Basal cell carcinoma of the skin: whole genome screening by comparative genome hybridization revisited

Basal cell carcinoma (BCC) of the skin is considered to be the most common malignancy in people of European ancestry. It is often not clinically aggressive and has been regarded as genetically stable. However, histopathologic subtypes of BCC differ in their ability to invade surrounding tissues and recur. The aim of this work was to present a comprehensive study of chromosomal imbalances of cutaneous BCC and to correlate the findings with their histopathologic and clinical features. In all, 101 tumor samples were classified according to the current microscopic classification of BCC and then analyzed by comparative genomic hybridization (CGH). Over 60% of BCC were found to carry chromosomal imbalances – partial or whole chromosome gains and losses. Different subtypes of BCC presented common chromosomal alterations. No single chromosomal imbalance was found to be characteristic of a particular subtype of BCC, although the frequency of some chromosomal changes differed from one group to the other. The significance of chromosome 2 gains as a phenomenon that does not coexist with the losses in 9q is discussed.     

Expression of claudin‐11 by tumor cells in cutaneous squamous cell carcinoma is dependent on the activity of p38δ

The incidence of cutaneous squamous cell carcinoma (cSCC) is rapidly increasing, and the prognosis of patients with metastatic disease is poor. There is an emerging need to identify molecular markers for predicting aggressive behaviour of cSCC. Here, we have examined the role of tight junction (TJ) components in the progression of cSCC. The expression pattern of mRNAs for TJ components was determined with RNA sequencing and oligonucleotide array‐based expression analysis from cSCC cell lines (n=8) and normal human epidermal keratinocytes (NHEK, n=5). The expression of CLDN11 was specifically elevated in primary cSCC cell lines (n=5), but low or absent in metastatic cSCC cell lines (n=3) and NHEKs. Claudin‐11 was detected in cell‐cell contacts of primary cSCC cells in culture by indirect immunofluorescence analysis. Analysis of a large panel of tissue samples from sporadic UV‐induced cSCC (n=65), cSCC in situ (n=56), actinic keratoses (n=31), seborrhoeic keratoses (n=7) and normal skin (n=16) by immunohistochemistry showed specific staining for claudin‐11 in intercellular junctions of keratinizing tumor cells in well and moderately differentiated cSCCs, whereas no staining for claudin‐11 was detected in poorly differentiated tumors. The expression of claudin‐11 in cSCC cells was dependent on the activity of p38δ MAPK and knock‐down of claudin‐11 enhanced cSCC cell invasion. These findings provide evidence for the role of claudin‐11 in regulation of cSCC invasion and suggest loss of claudin‐11 expression in tumor cells as a biomarker for advanced stage of cSCC.     

Controversial role of mast cells in skin cancers

Cancer development is a multistep process characterized by genetic and epigenetic alterations during tumor initiation and progression. The stromal microenvironment can promote tumor development. Mast cells, widely distributed throughout all tissues, are a stromal component of many solid and haematologic tumors. Mast cells can be found in human and mouse models of skin cancers such as melanoma, basal and squamous cell carcinomas, primary cutaneous lymphomas, haemangiomas and Merkel cell carcinoma. However, human and animal studies addressing potential functions of mast cells and their mediators in skin cancers have provided conflicting results. In several studies, mast cells play a pro‐tumorigenic role, whereas in others, they play an anti‐tumorigenic role. Other studies have failed to demonstrate a clear role for tumor‐associated mast cells. Many unanswered questions need to be addressed before we understand whether tumor‐associated mast cells are adversaries, allies or simply innocent bystanders in different types and subtypes of skin cancers.     

