A possible chemopreventive role for photodynamic therapy in Gorlin syndrome: a report of basal cell carcinoma reduction and review of literature

Multiple basal cell carcinomas (BCC) are a significant cause of morbidity and disfigurement in patients with naevoid basal cell carcinoma syndrome (NBCCS). Chemopreventive strategies are needed to reduce the formation of new BCC in these patients. Appropriate evidence‐based guidelines for photodynamic therapy chemoprevention in NBCCS do not exist. We herein report one patient treated with methyl aminolevulinate PDT with red light (630 nm) activation to continue sustained chemoprevention following other BCC chemopreventive modalities and a relevant literature review.     

痣样基底细胞癌综合征一家系PTCH1基因突变分析

目的 对痣样基底细胞癌综合征一家系进行PTCH1基因突变分析。 方法 提取先证者（Ⅱ5）及Ⅱ1、Ⅱ3、Ⅲ4的DNA,以50例健康人为对照。应用聚合酶链反应（PCR）、DNA直接测序明确突变位点,根据突变位点设计特异性引物,用PCR来检测突变位点从而进一步确定该家系的致病原因。 结果 先证者PTCH1基因的1条等位基因第14号外显子上2137位胞嘧啶C被胸腺嘧啶T替代（c.2137C > T）,即CAG→TAG,导致终止密码产生（Q714X）,Ⅲ4也检测到相同突变。健康对照者中未检出该突变。 结论 PTCH1基因的无义突变（c.2137C > T）可能是引起该患者临床症状的特异性突变。     

Matrix metalloproteinases in keratinocyte carcinomas

The incidence of cutaneous keratinocyte‐derived cancers is increasing globally. Basal cell carcinoma (BCC) is the most common malignancy worldwide, and cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. BCC can be classified into subtypes based on the histology, and these subtypes are classified further into low‐ and high‐risk tumors. There is an increasing need to identify new therapeutic strategies for the treatment of unresectable and metastatic cSCC, and for aggressive BCC variants such as infiltrating, basosquamous or morpheaform BCCs. The most important risk factor for BCC and cSCC is solar UV radiation, which causes genetic and epigenetic alterations in keratinocytes. Similar gene mutations are noted already in sun‐exposed normal skin emphasizing the role of the alterations in the tumor microenvironment in the progression of cSCC. Early events in cSCC progression are alterations in the composition of basement membrane and dermal extracellular matrix induced by influx of microbes, inflammatory cells and activated stromal fibroblasts. Activated fibroblasts promote inflammation and produce growth factors and proteolytic enzymes, including matrix metalloproteinases (MMPs). Transforming growth factor‐β produced by tumor cells and fibroblasts induces the expression of MMPs by cSCC cells and promotes their invasion. Fibroblast‐derived keratinocyte growth factor suppresses the malignant phenotype of cSCC cells by inhibiting the expression of several MMPs. These findings emphasize the importance of interplay of tumor and stromal cells in the progression of cSCC and BCC and suggest tumor microenvironment as a therapeutic target in cSCC and aggressive subtypes of BCC.     

Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas

BACKGROUND: Basal cell carcinoma (BCC) of the skin is the most common human cancer. The genetic alterations underlying BCC development are only partly understood. OBJECTIVES: To investigate further the molecular genetics of sporadic BCCs, we performed mutation analyses of 10 skin cancer-associated genes in 42 tumours. METHODS: Single-strand conformational polymorphism analysis followed by DNA sequencing was used to screen for mutations in the sonic hedgehog pathway genes PTCH, SMOH, SUFUH and GLI1, in the TP53 tumour suppressor gene, and in the proto-oncogenes NRAS, KRAS, HRAS, BRAF and CTNNB1. Microsatellite markers flanking the PTCH, SUFUH and TP53 loci at 9q22, 10q24 and 17p13, respectively, were studied for loss of heterozygosity (LOH). RESULTS: PTCH mutations were found in 28 of 42 tumours (67%). Microsatellite analysis revealed LOH on 9q22 in 20 of 38 tumours investigated (53%), including 14 tumours with and six tumours without PTCH mutations. SMOH mutations were identified in four of the 42 BCCs (10%) while two tumours demonstrated mutations in SUFUH, including one missense mutation and one silent mutation. None of the BCCs showed LOH at markers flanking the SUFUH locus. Seventeen BCCs (40%) carried TP53 mutations, with only three tumours showing evidence of biallelic TP53 inactivation. TP53 mutations were present in BCCs with and without mutations in PTCH, SMOH or SUFUH. Interestingly, 72% of the TP53 alterations were presumably ultraviolet (UV)-induced transition mutations. In contrast, only 40% of the PTCH and SMOH alterations corresponded to UV signature mutations. No mutations were identified in GLI1, NRAS, KRAS, HRAS, BRAF or CTNNB1. CONCLUSIONS: Our data confirm the importance of PTCH, SMOH and TP53 mutations in the pathogenesis of sporadic BCCs. SUFUH alterations are restricted to individual cases while the other investigated genes do not appear to be important targets for mutations in BCCs.     

