Tp53/p53 status in keratoacanthomas.

BACKGROUND: Keratoacanthoma (KA) is a common keratinocytic skin neoplasm that typically develops rapidly and undergoes complete spontaneous regression. As the pro-apoptotic p53 protein may be involved in the lifecycle of KA, we studied the p53 status throughout the main stages of KA that include proliferation, maturation and regression in a large series of lesions. METHODS: One-hundred and twenty-four KAs were characterized with respect to age of the lesions both clinically and histopathologically, in addition to phenotypic characteristics such as cellular atypia, infiltration, inflammation and fibrosis. Tp53 mutations were detected by capillary electrophoresis, and p53 protein levels were assessed by immunohistochemistry. RESULTS: Tp53 mutations were detected in 49 cases (39.5%) and were associated with high p53 protein levels (p = 0.007) and histopathologic age of the lesions (p = 0.044). Significant association was also seen between high p53 protein levels and atypia (p = 0.036), whereas the association with infiltration showed borderline significance (p = 0.057). High p53 protein levels were significantly associated with gene mutations in transplanted, but not in non-transplanted patients. CONCLUSION: We show a high frequency of Tp53 mutations in KAs that is associated with increased p53 levels. The results indicate a role for the p53 protein in KA development.

COX-2 overexpression in resected pancreatic head adenocarcinomas correlates with favourable prognosis.

BACKGROUND: Overexpression of cyclooxygenase-2 (COX-2) has been implicated in oncogenesis and progression of adenocarcinomas of the pancreatic head. The data on the prognostic importance of COX expression in these tumours is inconsistent and conflicting. We evaluated how COX-2 overexpression affected overall postoperative survival in pancreatic head adenocarcinomas. METHODS: The study included 230 consecutive pancreatoduodenectomies for pancreatic cancer (PC, n = 92), ampullary cancer (AC, n = 62) and distal bile duct cancer (DBC, n = 76). COX-2 expression was assessed by immunohistochemistry. Associations between COX-2 expression and histopathologic variables including degree of differentiation, histopathologic type of differentiation (pancreatobiliary vs. intestinal) and lymph node ratio (LNR) were evaluated. Unadjusted and adjusted survival analysis was performed. RESULTS: COX-2 staining was positive in 71% of PC, 77% in AC and 72% in DBC. Irrespective of tumour origin, overall patient survival was more favourable in patients with COX-2 positive tumours than COX-2 negative (p = 0.043 in PC, p = 0.011 in AC, p = 0.06 in DBC). In tumours of pancreatobiliary type of histopathological differentiation, COX-2 expression did not significantly affect overall patient survival. In AC with intestinal differentiation COX-2 expression significantly predicted favourable survival (p = 0.003). In PC, COX-2 expression was significantly associated with high degree of differentiation (p = 0.002). COX-2 and LNR independently predicted good prognosis in a multivariate model. CONCLUSIONS: COX-2 is overexpressed in pancreatic cancer, ampullary cancer and distal bile duct cancer and confers a survival benefit in all three cancer types. In pancreatic cancer, COX-2 overexpression is significantly associated with the degree of differentiation and independently predicts a favourable prognosis.

Inhibitory effects of prostaglandin E2 on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma.

BACKGROUND: Several studies have described an increased cyclooxygenase-2 (COX-2) expression in pancreatic cancer, but the role of COX-2 in tumour development and progression is not clear. The aim of the present study was to examine expression of COX-2 in cancer cells and stromal cells in pancreatic cancer specimens, and to explore the role of PGE2 in pancreatic stellate cell proliferation and collagen synthesis. METHODS: Immunohistochemistry and immunofluorescence was performed on slides from whole sections of tissue blocks using antibodies against COX-2 and α-smooth muscle actin (αSMA). Pancreatic stellate cells (PSC) were isolated from surgically resected tumour tissue by the outgrowth method. Cells were used between passages 4 and 8. Collagen synthesis was determined by [(3)H]-proline incorporation, or by enzyme immunoassay measurement of collagen C-peptide. DNA synthesis was measured by incorporation of [(3)H]-thymidine in DNA. Cyclic AMP (cAMP) was determined by radioimmunoassay. Collagen 1A1 mRNA was determined by RT-qPCR. RESULTS: Immunohistochemistry staining showed COX-2 in pancreatic carcinoma cells, but not in stromal cells. All tumours showed positive staining for αSMA in the fibrotic stroma. Cultured PSC expressed COX-2, which could be further induced by interleukin-1ß (IL-1ß), epidermal growth factor (EGF), thrombin, and PGE2, but not by transforming growth factor-ß1 (TGFß). Indirect coculture with the adenocarcinoma cell line BxPC-3, but not HPAFII or Panc-1, induced COX-2 expression in PSC. Treatment of PSC with PGE2 strongly stimulated cAMP accumulation, mediated by EP2 receptors, and also stimulated phosphorylation of extracellular signal-regulated kinase (ERK). Treatment of PSC with PGE2 or forskolin suppressed both TGFß-stimulated collagen synthesis and PDGF-stimulated DNA synthesis. CONCLUSIONS: The present results show that COX-2 is mainly produced in carcinoma cells and suggest that the cancer cells are the main source of PGE2 in pancreatic tumours. PGE2 exerts a suppressive effect on proliferation and fibrogenesis in pancreatic stellate cells. These effects of PGE2 are mediated by the cAMP pathway and suggest a role of EP2 receptors.

Spindle proteins in resected pancreatic head adenocarcinomas: BubR1 is an independent prognostic factor in pancreatobiliary-type tumours.

