, is frequently found mutated in sunlight-induced skin tumours. OBJECTIVE: To investigate the immunoreactivity of the p53 gene in non-melanoma skin cancers and to correlate its expression with apoptotic and cell proliferation markers. METHODS: We analysed 35 non-melanoma tumours including 19 BCCs and 16 SCCs from sun-exposed skin areas. p53 protein expression was studied immunohistochemically using the DO7 monoclonal antibody against wild-type and mutant p53 forms. The percentage of p53-immunopositive nuclei was measured by image analysis. Cell proliferation and apoptosis were also assessed by image analysis following Ki-67 immunostaining and application of the TUNEL method on paraffin sections, respectively. RESULTS: The percentage of p53-expressing cells varied from 3.5 to 90 in BCCs (median value 54.4%) and from 3.7 to 94 in SCCs (median value 40.3%). The mean value of Ki-67-positive cells was comparable in both groups of tumours with a mean value of 40.6% in BCCs and 34.6% in SCCs. Conversely, the TUNEL assay showed sporadic staining of apoptotic cells within the tumours with a mean value of 1.12% in BCCs and 1.8% in SCCs. p53 protein expression was correlated positively with cell proliferation (r = 0.75, P = 0.000001) and negatively with apoptosis (r = -0.23, P = 0.05). CONCLUSION: p53 immunoreactivity was high in the majority of the skin carcinomas examined and correlated positively with cell proliferation and negatively with apoptosis. The p53 protein overexpression appears to be related to an inactivated protein resulting from mutations of the p53 gene or other unclear molecular mechanisms.     

Cutaneous lymphadenoma

Lymphoepithelial neoplasms are biphasic tumours that contain both epithelial and lymphoid components. This heterogeneous group includes benign cutaneous lymphadenoma (CL), malignant lymphoepithelioma-like carcinoma of the skin and dermal thymus. We present two cases of CL in male subjects of 14 and 64 years of age. The latter man had a history of multiple basal cell carcinomas (BCCs) and solar keratoses. Histological sections of both tumours revealed similar features of an invasive non-ulcerated tumour with a mixed architecture of BCC and trichoepithelioma. Immunocytochemical examination revealed a biphasic epithelial tumour of follicular differentiation, possibly a variant of trichoepithelioma or a BCC. Within the epithelial islands there was a heavy infiltration that was confirmed as CD3-positive T cells and S-100-positive dendritic cells by immunocytochemistry.     

CD44 and melanocytic tumors: a possible role for standard CD44 in the epidermotropic spread of melanoma

CD44 is a polymorphic family of cell membrane glycoproteins that mediate cell-matrix and cell-cell interactions involved in the mechanisms of tumor invasion and metastasis, and are subject to differential regulation during normal and malignant cell growth. We have investigated immunohistochemically the expression of CD44S and the variant isoforms CD44v3 and CD44v6 in paraffin-embedded tissue from 5 Spitz nevi, 3 compound melanocytic nevi, 2 blue nevi, 6 primary melanomas, 15 cutaneous metastases (three epidermotropic, nine dermal and three ulcerated) and 10 lymph node metastases of melanoma. Melanocytes were extensively positive for CD44S in primary melanomas and benign melanocytic proliferations. Among 15 cases of cutaneous metastases of melanoma, the three epidermotropic metastases, as well as one of the three ulcerated ones were positive for CD44S. CD44S expression was diminished or totally absent in six of the nine dermal metastases, in two of the ulcerated metastases and in seven of the ten lymph node metastases. CD44v3 and CD44v6 melanocytic expression was absent in all the lesions studied.
According to our results, selective retention of CD44S expression by melanocytes in epidermotropic metastases of melanoma seems to indicate that preservation of CD44S may contribute to the intraepidermal spread of melanoma.     

Changes in CD44 isoform expression during inflammatory skin disease

The CD44 family of cell surface glycoproteins is widely-expressed in epithelial, mesothelial and haemopoietic tissues and is thought to function primarily as adhesion molecules. The molecule has an intracellular, a trans-membrane and an extracellular domain. The membrane proximal region of the extracellular domain is of variable structure depending on which of 10 variable exons are involved in coding for this region. Both in vitro stimulated T cells and cytokine stimulated keratinocytes are known to express certain isoforms. In this study we have investigated whether specific isoforms of the CD44 molecule are expressed on epithelial cells and lymphocytes in the course of two inflammatory skin diseases, namely lupus erythematosus and lichen planus. Monoclonal antibodies, specific to the epitopes of the CD44 molecule encoded by v3, v4/5, v6 and v8/9 variable exons and a pan CD44 marker, were used on 10 lupus and 8 lichen planus frozen skin samples and compared with normal skin from 9 different body sites. Results failed to show detectable levels of variant isoforms of CD44 on lymphocytes in either inflammatory skin disease, despite evidence of T cell activation. All CD44 variant isoforms were reduced on the keratinocytes in some sections of lupus and lichen planus. The results are discussed in the context of the current models for the role of CD44 in the immune response.     

CD44 expression in melanocytic lesions: a marker of malignant progression?

CD44 is the major human cell surface receptor for hyaluronate and functions in a diverse range of physiological processes. Alternative splicing of a single gene generates a lamily of splice variants (CD44v1-10) in addition to the standard isoform. CD44H. Expression of CD44. particularly CD44v6. has heen descrihcd to correlate with metastasis formation in various tumours, although evidence in malignant melanoma is inconclusive. In this study, we explored the immunohisto-chemical pattern of CD44 expression in a range of melanocytic lesions using a panel of monoclonal antibodies raised to CD44H and the variants v 3, v4/5. v6and v8/9. Skin biopsies of 106 lesions from 100 patients were assessed and included benign and dysplastic naevi. melanoma in situ, malignant melanomas in horizontal and vertical growth phase, and cutaneous and lymph node metastases. CD44H was highly expressed in benign and dysplastic naevi and in melanoma in situ. However, expression within melanomas diminished with increasing invasiveness. and the pattern of expres-sion observed correlated significantly with the growth phase of the lesion rather than its Brcslow thickness. CD44 splice variants were not detected in any lesions. These results suggest a possible role for downregulation of CD44H in modulating the biological behaviour of malignant melanoma.