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A 10-year-old female Papillon dog that had previously developed a mammary tumor was admitted for treatment of a hypoglycemic attack. Blood examination showed severe hypoglycemia and decreased blood insulin concentration. Computed tomography indicated multiple tumors in the cranial and caudal lobes of the right lung. These tumors were resected surgically and diagnosed as pulmonary adenocarcinomas by histopathologic examination. Hypoglycemia was temporarily improved after the resection, but a hypoglycemic event occurred 2 months after the surgery. Immunohistochemistry of the tumor demonstrated the expression of insulin-like growth factor 2 in tumor cells. Western blot analysis revealed the expression of high-molecular-weight (big)–insulin-like growth factor 2 in the tumor region. Insulin-like growth factor 2 mRNA expression was also confirmed in the tumor using reverse transcription–polymerase chain reaction. These findings indicate the diagnosis of non–islet cell tumor-induced hypoglycemia caused by big-insulin-like growth factor 2 produced by the tumor in the dog. This report provides information on differentiating tumors that cause paraneoplastic hypoglycemia.

Summary A 12-year-old castrated male Jack Russell Terrier presented with intermittent vomiting. Abdominal ultrasonographic examination detected a thickened stomach wall with a mass measuring approximately 1.5 cm in diameter. Computed tomography revealed a solitary mass measuring approximately 2.1 cm in diameter between the submucosa and muscle layers in the greater curvature the pyloric region of the stomach, and a swelling in the hepatic lymph node. The gastric mass was composed of round neoplastic cells arranged in a diffuse pattern. The neoplastic cells had a round nucleus and a pale abundant cytoplasm. Multinucleated giant cells were often found. Hyalinized eosinophilic material, which did not stain with Congo red and had no affinity for thioflavin T, was also observed. Neoplastic cells were immunopositive for MUM1, CD79a and Ig lambda light chain but negative for CD3, CD20, BLA36, IgG and Ig kappa light chain. Stromal eosinophilic material was positive for Ig lambda light chain. The neoplasm was therefore diagnosed as a gastric plasmacytoma with non-amyloid Ig lambda light chain deposition.

Background Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder mainly caused by heterozygous mutations of PTCH1 . In addition to characteristic clinical features, detection of a mutation in causative genes is reliable for the diagnosis of NBCCS; however, no mutations have been identified in some patients using conventional methods. Objective To improve the method for the molecular diagnosis of NBCCS. Methods We performed targeted exome sequencing (TES) analysis using a multi-gene panel, including PTCH1 , PTCH2 , SUFU , and other sonic hedgehog signaling pathway-related genes, based on next-generation sequencing (NGS) technology in 8 cases in whom possible causative mutations were not detected by previously performed conventional analysis and 2 recent cases of NBCCS. Subsequent analysis of gross deletion within or around PTCH1 detected by TES was performed using chromosomal microarray (CMA). Results Through TES analysis, specific single nucleotide variants or small indels of PTCH1 causing inferred amino acid changes were identified in 2 novel cases and 2 undiagnosed cases, whereas gross deletions within or around PTCH1 , which are validated by CMA, were found in 3 undiagnosed cases. However, no mutations were detected even by TES in 3 cases. Among 3 cases with gross deletions of PTCH1 , deletions containing the entire PTCH1 and additional neighboring genes were detected in 2 cases, one of which exhibited atypical clinical features, such as severe mental retardation, likely associated with genes located within the 4.3Mb deleted region, especially. Conclusion TES-based simultaneous evaluation of sequences and copy number status in all targeted coding exons by NGS is likely to be more useful for the molecular diagnosis of NBCCS than conventional methods. CMA is recommended as a subsequent analysis for validation and detailed mapping of deleted regions, which may explain the atypical clinical features of NBCCS cases.

Cowden syndrome (CS) is an autosomal dominant inherited disorder characterized by macrocephaly and multiple hamartomas. The responsible gene is PTEN (phosphate and tensin homolog detected on chromosome 10), which negatively regulates cell proliferation and survival. We herein present a 46-year-old woman with the typical clinical features of CS. A DNA sequencing analysis of the coding regions and flanking introns of the PTEN gene revealed a novel heterozygous mutation (c.403A > G, p.Ile135Val) in exon 5 that had not been previously reported in CS. J. Med. Invest. 67 : 200-201, February, 2020.

The aim of this study was to investigate the injuries in Japanese collegiate sepak takraw players. We primarily focused on hamstring strain injury (HSI), and investigated the associated physical characteristics. The study included 77 Japanese collegiate sepak takraw players who were interviewed; data were collected regarding injuries sustained by them during the game within the past year. The hip range of motion (ROM) was measured. The total number of injuries was 48 in a year. The rate of HSI was the highest (31.3%) among all the injuries. All HSIs occurred in the dominant leg because of the sunback spike. Using the Mann-Whitney U test, significant differences in age and sport-related experience were observed between the injured group and uninjured group. Upon using logistic regression analysis, the presence of a HSI was found to be associated with the sport-related experience (adjusted odds ratio [OR], 0.30; 95% confidence interval [CI], 0.12-0.77) and the hip extension ROM (adjusted OR, 0.81; 95% CI, 0.66-0.99) after adjusting for sex, sport-related experience, and the hip ROM. HSI is the most common injury in Japanese collegiate sepak takraw players. Short sport-related experience and small hip extension ROM are related with the occurrence of HSI.