AIMS: Spindle proteins such as Aurora A, Mad2 and BubR1 are important for chromosome segregation during mitosis. Dysfunction of these proteins is implicated in the development of many cancers. The aim was to examine their possible prognostic impact in resected adenocarcinomas in the pancreatic head. METHODS AND RESULTS: Two hundred and eighteen consecutively resected pancreatobiliary-type (n=145) and intestinal-type (n=73) adenocarcinomas involving the pancreatic head were examined for expression of Aurora A, Mad2 and BubR1 by immunohistochemistry on tissue microarrays. Aurora A (P<0.001) and Mad2 (P=0.003) were expressed more often and at higher levels in intestinal-type compared with pancreatobiliary-type tumours, whereas BubR1 was equally expressed in both histological types. Expression of BubR1, Aurora A and Mad2 was not associated with ploidy status. None of the spindle proteins was significantly associated with prognosis in intestinal-type tumours. In pancreatobiliary-type tumours, any BubR1 expression was sufficient to predict poor prognosis (P=0.006), whereas Aurora A and Mad2 expression was not significantly associated with prognosis (P=0.86 and P= 0.87, respectively). On adjusted Cox regression analysis, BubR1 expression independently predicted poor prognosis [P=0.002; hazard ratio (HR) 1.87, 95% confidence interval (CI) 1.26, 2.79)], particularly in small tumours (P=0.001; HR 2.93, 95% CI 1.53, 5.62). CONCLUSION: BubR1 expression is a novel, independent adverse prognostic factor after pancreatoduodenectomy of pancreatobiliary-type adenocarcinomas.

Spindle proteins are differentially expressed in the various histological subtypes of testicular germ cell tumors.

BACKGROUND: Testicular germ cell tumors (TGCTs) are characterized by an aneuploid DNA content. Aberrant expression of spindle proteins such as the Aurora kinases and the spindle checkpoint proteins MAD2 and BUB1B, are thought to contribute to the development of chromosomal instability and DNA aneuploidy in cancer. The importance of these spindle proteins remains unknown in the development of TGCTs, thus we have explored the expression levels of these proteins in normal and malignant testicular tissues. MATERIALS AND METHODS: Using tissue microarrays the expression levels of Aurora kinase A (AURKA), Aurora kinase B (AURKB), BUB1B and MAD2 were measured in normal, preneoplastic and malignant testicular tissues of different histological subtypes from 279 orchidectomy specimens by means of immunohistochemistry. RESULTS: All the spindle proteins except for AURKB were expressed in normal testis. Sixty-eight and 36%, respectively, of the primary spermatocytes in the normal testis were positive for BUB1B and MAD2, while only 5% of the cells were positive for AURKA. There was a significantly lower expression of the spindle checkpoint proteins in carcinoma in situ compared to normal testis (P=0.008 and P=0.043 for BUB1B and MAD2, respectively), while the level of AURKA was increased, however, not significantly (P=0.18). The extent of spindle protein expression varied significantly within the different histological subtypes of TGCTs (P<0.001 for AURKB, BUB1B and MAD2, P=0.003 for AURKA). The expression of AURKA was significantly elevated in both non-seminomas (P=0.003) and seminomas (P=0.015). The level of BUB1B was significantly decreased in non-seminomas (P<0.001). A similar tendency was observed for MAD2 (P=0.11). CONCLUSIONS: In carcinoma in situ of TGCTs the spindle checkpoint proteins MAD2 and BUB1B are significantly less expressed compared to normal testis, while the expression of AURKA is increased. We suggest that these changes may be of importance in the transition from in situ to invasive testicular cancer.

Role of the Wnt signaling pathway in keratoacanthoma.

BACKGROUND: Keratoacanthoma (KA) has a unique life cycle of rapid growth and spontaneous regression that shows similarities to the hair follicle cycle, which involves an active Wnt signaling during physiological regeneration. We analyzed the expression of the Wnt signaling proteins ß-catenin, Lef1, Sox9, and Cyclin D1 in young and old human KAs to investigate a possible role for Wnt signaling in KAs. AIM: To investigate the role of the Wnt/ß-catenin signaling pathway in human KAs. METHODS AND RESULTS: Formalin-fixed, paraffin-embedded tissue samples of 67 KAs were analyzed for protein expression using immunohistochemistry. The majority of KAs were positive for Sox9 and Cyclin D1 but not for nuclear-localized ß-catenin or Lef-1. No significant differences in protein expressions were seen between young and old KAs. However, we found a significant association between Ki67 and Cyclin D1 proteins (P= .008). CONCLUSIONS: The Wnt signaling pathway does not appear to play a significant role in the biogenesis of human KA. Sox9 overexpression may be indicative of inhibition of Wnt signaling. Sox-9 and Cyclin D1 are proliferation markers that are most likely transactivated by alternate signaling pathways.

Differentiation markers in pancreatic head adenocarcinomas: MUC1 and MUC4 expression indicates poor prognosis in pancreatobiliary differentiated tumours.

AIMS: To examine how accurately immunohistochemical markers discriminate between pancreatobiliary and intestinal-type adenocarcinomas in the pancreatic head and to explore the prognostic importance of these markers among each of these histological types. METHODS AND RESULTS: Histopathological features of 114 consecutively resected adenocarcinomas of pancreatobiliary (n = 67) and intestinal (n = 47) type of differentiation were recorded according to a standardized protocol. Immunohistochemistry for cytokeratin (CK) 7, CK20, MUC1, MUC2, MUC4 and CDX2 was performed on tissue microarrays. Classification of the adenocarcinomas based on immunohistochemistry was compared with the morphological evaluation of histological type. Presence of CK7 and MUC4, and absence of CDX2, were independent predictors of pancreatobiliary versus intestinal type. