A single nucleotide polymorphism in the 3'untranslated region of the CDKN2A gene is common in sporadic primary melanomas but mutations in the CDKN2B, CDKN2C, CDK4 and p53 genes are rare.

In this report we present the results of mutational analysis of the CDKN2B, CDKN2C, CDK4, p53 genes and 5'UTR of the CDKN2A gene in a set of 44 sporadic primary melanomas, which had been earlier analysed for mutations in the CDKN2A (p16/p14(ARF)) gene. No tumour-associated mutations were detected except in 1 melanoma where we found a CC>T* deletion-mutation in the codon 151-152 (exon 5) of the p53 gene. On the basis of our preliminary results, we did extended genotyping of the 500 C>G and 540 C>T polymorphisms in the 3'UTR of the CDKN2A gene in 229 melanoma cases and 235 controls. The T-allele frequency (for 540 C>T polymorphism) in melanomas was significantly higher than in controls (0.14 vs. 0.08; chi(2) = 5.95, p = 0.01; OR = 1.71, 95%CI = 1.11-2.66). The heterozygote frequency for this polymorphism was 0.26 (59/229) in melanomas compared to 0.13 (30/235) in healthy controls (chi(2) = 11.4; p = 0.0007; OR = 2.34, 95% CI = 1.40-3.92). The frequency of the 500 C>G polymorphism in the 3'UTR in the CDKN2A gene was not significantly higher in melanomas compared to healthy controls. The 500 C>G polymorphism, however, was in linkage disequilibrium with approximately 50 kb apart the C>A intronic polymorphism in the CDKN2B gene (determined in 44 melanomas and 90 controls; Fisher exact test, p<0.0001). Finally, the sequence analysis of genomic DNA isolated from T cell lymphocytes of healthy individuals exhibited that the codon reported as last of exon 2 of the CDKN2C gene is rather the first codon of exon 3.

Increased frequency of LOH on chromosome 9 in sporadic primary melanomas is associated with increased patient age at diagnosis.

We carried out statistical analysis of the frequency of loss of heterozygosity (LOH) at 10 microsatellite markers on chromosome 9. In 44 microdissected sporadic primary melanomas a comparison of LOH frequency data with other patient data, like age at diagnosis and tumour thickness, showed an interesting correlation between patient age at diagnosis and frequency of LOH on chromosome 9. The patient group with age >72 years at diagnosis (n = 22, mean age 82.3 +/- 6.0 years, mean LOH 3.4 +/- 2.3) showed significantly increased LOH frequency (OR 3.1, 95% CI 1.8-5.3; chi(2) test, P < 0.0001) compared with age group

Prognostic value of serial tissue prostate-specific antigen measurements during different hormonal treatments in prostate cancer patients.

To reveal the effects of different hormonal treatments directly on the prostate during treatment, the concentration of prostate-specific antigen in the tissue (T-PSA) was studied in 63 patients with untreated newly diagnosed carcinoma of the prostate (CaP). T-PSA measurements were performed in fine-needle aspiration biopsies at the time of diagnosis and 6, 12, and 24 months after initiation of treatment. Treatments modalities were bilateral orchidectomy, gonadotropin-releasing hormone (GnRH) agonists, or parenteral estrogens. Thirty-one (49%) of the patients died of CaP and 18 (29%) of other diseases. Fourteen of the patients (22%) were still alive at the end of the observation period (median follow-up time, 111.5 months; range, 98-128 months). In all of the 31 patients who died of CaP, T-PSA values increased during treatment. This increase was observed long before clinical signs of progression appeared (median of interval, 14 months). Twenty of these 31 patients showed an increase in T-PSA from pretreatment values at 6 months. At 12 months this increase was observed in 30 of 31 patients. In contrast, in all of the patients who responded to the hormonal regimen, T-PSA values decreased and remained low during treatment. Furthermore, the patients who did not die of CaP and received estrogen treatment had significantly higher T-PSA values compared with those who were treated with bilateral orchidectomy or GnRH agonists. This indicates that estrogens may stimulate PSA synthesis in tumor tissue in vivo in the presence of castration levels of testosterone. Statistical evaluation showed that the T-PSA ratio between month 12 and month 0 had the most significant prognostic value for predicting the clinical outcome. This ratio was superior to clinical classifications, e.g., tumor stage and cytological grade, and also was higher than T-PSA at the time of diagnosis. This study has shown that aspiration biopsy material can be used to reveal biochemical changes in the tissue during treatment and that one specific marker (T-PSA) can predict the clinical outcome of endocrine treatment of CaP patients better than previously used methods. We believe that selected tissue markers or the protein pattern can help us to characterize the tumors and predict the clinical outcome so an optimal treatment can be chosen for every patient.

