Aims-To study the expression and importance (if any) of p53 protein in gall bladder carcinoma and its precursor lesions. Methods-Immunohistochemical staining was performed on formalin fixed, paraffin wax embedded histological sections with an anti-human p53 monoclonal antibody (DO-7; Dako Corporation M7001) (24 carcinomas, one adenocarcinoma in situ, six dysplasias, three adenomas and four cases of chronic cholecystitis). Invasive, in situ, and dysplastic areas as well as normal-looking epithelium were sought. Nuclear Carcinogenesis is a multistep process involving several genetic changes which include activation of cellular proto-oncogenes and inactivation of tumour suppressor genes.7Invasive carcinomas of the gall bladder are often associated with dysplasia and adenocarcinoma in situ changes in the neighbouring epithelium. This association implies a temporal progression or radical change in the process of carcinogenesis and provides a good sequential model for studying the evolution of p53 protein expression in the malignant transformation of gall bladder epithelium. MethodsTwenty four carcinomas of the gall bladder, one adenocarcinoma in situ, six epithelial dysplasias, three adenomas and four cases of chronic cholecystitis were retrieved from the files of the department of pathology, National University Hospital. All the tissue had been fixed in 10% buffered formalin and embedded in paraffin wax. Slides stained with haematoxylin and eosin and periodic acid-Schiff before and after diastase digestion were evaluated. The cases were analysed for invasive, in situ, and dysplastic areas as well as normallooking epithelium.Immunohistochemical staining was performed using a streptavidin-biotin immunoperoxidase method. Waxed sections (4 im) were treated with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase. The sections were then incubated with 5% bovine serum albumin (BSA) in TRISbuffered saline (TBS) for 30 minutes to eliminate any non-specific staining. Excess BSA was drained from the slides before incubation with an anti-human p53 monoclonal antibody (DO-7; Dako Corporation M7001) at an optimal dilution of 1 in 50 in 0-1% BSA overnight at 4°C. The tissue sections were subsequently washed with TBS and incubated with biotinylated rabbit-anti-mouse immunoglobulin (Dako; E354), 1 in 200 in 0-1% BSA

Background. Expression of p53 protein has been described in a variety of human malignant tumors. Recent reports have also demonstrated its presence in benign and reactive lesions. The significance of p53 expression is unclear. Methods. This study examines the p53 expression in proliferative lesions of skin, including 6 pseudoepitheliomatous hyperplasia, 33 keratoacanthoma, and 45 squamous cell carcinoma, and to evaluate its significance. Results. p53 expression was observed in all of the six cases of pseudoepitheliomatous hyperplasia, 78.8% of keratoacanthoma, and 75.5% of squamous cell carcinoma. The staining pattern of pseudoepitheliomatous hyperplasia and keratoacanthoma was generally less intense and extensive compared with that of squamous cell carcinoma. A keratoacanthoma with nuclear atypia that showed strong and extensive p53 staining was also encountered. The perilesional skin in sun‐exposed sites often showed the presence of p53‐positive keratinocytes. Control skin taken from the buttock was negative for p53 protein. Conversely, p53 was often expressed in carcinomas arising from sun‐exposed as well as sun‐protected sites. p53 positivity involved mainly the undifferentiated cells at the base of the epidermis or periphery of tumor cords. Differentiated keratinized cells were not stained. p53‐positive fibroblasts were also noted in the inflammatory and granulation tissues of pseudoepitheliomatous hyperplasia. Conclusions. p53 expression in skin is common and appears to be an early event in a series of genetic alterations reflecting underlying actinic damage, which may lead to but does not necessarily indicate neoplastic or malignant transformation. Because p53 staining is seen in proliferative and undifferentiated cells and ceases to be expressed when the cells differentiate, it appears that the expression of p53 protein, mutant or wild‐type, is an indicator of immaturity and proliferative capacity of the cell rather than one of neoplasia or malignancy.

