MMP‐10 (Stromelysin‐2) and MMP‐21 in human and murine squamous cell cancer

Abstract: The squamous cell cancers (SCC) of renal transplant recipients are more aggressive and metastasize earlier than those of the non‐immunocompromised population. Matrix metalloproteinases (MMPs) have a central role in tumor initiation, invasion and metastasis. Our aim was to compare the expression of MMPs‐10, ‐12 and ‐21 in SCCs from immunosuppressed (IS) and control patients and the contribution of MMPs‐10 and ‐21 to SCC development in the FVB/N‐Tg(KRT5‐Nfkbia)3Rto mouse line. Immunohistochemical analysis of 25 matched pairs of SCCs, nine of Bowen’s disease and timed back skin biopsies of mice with selective inhibition of Rel/NF‐κB signalling were performed. Semiquantitatively assessed stromal MMP‐10 expression was higher (P = 0.009) in the control group when compared with IS patients. Tumor cell‐derived MMP‐10, ‐12 and ‐21 expression did not differ between the groups but stromal fibroblasts of the control SCCs tended to express MMP‐21 more abundantly. MMP‐10 expression was observed already in Bowen’s disease while MMP‐21 was absent. MMP‐10 and ‐21 were present in inflammatory or stromal cells in ageing mice while dysplastic keratinocytes and invasive cancer were negative. Our results suggest that MMP‐10 may be important in the initial stages of SCC progression and induced in the stroma relating to the general host‐response reaction to skin cancer. MMP‐21 does not associate with invasion of SCC but may be involved in keratinocyte differentiation.     

MMP13 can be a useful differentiating marker between squamous cell carcinoma and benign hyperkeratotic lesions in recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe hereditary mechanobullous disease resulting from mutations in the COL7A1 gene, coding for type VII collagen. Patients with RDEB tend to develop squamous cell carcinomas (SCCs) at sites of chronic ulceration or scarring on the whole body. Distinguishing SCC from benign hyperkeratotic lesions is often difficult, not only clinically but also histologically in patients with RDEB. We investigated several matrix metallopeptidase (MMP) subtypes by comparing the DNA amplification microarray findings between evident SCCs and benign hyperkeratotic lesions in the same patient with RDEB. We report that MMP13 was found to be strongly positive in SCCs but negative in benign hyperkeratotic lesions. We found that there is an evident difference in the transitional area between SCCs and benign hyperkeratotic lesions. We propose that MMP13 may be a useful differentiating marker between SCC and benign hyperkeratotic lesions in RDEB.     

Possible pro‐inflammatory role of heparin‐binding epidermal growth factor‐like growth factor in the active phase of systemic sclerosis

Heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) is a member of the EGF family growth factors, which affects multiple aspects of the wound healing process such as epithelialization, wound contraction and angiogenesis. In our study, we measured the serum HB‐EGF levels of 51 systemic sclerosis (SSc) patients, which showed a significant increase compared with those of 20 normal subjects. Further analysis revealed a positive correlation between the HB‐EGF level and pulmonary ground‐glass score but no correlation between the former and pulmonary fibrosis score. Other findings include: a significant increase of serum sialylated carbohydrate antigen KL‐6 levels and significant shortness of disease duration in the diffuse cutaneous SSc patients with elevated HB‐EGF levels; and significantly higher HB‐EGF levels in the presence of Raynaud's phenomenon, in that of telangiectasia, and in the absence of contracture of phalanges in all SSc patients. We then evaluated HB‐EGF mRNA levels of fibroblasts harvested from skin samples of the SSc patients and those of foreskin‐derived fibroblasts treated with transforming growth factor‐β, both of which were significantly higher than each control. In conclusion, we speculate that HB‐EGF plays a pro‐inflammatory role in the active skin and lung lesions of SSc.     

