Abstract: Basal cell carcinoma (BCC) is characterized by slow growth, virtual absence of metastases, and strong stroma-dependency. Cancer-associated fibroblasts (CAFs) in the tumor stroma influence tumor growth, invasion, and metastasis. To comprehensively characterize CAFs of BCC in their in situ cancer environment, laser capture microdissection, linear gene amplification, microarray analysis, and quantitative real-time PCR (qRT-PCR) were combined. Pair-wise comparison of gene expression of microdissected CAFs and corresponding normal perifollicular fibroblasts identified 65 genes that were significantly upregulated in at least two of three different patients. Among the annotated genes, as many as 13 genes encoded secreted proteins, of which six were previously implicated as CAF-associated proteins in various tumor types. Four of the seven novel CAF genes--matrix Gla-protein, secreted frizzled-related protein 2, angiopoietin-related protein-2, and platelet-derived growth factor receptor-like protein--were selected for further analyses by qRT-PCR and were found to be frequently upregulated in CAFs of three independent BCC tissues. Analyses of CAFs from squamous cell cancer, prostate cancer, and colon cancer did not indicate that these genes were upregulated in these cancers. This study thus validates a novel approach for comprehensive characterization CAFs in their in situ environment of BCC. The results suggest a specific expression profile of CAFs in BCC possibly accounting for disease-specific pathological roles.

Abstract: Here we report the development of a publicly available web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed towards human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas, to discover potential protein biomarkers. Such biomarkers include tissue specific markers, cell type specific markers, tumor type specific markers, markers of malignancy and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high-resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies employing more classical proteomics tools.

Abstract: RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.

Abstract: Completion of the Human Genome Project and recent developments in proteomics make it possible to systematically generate affinity reagents to a large portion of the proteome. Recently an antibody-based human protein atlas covering many organs including four areas of the brain has been released (www.proteinatlas.org). Due to the heterogeneity, size, and availability of tissue a more thorough analysis of the human brain is associated with considerable difficulties. Here we applied 120 antibodies raised against 112 human gene products to the smaller rat brain, a rodent animal model, where a single section represents a 'superarray' including many brain areas, and consequently allowing analysis of a huge number of cell types and their neurochemicals. Immunoreactive structures were seen in the investigated brain tissue after incubation with 56 antibodies (46.6%), of which 25 (20.8%) showed a clearly discrete staining pattern that was limited to certain areas, or subsets of brain cells. Bioinformatics, pre-adsorption tests and Western blot analysis were applied to identify non-specific antibodies. Eleven antibodies, including such raised against four 'ambiguous' proteins, passed all validation criteria, and the expression pattern and subcellular distribution of these proteins were studied in detail. To further explore the potential of the systematically generated antibodies, all 11 antibodies that passed validation were used to analyze the spinal cord and lumbar dorsal root ganglia after unilateral transection of the sciatic nerve. Discrete staining patterns were observed for four of the proteins, and injury-induced regulation was found for one of them. In conclusion, the study presented here suggests that a significant portion (10%) of the antibodies generated to a human protein can be used to analyze orthologues present in the rodent brain and to produce a protein-based atlas of the rodent brain. It is hoped that this type of antibody-based, high throughput screening of brain tissue from various rodent disease models will provide new information on the brain chemical neuroanatomy and insights in processes underlying neurological pathologies.

Abstract: In a previously published insertional mutagenesis screen for candidate brain tumor genes in the mouse using a Moloney mouse leukemia virus encoding platelet-derived growth factor (PDGF)-B, the Sox10 gene was tagged in five independent tumors. The proviral integrations suggest an enhancer effect on Sox10. All Moloney murine leukemia virus/PDGFB tumors had a high protein expression of Sox10 independently of malignant grade or tumor type. To investigate the role of Sox10 in gliomagenesis, we used the RCAS/tv-a mouse model in which the expression of retroviral-encoded genes can be directed to glial progenitor cells (Ntv-a mice). Both Ntv-a transgenic mice, wild-type, and Ntv-a p19Arf null mice were injected with RCAS-SOX10 alone or in combination with RCAS-PDGFB. Infection with RCAS-SOX10 alone did not induce any gliomas. Combined infection of RCAS-SOX10 and RCAS-PDGFB in wild-type Ntv-a mice yielded a tumor frequency of 12%, and in Ntv-a Arf-/- mice the tumor frequency was 30%. This indicates that Sox10 alone is not sufficient to induce gliomagenesis but acts synergistically with PDGFB in glioma development. All induced tumors displayed characteristics of PNET-like structures and oligodendroglioma. The tumors had a strong and widely distributed expression of Sox10 and PDGFR-alpha. We investigated the expression of Sox10 in other human tumors and in a number of gliomas. The Sox10 expression was restricted to gliomas and melanomas. All glioma types expressed Sox10, and tumors of low-grade glioma had a much broader distribution of Sox10 compared with high-grade gliomas.

Abstract: Advances in antibody production render a growing supply of affinity reagents for immunohistochemistry (IHC), and tissue microarray (TMA) technologies facilitate simultaneous analysis of protein expression in a multitude of tissues. However, collecting validated IHC data remains a bottleneck problem, as the standard method is manual microscopical analysis. Here we present a high-throughput strategy combining IHC on a recently developed cell microarray with a novel, automated image-analysis application (TMAx). The software was evaluated on 200 digital images of IHC-stained cell spots, by comparing TMAx annotation with manual annotation performed by seven human experts. A high concordance between automated and manual annotation of staining intensity and fraction of IHC-positive cells was found. In a limited study, we also investigated the possibility to assess the correlation between mRNA and protein levels, by using TMAx output results for relative protein quantification and quantitative real-time PCR for the quantification of corresponding transcript levels. In conclusion, automated analysis of immunohistochemically stained in vitro-cultured cells in a microarray format can be used for high-throughput protein profiling, and extraction of RNA from the same cell lines provides a basis for comparing transcription and protein expression on a global scale.

Abstract: Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy for which better treatment strategies are needed. To identify potential diagnostic and therapeutic targets, a signature consisting of MCL-associated genes was selected based on a comprehensive gene expression analysis of malignant and normal B cells. The corresponding protein epitope signature tags were identified and used to raise monospecific, polyclonal antibodies, which were subsequently analyzed on paraffin-embedded sections of malignant and normal tissue. In this study, we demonstrate that the initial selection strategy of MCL-associated genes successfully allows identification of protein antigens either uniquely expressed or overexpressed in MCL compared with normal lymphoid tissues. We propose that genome-based, affinity proteomics, using protein epitope signature tag-induced antibodies, is an efficient way to rapidly identify a number of disease-associated protein candidates of both previously known and unknown identities.

Abstract: Molecular tools for tissue profiling, such as expression microarrays and real-time PCR, generally require collection of fresh frozen tissues as sources of high-quality RNA. The fragile nature of RNA prompted us to examine the effects of storage time and transport conditions with regard to RNA integrity and gene expression in nonfixed surgical human specimens. At surgery, fresh normal tonsil and colon tissue was cut into pieces and snap frozen. Additional fresh tissue pieces were (i) left at room temperature, (ii) kept on ice, (iii) in normal saline or (iv) in a commercial RNA-stabilizing buffer (RNAlater) and snap frozen after 0.5, 1, 3, 6 and 16 h. Structural RNA integrity was analysed by microchip electrophoresis. Surprisingly, RNA remained stable in both tissue types under all conditions tested for up to 6-16 h. Gene expression by real-time PCR of cfos, HIF1alpha, Bcl2, PCNA, TGFbeta1 and SMAD7 was analysed at different storage time points in tonsil tissue. Expression levels were essentially stable when samples were kept on ice, while marked regulation of single genes was observed during storage at room temperature, in normal saline and in RNAlater. Furthermore, we analysed selected tissue types from the local biobank representing 47 normal and malignant tissues transported on ice for up to 2-3 h before biobanking. RNA prepared from 45 of the 47 samples exhibited distinct ribosomal peaks indicating intact RNA. This study shows that RNA degradation is a minor problem during handling of fresh human tissue before biobanking. Our data indicate that nonfixed tissue specimens may be transported on ice for hours without any major influence on RNA quality and expression of the selected genes. However, further studies are warranted to clarify the impact of transport logistics on global gene expression.