Multiple large surface photodynamic therapy sessions with topical methylaminolaevulinate in PTCH heterozygous mice

BACKGROUND: Photodynamic therapy (PDT) combines the administration of a photosensitizer with its subsequent activation by light of the appropriate wavelength. Methylaminolaevulinate (MAL) is a photosensitizer precursor, transformed by cells into protoporphyrin IX. The PTCH gene plays a central role in the genesis of basal cell carcinoma (BCC). The PTCH transgenic mouse develops microscopic BCCs when chronically exposed to ultraviolet (UV) or ionizing radiation. OBJECTIVES: The aim of this study was to explore the ability of multiple large surface MAL-PDT to prevent BCC, using the PTCH heterozygous mouse as a model. METHODS: Thirty-five mice were exposed to UV radiation for a total of 20 weeks. Group 1 (20 mice) was exposed only to UV whereas group 2 (15 mice) was exposed to UV and weekly to MAL-PDT. At 28 weeks the mice were killed and the skin of the back processed for standard histopathology. Assessment was blind and any slide showing the presence of BCC was counted as a single BCC. The number of mice in groups 1 and 2 showing BCC were compared using Fisher's exact test. RESULTS: Nineteen BCCs in nine mice from group 1 were found, but no BCCs in mice from group 2. The difference was statistically significant (P = 0.001). CONCLUSIONS: Weekly suberythematous PDT sessions with topical MAL were able to delay the development of microscopic BCCs in PTCH mice chronically exposed to UV radiation.     

UV-specific p53 and PTCH mutations in sporadic basal cell carcinoma of sun-exposed skin

UVB irradiation is known to produce DNA damage at mutation hotspots in the p53 tumor suppressor gene, leading to the development of skin cancers. Mutations in the PTCH tumor suppressor gene, which is known to be responsible for the development of nevoid basal cell carcinoma syndrome, have also been identified in sporadic basal cell carcinomas (BCCs). We describe the case of an 80-year-old welder in whom 3 novel p53 mutations, as well as UV-specific PTCH mutations, were detected in two BCC samples from sun-exposed skin. The simultaneous presence of UV-specific p53 and PTCH mutations in the same BCC sample has not previously been reported.     

Germline mutations of the PTCH gene in Japanese patients with nevoid basal cell carcinoma syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental and skeletal anomalies, palmo-plantar pits, odontogenic keratocysts, ectopic calcification, and occurrence of various types of tumors including basal cell carcinoma. Recent evidence has indicated that the human homologue of a Drosophila segment polarity gene, PTCH, is a NBCCS susceptibility gene. In the study presented here, we detected two novel mutations of the PTCH gene, I805X/2395delC and Y93X/C297A, in two unrelated Japanese patients. Early protection of the skin from the sunlight is important to the prevention of BCC development in NBCCS patients. Genetic analysis of the PTCH gene is essential for the early, definitive diagnosis of NBCCS, especially before the expression of clinical manifestations is complete.     

Linear basal cell nevus with a novel mosaic PTCH1 mutation

The patched tumor suppressor gene (PTCH1) encodes a receptor, which is a key component of the hedgehog signalling pathway. Mutations in PTCH1 are implicated in the development of sporadic basal cell carcinomas (BCC), as well as those in Gorlin Syndrome. Rarely, BCCs may develop in a linear pattern along lines of Blaschko due to cutaneous mosaicism. In cases in which there are other features of Gorlin syndrome, genomic analysis has demonstrated lesional mutations in the Hedgehog signalling pathway. Causative mutations, however, have not been firmly demonstrated in the cases of linear and segmental BCCs in otherwise healthy individuals. Herein, we report a case of a 31 year-old Caucasian woman with linear development of multiple superficial BCCs in a Blaschkoid distribution without other characteristic findings of Gorlin syndrome. Genomic analysis of lesional skin by whole-exome sequencing identified a novel heterozygous mutation PTCH1: NM_000264.3, Exon 15, c.2336-2337insGGTAGGA, p.Asp779Glufs*13 in PTCH1, shared by two discrete samples within the lesion, while no mutations were found in the non-lesional skin or peripheral blood. Given the young age of our patient and linear distribution of BCCs on non-sun exposed skin, our findings suggest segmental mosaicism. The patient was treated with topical 5% imiquimod with histologically confirmed clearance of BCCs in 2 months.     

