BRM and BRG1 subunits of the SWI/SNF chromatin remodelling complex are downregulated upon progression of benign skin lesions into invasive tumours.

Background Nonmelanoma skin cancer is caused by exposure to ultraviolet radiation within sunlight. Actinic keratoses (AKs) are benign precursor lesions that can develop into invasive squamous cell carcinoma (SCC). Little is known about the molecular events that lead to human skin cancer progression from benign to invasive. Objectives To determine novel genes that may be involved in skin cancer progression based on data from an initial microarray screen of human skin cancers. Methods The SWI/SNF chromatin remodelling ATPase subunit BRM was identified as being downregulated in SCC but not AK compared with normal skin in our microarray screen. Therefore reverse transcription-polymerase chain reaction, gene methylation and protein expression was used to study BRM and its alternative ATPase subunit BRG1 in a range of human skin cancers. Results We found reduced levels of mRNA coding for BRM but not BRG1 in SCC. BRM mRNA levels in AK were similar to those in normal skin. Deregulation of BRM did not result from hypermethylation of CpG regions in the promoter of these genes. Both BRM and BRG1 protein was reduced by about 10-fold in 100% of SCC and basal cell carcinoma, but not in AK specimens examined. Conclusions BRM protein may be decreased due to low levels of mRNA, while BRG1 protein loss appears to be post-translational. BRM and BRG1 may be novel tumour suppressor genes for human skin cancer. They appear to be involved after development of benign lesions, and are downregulated during progression towards invasion.

A large-scale meta-analysis to refine colorectal cancer risk estimates associated with MUTYH variants.

BACKGROUND: defective DNA repair has a causal role in hereditary colorectal cancer (CRC). Defects in the base excision repair gene MUTYH are responsible for MUTYH-associated polyposis and CRC predisposition as an autosomal recessive trait. Numerous reports have suggested MUTYH mono-allelic variants to be low penetrance risk alleles. We report a large collaborative meta-analysis to assess and refine CRC risk estimates associated with bi-allelic and mono-allelic MUTYH variants and investigate age and sex influence on risk. METHODS: MUTYH genotype data were included from 20 565 cases and 15 524 controls. Three logistic regression models were tested: a crude model; adjusted for age and sex; adjusted for age, sex and study. RESULTS: all three models produced very similar results. MUTYH bi-allelic carriers demonstrated a 28-fold increase in risk (95% confidence interval (CI): 6.95-115). Significant bi-allelic effects were also observed for G396D and Y179C/G396D compound heterozygotes and a marginal mono-allelic effect for variant Y179C (odds ratio (OR)=1.34; 95% CI: 1.00-1.80). A pooled meta-analysis of all published and unpublished datasets submitted showed bi-allelic effects for MUTYH, G396D and Y179C (OR=10.8, 95% CI: 5.02-23.2; OR=6.47, 95% CI: 2.33-18.0; OR=3.35, 95% CI: 1.14-9.89) and marginal mono-allelic effect for variants MUTYH (OR=1.16, 95% CI: 1.00-1.34) and Y179C alone (OR=1.34, 95% CI: 1.01-1.77). CONCLUSIONS: overall, this large study refines estimates of disease risk associated with mono-allelic and bi-allelic MUTYH carriers.

Breast cancer immunohistochemistry can be useful in triage of some HNPCC families.

Immunohistochemistry of tumour samples is increasingly used in the triage of families where hereditary non-polyposis colorectal cancer (HNPCC) due to mismatch repair defects is suspected. Usually, this is undertaken in tumours that are a recognised part of the spectrum of HNPCC-related cancers e.g. colon or endometrial cancers. Although breast cancers are not classed as part of this spectrum, this study examined the extent to which some breast tumours do arise by the mismatch repair pathway in these families. This may have clinical utility in families where an individual with a 'classic HNPPC-related' tumour is not available for evaluation. Immunohistochemistry of a breast tumour may identify an individual in whom germline mutation testing is worthwhile.

Germline mutation prevalence in the base excision repair gene, MYH, in patients with endometrial cancer.

Germline mutations in the base excision repair gene, MutY human homolog (MYH), have recently been associated with a recessively inherited multiple adenoma polyposis syndrome and colorectal cancer. The spectrum of extracolonic lesions is still being characterized, although preliminary reports suggest that bi-allelic mutation carriers may share some of the clinical features of other hereditary colon cancer syndromes. Of 225 endometrial cancer patients, we identified one individual as a compound heterozygote, carrying mutations Y165C and G382D of MYH, and five individuals with heterozygous defects (three G382D and two Y165C). The patient with the bi-allelic Y165C/G382D mutation also had a sebaceous carcinoma, a feature of Muir-Torre syndrome. Although several intronic polymorphisms were detected in the heterozygous carriers, no other pathogenic variants were identified. While not conclusive, this novel and interesting finding provides evidence that bi-allelic germline mutations in MYH may increase susceptibility to endometrial cancer.