Conclusion These findings suggest that low dose irradiation with 310 nm NB-UVB specifically suppressed the up-regulation of H1R gene expression without inducing apoptosis and that UVB of shorter or longer wavelength than 310 nm NB-UVB had no such effects. Objective To develop a narrowband-ultraviolet B(NB-UVB) phototherapy for allergic rhinitis, this study investigated the effects of irradiation with NB-UVB at wavelength of 310 nm on phorbol-12-myristate-13-acetate (PMA)-induced up-regulation of histamine H1 receptor (H1R) mRNA in HeLa cells. Methods The mRNA levels of H1R in HeLa cells were measured using real-time RT-PCR. Apoptosis were evaluated with DNA fragmentation assay. Results PMA induced a significant increase in H1R mRNA expression in HeLa cells. Irradiation with 305 nm UVB and 310 nm NB-UVB, but not with 315 nm UVB at doses of 200 and 300 mJ/cm(2) significantly suppressed PMA-induced up-regulation of H1R mRNA. At a dose of 200 mJ/cm(2), irradiation with 305 nm UVB, but not with 310 nm NB-UVB, induced apoptosis, although exposure of the cells to both 305 and 310 nm UVB induced apoptosis at a dose of 300 mJ/cm(2) after PMA treatment in HeLa cells. Conversely, irradiation with 315 nm UVB at doses of 200 and 300 mJ/cm(2) did not induce apoptosis.

Nivolumab, an anti-programmed death 1 antibody, produces antitumor effects by activating host immunity, which also causes immune-related adverse events (irAEs). The aim of this study was to analyze the association between antitumor effect and irAEs induced by nivolumab in patients with melanoma.Fifteen patients with melanoma who had received nivolumab at Tokushima University Hospital or Ehime University Hospital between January 2015 and December 2016 were enrolled in this study. Patients who had and did not have irAEs during nivolumab treatment were classified into an irAEs-positive group (n = 8) and an irAEs-negative group (n = 7), respectively. We compared the disease control rate (DCR) and overall survival (OS) between the 2 groups. Data on blood cell counts were also analyzed.After a median of 4 cycles of nivolumab treatment, irAEs occurred. The DCRs were 75% and 14% in the irAEs-positive and irAEs-negative groups, respectively (p 0.05). OS in the irAEs-positive group was higher than that in the irAEs-negative group (p 0.05). Multivariable Cox proportional hazards regression analysis revealed that irAE occurrence affected OS with nivolumab treatment. Moreover, the increase in baseline peripheral lymphocyte count at the time of onset of irAEs was significantly greater in the irAEs-positive group than in the irAEs-negative group after 4 cycles of nivolumab treatment (p 0.05).Our study indicated that clinical response with nivolumab treatment improves with irAE occurrence in patients with melanoma. Moreover, the early increase in peripheral lymphocyte count may act as a biomarker for predicting the occurrence of irAEs induced by nivolumab.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) control a wide range of biological functions ; however, their involvement in the pathogenesis of dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP) is currently unknown. In this study, we first confirmed the histological diagnosis by detecting fusion COL1A1-PDGFB transcripts in DFSP, and examined the expression of all FGFRs (FGFR1-4), some of their ligands (FGF1, 2, 9), and forkhead box N1 (FOXN1) as a downstream target of FGFR3 in DF and DFSP by immunohistochemical analysis. Although we failed to detect the expression of FGF1 and FGF9 as specific ligands for FGFR3 in DF, overexpression of FGFR3 and FOXN1 was observed in the epidermal regions of DF, suggesting that the epidermal regions of DF were similar to seborrhoeic keratosis both in terms of histological features and the activation of FGFR3/FOXN1. In addition, strong expression of FGF2 and FGFR4 was observed in the tumor lesions of DF. Expression patterns of FGFR3/FOXN1 and FGF2/FGFR4 in DF were in contrast with those of DFSP. The activation of FGFR signaling pathways may be not only relevant to the pathogenesis of DF, but also very useful in the differential diagnosis of DF and DFSP.

A 43-year-old Japanese man presented with a 13-year history of a grayish macule measuring 7 cm in diameter with sparse hairs on the vertex. Histopathological examination demonstrated two types of melanocytes, spindle-shaped and ovoid cells, with abundant melanin aggregated around the upper part of the pilosebaceous apparatus. Fibrous, thick collagen bundles were also seen surrounding the upper part of the small hair follicles. There was no infiltration of melanocytes or lymphocytes in the lower dermis or adipose tissue. Based on these findings, a diagnosis of blue nevus, cellular type, was made. Giant cellular blue nevi on the scalp are rare, and 11 cases reported in the published work have shown characteristic features such as hair loss and cranial invasion of nevus cells. It should be noted that melanocytes of giant blue nevi have a high potential to damage other organs such as underlying bone and hair follicles. The patient also showed a typical lesion of alopecia areata on the left temporal which was successfully treated with topical corticosteroid.