Protection against 12-O-tetradecanoylphorbol-13-acetate induced skin-hyperplasia and tumor promotion, in a two-stage carcinogenesis mouse model, by the 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-[2,3-b]quinoxaline analogue of ellipticine.

The effects of topical applications of 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-[2,3-b]quinoxaline (B-220), on 12-O-tetradecanoylphorbol-13-acetate (TPA) or benzoylperoxide (BPO) induced promotion of skin tumors and hyperplasia were studied in female SENCAR mice. Papillomas were induced by initiation with 7,12-dimethylbenz[a]anthracene (DMBA), followed by promotion biweekly with TPA or BPO. Administration of B-220 1 h before TPA promotion resulted in a prolonged latency period of tumor appearance and a significantly reduced (up to 15% of positive controls) papilloma yield at 20 weeks. Moreover, if B-220 treatment was terminated after 20 weeks and TPA treatment continued, papilloma development resumed indicating that initiated tumor cells were still present but were unable to grow with B-220 present. If B-220 pretreatment was not given during the first 10 weeks of TPA promotion, incidence at 20 weeks was not reduced but tumor multiplicity was still decreased. In addition a marked reduction of the TPA induced sustained epidermal hyperplasia was observed in the long term experiment. Neither the inflammatory response nor the increase in the number of apoptotic cells seen in short term experiment after a single TPA treatment were inhibited by B-220. B-220 administration before BPO promotion had no effect on the appearance of BPO induced papillomas or epidermal hyperplasia, suggesting that TPA and BPO promote tumor formation via at least partially different mechanisms. In experiments where B-220 was applied topically 1 h before DMBA initiation, little or no effect was seen. No morphological changes in mouse skin due to long term exposure (two times/week, 39 weeks) to B-220 were found. In conclusion, we present evidence that B-220 is a potent inhibitor of mouse skin tumor promotion by TPA, but has little effect on the initiation step or the survival of initiated cells.

Squamous cell carcinomas and increased apoptosis in skin with inhibited Rel/nuclear factor-kappaB signaling.

The Rel/nuclear factor-kappaB (Rel/NF-kappaB) transcription factors have been implicated previously in control of apoptosis, cell proliferation, and oncogenesis. Here we show that selective inhibition of Rel/NF-kappaB signaling in murine skin, by targeted overexpression of a super-repressor form of IkappaB-alpha, results in an increased basal frequency of apoptotic cells and the spontaneous development of squamous cell carcinomas. Presence of hyperplasia and hair follicle degeneration demonstrate an important role for Rel/NF-kappaB signaling in normal epidermal development and homeostasis. Transgenic skin, in addition, showed an enhanced sensitivity to UV-induced apoptosis. These data suggest an involvement of the Rel/NF-kappaB signaling pathway in apoptosis and cancer development of the skin.

A comparison between open and percutaneous needle biopsies in men with azoospermia.

Open testicular biopsy is a classic method of investigation in men with azoospermia. Recently, percutaneous needle biopsy of the testis has been used in attempts to obtain material for histopathological diagnosis in such cases and to retrieve spermatozoa for intracytoplasmic sperm injection (ICSI). To determine whether a 19 gauge (G) and a 21G butterfly needle could be used for percutaneous aspiration of testicular tissue to determine the presence of mature spermatids and assess spermatogenesis, 10 patients (16 testes) and 12 patients (17 testes) underwent 19G or 21G needle biopsy respectively, immediately followed by open testicular biopsy, with both procedures under local anaesthesia. Biopsy with each needle size was compared with open biopsy. With the 19G needle, in the 14 cases where material was obtained there was full agreement with open biopsy regarding the presence or absence of mature spermatozoa, whereas with the 21G needle only nine of the 13 biopsies yielding material were predictive in this respect. Each needle size correlated poorly with open biopsy regarding evaluation of spermatogenesis. We conclude that percutaneous biopsy with a 19G butterfly needle is a quick and reliable method for demonstrating spermatozoa for ICSI. But for a detailed histopathological diagnosis, however, the needle biopsies gave poor results, whereas the material from the open testicular biopsies was assessable.