Squamous cell carcinoma growth in mice and in culture is regulated by c-Jun and its control of matrix metalloproteinase-2 and -9 expression.

Squamous cell carcinoma (SCC) is an invasive malignancy of epidermal keratinocytes. Surgical excision is currently the main treatment; however, this can cause scarring and disfigurement. There is accordingly, an acute need for alternative strategies to treat SCC. The transcription factor c-Jun is expressed in human SCC and another common form of invasive skin cancer, basal cell carcinoma together with the mitogenic marker-proliferating cell nuclear antigen. Here, we have employed DNAzymes (catalytic DNA molecules) targeting c-Jun (Dz13) to inhibit c-Jun expression in SCC cells. Dz13 inhibits SCC proliferation and suppresses solid SCC tumor growth and tumor angiogenesis in severe combined immunodeficient mice. We further demonstrate that Dz13 inhibits c-Jun, together with matrix metalloproteinase (MMP)-2 and MMP-9 expression in the tumors, consistent with DNAzyme inhibition of MMP-2 and MMP-9 gelatinolytic activity by zymography. Dz13 also suppressed the expression of vascular endothelial growth factor and fibroblast growth factor-2 in the tumors. These findings demonstrate that c-Jun regulates SCC growth and suggest that DNAzymes targeting this transcription factor may potentially be useful as inhibitors of cutaneous carcinoma.

Association of MUTYH and colorectal cancer.

Mutations in the MUTYH gene have been reported to be associated with increased risk of developing colorectal cancer. In this study, we confirmed this association using original data on 928 colorectal cancer cases and 845 healthy controls from Scotland. We then conducted a meta-analysis from published data on the association between mutations at MUTYH and colorectal cancer risk. We show for the first time a small but significant mono-allelic effect with a genotype relative risk (GRR) of 1.27 (95% confidence interval (CI): 1.01-1.61), and confirm and give a more precise estimate of the strong bi-allelic effect with an estimated GRR of 117 (95% CI: 74-184). This study underscores the need for large sample sizes in order to identify small gene effects when the disease allele frequency is low.

Characteristics of 5-aminolaevulinic acid-induced protoporphyrin IX fluorescence in human skin in vivo.

BACKGROUND/PURPOSE: Topical photodynamic therapy (PDT) is increasingly being used to treat skin cancers. Knowledge of the detailed characteristics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence in diseased and normal skin is incomplete. Understanding the characteristics of PpIX fluorescence in normal skin may facilitate optimization of PDT regimes while minimizing side effects in the surrounding normal skin. We investigated the characteristics of ALA-induced PpIX fluorescence in normal skin. METHODS: ALA was applied to the arm, back and leg skin of 21 healthy volunteers for 1-6 h, and PpIX fluorescence was measured for up to 24 h after ALA application using a fluorescent spectrometer. The effect of tape stripping on fluorescence was also examined. RESULTS: Considerable inter-subject variation was observed in the time to reach peak PpIX fluorescence. Intra-subject variation in the time to peak fluorescence was dependent on ALA application time. Six hours after ALA application, no significant difference was observed in the degree of fluorescence achieved irrespective of ALA application times ranging between 1 and 6 h. PpIX fluorescence was reduced on the leg and increased by tape stripping. CONCLUSIONS: Marked inter- and intra-subject variation in ALA-induced PpIX fluorescence occurs in normal human skin. ALA application time, body site and the state of the stratum corneum are all determinants of PpIX fluorescence within subjects and these factors need to be taken into account in optimization of PDT regimes.

Imiquimod-induced regression of actinic keratosis is associated with infiltration by T lymphocytes and dendritic cells: a randomized controlled trial.