Expression of the p16Ink4a tumor suppressor gene, a sensor of oncogenic stress, is up-regulated by a variety of potentially oncogenic stimuli in cultured primary cells. However, because p16Ink4a expression is also induced by tissue culture stress, physiological mechanisms regulating p16Ink4a expression remain unclear. To eliminate any potential problems arising from tissue culture–imposed stress, we used bioluminescence imaging for noninvasive and real-time analysis of p16Ink4a expression under various physiological conditions in living mice. In this study, we show that oncogenic insults such as ras activation provoke epigenetic derepression of p16Ink4a expression through reduction of DNMT1 (DNA methyl transferase 1) levels as a DNA damage response in vivo. This pathway is accelerated in the absence of p53, indicating that p53 normally holds the p16Ink4a response in check. These results unveil a backup tumor suppressor role for p16Ink4a in the event of p53 inactivation, expanding our understanding of how p16Ink4a expression is regulated in vivo.

Although the role of p21 Waf1/Cip1 gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the role of p21 Waf1/Cip1 gene expression in vivo , we attempted to visualize the dynamics of p21 Waf1/Cip1 gene expression in living animals. In this study, we established a transgenic mice line (p21- p -luc) expressing the firefly luciferase under the control of the p21 Waf1/Cip1 gene promoter. In conjunction with a noninvasive bioluminescent imaging technique, p21- p -luc mice enabled us to monitor the endogenous p21 Waf1/Cip1 gene expression in vivo . By monitoring and quantifying the p21 Waf1/Cip1 gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p21 Waf1/Cip1 gene expression in the aging process. We also applied this system to chemically induced skin carcinogenesis and found that the levels of p21 Waf1/Cip1 gene expression rise dramatically in benign skin papillomas, suggesting that p21 Waf1/Cip1 plays a preventative role(s) in skin tumor formation. Surprisingly, moreover, we found that the level of p21 Waf1/Cip1 expression strikingly increased in the hair bulb and oscillated with a 3-week period correlating with hair follicle cycle progression. Notably, this was accompanied by the expression of p63 but not p53. This approach, together with the analysis of p21 Waf1/Cip1 knockout mice, has uncovered a novel role for the p21 Waf1/Cip1 gene in hair development. These data illustrate the unique utility of bioluminescence imaging in advancing our understanding of the timing and, hence, likely roles of specific gene expression in higher eukaryotes.

Summary Background Aberrant methylation of CpG islands in the promoter regions of cancer-related genes has been demonstrated in many human tumours. However, the methylation profile of these regions in cutaneous squamous cell carcinomas (SCCs) has not been well studied. Objectives To examine epigenetic abnormalities of a wide range of cancer-related genes in SCCs. Methods We investigated the methylation status of 11 candidate cancer-related genes (CDH1, p16INK4a, p14ARF, DAPK1, MGMT, RB1, RASSF1, p15INK4b, PTEN, PRDM2 and p53) in 20 cases of SCC by methylation-specific polymerase chain reaction, and comparatively examined the protein production of E-cadherin (CDH1), p16, RB1, p14, BMI1 and cyclin A by immunohistochemical analysis. Results The frequency of cancer-related gene methylation in SCCs was: CDH1 (95%), p16 (20%), p14 (15%), DAPK1 (15%), MGMT (15%), RB1 (5%), RASSF1 (5%), p15 (0%), PTEN (0%), PRDM2 (0%) and p53 (0%). Almost all cases with hypermethylation of CDH1, p16, RB1 and p14 showed no obvious production of each protein, suggesting that promoter hypermethylation of these genes contributes to the loss of protein production. The results of methylation analysis, in combination with the results of our previous mutation analysis of CDKN2A locus and p53, revealed that 70% of SCCs have alterations in the RB1/p16 or p53 pathway. Conclusions Our findings indicate that the promoter hypermethylation of cancer-related genes, especially CDH1, is frequently shown in SCCs, and dysregulation of the RB1/p16 and/or p53 pathway through either genetic or epigenetic mechanisms, except for epigenetic abnormalities of p53 itself, should contribute to the carcinogenesis of SCCs.

E2F/DP complexes were originally identified as potent transcriptional activators required for cell proliferation. However, recent studies revised this notion by showing that inactivation of total E2F/DP activity by dominant-negative forms of E2F or DP does not prevent cellular proliferation, but rather abolishes tumor suppression pathways, such as cellular senescence. These observations suggest that blockage of total E2F/DP activity may increase the risk of cancer. Here, we provide evidence that depletion of DP by RNA interference, but not overexpression of dominant-negative form of E2F, efficiently reduces endogenous E2F/DP activity in human primary cells. Reduction of total E2F/DP activity results in a dramatic decrease in expression of many E2F target genes and causes a senescence-like cell cycle arrest. Importantly, similar results were observed in human cancer cells lacking functional p53 and pRB family proteins. These findings reveal that E2F/DP activity is indeed essential for cell proliferation and its reduction immediately provokes a senescence-like cell cycle arrest.