Therapeutic and immune effects of 5‐aminolevulinic acid photodynamic therapy on UVB‐induced squamous cell carcinomas in hairless mice

The therapeutic effects of 5‐aminolevulinic acid (ALA)‐mediated photodynamic therapy (PDT) on cutaneous squamous cell carcinoma (SCC) are not fully understood, and the usefulness of topical PDT in the treatment of SCC is still debatable. The most interesting aspect in SCC PDT is perhaps its potential in inducing antitumor immune responses. In this study, cutaneous SCCs were established by UVB irradiation of hairless mice and treated with multiple ALA PDT. Immunohistochemistry assays showed that ALA PDT could induce quick apoptosis, overexpression of TNFα and marked increases in DCs, CD4⁺ and CD8⁺ cells in tumor interstitium and subcutaneous connective tissues. However, a complete response was only achieved for small SCCs. The clinical value of ALA PDT‐induced specific antitumor immune responses in long‐term control of SCCs deserves further study.     

T‐cadherin loss induces an invasive phenotype in human keratinocytes and squamous cell carcinoma (SCC) cells in vitro and is associated with malignant transformation of cutaneous SCC in vivo

Background Cadherins play important roles in controlling keratinocyte growth, differentiation and survival. Atypical glycosylphosphatidylinositol‐anchored T‐cadherin (T‐cad) is highly expressed in the basal keratinocyte layer of skin. The role of T‐cad in keratinocyte biology and pathology is unclear. Objectives To define the role of T‐cad in the pathogenesis of cutaneous squamous cell carcinoma (SCC) through gain‐of‐function and loss‐of‐function studies in vitro and through examination of T‐cad expression patterns in human cutaneous SCC specimens in relation to histological classification of degree of tumour differentiation. Methods In vitro studies employed lentiviral‐mediated overexpression/silencing of T‐cad in normal human keratinocyte (HaCaT) and SCC (A431) cell lines, monolayer and multicellular spheroid culture models, cell morphology analyses and assays of random motility and invasion. Immunohistochemistry was performed on skin specimens from patients with actinic keratosis, Bowen disease or SCC. Results In vitro, silencing of T‐cad induced a morphologically elongated and disorganized cell phenotype, increased random motility and markedly enhanced invasive potential. Overexpression of T‐cad induced a morphologically spread and compact cell phenotype and blunted invasive potential. In vivo, regional loss of T‐cad expression was more frequent and prominent in SCC classified as moderately‐to‐poorly differentiated than in SCC classified as well differentiated. However, in both categories aberrant and/or absence of T‐cad expression was associated with histological features of a potentially more malignant and invasive phenotype of cutaneous SCC. Conclusions T‐cad is a controlling determinant of SCC phenotype and invasive behaviour and its loss is associated with the process of malignant transformation from noninvasive to invasive SCC.     

The haptoglobin phenotype influences the risk of cutaneous squamous cell carcinoma in kidney transplant patients

Background Cutaneous squamous cell carcinoma (SCC) is the most frequent skin cancer after organ transplantation. Currently, the pre‐identification of transplant patients at increased risk for non‐melanoma skin cancer remains difficult. Objective To investigate the Hp polymorphism as a marker for the identification of a subset of patients with an increased susceptibility to develop SCC/Bowen’s disease. Methods Haptoglobin phenotyping was performed with haemoglobin‐supplemented starch gel electrophoresis in 300 kidney transplant patients. High‐performance gel permeation chromatography was used in case of low serum haptoglobin concentration. Results Cox regression analysis (adjusted for age, gender and Mediterranean origin) showed a significant association of the Hp 1‐1 phenotype with a higher risk of SCC/Bowen’s disease (P = 0.035) and multiple primary SCCs (P = 0.002). No significant difference between the Hp phenotypes was found for the development of Bowen’s disease and SCCs in the first 10 years following renal transplantation. However, after a follow‐up of >10 years, a significant association between the Hp 1‐1 phenotype and the occurrence of Bowen’s disease and SCC was reported (P = 0.002 and P = 0.001 respectively). Conclusions This study shows an increased risk for the development of (multiple) SCCs in kidney transplant patients with the Hp 1‐1 phenotype. This finding points to the role of Hp 1‐1 phenotype as an important predictor in identifying a subset of patients with an increased need for preventive measures and is in agreement with the decreased anti‐inflammatory capacity of this phenotype.     

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.     