Abstract: Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.

Abstract: The production of IL-8 can be induced by LPS via TLR4 signaling pathway. In this study, we tested the effect of a herbal melanin (HM) extract, from black cumin seeds (Nigella sativa L.), on IL-8 production. We used HM and LPS in parallel to induce IL-8 production by THP-I, PBMCs, and TLR4-transfected HEK293 cells. Both HM and LPS induced IL-8 mRNA expression and protein production in THP-1 and PBMCs. On applying similar treatment to HEK293 cells that express TLR4, MD2, and CD14, both HM and LPS significantly induced IL-8 protein production. We have also demonstrated that HM and LPS had identical effects in terms of IL-8 stimulation by HEK293 transfected with either TLR4 or MD2-CD14. Melanin extracted from N. sativa L. mimics the action of LPS in the induction of IL-8 by PBMC and the other used cell lines. Our results suggest that HM may share a signaling pathway with LPS that involves TLR4.

Abstract: BACKGROUND: The PTCH tumour suppressor gene is involved in the development of nearly all basal cell carcinomas (BCCs) of the skin and a fraction of squamous cell carcinomas (SCCs). A nonconservative Pro/Leu nucleotide polymorphism within PTCH exon 23 at codon 1315 was recently reported to be potentially important for the development of breast epithelial cell cancers. Objectives Accordingly, the status of PTCH codon 1315 was analysed for a possible association with the development of nonmelanoma skin cancers (NMSCs) in a pilot study. Because skin cancer risk is affected by specific population-dependent phenotypes such as skin and hair colour, codon 1315 was also analysed for normal allele frequency variation in human populations having differing extents of eumelanin vs. phaeomelanin. METHODS: The single nucleotide polymorphism in codon 1315 of the human PTCH gene was analysed in genomic DNA from six different populations comprising 472 blood samples and from 170 patients in four different categories with NMSC. Polymerase chain reaction and pyrosequencing were used to determine the allele frequencies. Allelic loss was furthermore determined in tumours following microdissection. RESULTS: The Pro/Pro genotype frequency ranged from 30% to 65% between populations, with a significant trend for a reduced frequency of the Pro/Pro genotype in populations having lighter pigmentation (P = 0.020). Pro/Pro frequency showed an increasing trend with increasing tumour case severity (P = 0.027). In 260 samples from 180 Swedish patients with NMSC and a control group of 96 healthy ethnically matched volunteers, no statistically significant pairwise differences between groups were detected in the PTCH codon 1315 allelic distribution, neither was a difference seen for multiple or early onset cases of BCC in the Swedish population. In Swedish patients with single tumours, allelic loss (loss of heterozygosity) was observed in 20 of 30 (67%) patients with BCC and four of 22 (18%) patients with SCC, with no preference in the allele lost. In contrast, the Pro/Pro genotype was frequent in seven U.S. patients having multiple independent BCCs. One of these patients was heterozygous, enabling allelic loss studies. Of 20 independent tumours, 11 had lost an allele; 10 of the 11 had lost Leu, suggesting nonrandom loss that favoured retention of Pro (P = 0.0059). CONCLUSIONS: Our results indicate an association between the eumelanin-to-phaeomelanin shift and a shift from the Pro/Pro genotype to Leu-containing genotypes. Failure to lose Pro during the shift to phaeomelanin may be associated with an increased population risk for BCC and increased individual risk for multiple BCC. During development of a tumour, the effect of Pro may be magnified by loss of the Leu allele.

Abstract: Carcinogenesis is a multi-step series of somatic genetic events. The complexity of this multi-hit process makes it difficult to determine each single event and the definitive outcome of such events. To investigate the genetic alterations in cancer-related genes, sensitive and reliable detection methods are of major importance for generating relevant results. Another critical issue is the quality of starting material which largely affects the outcome of the analysis. Microdissection of cells defined under the microscope ensures a selection of representative material for subsequent genetic analysis. Skin cancer provides an advantageous model for studying the development of cancer. Detectable lesions occur early during tumor progression, facilitating molecular analysis of the cell populations from both preneoplastic and neoplastic lesions. Alterations of the p53 tumor suppressor gene are very common in non-melanoma skin cancer, and dysregulation of p53 pathways appear to be an early event in the tumor development. A high frequency of epidermal p53 clones has been detected in chronically sun-exposed skin. The abundance of clones containing p53 mutated keratinocytes adjacent to basal cell (BCC) and squamous cell carcinoma (SCC) suggests a role in human skin carcinogenesis. Studies using p53 mutations as a clonality marker have suggested a direct link between actinic keratosis, SCC in situ and invasive SCC. Microdissection-based studies have also shown that different parts of individual BCC tumors can share a common p53 mutation yet differ with respect to additional alterations within the p53 gene, consistent with subclonal development within tumors. Here, we present examples of using well-defined cell populations, including single cells, from complex tissue in combination with molecular tools to reveal features involved in skin carcinogenesis.

Abstract: Here, we describe the use of antibody-based proteomics involving the generation of protein-specific antibodies to functionally explore the human proteome. The antibodies can be used for analysis of corresponding proteins in a wide range of assay platforms, including i) immunohistochemistry for detailed tissue profiling, ii) specific affinity reagents for various functional protein assays, and iii) capture ("pull-down") reagents for purification of specific proteins and their associated complexes for structural and biochemical analyses. In this review, the use of antibodies for such analysis will be discussed with focus on the possibility to create a descriptive and comprehensive protein atlas for tissue distribution and subcellular localization of human proteins in both normal and disease tissues.

Abstract: Human basal cell cancer (BCC) shows unique growth characteristics, including a virtual inability to metastasize, absence of a precursor stage and lack of tumour progression. The clonal nature of BCC has long been a subject for debate because of the tumour growth pattern. Despite a morphologically multifocal appearance, genetic analysis and three-dimensional reconstructions of tumours have favoured a unicellular origin. We have utilized the X-chromosome inactivation assay in order to examine clonality in 13 cases of BCC. Four parts of each individual tumour plus isolated samples of stroma were analysed following laser-assisted microdissection. In 12/13 tumours, the epithelial component of the tumour showed a monoclonal pattern suggesting a unicellular origin. Surprisingly, one tumour showed evidence of being composed of at least two non-related monoclonal clones. This finding was supported by the analysis of the ptch and p53 gene. Clonality analysis of tumour stroma showed both mono- and polyclonal patterns. A prerequisite for this assay is that the extent of skewing is determined and compensated for in each case. Owing to the mosaic pattern of normal human epidermis, accurate coefficients are difficult to obtain; we, therefore, performed all analyses both with and without considering skewing. This study concludes that BCC are monoclonal neoplastic growths of epithelial cells, embedded in a connective tissue stroma at least in part of polyclonal origin. The study results show that what appears to be one tumour may occasionally constitute two or more independent tumours intermingled or adjacent to each other, possibly reflecting a local predisposition to malignant transformation.

Abstract: Laser-assisted microdissection has enabled the collection of morphologically defined cell populations from a tissue section. The PALM Robot MicroBeam laser microdissection system provides a robust system for the retrieval of specified cells (including single cells). Due to the fragile nature of DNA, and in particular RNA, robust protocols are required to obtain reliable data from a limited number of cells (1-10.000 cells). This chapter describes the application of the PALM MicroBeam system to isolate RNA and DNA from cells in a complex tissue for subsequent molecular analysis. Protocols for successful analysis of RNA from 500 to 1000 cells, including steps to produce cDNA for subsequent polymerase chain reaction analysis, are given. The cDNA could also be used as a template for linear amplification in order to perform gene array analysis. Furthermore, a protocol for genomic analysis of p53 mutations from single cells is given. The described procedures emphasize preparation of tissue, laser microdissection including catapulting of cells, and extraction of RNA and DNA. Downstream experiments for validation are also shown.