The nuclear factor NF-kappaB and oncogenic Ras can alter proliferation in epidermis, the most common site of human cancer. These proteins are implicated in epidermal squamous cell carcinoma in mice, however, the potential effects of altering their function are uncertain. Whereas inhibition of NF-kappaB enhances apoptosis in certain tumours, blockade of NF-kappaB predisposes murine skin to squamous cell carcinoma. Because therapeutics inhibiting Ras and NF-kappaB pathways are being developed to treat human cancer, it is essential to assess the effects of altering these regulators. The medical relevance of murine studies is limited, however, by differences between mouse and human skin, and by the greater ease of transforming murine cells. Here we show that in normal human epidermal cells both NF-kappaB and oncogenic Ras trigger cell-cycle arrest. Growth arrest triggered by oncogenic Ras can be bypassed by IkappaBalpha-mediated blockade of NF-kappaB, generating malignant human epidermal tissue resembling squamous cell carcinoma. Human cell tumorigenesis is dependent on laminin 5 and alpha6beta4 integrin. Thus, IkappaBalpha circumvents restraints on growth promotion induced by oncogenic Ras and can act with Ras to induce invasive human tissue neoplasia.

Ras acts with other proteins to induce neoplasia. By itself, however, strong Ras signaling can suppress proliferation of normal cells. In primary epidermal cells, we found that oncogenic Ras transiently decreases cyclin-dependent kinase (CDK) 4 expression in association with cell cycle arrest in G1 phase. CDK4 co-expression circumvents Ras growth suppression and induces invasive human neoplasia resembling squamous cell carcinoma. Tumorigenesis is dependent on CDK4 kinase function, with cyclin D1 required but not sufficient for this process. In facilitating escape from G1 growth restraints, Ras and CDK4 alter the composition of cyclin D and cyclin E complexes and promote resistance to growth inhibition by INK4 cyclin-dependent kinase inhibitors. These data identify a new role for oncogenic Ras in CDK4 regulation and highlight the functional importance of CDK4 suppression in preventing uncontrolled growth.

Genital Bowen disease (BD) has been linked to the high-risk types of human papillomavirus (HPV) infection. Recently, it has been recognized that HPV also can be associated with extragenital BD. HPV oncoproteins E6 and E7 interfere with the function of p53 and pRb, respectively, leading carcinogenesis. p16(INK4a) overexpression induced by inactivation of pRb is recognized as a surrogate marker for HPV-associated cervical cancer. In this study, we examined the presence of HPV DNA in 142 BD lesions by polymerase chain reaction (PCR), and determined the type of HPV by PCR restriction fragment length polymorphism or direct DNA sequencing. HPV DNA was detected in 66.7% of genital BD and 8.3% of extragenital BD. The types of HPV detected were HPV types 6, 16, 33, 52, 56, 58 and 59. We also investigated the expression of p16(INK4a) , pRb and p53 by immunohistochemistry. Positive expression was detected in 88.6% for p16(INK4a) , 25.2% for pRb, and 63.8% for p53. There was no significant difference in p16(INK4a) and pRb expression between HPV-positive and -negative BD. However, a strong correlation of HPV positivity with p53 negativity was found. A total of 66.7% of HPV-positive BD showed no p53 expression, whereas the corresponding rate was 32.8% of HPV-negative BD. This study demonstrated that HPV can participate in the development of BD, not only in the genital lesion, but also in extragenital lesion. p16(INK) (4a) overexpression is not a marker for HPV infection in BD. Instead, negative p53 expression is correlated with HPV-associated BD.

Granulomatous mastitis (GM) is a rare chronic inflammatory breast disease that can be mistaken for a variety of inflammatory and neoplastic disorders of the breast. Erythema nodosum (EN) is a common panniculitis, and is often associated with a variety of diseases, yet coexistence of EN and GM is very rare. In this paper, we present a young Japanese woman with EN associated with GM probably due to Corynebacterium infection. Both lesions of EN and GM were successfully treated with oral minocycline and surgical drainage. To the best of our knowledge, this is the first case of EN associated with GM due to Corynebacterium infection in the English-language published work.

Squamous cell carcinomas (SCCs) are supposed to develop through the multistep process that involves activation of proto-oncogenes and/or inactivation of tumor suppressor genes in the human epidermis. To elucidate the multistep process, we screened mutations in H-Ras, p53, PTEN, and the INK4a locus in 21 SCCs by meansof PCR-SSCP analysis. One, 3, and 7 SCCs were detected mutations in H-Ras, INK4a, and p53, respectively. No mutation was detected in PTEN in any SCC. Nine SCCs in total were shown to have mutations at least in one gene. Among them, we detected both mutations of H-Ras and INK4a in one SCC, and both mutations of p53 and INK4a in another SCC. In particular, activation of H-Ras and inactivation of both of p16INK4a and p14ARF were supposed in the former SCC. This finding indicates the possibility that the combination of mutations might contribute to the cacinogenesis in the SCC. [Skin Cancer (Japan) 2001; 16: 353-356]

A 71-year-old Japanese man was seen in May 1995 with a 3-year history of a tumor developing on his left cheek. Although he had the tumor excised in 1994 by a local doctor, it locally recurred and enlarged to a 3×3cm, domeshaped tumor. After a diagnosis of eccrine porocarcinoma was made on the basis of clinical and histological findings, excision of the tumor with 3cm of margin was performed together with superficial lobectomy of the left parotid gland and left radical neck dissection. The expression of carcinoembryonic antigen (CEA) was immunohistochemically observed in the cytoplasm of tumor cells. A serum level of CEA was elevated to 10.3ng/ml (normal