Electrical impedance and other physical parameters as related to lipid content of human stratum corneum.

BACKGROUND/AIMS: In previous studies we have shown that variations in the properties of the stratum corneum are reflected by alterations in electrical impedance. The aim of this study was to explore the ability of the electrical impedance technique to detect changes in the lipid content of the stratum corneum, and to compare It with the other non-invasive methods, measurement of transepidermal water loss and of skin moisture. METHODS: Twenty-two healthy test subjects were recruited. Transepidermal water loss was measured at standard sites on the forearms and wrists, followed by skin moisture estimation by electrical capacitance, and finally by the recording of electrical impedance spectra in the frequency range 1 kHz to 1 MHz. Readings by all three methods were taken before the start of each series of test procedures, as well as after cyclohexane swabbing, a skin stripping procedure and lipid extraction, and also during the recovery process. A mixture of hexane:isopro-panol was used for lipid extraction of the skin, and the extracts were evaluated using HPLC/LSD and GC/MS/FID analysis. Biopsy samples for light and electron microscopy were obtained after lipid extraction. RESULTS: Electrical impedance results showed greater changes after lipid extraction than either transepidermal water loss or skin moisture content. Baseline values varied from the cubital fossa to the wrist, both for the non-invasive methods and for lipid composition. CONCLUSIONS: The electrial impedance is dependent on the lipid content of the stratum corneum, as studied by lipid extraction experiments.

Electrical impedance. A method to evaluate subtle changes of the human oral mucosa.

In a previous study, we mapped the differences in electrical impedance between various anatomical locations in the oral mucosa. We now explore the ability of the impedance technique to detect mild reactions in the buccal mucosa induced by the irritant sodium lauryl sulphate. This substance was applied for 15 min at a concentration of 2% to the mucosa of 26 healthy subjects. A contralateral site was used as a control. Responses were evaluated by measuring electrical impedance before exposure and after removal of the irritant, and also by visual inspection and histology. Magnitude and phase of impedance were determined in the frequency range 1 kHz to 1 MHz at 5 depth settings, and 4 physically distinct indices were calculated from the impedance data. The results showed the response to be at its maximum 5 min after removal of the test chamber, for all indices. These changes were statistically significant, whereas visual and histological alterations were slight or negligible. We conclude that the electrical impedance technique is capable of detecting mucosal changes in the invisible or barely visible range, and that the mucosal response to sodium lauryl sulphate is well characterised by the 4 indices.

Allergic contact reactions in the skin assessed by electrical impedance - a pilot study.

BACKGROUND/AIMS: In our previous studies of the electrical impedance of the skin, we formulated a set of physical indices that could be used to distinguish between the cutaneous effects produced by different chemical irritants. We now describe an investigation of allergic contact reactions, using the same set of impedance indices for characterization. METHODS: Skin reactions were induced in the forearm of eight female patients who were allergic to nickel by exposure to nickel sulphate in petrolatum at various concentrations. The responses were evaluated by measurements of electrical impedance and transepidermal water loss, as well as by visual scoring and biopsy. Normal skin was used for controls. RESULTS: Different degrees of allergic contact reactions were produced, and the changes in value of the impedance indices were found to follow a particular pattern. This pattern diverged markedly from that obtained in controls, and the differences were statistically significant. CONCLUSIONS: Our results suggest that, by the application of a technique based on electrical impedance, it will be possible to characterize allergic skin reactions.

Differential allele loss on chromosome 9q22.3 in human non-melanoma skin cancer.