Abstract: Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Abstract: A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

Abstract: We present a detailed in vivo characterization of hepatocyte transcriptional regulation in HepG2 cells, using chromatin immunoprecipitation and detection on PCR fragment-based genomic tiling path arrays covering the encyclopedia of DNA element (ENCODE) regions. Our data suggest that HNF-4alpha and HNF-3beta, which were commonly bound to distal regulatory elements, may cooperate in the regulation of a large fraction of the liver transcriptome and that both HNF-4alpha and USF1 may promote H3 acetylation to many of their targets. Importantly, bioinformatic analysis of the sequences bound by each transcription factor (TF) shows an over-representation of motifs highly similar to the in vitro established consensus sequences. On the basis of these data, we have inferred tentative binding sites at base pair resolution. Some of these sites have been previously found by in vitro analysis and some were verified in vitro in this study. Our data suggests that a similar approach could be used for the in vivo characterization of all predicted/uncharacterized TF and that the analysis could be scaled to the whole genome.

Abstract: Laser microdissection and pressure catapulting has become a powerful tool to obtain homogeneous cell populations from tissue samples in nearly all fields of biomedical research. The isolated cells can be subsequently used for the analysis of proteins, DNA or RNA. However, the method requires physical access to the tissue surface and the sections therefore need to be air-dried and uncovered. The consequence is poor morphology, which severely reduces the potential of the technique, especially in non-homogeneous tissues or tissues with infiltrating immune cells. To overcome this limitation, a fluid cover medium was developed and the effects on frozen and paraffin wax-embedded tissue morphology were evaluated. The cover medium improved the morphology such that it was almost comparable to sections overlaid with glass coverslips. Moreover, the laser microdissection procedure was facilitated, since the medium allowed larger areas of tissues to be laser pressure-catapulted. Neither the isolation of proteins nor the extraction of genomic DNA was adversely affected by the use of the fluid cover medium. No significant differences in RNA quantity and integrity were detected by TaqMan real-time PCR for GAPDH, and microchip electrophoresis, between covered and uncovered tissue sections. In conclusion, this method provides considerably improved morphology for laser microdissection and pressure catapulting techniques without affecting RNA-dependent downstream applications. This not only facilitates established procedures, but will also extend the application to tissues that require superior morphological resolution.

Abstract: BACKGROUND: It is well accepted that ultraviolet radiation from the sun can induce and promote growth of skin tumours. Skin cancer develops as a consequence of multiple genetic hits, where an initial, important step includes proliferation of cells susceptible to malignant transformation. Foci of morphologically normal epidermal keratinocytes overexpressing p53 protein are common in chronically sun-exposed skin. Such foci have previously been shown to represent expanding clones of p53-mutated keratinocytes. Although several characteristics concerning epidermal p53 clones remain to be resolved, an important role in skin carcinogenesis is anticipated. The density of epidermal p53 clones in human skin is largely unknown. OBJECTIVES: To compare the occurrence of epidermal p53 clones in skin surrounding cancers with that in skin surrounding benign melanocytic naevi. To assess the influence of age on frequency and size of epidermal p53 clones in human facial skin. METHODS: We have analysed the number and sizes of epidermal p53 clones in skin specimens from patients with squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and benign melanocytic naevi. Cases included normal facial skin from four different age groups. Tissue sections were immunohistochemically stained and the presence of p53 clones was recorded. Approximately 1.4 m of epidermis from a total of 112 biopsies was analysed. RESULTS: We found 128 epidermal p53 clones in biopsy specimens from 112 patients. The results showed that the number and size of p53 clones increase with age. In normal skin adjacent to SCC p53 clones were significantly more numerous and greater in size in comparison with those in normal skin both adjacent to benign naevi and adjacent to BCC. Interestingly, normal skin in the close vicinity of BCC and melanocytic naevi showed similar results regarding both number and size of epidermal p53 clones. CONCLUSIONS: Our findings suggest a connection between development of epidermal p53 clones and SCC.

Abstract: The arginine variant of the p53 codon 72 polymorphism as well as anogenital and epidermodysplasia verruciformis (EV) types of human papilloma virus (HPV) are suggested to confer increased risk for developing cutaneous squamous cell carcinoma (SCC). In this pilot study, we analysed the p53 codon 72 genotype distribution in 106 microdissected samples from normal and tumour tissues of 53 cases of cutaneous SCC and 96 controls from Sweden. Both normal and tumour samples from cases of SCC were screened for anogenital and EV HPV. The p53Arg allele was not associated with the development of cutaneous SCC. Anogenital HPV (44%) was more prevalent than EV HPV (12%). Data also indicate that anogenital HPV is more common in tumour samples, but HPV infection was not identified as a significant risk factor for developing SCC. The presence of anogenital HPV, but not EV HPV might be a risk factor for development of cutaneous SCC.

Abstract:

Abstract: Foci of normal keratinocytes overexpressing p53 protein are frequently found in normal human skin. Such epidermal p53 clones are common in chronically sun-exposed skin and have been suggested to play a role in skin cancer development. In the present study, we have analyzed the prevalence of p53 mutations in epidermal p53 clones from normal skin surrounding basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Using laser-assisted microdissection, 37 epidermal p53 clones adjacent to BCC (21) and SCC (16) were collected. Genetic analysis was performed using a multiplex/nested polymerase chain reaction followed by direct DNA sequencing of p53 exons 2-11. In total, 21 of 37 analyzed p53 clones consisted of p53-mutated keratinocytes. The identified mutations were located in p53 exons 4-8, corresponding to the sequence-specific DNA-binding domain. All mutations were missense, and 78% displayed a typical ultraviolet signature. The frequency of p53 mutations was similar in skin adjacent to BCC compared to SCC. The presented data confirm and extend previous knowledge on the genetic background of epidermal p53 clones. The mutation spectra found in epidermal p53 clones resemble that of non-melanoma skin cancer. Approximately, 40% of the epidermal p53 clones lacked an underlying p53 mutation, suggesting that other genetic events in genes up- or downstream of the p53 gene can generate foci of normal keratinocytes overexpressing p53 protein.

Abstract: Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.

Abstract: Advantageous preservation of histology and detailed cellular morphology has rendered neutral buffered formalin (NBF) the most widely used fixative in clinical pathology. Despite excellent morphology for routine diagnostics, a major drawback of NBF fixation is its detrimental effect on DNA and RNA quality. In addition to complicating analysis of genes and transcripts in complex tissues, NBF denatures proteins and thereby hampers immunohistochemical visualization of certain antigens. In the present study, we evaluated a zinc-based fixative (ZBF) regarding its effects on tissue morphology, quality of genomic DNA, and preservation of protein immunoreactivity in a broad spectrum of tissues. Four different modes of fixation were analyzed: ZBF-paraffin embedding, NBF-paraffin embedding, ZBF-fixation prior to snap-freezing, and immediate snap-freezing. Laser-assisted microdissection, allowing retrieval of a defined number of cells for PCR, was used to study DNA quality. Genomic DNA was analyzed using primers for beta2-microglobulin and the transferrin receptor. Immunohistochemistry was performed using nine antibodies. Tissue microarray blocks were used for analysis of morphology and immunoreactivity. Only slight impairment of morphologic qualities was found after ZBF-paraffin embedding, whereas ZBF prior to freezing resulted in a more crisp morphology compared with routine cryosections. A significantly higher DNA yield was observed in samples isolated from ZBF-paraffin-embedded tissues compared with NBF-paraffin-embedded tissues. Both yield and quality of DNA was comparable in frozen tissues irrespective to prior ZBF fixation. Immunoreactivity in paraffin-embedded tissue was superior in ZBF-fixated tissue compared with NBF-fixated for a majority of tested antibodies. Furthermore, for seven out of nine antibodies, antigen retrieval pretreatment proved unnecessary in ZBF-fixated tissue. Thus, despite a slight impairment of morphology, ZBF preserves protein structures well. We conclude that ZBF is superior to NBF for analysis of DNA and protein expression. Fixation of tissues in ZBF may also be an alternative strategy to freeze storage of tissue specimens, eg, in future bio-banks.

Abstract: The p53 protein plays a key role in protecting cells from acquiring manifest mutations by inducing cell cycle arrest or apoptosis. The mechanisms for differences in epidermal responses to ultraviolet irradiation are unclear, although they have been shown to be related to both genetic events and environmental factors. In this study, we compared epidermal ultraviolet responses in chronically sun-exposed and non-sun-exposed skin using immunohistochemistry with antibodies recognizing thymine dimers and p53 protein. Six healthy volunteers were subjected to both artificial ultraviolet irradiation and natural sunlight, with and without photoprotection. A smaller number of thymine dimer-positive keratinocytes were detected 24 h after ultraviolet exposure in chronically sun-exposed skin compared to non-sun-exposed skin. Further, the p53 response was more variable in chronically sun-exposed skin. A significant correlation between total ultraviolet dose and number of p53-immunoreactive keratinocytes was found after natural sun exposure. Our findings suggest that repair of DNA damage is more efficient in chronically sun-exposed skin than in non-sun-exposed skin.