Pseudocarcinomatous hyperplasia mimicking squamous cell carcinoma in a case of CD56‐positive cytotoxic T‐cell lymphoma

We present the case of an 84‐year‐old patient with a cutaneous CD56 positive cytotoxic T‐cell lymphoma associated with substantial pseudocarcinomatous hyperplasia mimicking squamous cell carcinoma (SCC). The patient presented with a 7‐month history of several progressive, ulcerated plaques on his right forearm. An initial biopsy showed changes consistent with a diagnosis of SCC for which the patient underwent surgical treatment. Several months later, the patient developed recurrent ulcerated plaques on the right forearm of which several biopsies were performed. The biopsies repeatedly showed marked pseudocarcinomatous hyperplasia resembling SCC. Deeper punch biopsies, however, showed a dense superficial and deep infiltrate of markedly atypical lymphocytes. Immunohistochemical analysis revealed strong positive staining for CD3, CD8, CD56 with negative stains for CD30 and Epstein‐Barr virus‐encoded small non‐polyadenylated RNAs (EBER). Staining for beta F1 and gamma‐delta T‐cell receptor (γδ TCR) were both negative. This constellation was most consistent with a diagnosis of cutaneous peripheral T‐cell lymphoma, unspecified in association with marked pseudocarcinomatous hyperplasia. Our case adds cutaneous peripheral T‐cell lymphoma, unspecified to the list of conditions associated with pseudocarcinomatous hyperplasia (PCH) and illustrates once again the potential pitfalls of distinguishing marked pseudocarcinomatous hyperplasia from SCC.     

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.     

S100A7 acts as a dual regulator in promoting proliferation and suppressing squamous differentiation through GATA‐3/caspase‐14 pathway in A431 cells

S100A7 is expressed in many squamous cell carcinomas (SCCs), such as SCC of the skin, and well‐differentiated SCC always displays stronger staining of this protein. A431 cells, an epidermal cancer cell line, were selected as a cell model to investigate the roles and mechanism of S100A7 in SCC of the skin. In this study, we demonstrated that the overexpression of S100A7 in A431 cells significantly promoted cell proliferation in vitro and tumor growth in vivo, whereas it suppressed the expression of GATA‐3, caspase‐14 and three squamous differentiation markers, keratin‐1, TG‐1 and involucrin. Conversely, the overexpression of caspase‐14 not only significantly decreased cell proliferation and delayed tumor growth but also markedly induced the expression of three squamous differentiation markers, whereas S100A7 and GATA‐3 were not influenced. Further evidence showed that silencing GATA‐3 greatly inhibited the expression of caspase‐14 and three differentiation markers, while the expression of S100A7 was not changed; contrary results were obtained when overexpressing GATA‐3. Importantly, restoring the expression of GATA‐3 and caspase‐14 in A431‐S100A7 cells could bypass the ability of S100A7 to increase cell viability and repress squamous differentiation. These data suggested that S100A7 expression in SCC may play an important role in the maintenance of SCC cell dedifferentiation, at least in SCC of the skin.     

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.     

CK7 expression in primary cutaneous squamous cell carcinoma

Aim: To evaluate cytokeratin 7 (CK7) immunoreactivity in invasive primary cutaneous squamous cell carcinomas (SCCs). Methods: Twenty‐seven primary cutaneous SCCs from 25 patients were evaluated for tumor grade using hematoxylin and eosin‐stained slides and for percentage and intensity of immunoreactivity for CK7. All cases exhibited features of SCC with an in situ component. No glandular or tubular differentiation was present. Staining intensity was graded on a scale of 0–3, with 0 indicating no reaction. Of immunoreactive cases, percentage of tumor staining and distribution of immunoreactivity was documented. Results: Six of 27 SCCs (22%) exhibited immunostaining for CK7. Of those cases, three were poorly differentiated, exhibiting 2 to 3+ intensity in 5–15% of cells. Two were poorly differentiated, with 2 to 3+ intensity in 30–60% of cells. The remaining immunoreactive tumor was moderately differentiated, with 1+ intensity and 5% staining in an area of microinvasion. Conclusion: A subset of cutaneous SCCs, in particular, poorly differentiated tumors, may show focal‐to‐partial immunoreactivity for CK7. This is important to bear in mind when immunohistochemistry is used to distinguish SCC from simulants, such as porocarcinoma, or other adnexal carcinomas with squamous metaplasia. Pulitzer M, Desman G, Busam KJ. CK7 expression in primary cutaneous squamous cell carcinoma.     