We searched for the rat homologue of the human tuberous sclerosis (TSC2) gene mutations in loss of heterozygosity (LOH)-negative Eker rat renal carcinomas (RCs) by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis using 45 primer sets covering all 41 coding exons and one leader exon including splicing donor/acceptor sites. We have identified intragenic somatic mutations in 7 of 21 spontaneous RCs, including one cell line (33%), and in 3 of 9 (33%) N-ethyl-N-nitrosourea (ENU)-induced LOH-negative RCs. Interestingly, five mutations in the spontaneous RCs were either deletion or duplication (5/7 = 71%). In contrast, all three in ENU-induced RCs were base substitutions (3/3 = 100%), as expected. Thus, a qualitative difference in the second hit might exist between spontaneous and ENU-induced mutations (e.g., deletion or duplication versus point mutation). By a direct cloning approach utilizing the restriction length difference caused by germline insertional mutation or reverse transcriptase-PCR analysis in two applicable cases, we could clearly show the presence of intragenic somatic mutations in the second copy (wild-type) of the Tsc2 gene. This is the first demonstration at the DNA sequence level of the validity of Knudson's two-hits hypothesis in the Tsc2 gene.

We have recently identified on rat chromosome 10q a germline mutation in the tuberous sclerosis gene (Tsc2), the gene predisposing to renal carcinoma (RC) in the Eker rat. The homozygous mutant condition is lethal at around the 13th day of fetal life. In heterozygotes, RCs invariably develop in the first year of life. Histologically, RCs develop through multiple stages from early preneoplastic lesions (i.e., phenotypically altered tubules) to adenomas. The wild-type allele mutation has been found even in the earliest preneoplastic lesions, fitting Knudson's two-hit hypothesis and supporting the hypothesis that Tsc2 is a tumor suppressor gene. In this study, homozygous deletion of the Ink4 homologue on rat chromosome 5q was observed in 14 of 24 (58%) RC-derived cell lines. This may represent involvement of a second tumor suppressor gene, contributing to tumor progression. Considering previous results of studies of homozygous deletion of the Ifn alpha gene in five of 24 cases (21%) and the Ifn beta gene in one of 24 cases (4%), the order of the genes may be Ink4-Ifn alpha-Ifn beta. Microsatellite instability was not observed in 26 Eker rat tumors.

Hereditary renal carcinomas (RCs) develop in virtually all Eker rats by the age of one year. Investigation of extra‐renal primary tumors co‐occurring in Eker rats late in life (at 2 years) additionally revealed enhanced development of hemangiosarcomas of the spleen, uterine leiomyosarcomas and pituitary adenomas, although the demonstrated predilection for these extra‐renal tumors was not as complete as with RCs. We identified the germline mutated tuberous sclerosis (Tsc2) gene as the predisposing Eker gene and revealed the tumor suppressor nature of Tsc2 gene function in renal carcinogenesis. In the present study, we examined allelic loss at the Tsc2 gene locus in uterine leiomyosarcomas and pituitary adenomas developing in hybrid F1 rats carrying the Eker mutation as well as in pituitary adenomas from non‐carrier rats. We detected loss of heterozygosity in 4 of 11 uterine leiomyosarcomas (36%) and 11 of 31 pituitary adenomas (35%) from Eker rats but in none of 9 pituitary adenomas from non‐carrier rats (P

p53 expression was studied immunohistochemically to identify a precursor lesion of basal cell carcinoma (BCC) in the epidermis adjacent to BCC. With two different anti-p53 antibodies of CM1 and DO7, p53 expression was frequently detected in the epidermis adjacent to BCCs arising on the face and in the normal epidermis with usual sun exposure. In the epidermis adjacent to BCC, stained cells were occasionally clustered in a small area, but no cluster was found in the normal epidermis with usual sun exposure. The expression was less frequent in the normal epidermis with rare sun exposure. Ten cases of normal skin with usual sun exposure, showing CM1 staining in the epidermis, were screened for p53 gene mutations with polymerase chain reaction-single- strand conformation polymorphism analysis using DNAs obtained from the epidermis. No mutation was detected in exons 2 to 10 of the p53 gene in these 10 cases. The epidermis flanking three BCCs that was stained with CM1, on the other hand, carried a missense mutation of C to G transversIon at a dipyrimidine site of codon 249. This alteration replaced arginine with threonine. The mutation of codon 249 was not detected in the three BCCs. Our results first suggest that ultraviolet light irradiating the skin in a daily life induces p53 accumulation in the epidermis and secondly that the frequent clonal expansion of p53 mutant cells occurs in the epidermis adjacent to BCCs. This clonal expansion of mutant p53 may provide a molecular basis for high risk of developing subsequent new skin cancers in patients with BCC.