Abstract: Recombinant immunoconjugates constitute a novel class of immunoassay reagents produced by genetic fusion between an antigen recognizing moiety and a reporter enzyme or fluorescent protein, obviating the need for chemical coupling. In this work, we describe the construction, Escherichia coli production and characterization of recombinant beta-galactosidase (beta-gal)-based immunoconjugates directed to human immunoglobulin A (IgA). As the antigen recognizing moieties, either monovalent or dimeric (head-to-tail) versions of an IgA-specific affibody (Z(IgA1)) were used, previously selected in vitro from a protein library based on combinatorial engineering of a single staphylococcal protein A domain. To increase the likelihood of proper presentation on the assembled homotetrameric enzyme surface, the affibody moieties were linked to the N-terminus of the enzyme subunits via a heptapeptide linker sequence. The two resulting immunoconjugates Z(IgA1)-beta-gal and (Z(IgA1))(2)-beta-gal, containing four and eight affibody moieties per enzyme, respectively, could be expressed as soluble and proteolytically stable proteins intracellularly in E. coli from where they were purified to high purity by a single anion exchange chromatography step. The yields of immunoconjugates were in the range 200-400 mg/l culture. Biosensor-binding studies showed that both the Z(IgA1)-beta-gal and (Z(IgA1))(2)-beta-gal immunoconjugates were capable of selective IgA-recognition, but with an apparent higher binding affinity for the variant containing divalent affibody moieties, presumably due to avidity effects. The applicability of this class of recombinant immunoconjugates was demonstrated by IgA detection in enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. In addition, using human kidney biopsy samples from a nephropathy patient, IgA depositions in glomeruli could be detected by immunohistochemistry with low background staining of tissue.

Abstract: Ultraviolet radiation (UVR) plays an important role in the development of non-melanoma skin cancer. Most tumors develop in chronically sun-exposed skin, most often in cosmetically sensitive locations, where in vivo experiments may be difficult to perform. In this study, we describe a skin organ culture model with preserved normal morphology and intact response to UVR. Skin explants from chronically sun-exposed and non-sun-exposed skin were irradiated with artificial UVA+UVB with and without topical sunscreen. UV-induced DNA damage, epidermal p53 response and repair kinetics were analyzed using immunohistochemistry. Four hours after UV-irradiation epidermal keratinocytes showed a strong immunoreactivity for thymine-dimers. Gradual repair during an incubation time resulted in few residual thymine-dimers after 48 h. Repair appeared to be more efficient in chronically sun-exposed skin compared with non-sun-exposed skin. There was also an accumulation of p53 protein in epidermal keratinocytes, peaking at 4-24 h after irradiation. Large interindividual differences with respect to formation and repair of thymine-dimers as well as induction and duration of the p53 response were observed. Skin explants treated with topical sunscreen prior to UV-irradiation showed a clear reduction of thymine-dimers and p53 expression. The epidermal UV-responses and repair kinetics in organ-cultured skin were similar to what was found in vivo. Our data suggest that organ-cultured skin provides a valuable tool for studies of UV-induced epidermal responses in chronically sun-exposed skin.

Abstract: The paper describes the use of laser-assisted microdissection to retrieve microscopically defined cell populations including single cells from tissue sections for subsequent analysis of genomic DNA and mRNA. A general background is given on the techniques available and requirements for PCR based on minute templates. Different pre-PCR approaches are briefly described and possibilities and limitations of using archival material compared to fresh frozen tissue are discussed. In the article we give one example on how we have used the PALM laser microscopy system in combination with a nested, multiplex PCR system to analyze single normal keratinocytes as well as tumor cells from a case of basal cell cancer. We found that p53 mutations are common in normal, chronically sun-exposed skin. Widespread yet common mutations in the p53 gene that were unrelated to immunoreactivity for the p53 antibody were found in tumor cells. In addition there were rare mutations in occasional tumor cells that apparently did not result in selective growth advantage. Perspectives for the future are presented and the potential of laser assisted microdissection is highlighted within the fields of cancer research, developmental studies as well as studies of inflammatory and degenerative diseases. The combination of a method that allows careful selection of defined cells with powerful micro array based techniques, provides a setting with potential to uncover pathogenic mechanisms for large variety of human diseases.

Abstract: BACKGROUND: Sun exposure is accepted as the major risk factor for developing skin cancer, the most common cancer in the western world. Ultraviolet-B (UV-B) radiation is considered the causative agent, but recently several findings suggest a role also for ultraviolet-A (UV-A) radiation. Repeated suberythemal doses of ultraviolet-A1 (UV-A1) on healthy human skin induce an increase of p53 immunoreactive cells in epidermis, which may indicate cell cycle arrest and/or occurrence of p53 mutations. METHODS: We have investigated the possible mutagenic effect of UV-A1 on skin by sequencing exons 4-11 and adjacent intron sequence of the p53 gene in immunoreactive single cells from three healthy individuals. Previously unexposed buttock skin was irradiated three times a week for 2 weeks with physiological fluences (40 J/cm2) of UV-A1. Punch biopsies were taken before and at different time-points after the exposure, and from these single p53 immunoreactive cells were isolated by using laser-assisted microdissection. RESULTS: Three mutations--all being indicative of oxidative damage and most likely related to UV-A exposure--were found among the 37 single cells from exposed skin, whereas no mutations were found in the 22 single cells taken before exposure. CONCLUSIONS: The findings indicate a mutagenic effect of low-dose UV-A1 on healthy human skin, which further demonstrates the importance of considering UV-A when taking protective measures against skin cancer.

Abstract:

Abstract: During early development of the female embryo, one X-chromosome is randomly inactivated in each cell. As a result of growth, migration, and differentiation, the adult female becomes a mosaic of cells with either the paternal or the maternal X-chromosome inactivated. It is not known what structure the X-chromosome inactivation pattern has in skin of normal individuals. We investigated normal skin from four healthy females, heterozygous for the HUMARA microsatellite on the X-chromosome. Following careful microdissection, DNA from adjacent epidermal samples consisting of approximately 35 basal keratinocytes was digested with the methylation-sensitive enzyme HpaII. The inactivated X-chromosome remained intact due to extensive methylation. The enzyme-digested DNA was amplified using polymerase chain reaction and fragments were analyzed for size. Through examination of adjacent samples and consecutive sections, we found normal human skin to be composed of a fine mosaic of tiles with either maternal or paternal X-chromosome inactivated. The sizes of these tiles were between 20 and 350 basal cells. The method described has the potential to resolve the clonal status in normal as well as pathologic conditions.

Abstract: BACKGROUND: p53 protein plays an important role in the response to DNA damage, and radiotherapy can cause radiation dermatitis. p53 and p21 levels increase in vitro when DNA is damaged by UVA, UVB, or gamma-radiation. To determine whether this response occurs in human skin and predicts the level of radiation dermatitis, we investigated levels of p53 and p21 in skin exposed to different types of radiation as part of a randomized study of women with breast cancer to evaluate topical steroid or emollient cream treatments for radiation dermatitis of their irradiated breast. METHODS: After surgery but before receiving tangential 5-mV photo-beam radiotherapy (2 Gy and 54 Gy) to the affected breast parenchyma, multiple areas on the backs of 50 women were irradiated with UVA and other areas were irradiated with UVB. Skin biopsy samples were taken from areas of normal unirradiated skin and all irradiated areas, and p53 and p21 were detected immunohistochemically. All statistical tests are two-sided. RESULTS: In skin irradiated with UVA or UVB, medians of 4.4% (range = 0%-40.5%) or 45.5% (range = 5.3%-74.6%) p53-positive keratinocytes, respectively, were observed. Radiotherapy produced medians of 31.0% (range = 0%-79.3%) p53-immunoreactive cells after 2 Gy of radiation and 83.2% (range = 37.6%-95.2%) after 54 Gy of radiation. Despite large interindividual differences in p53 response, comparable increases in epidermal p53 response were independent of the type of radiation. A correlation between p53 and p21 was also evident (r(s) =.78). In breast skin, there was no association between the p53 response and the degree of erythema (a measure of radiation dermatitis) and no statistically significant difference between treatment arms and p21/p53 responses. CONCLUSIONS: Individual responses to radiation-induced DNA damage varied widely and may be independent of the type of radiation. The epidermal p53 response does not predict the degree of radiation dermatitis.