PTCH1 gene haplotype association with basal cell carcinoma after transplantation

Background Basal cell carcinoma (BCC) is 10 times more frequent in organ transplant recipients (OTRs) than in the general population. Factors in OTRs conferring increased susceptibility to BCC include ultraviolet radiation exposure, immunosuppression, viral infections such as human papillomavirus, phototype and genetic predisposition. The PTCH1 gene is a negative regulator of the hedgehog pathway, that provides mitogenic signals to basal cells in skin. PTCH1 gene mutations cause naevoid BCC syndrome, and contribute to the development of sporadic BCC and other types of cancers. Associations have been reported between PTCH1 polymorphisms and BCC susceptibility in nontransplanted individuals. Objectives To search for novel common polymorphisms in the proximal 5′ regulatory region upstream of PTCH1 gene exon 1B, and to investigate the possible association of PTCH1 polymorphisms and haplotypes with BCC risk after organ transplantation. Methods Three PTCH1 single nucleotide polymorphisms (rs2297086, rs2066836 and rs357564) were analysed by restriction fragment length polymorphism analysis in 161 northern Italian OTRs (56 BCC cases and 105 controls). Two regions of the PTCH1 gene promoter were screened by heteroduplex analysis in 30 cases and 30 controls. Results Single locus analysis showed no significant association. Haplotype T₁₆₈₆–T₃₉₄₄ appeared to confer a significantly higher risk for BCC development (odds ratio 2·98, 95% confidence interval 2·55–3·48; P = 0·001). Two novel rare polymorphisms were identified at positions 176 and 179 of the 5′UTR. Two novel alleles of the ‐4 (CGG)_n microsatellite were identified. No association of this microsatellite with BCC was observed. Conclusions Haplotypes containing T₁₆₈₆–T₃₉₄₄ alleles were shown to be associated with an increased BCC risk in our study population. These data appear to be of great interest for further investigations in a larger group of transplant individuals. Our results do not support the hypothesis that common polymorphisms in the proximal 5′ regulatory region of the PTCH1 gene could represent an important risk factor for BCC after organ transplantation.     

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.     

Expression profiles of cortisol‐inactivating enzyme, 11β‐hydroxysteroid dehydrogenase‐2, in human epidermal tumors and its role in keratinocyte proliferation

The enzyme 11β‐hydroxysteroid dehydrogenase (11β‐HSD) catalyzes the interconversion between hormonally active cortisol and inactive cortisone within cells. There are two isozymes: 11β‐HSD1 activates cortisol from cortisone and 11β‐HSD2 inactivates cortisol to cortisone. 11β‐HSD1 was recently discovered in skin, and we subsequently found that the enzyme negatively regulates keratinocyte proliferation. We verified 11β‐HSD1 and 11β‐HSD2 expression in benign and malignant skin tumors and investigated the role of 11β‐HSD in skin tumor pathogenesis. Randomly selected formalin‐fixed sections of skin lesions of seborrheic keratosis (SK), squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) were stained with 11β‐HSD1 and 11β‐HSD2 antibodies, and 11β‐HSD expression was also evaluated in murine epidermis in which hyperproliferation was induced by 12‐O‐tetradecanoylphorbol‐13 acetate (TPA). We observed that 11β‐HSD1 expression was decreased in all SK, SCC, and BCC lesions compared with unaffected skin. Conversely, 11β‐HSD2 expression was increased in SK and BCC but not in SCC. Overexpression of 11β‐HSD2 in keratinocytes increased cell proliferation. In the murine model, 11β‐HSD1 expression was decreased in TPA‐treated hyperproliferative skin. Our findings suggest that 11β‐HSD1 expression is decreased in keratinocyte proliferative conditions, and 11β‐HSD2 expression is increased in basal cell proliferating conditions, such as BCC and SK. Assessing 11β‐HSD1 and 11β‐HSD2 expression could be a useful tool for diagnosing and characterizing skin tumors.     