Update on the anti‐programmed cell death‐1 receptor antibodies in advanced cutaneous squamous‐cell carcinoma

Regarding the rising incidence and the not negligible mortality, the treatment of cutaneous squamous‐cell carcinoma (cSCC) has a high clinical relevance. Immune checkpoint inhibitors (ICI), especially anti‐programmed cell death‐1 receptor (anti‐PD‐1) antibodies such as pembrolizumab and cemiplimab have shown promising results in Phase 2 studies for patients with locally advanced and/or metastatic cSCC. We are presenting a review of the latest results in the treatment of cSCC with ICI. Patients with locally advanced or metastatic cSCC have been treated with cemiplimab 3 mg/kg every 2 weeks. For locally advanced cSCC, an objective response was observed in 44% of patients, 13% patients with a complete response, and 31% with a partial response. For metastatic patients, the overall response rate was 49.2%. The approved dose for cemiplimab in the United States and Europe is 350 mg every 3 weeks. These ICI seem to achieve higher response rates compared with epidermal growth factor receptor (EGFR) inhibitors, with a durable response superior to both chemotherapy and EGFR inhibitors. The side effect profile of anti‐PD‐1 antibodies appears to be favorable compared to chemotherapy. In this way, PD‐1 inhibitors are expected to become the new gold‐standard treatment for patients with locally advanced and metastatic cSCC.     

Giant neglected squamous cell carcinoma of the skin

Nonmelanoma skin cancers (NMSCs) are the most common type of skin tumor, representing about one‐third of all malignancies diagnosed worldwide each year. Cutaneous squamous cell carcinoma (cSCC) is the second most common form of NMSCs and the risk of cSCC invasiveness should be assessed on the basis of tumor size, anatomical location, and histological subtype. Although most cSCCs are early diagnosed and successfully treated, in a small percentage of patients with giant cSCC (maximum diameter >5 cm), metastases may occur; treatment options are limited and not really effective. We report the case of a giant metastatic cSCC that had been neglected for more than 20 years. Radiotherapy or surgery were not feasible and polichemotherapy (cisplatin, 5‐fluorouracil and paclitaxel) was not effective. Therefore, the patient was treated with palliative electrochemotherapy (ECT) achieving a partial reduction of cutaneous metastasis and pain relief but unfortunately the patient died 3 months after the second ECT treatment.     

Twist1 in tumor cells and α‐smooth muscle actin in stromal cells are possible biomarkers for metastatic giant basal cell carcinoma

We previously reported a case of giant basal cell carcinoma (BCC) in a 75‐year‐old Japanese man, who subsequently developed a pulmonary metastasis. With regard to the pathogenesis of metastasis of BCC, recently, it has been reported that high levels of expression of Twist1 and N‐cadherin in primary and metastatic tumor cells, suggesting that Twist1 expression and an epithelial–mesenchymal transition (EMT) of tumor cells are important for the promotion of tumor invasion and subsequent metastasis. In this report, we identified the expressions of Twist1 in tumor cells and α‐smooth muscle actin (α‐SMA) in stromal cells in the primary and metastatic sites of giant BCC. These results suggest that Twist1‐induced EMT of tumor cells might have been associated with distant organ metastasis in our case, and the presence of α‐SMA‐positive myofibroblasts surrounding a BCC nest can be one of hallmarks of the aggressiveness of BCC.     

Basal cell carcinoma progression correlates with host immune response and stromal alterations: a histologic analysis

Neoplastic progression is characterized in part by escape from immune surveillance and formation of growth-permissive stroma. Basal cell carcinoma (BCC) can be subclassified into low- and high-risk types for local recurrence. To determine whether these types of BCC correlate with alterations in local host immune response and stroma and whether these changes follow stepwise histologic progression from low- to high-risk subtypes, we assessed the clinicopathologic features in 175 consecutive primary (nonrecurrent) BCC excision specimens. BCCs exhibited a significantly higher frequency of mixed rather than homogeneous growth patterns (76% vs. 24%, P=0.0001). Nodular (84%) was the most common pattern identified followed by superficial (77%), infiltrative (27%), morpheic (5%) and micronodular patterns (4%). Only superficial (12% of all BCC) and nodular (12%) patterns were identified in BCC with a homogeneous histologic phenotype. Micronodular and infiltrative-morpheic patterns were not identified together in mixed patterned BCCs, and these high-risk types were contiguous with nodular BCC. Superficial predominant BCC (major growth pattern) was significantly associated with trunk and extremity location (76%) and skin without solar elastosis (82%), whereas BCC harboring a nodular growth pattern component was significantly associated with a head and neck location (63%) and the presence of adjacent solar elastosis (all P< or =0.03). Significant correlations were identified for BCC subtypes with inflammatory and stromal alterations: superficial BCC with old regression and moderate to dense peritumoral lymphocytic infiltrates; high-risk types correlated with active regression; infiltrative and morpheic BCC with fibrosing tumor stroma; and micronodular BCC with loss of both host inflammatory and stromal tumor responses. Evaluating the theoretical histologic stepwise model of BCC progression (superficial-to-nodular-to-micronodular, or superficial-to-nodular-to-infiltrative-to-morpheic BCC types) revealed significant linear correlations with host response and alterations of tumor stroma (r=0.54, P=0.0001). BCC exhibit distinct epithelial-stromal-inflammatory patterns that correlate with BCC subtype and tumor progression. This ostensible pathway of diminishing host response and gain of permissive tissue environment highlights neoplastic evolution from low to high risk for local recurrence of BCC and implicates a histologic continuum reflecting dynamic host-BCC interactions.     

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.     

Matrix metalloproteinases in tumor progression: focus on basal and squamous cell skin cancer

Many normal biological processes, such as reproduction, fetal development and wound healing, are critically dependent on controlled degradation of extracellular matrix (ECM) macromolecules. However, excessive degradation of matrix components occurs in pathologic tissue destruction, e.g. in atherosclerosis, rheumatoid arthritis, and cancer. Matrix metalloproteinases (MMPs) are degradative enzymes that play an important role in all aspects of tumor progression by enhancing tumor-induced angiogenesis and destroying local tissue architecture and basement membranes to allow tumor invasion and metastasis. Efficient breakdown of the ECM surrounding invasive cancer islands involves interplay between tumor cells, stromal cells, and inflammatory cells, all of which express a distinct set of MMPs. Besides the classical role of MMPs in degradation of ECM, MMPs may also indirectly influence the tumor microenvironment through the release of growth factors, cryptic sites or angiogenic factors, or through the generation of matrix fragments that inhibit tumor cell proliferation, migration and angiogenesis. This makes the contribution of MMPs to tumorigenesis much more complex than initially thought. Currently, a number of clinical studies have focused on testing MMP inhibitors as potential antineoplastic agents. In this review we discuss the present role of MMPs in the development and progression of cancer, focusing on non-melanoma skin cancers basal (BCC) and squamous (SCC) cell carcinoma, and the possible influence of MMPs in their differences.     

Collagens XV and XVIII show different expression and localisation in cutaneous squamous cell carcinoma: type XV appears in tumor stroma,while XVIII becomes upregulated in tumor cells and lost from microvessels

As the second most common skin malignancy, cutaneous squamous cell carcinoma (cSCC) is an increasing health concern, while its pathogenesis at molecular level remains largely unknown. We studied the expression and localisation of two homologous basement membrane (BM) collagens, types XV and XVIII, at different stages of cSCC. These collagens are involved in angiogenesis and tumorigenesis, but their role in cancer development is incompletely understood. Quantitative RT‐PCR analysis revealed upregulation of collagen XVIII, but not collagen XV, in primary cSCC cells in comparison with normal human epidermal keratinocytes. In addition, the Ha‐ras‐transformed invasive cell line II‐4 expressed high levels of collagen XVIII mRNA, indicating upregulation in the course of malignant transformation. Immunohistochemical analyses of a large human tissue microarray material showed that collagen XVIII is expressed by tumor cells from grade 1 onwards, while keratinocytes in normal skin and in premalignant lesions showed negative staining for it. Collagen XV appeared instead as deposits in the tumor stroma. Our findings in human cSCCs and in mouse cSCCs from the DMBA‐TPA skin carcinogenesis model showed that collagen XVIII, but not collagen XV or the BM markers collagen IV or laminin, was selectively reduced in the tumor vasculature, and this decrease associated significantly with cancer progression. Our results demonstrate that collagens XV and XVIII are expressed in different sites of cSCC and may contribute in a distinct manner to processes related to cSCC tumorigenesis, identifying these collagens as potential biomarkers in the disease.     