Abstract: Conflicting results regarding the association of a polymorphism at codon 72 of the p53 tumour suppressor gene and susceptibility to develop human papilloma virus (HPV)-associated cervical cancer have been published over the last year, implicating differences in ethnic background, sample origin, sample size and/or detection assay. The material for this study was collected in the identical geographical region as for 2 previous reports with contradictory results regarding the association of codon 72 genotype with squamous cell cancer (SCC). We have used an alternative detection assay, based on pyrosequencing technology, that interrogates the variable position by the accuracy of DNA polymerase. In addition to cervical clinical specimens from SCC, HPV16- and HPV18-infected adenocarcinoma cases as well as cervical intraepithelial neoplasia (CIN) were investigated. No significant association was found between p53 codon 72 genotype and the risk to develop adenocarcinoma, SCC or CIN in the Swedish population.

Abstract: Epidermal clones of p53-mutated keratinocytes are abundant in chronically sun-exposed skin and may play an important role in early development of skin cancer. Advanced laser capture microdissection enables genetic analysis of targeted cells from tissue sections without contamination from neighboring cells. In this study p53 gene mutations were characterized in single cells from normal, chronically sun-exposed skin. Biopsies were obtained from skin subjected to daily summer sun and skin totally protected from the sun by blue denim fabric. Using laser capture microdissection, 172 single-cell samples were retrieved from four biopsies and analyzed using single-cell polymerase chain reaction and direct DNA sequencing. A total of 14 different mutations were identified in 26 of 99 keratinocytes from which the p53 gene could be amplified. Mutations displayed a typical UV signature and were detected in both scattered keratinocytes and in a small cluster of p53-immunoreactive keratinocytes. This minute epidermal p53 clone had a diameter of 10 to 15 basal cells. Two missense mutations were found in all layers of epidermis within the p53 clone. The presented data show that p53 mutations are common in normal skin and that a clone of keratinocytes with a mutated p53 gene prevailed despite 2 months of total protection from ultraviolet light.

Abstract: A cellular p53 response, DNA repair enzymes and melanin pigmentation are important strategies utilized by skin keratinocytes against impairment caused by ultraviolet radiation (UVR). In this study a double-immunofluorescence technique was used to investigate UVR-induced thymine dimers and p53 protein simultaneously. Four healthy volunteers were irradiated on both sides of their buttock skin with a single dose of solar-simulating UVR. One side was pretreated with a topical sunscreen. Biopsies from different time-points were immunostained for visualization of thymine dimers, p53 and proliferation. One single physiological dose of UVR generated widespread formation of thymine dimers throughout the epidermis 4h after irradiation. The level of thymine dimers decreased over time and was followed by a p53 response in the same cells. A late proliferative response was also found. The formation of thymine dimers, the p53 response and the late proliferative response were partially blocked by topical sunscreen. Large inter-individual differences in the kinetics of thymine dimer formation and repair as well as in the p53 response were evident in both sunscreen-protected and unprotected skin.

Abstract: It is widely accepted that disruption of the hedgehog-patched pathway is a key event in development of basal cell cancer. In addition to patched gene alterations, p53 gene mutations are also frequent in basal cell cancer. We determined loss of heterozygosity in the patched and p53 loci as well as sequencing the p53 gene in tumors both from sporadic and hereditary cases. A total of 70 microdissected samples from tumor and adjacent skin were subjected to PCR followed by fragment analysis and DNA sequencing. We found allelic loss in the patched locus in 6/8 sporadic basal cell cancer and 17/19 hereditary tumors. All sporadic and 7/20 hereditary tumors showed p53 gene mutations. Loss of heterozygosity in the p53 locus was rare in both groups. The p53 mutations detected in hereditary tumors included rare single nucleotide deletions and unusual double-base substitutions compared to the typical ultraviolet light induced missense mutations found in sporadic tumors. Careful microdissection of individual tumors revealed genetically linked subclones with different p53 and/or patched genotype providing an insight on time sequence of genetic events. The high frequency and co-existence of genetic alterations in the patched and p53 genes suggest that both these genes are important in the development of basal cell cancer.

Abstract: Cervical cancers are considered to originate from a series of pre-malignant lesions (cervical intra-epithelial neoplasia, CIN). The mechanisms behind these events are unknown. In addition to HPV infection, deletions of chromosome 3p have been found to be a frequent event in cervical cancer and likely play an important role in the transition of CIN to invasive cancer. To classify the potential role of 3p deletions in early-stage cervical carcinogenesis, we analyzed LOH of 3p in cervical precancers. Thirty cases with single or multiple CIN lesions were selected for the study, including 20 cases without and 10 cases with synchronous invasive cancers. Allelic losses on 1 or more 3p loci were recorded in 33% (3/9) of CIN II and 36% (5/14) of CIN III lesions from 20 cases without co-existing invasive cancer, whereas an increasing percentage of LOH was observed in the 10 precancerous lesions synchronous with invasive cancer, with 71% (5/7) CIN II and 76% (13/17) CIN III lesions. This result implies that 3p deletions have selective roles in early transition of pre-malignancy to invasive cancer. Comparing the LOH patterns between the 2 groups, genetic deletions in cases with invasive cancers involved extensive regions of 3p but were more localized in precancer cases without concomitant invasive cancer. Two interstitial regions, 3p22-21.3 around marker D3S1260 and 3p21.1 around markers D3S1289 and D3S1076, were most frequently deleted in both groups, suggesting that these 2 regions are novel tumor-suppressor loci which may play a role in early transition of cervical precancer to invasive cancer. Identical LOH patterns between multiple CIN lesions and synchronous invasive cancer in the same case suggests that different cervical precancers and invasive cancer are genetically linked and most likely originate from a single precursor cell.

Abstract: Regulation of cell differentiation is most often impaired in malignant tumors and may represent a key mechanism for the progression of the disease. CCAAT-enhancer binding protein (C/EBP) is a family of transcription factors involved in the regulation of embryonic gut development in rodents, which has also been detected in various malignancies, e.g., liposarcomas and breast and ovarian epithelial tumors. We studied the relationship between C/EBP and tumor histology (Duke's invasive stage and pathological grade) in colorectal cancer. Immunoblotting techniques were used on microdissected fresh frozen tumor specimens, and expression of C/EBPalpha, C/EBPbeta and C/EBPzeta (CHOP) was analyzed in addition to that of the cell-cycle regulator p53 and the proliferation marker PCNA. Expression of C/EBPbeta (LAP isoforms) was markedly increased in all tumors compared with normal colon mucosa. Although the inter-patient variability was large, we found that LIP, the isoform of C/EBPbeta known to inhibit transcription, was expressed at higher levels in Duke's stage B tumors compared with Duke's stage A, whereas Duke's C tumors had the lowest LIP expression. A similar relationship was seen for CHOP. The cell-cycle regulator gene p53 was the only factor that clearly correlated with pathological grade: a decrease in p53 expression was demonstrated. Our data suggest that genetic and cellular events involving C/EBPbeta and CHOP are important for tumor invasion and that these events do not appear to be related to the pathological grade of the tumor.