基底细胞癌的分子遗传机制研究进展

基底细胞癌是一种最为常见的皮肤癌。近年来研究表明,SHH信号通路的变异在基底细胞癌的发生中起到一个关键的作用,SHH通路的重要成员包括SHH、PTCH1、SM0、GLI,SHH的失调与细胞的PTCH1基因突变相关,SMO的过度激活和转录因子GLI的异常导致多种细胞生长和增殖的基因转录表达上调,并促进了基底细胞癌的形成。SHH信号通路抑制剂对肿瘤的抑制作用为基底细胞癌的治疗提供了一个新的策略。     

Germline mutations of the <Emphasis Type="Italic">PTCH</Emphasis> gene in Japanese patients with nevoid basal cell carcinoma syndrome

We identified seven novel germline mutations of the PTCH gene in eight unrelated Japanese patients with nevoid basal cell carcinoma syndrome (NBCCS). In order to ensure genetic diagnosis, all 23 coding exons of the PTCH gene were amplified from genomic DNA by polymerase chain reaction (PCR) and sequenced. Mutations were found in all eight patients with NBCCS. The mutations detected in this study include one insertion/deletion mutation, one 1-bp insertion, two 1-bp deletions, one nonsense mutation and two missense mutations. None of the mutations have been previously reported. Five mutations caused premature stop codons that are predicted to result in a truncated protein. In the two missense mutations, the strong basic residue arginine was substituted by serine or glycine in highly conserved components of the putative transmembrane domain of PTCH, and these mutations may therefore affect the conformation and function of the PTCH protein. No phenotype-genotype relationships were found in the Japanese NBCCS patients, consistent with results of previous studies on NBCCS in African-American and Caucasian patients.M. Tanioka and K. Takahashi contributed equally to this work.     

Expression of GLTSCR2/Pict-1 in squamous cell carcinomas of the skin

The most important cause of cutaneous squamous cell carcinomas (SCC) is DNA damage induced by exposure to solar UV irradiation. DNA damage induced by UV irradiation is sensed by early DNA damage response (DDR) proteins. Recently, GLTSCR2 has been suggested to play a role in UV light-induced DDR. To explore the role of GLTSCR2 in the development of cutaneous SCC, we investigated the molecular mechanism underlying GLTSCR2 inactivation in response to UV irradiation. We analyzed cutaneous SCC (n = 42), basal cell carcinomas (BCC; n = 26), and normal skin tissue samples (n = 36) and compared GLTSCR2 expression between tumor and normal tissues, using immunohistochemistry. Next, to investigate the effects of UV irradiation on GLTSCR2, we performed immunocytochemistry, RT-PCR, immunoblotting, half-life assay for GLTSCR2, and comet assay after UV irradiation in primary keratinocytes. GLTSCR2 expression in SCC was significantly lower than that of normal skin tissue (p < 0.05), but not different between BCC and normal skin. In cultured primary keratinocytes, GLTSCR2 expression was decreased and translocated after UV irradiation. UV irradiation accelerated degradation of GLTSCR2 through proteasomal pathway. Knockdown of GLTSCR2 resulted in marked decrease in γH2AX foci after UV exposure. Furthermore, comet assay showed that DNA damage after UV exposure persists longer in GLTSCR2 knocked-down cells. Our data show that GLTSCR2 is downregulated in SCC of the skin and that UV light exposure decreases the stability of GLTSCR2 and sensitizes keratinocytes to DNA damage. Therefore, our data suggest that GLTSCR2 might be involved in the development and/or progression of SCC of the skin.     

Novel clinical and molecular findings in Spanish patients with nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin Syndrome, affects only 1 in 56 000 people. It is usually inherited from a parent with the condition and affects both males and females equally (autosomal dominant inheritance). People with NBCCS may show characteristic features such as extra fingers or toes, irregular ribs, an unusually shaped face and large head. Tumours sometimes develop, particularly jaw cysts and basal cell carcinomas (BCCs) of the skin from which the syndrome is named. NBCCS is caused by mistakes (mutations) in a gene called PTCH1 located on the long arm of chromosome 9. PTCH1 acts as a brake on a set of chemical processes in cells called the Hedgehog (HH)/GLI signalling pathway. In NBCCS, mutations inactivate PTCH1 allowing excessive Hedgehog signalling. This loss of control results in the abnormal bones and tumours. Within an affected family, people with the same genetic mutation can have very different abnormalities. This group from Spain studied 22 unrelated Spanish people with NBCCS to see if they could discern any relationship between specific clinical abnormalities and specific genetic mutations. BCCs were very common, occurring in 96% of patients with 43% having more than 50; 77% had jaw cysts. Two abnormalities had not previously been recognised in NBCCS: double uterus and a benign nerve tumour. They found 19 PTCH1 mutations that had not previously been reported. However, they could not find any pattern relating the type of mutation to the clinical abnormalities. Presumably other factors besides the PTCH1 mutation causes the clinical features.     

Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin

Background  Fluorescence lifetime imaging (FLIM) is a novel imaging technique that generates image contrast between different states of tissue due to differences in fluorescence decay rates.
Objectives  To establish whether FLIM of skin autofluorescence can provide useful contrast between basal cell carcinomas (BCCs) and surrounding uninvolved skin.
Methods  Unstained excision biopsies of 25 BCCs were imaged en face with FLIM following excitation of autofluorescence with a 355 nm pulsed ultraviolet laser.
Results  Using FLIM we were able to distinguish areas of BCC from surrounding skin in an ex vivo study. Significant reductions in mean fluorescence lifetimes between areas of BCC and areas of surrounding uninvolved skin were demonstrated ( P  <   0·0001). These differences were apparent irrespective of the decay model used to calculate the fluorescence lifetimes (single vs. stretched exponential) or the long-pass filter through which the emitted autofluorescence was collected (375 vs. 455 nm). Conversely, there was no significant difference between the BCC and uninvolved areas of each sample when mean autofluorescence intensities were examined. Moreover, wide-field false-colour images of fluorescence lifetimes clearly discriminated areas of BCC from the surrounding uninvolved skin.
Conclusions  We therefore believe that FLIM has a potential future clinical role in imaging BCCs for rapid and noninvasive tumour delineation and as an aid to determine adequate excision margins with best preservation of normal tissue.     

Photodynamic therapy of eyelid basal cell carcinoma

Background  Photodynamic therapy (PDT; i.e. selective destroying of malignant cells by exposing them to red light after photosensitization) has been increasingly used for non-melanoma skin cancers. Due to excellent cosmetic and functional results, especially in difficult-to-treat areas, it may offer a comprehensive alternative to previous treatment modalities.
Objective  A series of six patient cases was used to evaluate the efficacy of PDT in the treatment of lower eyelid basal cell carcinoma (BCC).
Methods  Histological confirmation of BCC, a detailed demonstration of the technique with an illustrative series of cases and a review of related literature.
Results  Six patients with lower eyelid BCC were treated with two treatment sessions of PDT within a week. Five patients had a nodular type and one patient had a superficial type of BCC. The follow-up after treatment continued for 20–36 (mean 26.5) months. No recurrences were observed during the follow-up. All the patients were satisfied with the good results and tolerated the treatment well without any harm to the eyeball or surrounding skin.
Conclusion  PDT, although experimental in the eyelid area because of the small amount of data and the lack of a long-term follow-up, may be considered a promising comprehensive alternative when treating BCC in the eyelid area.

Conflicts of interest

None declared     

PTCH promoter methylation at low level in sporadic basal cell carcinoma analysed by three different approaches

Please cite this paper as: PTCH promoter methylation at low level in sporadic basal cell carcinoma analysed by three different approaches. Experimental Dermatology 2010. Abstract: Basal cell carcinoma (BCC) is the most common form of skin cancer. Mutations of the PTCH hallmark gene are detected in about 50–60% of BCCs, which raises the question whether other mechanisms such as promoter methylation can inactivate PTCH. Therefore, we performed methylation analysis of the PTCH promoter in a total of 56 BCCs. The sensitivity of three different methods, including direct bisulphite sequencing PCR, MethyLight and high‐resolution melting (HRM), was applied and compared. We found that HRM analysis of DNA from fresh tissue [rather than formalin‐fixed and paraffin‐embedded tissue (FFPE)] was the most sensitive method to detect methylation. Low‐level methylation of the PTCH promoter was detected in five out of 16 analysed BCCs (31%) on DNA from fresh tissue but only in two (13%) samples on short‐time stored FFPE DNA from the very same tumors. In contrast, we were unable to detect methylation by HRM on long‐time stored DNA in any of the remaining 40 BCC samples. Our data suggest that (i) HRM on DNA extracted from fresh tissue is the most sensitive method to detect methylation and (ii) methylation of the PTCH promoter may only play a minor role in BCC carcinogenesis.