Stromal expression of cathepsin K in squamous cell carcinoma

Background Cathepsin K (CTSK), a cysteine protease with strong collagenolytic and elastolytic properties involved in extracellular matrix turnover, may be produced by neoplastic cells as well as stromal macrophages and fibroblasts. Its expression is suggested as associated with increased invasive and metastatic potential. Objectives The aim of this study is to examine stromal expression of cathepsin K in skin tumors. Methods A series of 13 normal skin and 109 skin tumours, including 51 benign and 58 malignant epidermal tumours were tested for CTSK and Ki‐67 expression by immunohistochemical analysis. Results Stromal CTSK expression and the tumoral Ki‐67 labelling index were significantly higher in invasive squamous cell carcinoma (SCC) than in other epidermal tumours. Conclusion Cathepsin K‐positive stromal fibroblasts may play a crucial role in SCC progression by promoting extracellular matrix degradation, thereby facilitating SCC growth and invasion into surrounding tissue and vasculature. CTSK inhibitors may be a potential novel therapeutic option to decrease SCC progression.     

Engineering melanoma progression in a humanized environment in vivo

To overcome the lack of effective therapeutics for aggressive melanoma, new research models closely resembling the human disease are required. Here we report the development of a fully orthotopic, humanized in vivo model for melanoma, faithfully recapitulating human disease initiation and progression. To this end, human melanoma cells were seeded into engineered human dermo-epidermal skin substitutes. Transplantation onto the back of immunocompromised rats consistently resulted in the development of melanoma, displaying the hallmarks of their parental tumors. Importantly, all initial steps of disease progression were recapitulated, including the incorporation of the tumor cells into their physiological microenvironment, transition of radial to vertical growth, and establishment of highly vascularized, aggressive tumors with dermal involvement. Because all cellular components can be individually accessed using this approach, it allows manipulation of the tumor cells, as well as of the keratinocyte and stromal cell populations. Therefore, in one defined model system, tumor cell-autonomous and non-autonomous pathways regulating human disease progression can be investigated in a humanized, clinically relevant context.     

CXC chemokine receptor CXCR4 expression enhances tumorigenesis and angiogenesis of basal cell carcinoma

BACKGROUND: Chemokines and their receptors, well known for their ability to attract leucocytes, also play important roles for tumour progression. OBJECTIVES: To investigate the possible involvement of chemokine receptors in the pathogenesis of cutaneous basal cell carcinoma (BCC). METHODS: We performed an expression analysis of chemokine receptors using a well-characterized human BCC cell line. Upon the finding of CXCR4 expression by BCC, retroviral transduction of BCC cells with the CXCR4 gene was employed to address its functional significance for BCC in vitro and in vivo. RESULTS: We found expression of the CXC chemokine receptor CXCR4 by a human cell line and a subset of tissue samples from BCC, especially in noduloulcerative and sclerosing types. Following treatment with CXCL12, the ligand for CXCR4, CXCR4-transduced BCC cells (CXCR4-BCC) showed increased proliferation under low serum concentration and resistance to apoptosis induced by ultraviolet B irradiation in vitro. Conditioned media from CXCR4-BCC preincubated with CXCL12 enhanced tubule formation of human endothelial cells in vitro. These responses of CXCR4-BCC were negated by cotreatment with either neutralizing antibodies or specific blocking peptides for CXCR4 in vitro. Moreover, xenograft tumour transplants produced by injection of CXCR4-BCC yielded significant tumour progression in nude mice, whereas additional serial injections of CXCR4-blocking peptides resulted in tumour regression. CONCLUSIONS: CXCR4 expression may play a critical role in tumour progression and angiogenesis of certain subtypes of BCC with more aggressive nature, and functional blockade of CXCR4 could be a potential therapeutic strategy for these tumours.     