Aims-To study the expression and clinical significance (ifany) ofp53 protein in hepatocellular carcinomas (HCC) Singapore, with a predominantly Chinese population, has a high incidence of HCC.'7 It also has a significant hepatitis B virus (HBV) carrier population with most cases of HCC arising in patients with HBV infection.'8 Integration of HBV DNA into the hepatocytic genome is frequently associated with the presence of HCC, with chromosome 17 being one of the targets.'9 In some cases the viral insert was mapped to chromosome 17p near the p53 gene chromosomal location,20 raising the possibility of a link between p53 gene mutation and HBV induced HCC. There have been some recent data showing that the prevalence of p53 positive HCC was significantly higher in patients with serological markers of hepatitis B or C viral infection than in those without.2'With this in mind, we decided to analyse immunohistochemically the frequency and clinical significance (if any) of p53 protein expression in HCC occurring in Singapore where HBV infection is endemic. MethodsHepatocellular carcinoma liver resection specimens taken between January 1988 and March 1994 were retrieved from the archives of the Department of Pathology, National University Hospital, Singapore. This retrospective review yielded 46 cases, all of whom were of Asian origin (41 Chinese, three Malay and two Indonesian). All of the HCC specimens had been fixed in 10% buffered formalin and routinely processed. Paraffin wax embedded histological sections stained with haematoxylin and eosin were evaluated. The tumour size was noted. HCC specimens were graded from I to IV according to Edmondson and Steiner's classification.22 Vascular invasion was recorded. Adjacent nontumorous liver parenchyma was examined for the presence of cirrhosis. p53 protein immunostaining was performed using a Streptavidin biotin immunoperoxidase method. Deparaffinised 4 gm sections were treated with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase. The sections were then incubated with 5% bovine serum albumin (BSA) in TRIS-buffered saline (TBS) for 30 minutes to eliminate any non-specific staining. Excess BSAwas drainedfromthe slides before the addition of an antihuman p53 monoclonal antibody (DO-7; Dako, Glostrup, Denmark; M7001) at an optimal dilution of 1 in 50 in 0-1% BSA and incubated overnight at 4°C. The tissue sections were subsequently washed with TBS and incubated with biotinylated 236 on 7 May 2018 by guest. Protected by copyright.

The activity of a previously identified hematopoietic stem cell marker, Runx1 enhancer element (eR1) (Ng et al, Stem Cells 28, 1869-1881, 2010), was found to mark tissue stem cells of multiple organs. In corpus of stomach, stem cells are known to reside at isthmus, upper part of gastric unit. They are undifferentiated cells. However, molecular approach to these stem cells has not been described. Recently, fully differentiated Pepsinogen expressing chief cells at the bottom of gastric unit were shown to have tissue regeneration activity, and these cell were named reserve stem cells (Stange et al, Cell 155, 357-368, 2013). In our study, eR1 was shown to mark stem cells of both types. Lineage tracing experiments demonstrated that both types of stem cells continuously gave rise to mature cells to maintain the gastric unit. Manipulation of gene expression in a stem cell-specific manner in the stomach, especially in undifferentiated stem cells, is a long sought-after approach to study gastric carcinogenesis. We crossed transgenic mouse carrying eR1-CreERT2 with LSL-K-rasG12D mice. After tamoxifen treatment, rapid differentiation from the stem cells in the isthmus was observed, mainly into Muc5ac+ cells (surface epithelial cells). As a result, pseudo-pyloric metaplasia was induced which is similar to that observed in human gastritis. Acid-producing parietal cells were eliminated during this process. Rapid generation of Muc5ac+ cells and other phenotype are similar, but not identical, to the phenotype described in Menetrier disease which is known to be caused by excessive production of TGF-α and hyper-stimulation of EGF receptor (Reviewed in Coffey et al, J Clin Invest. 117:70-80, 2007). Menetrier disease is considered to be pre-malignant state. Activation of K-rasG12D in eR1+ chief cells also induced metaplastic lesions. In this case, pepsinogen producing chief cells robustly expressed the marker of mucous neck cells that are considered to be a precursor to chief cells. Therefore, chief cells appeared to be de-differentiated to precursor cells and acquired the stem cell property. We are using eR1 to study step-wise carcinogenesis in stomach and other organs. Citation Format: Junichi Matsuo, Shunichi Kimura, Cai Ping Koh, Md Zakir Hossain, Akihiro Yamamura, Kazuyoshi Kohu, Michiaki Unno, Jimmy Bok Yan So, Feng Zhu, Supriya Srivastava, Teh Meng, Nicholas Barker, Khay Guan Yeoh, Motomi Osato, Yoshiaki Ito. Tissue stem cell specific enhancer element identifies two types of stem cells in the corpus epithelium of the stomach. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-139. doi:10.1158/1538-7445.AM2015-LB-139