Abstract: Two problems were the focus of this study. (1) Is precancer and/or invasive cancer of the human cervix a poly- or monoclonal proliferation of neoplastic cells? (2) Are simultaneously present precancers and cancers of the cervix clonally related, or do they arise independently? Microdissection of 37 neoplastic lesions with different degrees of histologic severity in 22 patients followed by polymerase chain reaction-based analysis of X-chromosome inactivation was used as a principal method. Invasive cancers were interpreted as monoclonal because samples invariably showed monoclonal signals. In two thirds of these cases, simultaneously present precursors had the identical X-chromosome inactivation pattern, but in one third the pattern was different. Polyclonality was seen in a subgroup of precursors, where there was no simultaneous presence of invasive cancer. In contrast, when invasive cancer was present, no precursor signaled polyclonality. Data taken together indicate that the pathogenesis of cervical cancer is probably even more complicated than that of other cancers involving selection of subclones from originally polyclonal precursors and possibility of coexistence of precursors of different monoclonal composition. The study also observed that a large field of normal cervical squamous epithelium (approximately 500 basal squamous epithelial cells) with nonrandom X-chromosome inactivation was present. It remains to be further investigated whether this phenomenon represents an embryologic lyonization pattern of X-chromosome inactivation or postembryologic clonal expansion of submorphologically transformed cells.

Abstract: We have previously reported on direct sequence analysis of the p53 gene in laser-dissected single cells from tissue sections, where each allele of two fragments (exons 7 and 8) could be accurately analyzed in only 14% of the cells due to the high frequency of exon and allele dropout. Here in an effort to minimize this problem, we have investigated various approaches for sample preparation and gene amplification. By pinpointing some critical steps in the procedure, we could increase the number of investigated exons and substantially improve the genetic analysis of single cells obtained from histochemically stained frozen tissue sections. The biggest improvement was achieved by minimizing DNA degradation using EDTA as a nuclease inhibitor in all sample preparation steps. Efforts to increase primer annealing, by increasing the concentration of template and primers, in addition to prolonging the annealing and extension times, also improved the amplification efficiency. With these measures we can now amplify six individual exons of the p53 gene (exons 4-9) in 70% of the cells and in 50% of these cells both alleles are amplified. This allows application of the method in various investigations such as within the field of tumor pathology.

Abstract: We have recently reported on the genetic organisation of a novel Krüppel-like zinc finger, ZNF189, located to 9q22-q31. In that study we found no mutations in the coding sequence when using ZNF189 as a candidate gene for sporadic basal cell cancer and squamous cell cancer. Here, by direct sequencing of the proximal promotor of ZNF189, mutations were found to appear in a small hot-spot region in over 50% of analysed tumour samples, the majority being G to A substitutions. The hot-spot region spans a 24bp G-rich region. Repeated analyses of the original sample lysates fail to confirm each of these mutations; and frequently new mutations appear at neighbouring positions. Subsequent analysis with serial dilutions of genomic DNA and a cosmid harbouring the wild-type ZNF189 gene demonstrate that these sequence-specific alterations arise in the outer PCR-amplification when 50 copies or less of template are used. Although the mechanism of how these context-specific alterations arise is not proven, the results demonstrate a previously unreported type of PCR-mediated sequence-specific alteration that easily could have been interpreted as being of clinical relevance. The phenomena observed show that mutations detected by direct sequencing can be caused by PCR-introduced alterations. Consequently, this should be of general caution in mutation analysis of disease gene candidates when using small amounts of template, such as microdissected biopsies.

Abstract: We microdissected 15 specimens of invasive cervical cancer co-existing with some of its precursors. Out of 15 cases, 10 carried HPV16, 2 HPV31, 1 HPV18 and 2 were HPV-negative. We found 3 HPV16 E6 variants among the 10 cases; one was A --> G in nt 131 (one case) and a second was A --> G in nt 276. The third, T;--> G in nt 350, was common, and was found in 5 of the 10 patients infected by HPV16. The type of HPV and the E6 variant were identical in all lesions within the same patient. Viral DNA present in normal epithelium was identical in type and E6 variant to HPV in the same patient's lesions. Multiple samples from invasive cancers with HPV were consistently positive. The data suggest that the originally infecting HPV, including its variant type in the E6 gene, persists unaltered in the whole series of CIN that precedes invasive cancer. Our data are compatible with an essential role of HPV manifested by persistence of the viral genome during the entire natural life history of cervical cancer. We did not confirm previous data on the specific association of invasive cancer with an HPV E6 variant (G at nt 350 rather than T). The discrepancy may depend on the relatively few cases investigated or selection of a special sub-set with progression from CIN to invasive cancer already manifest.

Abstract: Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.

Abstract: A patient with xeroderma pigmentosum group C was extensively examined for mutations in the p53 gene in normal skin exposed to varying degrees of sunlight and in excisional biopsies of basal cell cancer, squamous cell cancer, and squamous cell dysplasia. Seventy-three samples were analyzed by microdissection of small cell clusters, followed by PCR and direct DNA sequencing. In skin taken from areas that most likely had never been exposed to the sun, no mutations were found. However, in skin exposed to the sun, we observed a multitude of mutations in the p53 gene. UV light-induced mutations were found in all types of lesions, as well as in clusters of morphologically normal epidermal cells. Twenty-nine distinct mutations were found in exons 5-8, all missense or nonsense, of which 27 (93%) were UV-specific C --> T or CC --> TT transitions at dipyrimidine sites of the nontranscribed strand. Two types of normal skin areas containing p53 mutations were observed: areas that stain strongly with p53 antibody (p53 patches) and those that do not stain. Because no silent or intron mutations were found in these cell clusters, the alterations in the p53 gene of morphologically normal cells are likely to have resulted in a selective growth advantage. The poor correlation between mutations and morphological phenotypes demonstrates that p53 mutations alone do not determine the phenotypes observed.

Abstract: A 3-kb-long cDNA encoding a Krüppel-like human zinc finger protein was isolated and mapped to chromosome 9q22-q31. The ZNF189 gene encodes a protein with 16 zinc fingers at its C-terminus and belongs to the Krüppel-associated box (KRAB)-containing group of zinc finger proteins. Four differently spliced cDNA transcripts, differing at the 5' coding region where a KRAB A repressor domain is encoded, were isolated. In addition, Northern blot analysis indicates the presence of two additional unidentified splice variants. Comparison of cDNA and genomic sequences shows that the ZNF189 gene spans approximately 11 kb and is organized into at least four exons, the large 3'-end exon coding for the complete zinc finger domain and the 3' untranslated region. ZNF189 is expressed in all tissues and cell types currently investigated, at varying levels, but with a tissue- or cell-type-restricted expression pattern for the different splice variants. ZNF189 is conserved in the genome of several mammalian species. Direct sequencing of the ZNF189 gene in microdissected tumor biopsies of sporadic basal cell carcinoma and squamous cell carcinoma reveals no mutations in the coding sequence or at exon/intron boundaries.

Abstract: The high prevalence of p53 mutations in human cancers and the suggestion from several groups that the presence or absence of p53 mutations might have both prognostic and therapeutic consequences point to the importance of optimal methods for p53 determination. Several strategies exploring this have been described, based either on mRNA or genomic DNA as a template. However, no comparative study on the reliability of the two templates has been performed. The principal aim of this study was to study the concordance of RNA- and DNA-based direct sequencing methods in detecting p53 mutations in breast tumors. In 100 tumors, 22 mutations were detected by both methods. Furthermore, one stop mutation, two splice-site mutations, and one intron alteration were found only by genomic sequencing. In addition, the comparative study suggests that cells with missense mutations have increased steady-state concentrations of p53-specific mRNA, in contrast to cells with a gene encoding a truncated protein.

Abstract: We analyzed incidence trends of cutaneous melanoma in situ in Sweden in 1968-1992. Among men, age-standardized rates increased from 0.1/100,000 in 1968 to 2.9/100,000 in 1992, which corresponds to an average annual increase of 15.0%. Among women, rates increased from 0.3 to 3.7, and the annual increase was 12.8%. Age-specific rates increased since 1978, predominantly in men aged 45 years and older, whereas in women rates increased in all ages. Multivariate analysis showed that incidence rates could be explained by both cohort effects and period effects in addition to age. However, cohort effects seemed more important among men than among women, in whom period effects dominated. Thus, factors associated with the development of melanoma in situ may differ between the sexes. Melanoma in situ is an immediate precursor of invasive melanoma, and the increased detection and surgical excision of these tumors will prevent the occurrence of invasive melanoma.