Anaplastic neoplasms arising from basal cellcarcinoma xenotransplants into SCID-beige mice

BACKGROUND: An animal model for the study of basal cell carcinoma (BCC) is required to better understand its biology. Several attempts to grow BCC in immuno-incompetent animals have been only modestly successful. METHODS: To test the ability of BCC to grow in a mouse with complete and severe immuno-incompetence, 14 individual BCC were transplanted into the subcutaneous tissue of 18 SCID-beige mice (T, B and natural killer cell deficient). Light microscopy and immunophenotypic analyses were performed on primary BCC and first and seventh passage tumors. RESULTS: Transplantation of three BCC yielded rapidly growing anaplastic tumors for a tumor take of 18% (3/18). SCID-beige mice without tumor growth had mostly scars or epidermoid cysts at the transplant sites. The three patients whose BCC gave rise to the anaplastic tumors were significantly older than those without tumor growth (87 vs. 64, p = 0.001), but they did not differ with respect to BCC type or general health. These three anaplastic tumors were histologically and immunophenotypically similar, being composed of dyscohesive, pleomorphic cells that expressed vimentin and smooth muscle actin. In the first passage mice these tumors were locally invasive, tumor-forming nodules associated with an expansion of donor inflammatory cells (T and B lymphocytes and plasma cells), rare remnants of BCC epithelium and epidermoid cysts. By the seventh passage, the tumors were homogenous and metastasized widely throughout the mice. Changing transplantation location to the dermis to wound environment or supplementing the tumor with BCC-derived fibroblasts did not alter the phenotype or growth rate in SCID-beige mice. Anaplastic tumors also grew easily in SCID mice (T and B cell deficient). However, transplantation of the anaplastic tumors into normal mice (CB-17) or less severely immunodeficient mice (NCr and Balb/c: T and natural killer cell deficient) did not allow for growth. Furthermore, tumor growth could not be maintained in vitro. CONCLUSION:Empirically, these data suggest that BCC has the potential to become an aggressive metastatic neoplasm, given the right immune and stromal environment. Moreover, a functional B lymphocyte system appears to prevent this growth. As human lymphocytes also engraft in SCID-beige mice, the original host immune response could be responsible for the lack of tumor growth in the majority of xenografts. Furthermore, the anaplastic and metastatic phenotype of these BCC derived neoplasms may be the experimental equivalent of metastatic BCC and BCC associated with carcinosarcoma.     

MicroRNA expression differs in cutaneous squamous cell carcinomas and healthy skin of immunocompetent individuals

Cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers, but the influence of microRNA (miRNA) expression has only been sporadically analysed. We hypothesized that miRNAs are differentially expressed in cSCC and hence influence its development. We therefore isolated total miRNA from well‐differentiated cSCCs and from controls without SCC. Expression analyses of 12 miRNAs showed three significantly differentially expressed miRNAs. We identified a significant upregulation of the miR‐21 and the miR‐31, a proto‐oncogene like miR‐21. While the upregulated expression of miR‐21 has been known for some time, the increased expression of miR‐31 was never shown so clearly. Furthermore, we showed the upregulation of miRNA‐205, which has never been described before. The miR‐205 induces specific keratinocyte migration and could be a characteristic marker for cSCC. It has to be determined in following studies whether these upregulated expressions are specific for cSCC and if so, for which cSCC stages.     