Las células claras en el carcinoma espinocelular cutáneo

IntroductionAlthough few cases of squamous cell carcinoma (SCC) with clear cells have been published, we believe that these cells are often present in SCC.Material and methodsWe studied 249 SCCs, analyzing a number of clinical and histological variables. Various immunohistochemical techniques (immunoperoxidase method) were used to determine whether adnexal differentiation was present.ResultsThere were 96 SCCs with a proportion of clear cells of over 25 %. Advanced or established SCCs and SCCs associated with Bowen disease contained a larger proportion of clear cells. We defined 2 histological patterns: a) clear cells around the keratin pearls of SCCs arising from pre-existing actinic keratosis and with indirect signs of human papilloma virus infection in hair follicles; and b) clear cells that simulate adnexal differentiation in lesions arising on pre-existing Bowen disease lesions. There were also 19 carcinomas with true adnexal differentiation.DiscussionClear cells are frequently observed in SCC, though large numbers of clear cells are present only in certain SCCs. The appearance of clear cells in SCCs is progressive and they are only present in more advanced SCC. The presence of clear cells is suggestive of adnexal differentiation; however, in the majority of cases, their presence is due to infiltration of normal adnexal structures by the cells of pagetoid Bowen disease. True adnexal differentiation exists only in a small percentage of cases (7.6 % in our study). The histological pattern described as clear cells around keratin pearls practically rules out this differentiation.     

Cutaneous squamous cell carcinoma and human papillomavirus

The relationship between mucosal human papillomavirus (HPV) and cervical carcinoma or anogenital squamous cell carcinoma (SCC) is becoming increasingly evident, whereas a link between HPV and other cutaneous SCCs is less clear. Recent studies have reported links between epidermodysplasia-verruciformis-associated HPV and extragenital cutaneous SCC, particularly in immunosuppressed patients, although immunocompetent patients have also been affected. Mucosal HPV could also be linked to some types of Bowen disease and certain SCCs of the fingers, oropharyngeal mucosa, etc. We review the possible oncogenic mechanisms involving mucosal HPV and epidermodysplasia-verruciformis-associated HPV. Most SCCs could be explained by the combined action of HPV, immunosuppression, and the oncogenic and immunosuppressive effect of UV radiation. HPV might be associated with worse prognosis of SCC, with implications for clinical practice including greater risk of metastasis.     

Update on the pathogenesis of squamous cell carcinoma development in recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a recessively inherited blistering disorder due to mutations in the collagen VII gene, COL7A1. Patients with RDEB have up to a 50 fold increased incidence of cutaneous squamous cell carcinoma (cSCC) which is independent of ultraviolet (UV) exposure. The pathogenesis of cSCC development and its aggressive nature in RDEB remain unclear. To date there is no convincing evidence that RDEB SCC and UV SCC are distinct. A recent discovery of a remarkably distinct tumour microenvironment in part explains the aggressive nature of RDEB SCC and the importance of collagen VII expression as a potential therapeutic measure. The role of gram negative flagellate bacteria increasing the risk of SCC development in chronic wounds has recently been elucidated in RDEB.     

A novel homozygous splice site mutation in COL7A1 in a Chinese patient with severe recessive dystrophic epidermolysis bullosa and squamous cell carcinoma

Background Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils in the dermo–epidermal junction. The development of cutaneous squamous cell carcinoma (SCC) is one of the most serious complications of this disease. We report herein a Chinese patient with the severe generalized subtype of RDEB (RDEB‐sev gen) complicated by SCC. Methods Skin biopsies were examined for histology, basement membrane ultrastructure, and type VII collagen expression. Genomic DNA was extracted from the peripheral blood samples and subjected to polymerase chain reaction amplification and direct automated DNA sequencing. Results Histopathological examination of the patient’s skin revealed an undetectable expression of type VII collagen polypeptides in the basement membrane zone. Mutation analysis identified a novel splice site mutation in intron 64 (IVS64+5g‐>a) of COL7A1 gene, which resulted in an in‐frame deletion of exon 64 in both alleles. Conclusions This report contributes to the expanding database of COL7A1 mutations and emphasizes the need to elucidate the underlying genetic mechanisms associated with the increased incidence of SCC in RDEB patients.     