Abstract: UV-induced DNA damage appears to play an essential role in skin carcinogenesis. Following acute UV irradiation, there is an overexpression of normal p53 protein in epidermal keratinocytes, representing a physiological response to DNA damage. Sun protection through topical sunscreens or clothing is believed to reduce the hazardous effects of UV irradiation and subsequently the risk of skin cancer. We have examined the effect of an SPF 15 topical sunscreen and blue denim fabric (SPF 1700) in chronically sun-exposed human skin after sun exposure during a normal summer. Skin biopsies from sun-protected and sun-exposed skin were compared with respect to immunohistochemically detectable p53. This method provides a model for assessing the significance of different degrees of UV protection under physiological conditions. Our results show a significant reduction of p53-positive cells in sun-protected skin as compared with sun-exposed skin. The reduction of p53-positive keratinocytes differed between topical sunscreen (33% reduction) and blue denim fabric (66% reduction). Interindividual variations were large, possibly because of variations in sun exposure. These variations also suggest that mechanisms determining UV damage at the cellular level are complex. The role of residual p53-positive keratinocytes after 2 months of total sun-protection (i.e., SPF 1700) is discussed.

Abstract: Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.

Abstract: Ultraviolet light, which is the major etiology of human skin cancer, will cause mutations in the p53 gene. We and others have found that such mutations occur in more than one-half of non-melanoma squamous cell cancer and precancer. Immunostaining for p53 has disclosed a characteristic compact pattern not only in cancer/precancer but also in areas of microscopically normal epidermis termed p53 patches. By microdissection, sequence analysis of the p53 gene, and analysis of loss of heterozygosity (LOH) at the site of this gene, we have now extended previous data to ascertain whether these p53 patches are precursors of simultaneously present squamous cell cancer or its morphologically recognized precancerous stages (dysplasia, carcinoma in situ). In none of 11 instances with co-existence of a p53 patch with dysplasia or in situ or invasive cancer were the mutations identical. We conclude that p53 patches, estimated to be approximately 100,000 times as common as dysplasia, have a very small or even no precancerous potential. Their common presence demonstrates that human epidermis contains a large number of p53 mutations apparently without detrimental effect. The only result of the mutation may be a clandestine benign clonal keratinocyte proliferation. The importance of p53 mutations for such benign cell multiplication on one band and malignant transformation on the other is unclear. Although the spectrum, type, and multiplicity of mutations were similar in both types of proliferative responses, there was a clear difference with respect to LOH. No LOH was found in 17 p53 patches. By contrast 11 of 30 precancers/cancers had LOH.

Abstract: We report a 32-year-old man with an unusual combination of congenital ichthyosis, sclerosing palmoplantar keratoderma with pseudoainhum, and bizarre striate hyperkeratosis in the fixtures, but no systemic involvement. The condition, which improved on oral etretinate therapy, had not appeared previously in the family. On light microscopy the involved epidermis showed marked acanthosis with hypergranulosis and hyperkeratosis. Electron microscopy disclosed numerous large keratohyaline granules in superficial keratinocytes. The clinical picture and histology are virtually identical to those of a Spanish family suffering from an autosomally recessive skin disease of unknown etiology. We hypothesize that the condition is due to a genetic defect in the formation of keratohyaline granules.

Abstract: Human basal cell cancer (BCC) has unique growth characteristics with virtual inability to metastasize. We investigated clonality and genetic progression using p53 mutations as marker. Sampling was done through microdissection of frozen immunohistochemically stained 16 microm slices of tumors. From 11 BCC tumors 78 samples were analysed. Direct DNA sequencing of exons 5-8 was performed, haplotypes were determined after cloning of p53 exons and loss of heterozygosity (LOH) ascertained by microsatellite analysis. All tumors had p53 mutations and in a majority both p53 alleles were affected, commonly through missense mutations. Microdissection of small parts (50-100 cells) of individual tumors showed BCC to be composed of a dominant cell clone and prone to genetic progression with appearance of subclones with a second and even third p53 mutation. Samples from normal immunohistochemically negative epidermis always showed wild type sequence, except for a case of previously unknown germline p53 mutation. Our analysis also included p53 immunoreactive patches i.e. morphologically normal epidermis with a compact pattern of p53 immunoreactivity. Mutations within those were never the same as in the adjacent BCC. This detailed study of only one gene thus uncovered a remarkable heterogeneity within a tumor category famous for its benign clinical behavior.

Abstract: In this study, we show that direct mutational analysis of genomic DNA can be performed on single somatic cells extracted from a frozen, immunohistochemically stained tissue section using laser-assisted capture microscopy. Eighty-nine single tumor cells were separately dissected from one case of human basal cell cancer (BCC) and p53 mutations were analyzed by direct semi-automated sequencing of PCR fragments. Amplification was obtained for at least one of the two analyzed exons from approximately 50% of the single tumor cells. Identical p53 mutations were found in widely spread areas of the tumor, suggesting a clonal proliferation originating from one cell. Interestingly, comparison between results of immunohistochemistry and genetic analysis of the single cells revealed the same p53 mutations irrespective of the p53 immunoreactivity. We propose that this approach has a great potential to allow investigation of genotypic differences in single cells and more specifically to resolve important and fundamental questions determining cancer heterogeneity.

Abstract: ERV3 (HERV-R) is a complete human endogenous retrovirus located on the long arm of chromosome 7. Long terminal repeat-envelope (env) gene spliced mRNAs of 9 and 3.5 kb are widely expressed in human tissues and cells, but gag-pol mRNAs have not been found. Furthermore, the env gp70 gene contains an open reading frame throughout its length. The highest expression of ERV3 mRNA detected so far is in placenta and the lowest in choriocarcinoma cell lines. We have previously shown that the human monoblastic cell line U-937 and some normal and neoplastic tissues also express high levels of ERV3 env message by Northern blot analysis; however, this method does not distinguish between mRNA expression in different cell types in tissues. In this report, we have studied the ERV3 mRNA expression in specific cell types of human skin by in situ hybridization. We found high levels expression of ERV3 env mRNA in human sebaceous glands in normal skin and dermoid cysts of the ovary. In all glands, the expression is maximal in the periphery of the lobule and ceases towards the center in the region of characteristic holocrine secretion. Since it is known that the regulation of sebaceous glands is primarily via steroid hormones, particularly androgens, it is possible that expression of ERV3 is hormone dependent.

Abstract: Microdissection of biopsies with sequencing of exons 4-8 of the p53 gene permitted precise morphological identification of correlation between mutations and/or loss of heterozygosity, immunoreactivty of p53 and type of squamous neoplasia. Seventy-two specimens from ten lesions of sun-exposed sites including normal epidermis were analysed. Irrespective of p53 immunoreactivity and morphological grade dysplasia, in situ or invasive cancer, in each case, carried the identical mutation indicating that invasive skin cancer and its precursors derive from the same original neoplastic clone. Additionally, morphologically normal epidermis showed some sharply demarcated immunoreactive areas. These never had the same p53 mutation as that of the adjacent tumor, indicating that their mutations were separate events and ruling them out as common precursors of cancer. Non-immunoreactive normal epidermis did not show p53 mutations. Our findings indicate that a large fraction of keratinocytes in sun-exposed human skin carry mutations of p53 and suggest that at least two options exist for such cells (i) innocuous clonal expansion with preserved morphology and normal differentiation or (ii) malignant transformation with the p53 mutation as an early event. Suggestive evidence existed that the p53 mutations were qualitatively different in the two respective groups of lesions.

Abstract: Specimens of squamous-cell neoplasms (81 invasive cancers, 36 in situ cancers, 70 dysplasias, 5 keratoacanthomas, 19 papillomas) and normal skin were immunostained with p53 antibody. Nuclear accumulation of p53 was visualized as following 2 distinct patterns: dispersed or compact. The former is interpreted as a reversible reaction to sunlight, whereas the latter, after microdissection and sequencing of DNA, has been shown to reflect clonal multiplication of keratinocytes with mutated p53. The dispersed pattern was diffusely distributed and usually only involved a small proportion of epidermal cells. The compact pattern was characterized as a contiguous area of homogeneously stained cells sharply demarcated from its surroundings. It involved patches of normal epidermis or large areas of dysplastic or malignant squamous epithelium. Immature cells were always stained, whereas immunoreactivity was variably present in differentiating keratinocytes. Dispersed patterns occurred in 94.7% of strongly UV-exposed skin (mainly face) and to a lesser extent in less exposed parts of the body. It showed no correlation to the age of the individual. About two-thirds of biopsies from individuals over age 50 displayed compact patterns in sun-exposed, otherwise normal, epidermis. About 65% of pre-malignant and malignant squamous-cell neoplasms had a compact pattern. The presence of p53 immunoreactivity as a compact pattern supports the idea that mutations of the p53 gene are early events in the sequence from dysplasia to invasive squamous-cell cancer of the skin. Also, even in the absence of cellular atypia, patches of epidermal cells can accumulate p53 in a way that is indistinguishable from that of cancer and pre-cancer.