BACKGROUND: Imiquimod 5% cream is a topically applied immune response modifier that has been shown to give effective treatment of actinic keratosis (AK). The therapeutic effects of imiquimod are likely to involve the provocation of a cutaneous immune response against abnormal cells, an assumption based on a strong correlation between complete clearance rates and the severity of the local skin reactions (erythema, oedema, erosion/ulceration, weeping/exudation and scabbing/crusting); however, no clinical studies have conclusively proved this mechanism. OBJECTIVES: To determine the nature of cellular infiltrates induced by the application of imiquimod to AK lesions and to study cells involved in the cutaneous immune response. METHODS: Eighteen patients participated in this phase I, randomized, double-blind, parallel group, vehicle-controlled study. Enrolled patients were randomized in a 2 : 1 ratio to receive imiquimod cream or vehicle cream and applied study cream to five lesions on the scalp, forearm or upper trunk once daily, three days per week for up to 16 weeks. Each patient had punch biopsies of two distinct AK lesions: a lesion was biopsied before treatment to obtain baseline biomarker levels, and a different lesion was biopsied after 2 weeks of treatment. Biopsy specimens were examined using routine and immunohistochemical staining. RESULTS: The imiquimod group showed statistically significant increases from baseline to week 2 in tissue biomarker levels for CD3, CD4, CD8, CD11c, CD86/CD11c, CD68, HLA-DR and TUNEL. No significant differences were seen for the vehicle group. Complete clearance of all treated AK lesions was achieved in five of 11 (45%) imiquimod patients and in none of six vehicle patients. CONCLUSIONS: Imiquimod stimulates a cutaneous immune response characterized by increases in activated dendritic cells and CD4+ and CD8+ T cells.

Imiquimod induced regression of clinically diagnosed superficial basal cell carcinoma is associated with early infiltration by CD4 T cells and dendritic cells.

Imiquimod is presumed to clear basal cell carcinoma (BCC) through apoptosis mediated by cytokines and lymphocytes, with erosion often observed correlating with complete clearance. The objective was to determine the cellular immune response early in the course of treatment in order to examine whether cell mediated immunity could be responsible for imiquimod mediated regression of BCC. Sixteen adults with clinically diagnosed BCC were openly assigned to 5 days per week of drug (1, 2 or 4 weeks) or placebo (2 weeks) in groups of four. No baseline biopsy was performed. Post-treatment excision specimens were examined by routine and immunohistochemical staining. Treatment was associated with the early appearance of CD4 cells, activated dendritic cells and macrophages, with later infiltration by CD8 T cells. Dendritic cells continually increased with time, while macrophages reached a maximum at 1 week and then declined slightly. There were comparatively few neutrophils or gammadelta T cells. Early infiltrates were most prominent in the tumour and upper dermis. The results are consistent with a cell mediated immune response being responsible for the clearance of the BCC. Several immune-mediated tumour destruction mechanisms are likely to be involved.

Prognosis in DNA mismatch repair deficient colorectal cancer: are all MSI tumours equivalent?

Microsatellite instability (MSI) in colorectal tumours is the hallmark of defective DNA mismatch repair (MMR) and high level MSI can be detected in up to 15% of incident colorectal cancers. MSI in sporadic colorectal tumours is primarily due to epigenetic silencing of MLH1 while MSI is almost universal in tumours from HNPCC family members due to germline MMR gene mutation with loss or mutational inactivation of the second copy as a somatic event. There is evidence that tumour MSI is associated with a better outcome than the generality of large bowel malignancy. However, although MSI occurs in both sporadic colorectal cancer and in tumours arising in patients with germline MMR gene mutations, cancer survival should not be considered to be equivalent for these two groups with MSI tumours simply because both exhibit similarities in molecular phenotype. Here, we review the evidence on prognosis in patients with sporadic MSI tumours compared to those who have inherited a germline DNA MMR repair gene defect. In addition, we explore whether there are variables that afford opportunity to distinguish three groups on the basis of MSI status, namely: sporadic MSI tumours; MSI tumours in carriers of germline MMR gene defects; microsatellite stable (MSS) tumours. Differences in prognosis between these three groups is important because it underpins the rationale for surveillance and early identification of tumours in MMR gene carriers, as well as refining understanding of the influence of MSI on cancer progression. Furthermore, we discuss the effect of MSI on the effectiveness of chemotherapy regimens.

Increased Fas ligand expression by T cells and tumour cells in the progression of actinic keratosis to squamous cell carcinoma.