In‐situ‐topoproteome analysis of cutaneous lymphomas: Perspectives of assistance for dermatohistologic diagnostics by Multi Epitope Ligand Cartography (MELC)

Background: Immunophenotyping is essential for diagnostics of cutaneous lymphomas. In this regard we present a skin tissue‐adapted application platform of MELC technology. Patients and Methods: This topoproteome analysis allows the subcellular colo‐calization of at least n = 100 epitopes in situ. For this purpose the specimen is processed by a Toponome Imaging Cycler^® for a n‐fold repetition of the following cycle: 1) staining with a fluorophore‐labeld antibody, 2) fluorescence‐imaging, and 3) photobleaching. Overlay and binarization of fluorescence images lead to combinatorial molecular phenotypes (CMP), which relate to a pixel or microtopographic unit (450 × 450 nm², 20× objective). Skin biopsies were derived from patients with mycosis fungoides (patch/plaque lesions), psoriasis, atopic eczema and from healthy skin donors. Results: In orientation to the WHO‐EORTC‐classification of cutaneous lym‐phomas a MELC‐library of 23 markers was established. According to an inaugurative detailed procedure the CMP frequency was determined in a normalization to 100 μm horizontal skin width. By a TopoMiner strategy mycosis fungoides could be separated from the other states with a maximum of significance (p ≤ 0.03) by at least 10‐fold overexpression of the following tumor cell‐representative CMP‐motif: CD3+/CD4+/CD1a‐/CD7‐/CD8‐/CD45R0+/CD45RA‐/CD11a+. Conclusions: The skin tissue‐adapted MELC‐application‐platform extends substantially conventional lymphoma diagnostics by an unprecedented dimension of in‐situ‐analysis of marker combinatorics including its exact quantification and visualization.     

Immunohistochemical staining of p16 in squamous cell carcinomas of the genital and extragenital area

Background and objectivesPositive immunostaining for the tumor suppressor protein p16 is associated with the presence of mucosal or αsubtypes of human papillomavirus (HPV) in cervical and genital squamous cell carcinoma (SCC). The aim of this study was to determine whether p16 immunostaining is also associated with mucosal HPV in extragenital SCC.Material and methodsParaffin sections of lesions located in the genital region (8 genital warts, 3 intraepidermal SCCs, and 7 invasive SCCs) and extragenital area (29 intraepidermal SCCs corresponding to Bowen disease and 10 invasive SCCs) were stained for p16 by immunohistochemistry. Mucosal HPV was detected by polymerase chain reaction (PCR).ResultsIn the genital area, p16 immunostaining was negative in genital warts and positive in all 3 intraepidermal SCCs and 2 invasive SCCs (29%). Mucosal HPV was detected in 6 genital warts and 2 intraepidermal SCCs (100% after exclusion of 3 lesions that could not be analyzed by PCR) and in the 2 invasive SCCs that were positive for p16. In the extragenital area, 19 intraepidermal SCCs (95%) and 2 invasive SCCs (20%) were immunopositive for p16. Mucosal HPV was detected in 4 intraepidermal SCCs (p16 immunopositive) and 1 invasive SCC (p16 immunonegative). In intraepidermal SCCs, p16 immunostaining facilitated the identification of dermal microinfiltration or invasion of normal skin appendages.ConclusionsAccording to our results, unlike in genital SCCs, p16 immunopositivity is independent of the presence of HPV in extragenital SCCs. Compared with intraepidermal SCCs, the absence of p16 protein in invasive SCCs in the extragenital area would indicate progression of the disease.