Abstract:

Abstract: It has been suggested that p53 plays an important role in skin carcinogenesis. The p21 molecule acts as a downstream effector of wild-type p53 by enacting cell cycle arrest. We studied p53 and p21 expression in sun-exposed skin. Healthy volunteers were exposed to ultraviolet irradiation (UVA + UVB) in normal, previously non-sun-exposed skin, and skin biopsies were taken. Immunohistochemically detectable p53 and p21 were quantified, and the pattern of distribution was recorded. p53 was induced in epidermal cells 4 h after irradiation and returned to nearly normal levels after 120 h. Suprabasal cells showed a peak at 4 h, whereas basal cells peaked later, at 48 h. In epidermis, the expression of p21 was induced with a pattern that mirrored that of p53. In addition, p21 was induced in mesenchymal cells of the upper dermis, where there was no p53, suggesting an alternative pathway for p21 induction. Topical sunscreen and pigmentation (skin type 5) nearly eliminated UV-induced expression of p53 and p21. In contrast to the complete absence of p53 in skin never exposed to UV radiation, p21 reactivity was found in sharply demarcated areas of anagen hair follicles and sebaceous glands, as well as in scattered epidermal cells. The prevalence and distribution suggest a physiologic role of p21 in stopping the cell cycle in terminally differentiating skin epithelium. Archival skin material from the vicinity of skin lesions with variable sun exposure were also stained for p53. There was an increased "disperse" reactive staining pattern in skin samples excised in the summer as compared with less sunny seasons. Intensely stained p53 foci were detected as "compact bands" in morphologically normal epidermis, predominantly in sun-exposed areas of the skin, suggesting the existence of clonal proliferation of p53 mutated keratinocytes. These data show that p53 and p21 play a role in the human skin response to UV exposure and that p21 is implicated in the homeostasis of differentiating skin appendages.

Abstract: Among the candidate cancer-prognostic genes is the p53 tumor suppressor gene, which, when mutated, plays an important role in the development of many types of cancers. To facilitate robust large-scale DNA analysis of microdissected tumor biopsies, we describe a multiplex/nested PCR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, involving a total of 14 primers. This approach reduces the required number of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification step. The HLA sequencing allows sample identification because the DQB1 locus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomated format. To improve the DNA sequence quality, we used 2'-deoxyribonucleoside 5'-O-1-thiotriphosphates in the sequencing reactions.

Abstract: A proposed progenitor cell for basal cell carcinoma is a stem cell located in the bulge of the hair follicle. Previous investigations have shown that basal cell carcinoma has a specific stroma requirement for its growth. Likewise the development of a normal hair follicle requires the inductive force of a specialized structure with condensed mesenchyme that eventually forms the dermal hair papilla. Investigations in mouse embryos also strongly indicate that induction/growth of skin structures is dependent on platelet-derived growth factor (PDGF) alpha-receptor expression in the mesenchyme. We therefore investigated the expression of PDGF A and B chain and PDGF alpha and beta receptors in basal cell carcinoma and in normal skin by immunohistochemistry and in situ hybridization. alpha and beta receptors were found in the specific stroma components of basal cell carcinoma, dermal hair papilla, and sweat glands, but not in the epithelial structures. The A and B chains, on the other hand, were mainly found in basal cell carcinoma cells, in hair matrix, and in sweat gland epithelium. This "appositional" expression of PDGF/PDGF receptor closely resembles that found in epithelial/mesenchymal structures during normal development. The findings also suggest that PDGF receptor expression is one of the characteristics of the specific stroma that is necessary for basal cell carcinoma growth.

Abstract: BACKGROUND: Previous malignant melanoma studies regarding prognostic factors have often selected their patients from hospitals. Unfortunately, most of these studies have had small numbers of patients, consisted of short-term data sets, omitted important factors, did not optimize histopathologic classification, had too short or inadequate follow-up, and did not test their predictive models. PURPOSE: Our study goals were to identify independent clinical and histopathologic determinants of survival in malignant melanoma, to analyze changes in prognostic value over follow-up time, and, finally to construct a prognostic index. METHODS: A random sample from the Swedish Cancer Registry of the records of 498 (246 men and 252 women) patients defined by gender, five 5-year time periods of diagnosis from 1960 through 1984, and five anatomic sites formed the cohort on whom data were analyzed by univariate analyses. Multivariate analyses were based on data on 476 patients with complete information about all variables. All patients in the cohort had complete follow-up through December 31, 1989. Clinical information was abstracted and recorded as: date of diagnosis, stage at diagnosis, sex, age, anatomic site of primary tumor, date of death, and cause of death. Histopathologic slides were re-examined and classified with regard to histogenetic type, level of invasion, tumor thickness, ulceration, vascular invasion, regression, lymphocytic reaction, pre-existing nevus, and cell type. RESULTS: All variables, except pre-existing nevus and cell type, were significant predictors of survival. In the multivariate analyses including all variables, women still had a significant, 33% lower relative hazard than men. The prognosis was poor in the youngest age group. Patients with external ear, scalp-neck, and trunk-located melanoma had increasing relative hazard when all variables were included. Regional metastases and tumor thickness remained independent prognostic factors. No significant association between histogenetic type or level of invasion persisted. Patients whose tumors showed ulceration or vascular invasion had lower relative hazard when all variables were included. Level of invasion, tumor thickness, ulceration, and vascular invasion were significantly associated with the prognosis during both short- and long-term follow-up. The patients were subgrouped according to percentage fractions of their score on the prognostic index. Survival curves for these groups of patients were well separated, thus identifying patients with a low or high risk of death from malignant melanoma. CONCLUSION: The present population-based study identifies independent clinical and histopathologic predictors of survival in cutaneous malignant melanoma and emphasizes the role of histopathologic characteristics such as tumor thickness, ulceration, and vascular invasion besides anatomic site.

Abstract: In Sweden, improvement in survival rates of patients with cutaneous malignant melanoma has counteracted the increase in incidence to produce a moderate rise in mortality. Our aim was to determine the possible impact of drift in diagnostic criteria, earlier diagnosis and changing biological features of the tumours upon trends in survival. We studied a stratified sample of 528 patients diagnosed between 1960 and 1984 in a strictly defined geographical region. No evidence of drift in diagnostic criteria was found. The proportion of patients with invasion level Clark II increased from 3.2% in 1960-64 to 22.5% in 1980-84, the proportion of thin melanomas (< or = 0.75 mm) increased from 9.4% to 31.5% and the tumour thickness decreased significantly between each 5 year period of diagnosis. These changes are most likely the results of earlier diagnosis. However, changes in tumour characteristics have occurred, since the proportion of superficially spreading malignant melanoma increased from 35% in 1960-64 to 51% in 1980-84 and the proportion of acral lentiginous melanoma decreased from 11% to 2%. The proportion of nodular melanomas remained fairly constant. The proportion of tumours with lymphocytic reaction did not change, whereas those with histological regression increased slightly. Proportional hazards analyses showed a significantly lower survival in patients diagnosed in 1960-64 but no apparent trend after 1965. This finding remained after adjustment for all studied clinical and histopathological factors which point towards changes in unmeasured biological features of the disease.

Abstract: A procedure for direct sequencing of the human p53 gene based on micro-dissection of frozen tumor tissue stained with methylene blue without prior fixation is described. The approach, which is suitable for large-scale analysis, is based on solid-phase DNA sequencing of amplified genomic DNA and does not involve preparative electrophoresis, precipitations, extractions or centrifugations. Here we show that basal cell carcinoma tissue isolated from human skin can be analyzed in a semiautomated fashion using a robotic workstation and a laser fluorescent electrophoresis unit. The results of analyses of patient material displaying various immunohistochemical staining patterns using a p53-specific antibody are presented.

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