Ultraviolet responses of Gorlin syndrome primary skin cells

Background  Gorlin syndrome, or naevoid basal cell carcinoma syndrome (NBCCS), is an autosomal dominant disorder associated with mutations in the PTCH1 gene, which encodes the receptor of SONIC HEDGEHOG. In addition to developmental abnormalities, patients with NBCCS are prone to basal cell carcinoma (BCC), the most frequent type of nonmelanoma skin cancer in humans.
Objectives  As ultraviolet (UV) exposure plays a prominent role in the development of sporadic BCC, we aimed to determine whether primary NBCCS skin cells exhibit differential responses to UV exposure compared with wild-type (WT) skin cells.
Methods  Primary fibroblast and keratinocyte strains were isolated from nonlesional skin biopsies of 10 patients with characteristic NBCCS traits. After identification of PTCH1 mutations, capacities of NBCCS cells to repair UV-induced DNA lesions and to survive after UV irradiation, as well as p53 responses, were compared with those of WT skin cells.
Results  The c1763insG PTCH1 mutation is described for the first time. DNA repair and cell survival analyses following UV irradiation revealed no obvious differences between responses of NBCCS and WT fibroblasts and keratinocytes. However, p53 accumulation after UV irradiation was abnormally persistent in all NBCCS primary keratinocyte strains compared with WT keratinocytes.
Conclusions  Our observations that NBCCS cells harbour normal DNA repair and survival capacities following UV irradiation better explain that BCC proneness of patients with NBCCS does not solely concern body areas exposed to sunlight and suggest rather that it might be due to cell cycle alterations.     

Fibroblasts support migration of monocyte‐derived dendritic cells by secretion of PGE2 and MMP‐1

The outcome of a cutaneous immune response is critically dependent upon the ability of dendritic cells (DC) to migrate from skin to the draining lymph nodes – a process that is influenced by the cutaneous tissue microenvironment. Here, the role of fibroblasts – a major component of the dermal microenvironment – on the migratory capacity of monocyte‐derived DC (MoDC) was investigated in a 3D collagen I matrix. Indeed, dermal fibroblasts supported the migration of pre‐activated MoDC through a 3D collagen I matrix. Activation of human MoDC resulted in the release of TNFα and IL‐1β that in turn stimulated MMP‐1 (human collagenase) and PGE₂ secretion by human dermal fibroblasts. Transmigration assays confirmed the importance of fibroblast‐derived MMP‐1 and PGE₂ for the migration of MoDC through a 3D collagen I matrix. Finally, in mice initiation of inflammation by induction of an irritant contact dermatitis or a psoriasis‐like skin inflammation, the expression of the PGE₂ generating cox‐2 and the mouse collagen I degrading enzyme matrix metalloproteinases (MMP)‐13 was strongly up‐regulated. Our study indicates that MoDC are able to instruct dermal fibroblasts resulting in enhanced migratory capability of MoDC, thus highlighting the role of a crosstalk of DC with their stromal microenvironment for the control of cutaneous immune responses.     

Crosstalk among p53 family members in cutaneous carcinoma

Cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer with a frequency increasing worldwide. The risk of developing cSCC has been strongly associated with chronic sun exposure, especially in light skin people. The aim of this viewpoint is to discuss the contribution of the tumor suppressor p53 and its homologues p63 and p73 in the formation and progression of cSCC. Mutations in the p53 gene are early and frequent events in skin carcinogenesis mainly as a consequence of UV light exposure, often followed by loss of function of the second allele. Although rarely mutated in cancer, p63 and p73 play key roles in human cancers, with their truncated isoforms lacking the N‐terminal transactivating domain (?N) being often upregulated as compared to normal tissues. ?Np63 is abundantly expressed in cSCC, and it is likely to favour tumor initiation and progression. The function of p73 in cSCC is more enigmatic and awaits further studies. Interestingly, an intimate interplay exists between both p53 and p63, and the Notch signalling pathway, often inactivated in cSCC. Here, we summarize our current knowledge about the biological activities of p53 family members in cSCC and propose that integration of their signalling with Notch is key to cSCC formation and progression.