Objective: Although monoammonium glycyrrhizinate/glycine/DL-methionine (MG) combination tablets have been widely used widely for the treatment of alopecia areata (AA), there are few studies on efficacious combinations with MG. This study was conducted to determine the efficacy and safety of MG plus 5% carpronium chloride (CC). Methods: In the present interventional study, MG tablets plus 5% carpronium chloride (CC) were compared with CC monotherapy in 31 patients with AA. Results: There were no significant differences in efficacy between the two groups, and the AA area at 8 and 12 weeks was significantly reduced in both. The results of subanalysis stratified by the presence of allergic factors as determined by IgE level showed that there were also significant decreases in the areas of AA 8 and 12 weeks after the start of the combination therapy in patients with allergic factors (p

Summary The aim of this study was to investigate the initiation and progression of autoimmune damage in the lesions of labial salivary glands (LSGs) from primary Sjögren's syndrome (SS) patients by examining the selective localization of T helper (Th) subsets such as Th1, Th2, Th17 regulatory T cells (Tregs) and follicular T helper cells (Tfh). The expression of cytokines and transcription factors associated with these Th subsets in the LSGs from 54 SS patients and 16 healthy controls was examined using real-time polymerase chain reaction (PCR) and immunostaining. Additionally, infiltrating lymphocytes without germinal centre (GC-) and with GC (GC+) in the LSGs specimens from eight SS patients were extracted selectively by laser capture microdissection (LCM). The mRNA expression of these molecules was compared between the two sample groups of GC- and GC+ by real-time PCR. The mRNA expression of cytokines and transcription factors of all T helper (Th) subsets in the LSGs from the SS patients was increased significantly in comparison with controls. In LSGs from the SS patients, Th2 and Tfh was associated closely with strong lymphocytic infiltration; however, Th1, Th17 and Tregs was not. In the selectively extracted lesions of LSGs, Th1 and Th17-related molecules were detected strongly in the GC-, while Th2 and Tfh-related molecules were detected in the GC+. In contrast, no significant association with strong lymphocytic infiltration was observed in Treg-related molecules. These results indicate that SS has selective localization of Th subsets such as Th1, Th2, Th17 and Tfh in the LSGs, which is associated closely with disease severity and/or status. SS might be initiated by Th1 and Th17 cells, and then progressed by Th2 and Tfh cells via GC formation.

Mikulicz’s disease (MD) was considered to be a subtype of Sjogren’s syndrome (SS), based on histopathological similarities. However, recent studies have indicated that patients with MD show high serum IgG4 concentration, and suggested that MD is one of “IgG4-related disease” and distinguishable from SS. Therefore, we clinically and histopathologically examined the disease states of MD and SS in detail. Twenty patients with Mikulicz’s disease and 18 with SS were comparatively studied to determine clinical characteristics in MD patients. Sialography in MD patients did not show the “apple-tree sign” typically seen in SS. Serologically, high serum IgG4 levels but not anti-SS-A or anti-SS-B antibodies were observed in MD. SS showed lymphocytic infiltration of various subsets with atrophy or severe destruction of the acini, while MD showed selective infiltration of IgG4+ plasma cells with hyperplastic germinal centers and mild acini destruction. Corticosteroid treatment of MD reduced IgG and IgG4 levels and improved salivary function. A negative correlation between disease duration and increasing rate of salivary flow was observed in MD. These results suggested that the pathogenesis of MD might be different from those of SS. Clinical Relevance: early diagnosis and treatment of MD is important for the improvement of salivary function.

Hereditary renal carcinomas (RCs) develop in virtually all Eker rats by the age of one year. Investigation of extrarenal primary tumors occurring in Eker rats late in life (at 2 years) additionally revealed pituitary adenomas (17/31 = 54.8% (carrier) vs 7/32 = 21.9% (non-carrier siblings of affected animals), P < 0.01), sarcomas of the spleen (21/31 = 67.7% vs. 0/32 = 0%, P < 0.001) and sarcomas (of probable stromal origin) of the uterus (8/17 = 47.1% vs. 0/15 = 0%, P < 0.01). Thus, the Eker rat might provide a novel animal model of cancer predisposition syndromes.

Mutations of exons 3 through 9 of the p53 gene in skin lesions were screened in 23 cases of squamous cell carcinoma (SCC), 25 cases of basal cell carcinoma (BCC), two cases of Bowen's disease, 10 cases of solar keratosis, and five cases of keratoacanthoma by polymerase chain reaction--single strand conformation polymorphism analysis. Mutations of the p53 gene were detected in seven of 23 SCCs (30%), three of 25 BCCs (12%), and none in all cases of Bowen's disease, solar keratosis, or keratoacanthoma. Of 23 cases of SCC, mutations were detected in four of 15 SCCs (27%) that originated in the sunlight-exposed skin region, in two of three SCCs (67%) that originated in the scar tissue, and in one of three SCCs (33%) that originated in radiation dermatitis. Mutations of C-->T transition predominated in SCC and BCC that originated in the sunlight-exposed skin region. Mutations of C-->A or CC-->AT observed in tumors that originated in the predisposed conditions, presumably unrelated to UV light, are different from those found in UV light-related SCC or BCC. Twelve cases of SCC were comparatively analyzed with the immunohistochemical staining with anti-p53 antibody. Two of four cases with positive staining had missense mutations, and three of eight cases with negative staining had nonsense mutations. Based on these findings, immunohistochemical results do not necessarily mean the presence or absence of p53 gene mutations in skin tumors, and sequence analysis is essential for determining whether the gene is mutated.