BACKGROUND: In the counterattack model of tumorigenesis, it has been proposed that tumours develop resistance to attack from Fas ligand (FasL)-expressing cytotoxic T cells by downregulating Fas (immune escape), while at the same time upregulating FasL expression to induce apoptosis in Fas-expressing T cells (counterattack). OBJECTIVES: The aim of this study was to examine Fas and FasL expression on tumour cells and infiltrating T cells during the progression of actinic keratoses (AK), the benign precursor lesion, to squamous cell carcinoma (SCC). PATIENTS AND METHODS: Samples of AK (n = 20) and SCC (n = 20) were collected from immunocompetent patients attending dermatology clinics. Double-label immunohistochemistry was performed on frozen sections using mouse monoclonal antibodies to Fas or FasL, simultaneously with a rabbit polyclonal antibody to either CD3 or cytokeratin, markers of T cells and keratinocytes, respectively. Cell densities and the optical density of tumour Fas expression were measured using image analysis. RESULTS: FasL-expressing T cells were observed in nine of 19 SCCs, compared with three of 20 AKs (P < 0.05). FasL-expressing tumour cells were found in nine of 18 SCCs, compared with only one of 20 AK specimens (P < 0.005). There was no difference in the number of Fas-expressing T cells infiltrating AK and SCC. Fas expression by keratinocytes, measured by optical density, was lower in SCC (range 0.1-40, median 17) compared with AK (range 4-62, median 25) (P < 0.05). CONCLUSIONS: These results suggest that the greater numbers of FasL-expressing T cells infiltrating into SCC compared with AK are targeting Fas-expressing tumour cells. As AK cells progress to SCC, they subvert this T-cell-mediated killing of tumour cells by downregulating their Fas expression (immune escape). Furthermore, tumour cells upregulate their expression of FasL, possibly as a counterattack measure to induce apoptosis in the increased number of tumour-infiltrating T cells. Thus changes in Fas/FasL-mediated interactions between T cells and tumour cells occur during the progression of AK into SCC.

Does solitary morphoea profunda progress?

Solitary morphoea profunda (SMP) is an unusual form of scleroderma and is rarely mentioned in the literature. The back of the trunk is described as the commonest site of involvement by SMP. This disease has been recognized as a nonprogressive condition. We report three cases of SMP seen at our department within a 1-year period. Interestingly, all three patients were females and the lesions were situated on the right upper buttock. In one patient the lesion extended despite using topical tacrolimus but subsequently the lesion was kept under control with topical clobetasol propionate.

Mechanisms in cutaneous drug hypersensitivity reactions.

Up to 3% of all hospital admissions are due to adverse drug reactions (ADRs), and between 10% and 20% of hospital inpatients develop ADRs. Individual susceptibility to becoming 'sensitized' or allergic to a drug is thought to result from altered metabolic handling of the drug. Reactive intermediate compounds form haptens, bind to proteins and induce immune responses. Depending on whether the immune system generates antibodies or sensitized T cells, different clinical patterns of hypersensitivity may result. At present, both in vivo or in vitro tests to identify the culprit drug or to confirm the presence of hypersensitivity are not widely used because they are either not generally robust or not readily accessible. In vitro tests require the true immunogen/antigen to detect antibodies or sensitized T cells. As the metabolic basis underlying susceptibility to adverse drug reactions is elucidated, the resolution of immunological mechanisms and development of reliable tests will ensue. This will also become of great value for prediction of individuals at risk of becoming sensitized by a particular drug.

A novel technique for the examination of skin biopsies by laser capture microdissection.

BACKGROUND: Skin is an inherently heterogeneous tissue, thus the procurement of pure cell populations is critical for the accurate correlation of a molecular profile to a particular cell type or histological location. Laser Capture Microdissection (LCM) permits the efficient procurement of cells and mapping of genetic changes from histologically prepared samples. METHODS: This paper describes a robust LCM protocol established in our laboratory for the extraction of high quality DNA which sequenced from 100% of microdissected samples without the need for cloning. The unique properties of skin, in particular its strong intercellular adhesive forces, have dictated a significant modification to the normal procedure of tissue preparation to ensure reliable cell procurement. RESULTS: Using the methods outlined below we were able to precisely map the pattern of genomic mutations in our target gene of interest in normal skin, actinic keratosis and squamous cell carcinoma. CONCLUSIONS: The capability to select pure cell populations from the skin will revolutionise our ability to understand the processes involved in cutaneous tumourigenesis.

Inflammation is associated with progression of actinic keratoses to squamous cell carcinomas in humans.

BACKGROUND: Squamous cell carcinoma (SCC) is a common skin tumour that may metastasize and lead to death. We have observed that before actinic keratoses (AK) progress to SCCs they may become tender and inflamed. In some of these, histological examination shows that they are, in fact, SCCs. OBJECTIVES: To study the progression of AK to SCCs. METHODS: We studied skin tumours from 50 patients with either asymptomatic AK, inflamed AK or SCCs, using immunocytochemistry. The diagnosis of each tumour was confirmed by histological examination. RESULTS: Studies of differentiation using heat shock protein 27 showed a stepwise loss of differentiation as the tumours progressed from asymptomatic AK, through inflamed AK to SCCs. During the inflamed AK phase, there was a marked increase in T lymphocytes and Langerhans cells: the number of infiltrating cells diminished as progression to SCC occurred. There was an increase in immunoreactive p53 and the apoptosis inhibitor bcl-2 as tumours progressed from AK to SCCs, and a decrease in Fas and Fas ligand. CONCLUSIONS: These studies have shown that progression from benign to malignant tumours may be associated with an inflammatory response, which appears to drive malignant conversion, but subsides rapidly following this conversion.