Familial predisposition to basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) of the skin are apparent in the autosomal dominant syndromes naevoid basal cell carcinoma syndrome (NBCCS) and multiple self-healing squamous epitheliomata (MSSE) respectively. The gene responsible for NBCCS has been proposed to be a tumour-suppressor gene and is mapped to the same 2 Mb interval on 9q22.3 as the MSSE gene ESS1. In an attempt to further map the NBCCS gene, we have examined loss of heterozygosity (LOH) in 16 sporadic BCCs and two familial BCCs using microsatellite markers located within the candidate gene region. The overall frequency of LOH observed was 67% in the BCCs and partial or interstitial deletions were found in eight tumours, with the highest LOH frequency at markers D9S280, D9S287 and D9S180. To determine if the same genomic region also shows frequent LOH in tumours with a squamous phenotype, we have examined 11 SCCs, four actinic keratoses and 13 cases of Bowen's disease for LOH at 9q22.3. An overall LOH frequency of 50% was observed at D9S180, and occurred in all types of squamous tumours. In contrast, a much lower LOH frequency of only 6% was found at the D9S287 locus. Our observation of different patterns of LOH at 9q22.3 in sporadic BCCs and SCCs implies that more than one tumour-suppressor gene might be located in this genomic region.

Cytomegalovirus-induced CD13-specific autoimmunity--a possible cause of chronic graft-vs-host disease.

Cytomegalovirus (CMV) infection has been suggested to be associated with certain autoimmune phenomena as well as with the development of chronic graft-versus-host disease (cGVHD) following allogeneic bone marrow transplantation. Earlier we found that the CMV-associated host protein CD13 is immunogenic during CMV infection in allogeneic bone marrow transplant patients, resulting in production of CD13-specific antibodies (7). Here we found a close correlation between CD13-specific immunity and later development of cGVHD in 26 of 33 patients who could be evaluated for this disease. Of seven patients with CMV disease, six developed extensive cGVHD, all of whom had CD13 specific antibodies (P=0.0002). All 14 patients who were CD13-immune later developed either limited or extensive cGVHD (P=0.0008). Antibodies in sera from the CD13-immune patients suffering from cGVHD recognized normal structures in cryosectioned skin biopsies from control individuals, producing a staining pattern similar to that of CD13-specific monoclonal antibodies. The antibody binding could be specifically blocked by preincubation of the skin sections with a mixture of monoclonal antibodies against CD13, and was also abolished after preabsorption of sera to mouse cells expressing human CD13. No other common autoantibodies were identified in more than single patients. Furthermore, in vivo binding of IgM antibodies in a CD13-like fashion was preferentially demonstrated in skin and oral mucosa biopsies from the CD13-immune patients suffering from cGVHD. Thus, we suggest that CMV-induced CD13-specific autoimmunity contribute to tissue damage in chronic graft-versus-host reactions.

Electrical impedance measured to five skin depths in mild irritant dermatitis induced by sodium lauryl sulphate.

The non-invasive electrical impedance technique used in this study reflects structural changes in a tissue, and provides an estimate of the level of oedema by a simple impedance index. Sodium lauryl sulphate (SLS), dissolved in water at concentrations of 0.1, 0.5 and 2.0%, was applied for 24 h in 12 mm Finn chambers on both volar forearms of 12 healthy volunteers. An unoccluded area was used as a reference site. Readings from all sites were taken before the application of the irritant, and 24 h after its removal. After the last reading, a 3-mm punch biopsy was taken from each test site for histological examination. The results obtained from electrical impedance measurements at five different skin depths were correlated with those obtained from histological examination, visual scoring and transepidermal water loss (TEWL). For all of the methods used the responses were proportional to the concentration of the irritant. Statistically significant changes of electrical impedance were found for all depths and concentrations, except for 0.1% SLS at the most superficial depth. The histological changes were focused in the epidermis, and mainly consisted of oedema. Alterations in the thickness of the epidermis due to oedema were used as a quantitative parameter for correlation with the assessment of irritation using the electrical impedance technique. For the detection of irritant reactions, TEWL and electrical impedance are more sensitive than visual scoring, and selection of the optimum depth penetration further increases the sensitivity of the electrical impedance measurement.

Histopathological and immunohistochemical changes in psoriatic skin during peptide T treatment.

Ten patients with plaque-type psoriasis were treated with 2 mg peptide T i.v. for 28 days. Six patients responded with a substantial clinical improvement. Sequential biopsies from skin lesions were taken before, during and after treatment. The histological score (defining the activity of the psoriasis), the epidermal thickness and the number of infiltrating dermal lymphocytes were all reduced in the six patients who responded to the treatment. An increase in the number of CD1+ dendritic cells was detected immunohistochemically in the epidermis of the responders. The nonresponders did not display any pronounced changes.