Objective Mikulicz disease has been considered to be a subtype of Sjogren's syndrome (SS). However, recent studies have suggested that Mikulicz disease is an IgG4-related disease and is distinguishable from SS. In addition, it has been reported that both interleukin-4 (IL-4) and IL-10 induce IgG4 production and inhibit IgE. This study was undertaken to examine the expression of these cytokines in patients with Mikulicz disease and patients with SS. Methods Labial salivary gland (LSG) sections from 15 patients with Mikulicz disease and 18 patients with SS were examined for subsets of the infiltrating lymphocytes, expression patterns of messenger RNA (mRNA) for cytokines/chemokines, and relationships between the IgG4:IgG ratio and the expression of mRNA for IL-4 or IL-10. Results Immunohistochemical analysis showed lymphocyte infiltration of various subsets in the LSGs of SS patients, and the selective infiltration of IgG4-positive plasma cells and Treg cells in the LSGs of Mikulicz disease patients. The levels of mRNA for both Th1 and Th2 cytokines and chemokines in LSGs from patients with SS were significantly higher than in controls, while the expression of both Th2 and Treg cells was significantly higher in the patients with Mikulicz disease than in controls. Furthermore, the expression of IL-4 or IL-10 in the LSGs was correlated with the IgG4:IgG ratio. Conclusion These results suggest that the pathogenesis of Mikulicz disease is different from that of SS. Mikulicz disease is a unique inflammatory disorder characterized by Th2 and regulatory immune reactions that might play key roles in IgG4 production.

Summary Background The activation of oncogenes is an important step in tumorigenesis, and recently, oncogene-induced senescence (OIS) was proposed as a critical barrier against malignant transformation in normal primary cells. Objectives The aim of this study was to examine the activation of fibroblast growth factor receptor 3 (FGFR3) as an oncogene product and OIS in human skin tumours. Methods We investigated the activation of FGFR3 and OIS by mutation and immunohistochemical analysis in skin tumours, including seborrhoeic keratosis, actinic keratosis (AK), Bowen’s disease (BD), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Results Activated point mutations of FGFR3 were identified in four of 22 cases (18%) of seborrhoeic keratosis, but no mutation was detected in the other skin tumours. Twenty-seven of 31 cases (87%) of seborrhoeic keratosis showed moderately to strongly positive expression of the FGFR3 protein, but almost all the other skin tumours were negative. On the other hand, almost all the seborrhoeic keratoses showed negative immunoreactivity for antiphoshohistone H2AX (γ-H2AX) as a marker of OIS, but 17 of 22 cases (77%) of AK were moderately to strongly positive. Immunoreactivity for γ-H2AX was significantly greater in AK than in seborrhoeic keratosis, BD, BCC and SCC. Conclusions The activation of FGFR3 might be a common feature in the tumorigenesis in seborrhoeic keratosis, although the activation does not induce a typical oncogenic signal in keratinocytes. In addition, OIS due to some oncogenic signals rather than activation of FGFR3 might be involved in the early skin carcinogenesis related to chronic ultraviolet radiation exposure.

Summary Background Ephrins are cell-membrane-bound ligands for Eph receptor tyrosine kinases (Eph). Although ephrins are known to regulate a variety of developmental processes, little is known of their role in hair development. Previously, we studied the gene expression of dermal papilla cells from androgenetic alopecia and found that ephrin-A3 was significantly down-regulated. Objective To characterize the expression of ephrin-A3 in the hair cycle and evaluate the effect of ephrin-A3 on hair growth. Methods We investigated gene expression and protein expression of each ephrin-As and EphAs in the skin of neonatal mice through the first and second hair cycle using quantitative PCR and immunohistochemical analysis, respectively. We also injected ephrin-A3 protein into the skin of neonatal mice and demonstrated the effect of ephrin-A3 on hair follicle development. Results Expression of ephrin-A3 revealed a rapid increase at the beginning of the anagen phase, a peak during the mid-anagen, and a rapid fading during the telogen phase. In addition, we found ephrin-A3 protein was expressed in the developing hair follicles with a characteristic spatiotemporal localization. Furthermore, injection of ephrin-A3 into the skin of neonatal mice markedly accelerated the differentiation process of hair follicles. In addition, injection of ephrin-A3 unexpectedly increased the number of hair follicles. Conclusion These findings demonstrated that ephrin-A3 not only accelerates anagen development but also increases the density of hair follicles, and also suggested that an ephrin-A–EphA signal pathway is closely involved in hair follicle development.

The tumor suppressor PTEN is mutated in many human cancers. We previously used the Cre-loxP system to generate mice (LckCrePten mice) with a Pten mutation in T-lineage cells. Here we describe the phenotype of Pten-deficient Valpha14iNKT cells. A failure in the development of Valpha14iNKT cells occurs in the LckCrePten thymus between stage 2 (CD44(high)NK1.1(-)) and stage 3 (CD44(high)NK1.1(+)), resulting in decreased numbers of peripheral Valpha14iNKT cells. In vitro, Pten-deficient Valpha14iNKT cells show reduced proliferation and cytokine secretion in response to alphaGalCer stimulation but enhanced inhibitory Ly49 receptor expression. Following interaction with dendritic cells (DCs) loaded with alphaGalCer, Pten-deficient Valpha14iNKT cells demonstrate activation of PI3K. Indeed, the effects of the Pten mutation require intact function of the PI3K subunits p110gamma and p110delta. In vivo, LckCrePten mice display reduced serum IFNgamma after alphaGalCer administration. Importantly, Valpha14iNKT cell-mediated protection against the metastasis of melanoma cells to the lung was impaired in the absence of Pten. Thus, the Pten/PI3K pathway is indispensable for the homeostasis and antitumor surveillance function of Valpha14iNKT cells.

The class III histone deacetylase (HDAC), SIRT1, is a mammalian homologue of the Saccharomyces cerevisiae chromatin-silencing factor Sir2 that regulates longevity. SIRT1 regulates cell survival via deacetylation of p53 and forkhead transcription factors, and overexpression of SIRT1 is reported to be essential for cell growth and survival in some kinds of cancer. To elucidate the role of SIRT1 in human skin carcinogenesis, we have examined SIRT1 protein expression in 20 cases each of squamous cell carcinoma (SCC), basal cell carcinoma (BCC), Bowen's disease (BD), and actinic keratosis (AK) by immunohistochemical analysis. Overexpression of SIRT1 is frequently observed in all kinds of non-melanoma skin cancers included in this study. In particular, strong expression was observed in all cases of BD. In addition, no obvious difference between AK and SCC was observed in the expression of SIRT1, suggesting that overexpression of SIRT1 may have some relevance to the early stage of skin carcinogenesis. We suppose that SIRT1 could be one of the critical targets for future therapy with the aim of inhibiting cell proliferation and promoting apoptosis in non-melanoma skin cancers.

We report a case of Bowen's disease on the sole presenting clinically as an exophytic, blackish-grey, verrucous tumour, and showing human papillomavirus (HPV) type 16 on analysis with polymerase chain reaction. Positive stains for HPV particles by immunohistochemical analysis were limited to several cell nuclei at the upper stratum Malpighii. However, all the tumour cells in the epidermis exhibited strong and diffuse nuclear and cytoplasmic stains for the tumour suppressor protein p16INK4a. We speculate that dysregulation of the retinoblastoma/p16INK4a pathway may be involved in the pathogenesis of the lesion, and p16INK4a overexpression might serve as a useful surrogate marker for identifying Bowen's disease harbouring high-risk types of HPV infection.

Cowden disease (CD) is an autosomal dominant syndrome characterized by multiple hamartomatous lesions and an increased risk for malignancies. Recent evidence has indicated that the PTEN gene, encoding a protein tyrosine phosphatase, is the CD susceptibility gene. However, another line of evidence has suggested that CD might be genetically heterogeneous. Clinical features of CD are variable, and there are interfamilial differences in the expression of skin lesions . Therefore, information on PTEN mutations in CD patients should be accumulated to clarify the genotype–phenotype correlation. In the present study, we found heterozygous germline mutations of PTEN in all of three Japanese patients with CD examined, indicating no genetic heterogeneity among our patients. The mutations included two non-sense mutations of R335X and R130X, and a mis-sense mutation of C136R. To the best of our knowledge, the C136R mutation has not previously been reported in CD patients. This novel mutation was located outside the core motif of the phosphatase domain of PTEN protein, where most of the missense mutations previously reported in CD patients were clustered. Mucocutaneous manifestations were far fewer in the patient with this mutation than in the patients with nonsense mutations. Whether the phenotypic difference in mucocutaneous features was due to the different mutations remains unclear.

The tumor suppressor gene PTEN is deleted and/or mutated in a variety of tumors and the susceptibility gene for Cowden disease. Loss of heterozygosity of chromosome 10q23, where PTEN resides, in squamous cell carcinomas (SCCs) of human skin and the association of SCC with Cowden disease were reported previously. In the present study, we screened for mutations of PTEN in SCCs by polymerase chain reaction–single strand conformation polymorphism analysis to examine whether PTEN is involved in the carcinogenesis of SCC. None of 21 SCCs showed somatic mutations in the coding regions of PTEM . Instead the same allelic variation was detected in two cases without any clinical features of Cowden disease. Our results indicate that inactivation of PTEN does not play an important role in the carcinogenesis of SCC.

The INK4a locus encodes two different, cell cycle-regulating proteins, p16INK4a and p19ARF. In this study, we screened mutations in all coding regions of the INK4a locus (exons 1 beta, 1 alpha, 2, and 3) in 21 squamous cell carcinomas (SCCs) of human skin by polymerase chain reaction-single strand conformation polymorphism analysis. Mutations were detected in 3 SCCs in exon 2, which is common to both p16INK4a and p19ARF. These included an in-frame deletion of 21 base pairs from codon 84 to 90, a frameshift mutation of CCC-->TC at codon 75, and a nonsense mutation of CGA-->TGA at codon 80 of the p16INK4a gene. These results suggest that inactivation of the INK4a locus has some relevance to the carcinogenesis in at least some of SCCs of human skin. This is the first demonstration of aberrations in the INK4a locus in SCCs of human skin.

The Eker rat hereditary renal carcinoma (RC) is an excellent example of a mendelian dominant predisposition to a specific cancer in an experimental animal. We have previously established a new conserved linkage group on rat chromosome 10q and human chromosome 16p13.3, and shown that the Eker mutation is tightly linked to the tuberous sclerosis (Tsc2) gene. We now describe a germline mutation in the gene encoding Tsc2 caused by the insertion of an approximately 5 kilobase DNA fragment in the Eker rat, resulting in aberrant RNA expression from the mutant allele. The phenotype of tuberous sclerosis in humans differs from that of the Eker rat, except for the occurrence of renal tumours. The Eker rat may therefore provide insights into species-specific differences in tumourigenesis and/or phenotype-specific mutations.