Effects of low-dose ultraviolet radiation on in vivo human cutaneous recall responses.

Relatively few studies have examined the effects of low-dose ultraviolet (UV) radiation on in vivo human cutaneous immunity, or the ability of sunscreens to prevent UV-induced immunosuppression. We have studied the effects of solar-simulated UV radiation on nickel contact hypersensitivity (CHS) in nickel-allergic volunteers, and on delayed type hypersensitivity responses in Mantoux-positive volunteers. Nickel CHS and Mantoux responses were significantly suppressed by acute, suberythemal UV exposures equivalent to less than 8 min summer sunlight. Both UVA and UVB wavebands were immunosuppressive, but UVA-induced immunosuppression was transient, whereas UVB had a more sustained effect. Dose-responses for UV immunosuppression were determined using the nickel method, enabling calculation of in vivo sunscreen immune protection factors in a manner analogous with sun protection factor measurement. Sunscreens were found to confer significantly less protection against UV-induced immunosuppression than against UV-induced erythema.

Treatment of refractory hand warts by isolated limb infusion with melphalan and actinomycin D.

We present an immunocompetent man with extensive warts on the hands, refractory to a number of conventional treatment modalities and causing substantial morbidity and impairment of normal function. Isolated limb infusion (regional intra-arterial chemotherapy) with melphalan and actinomycin D was performed, with substantial clearing of the warts within 2 months. Treatment-induced morbidity was limited to mild local erythema and oedema which resolved within 3 weeks. After 9 months' follow up, the patient had only a few residual warts and was able to resume normal activities.

Mutations associated with microsatellite unstable colorectal carcinomas exhibit widespread intratumoral heterogeneity.

Although microsatellite instability (MSI) has been shown to be present in 15% of sporadic colorectal carcinomas, the genetic events underlying the development of these tumors have not been well described. By investigating intratumoral heterogeneity, this study attempts to elucidate whether MSI-positive colorectal carcinomas develop as the result of a random accumulation of mutations or as an ordered, stepwise sequence of genetic alterations. Eighty-six regions from 16 MSI-positive sporadic colorectal carcinomas were examined for mutations in repeat nucleotide sequences of the tumour suppressor genes transforming growth factor beta type II receptor (TGFBRII), insulin-like growth factor II receptor (IGFIIR), and BAX, and the mismatch repair genes MSH3 and MSH6. At least 2 and up to 5 of these genes were mutated in each tumour, and widespread intratumoral heterogeneity was observed for each gene. Regions of tumour with TGFBRII mutations were correlated with a poorly differentiated histology. Unlike the situation in microsatellite stable colorectal carcinomas, the findings of the present study did not suggest that a particular sequence of tumour suppressor and mismatch repair genes are mutated during colorectal tumorigenesis. It seems likely that a random accumulation of mutations, as a result of a defect in the mismatch repair pathway, drives tumour progression in this type of colorectal carcinoma.

Cytokine profiles in spontaneously regressing basal cell carcinomas.

BACKGROUND: Basal cell carcinomas (BCCs) can cause considerable morbidity due to their ability to enlarge progressively and to destroy underlying tissues. However, some BCCs may undergo spontaneous regression in the absence of therapy capable of inducing antineoplastic effects. Histological criteria for this process have been described, and previous studies have suggested that it may be mediated by infiltrating activated CD4-positive T cells. OBJECTIVES: The purpose of this study was to compare the expression of cytokines in actively regressing and non-regressing BCCs, to ascertain if active regression is associated with a particular cytokine profile. METHODS: Reverse transcriptase-polymerase chain reaction, a sensitive, quantitative technique allowing analysis of multiple cytokines from small tumour samples, was used. RESULTS: Interferon (IFN)-gamma was significantly elevated in actively regressing BCCs compared with non-regressing BCCs. Furthermore, interleukin (IL)-2, tumour necrosis factor (TNF)-beta and CD3 delta tended to be elevated in actively regressing tumours, although not to statistically significant levels. IFN-gamma, IL-2, IL-10, TNF-beta, granulocyte-macrophage colony-stimulating factor and Fas ligand showed strong positive correlations with CD3 delta, indicating an association between infiltrating T cells and these cytokines. CONCLUSIONS: These findings support a role for T-helper 1 type cytokines in mediating spontaneous regression of BCCs.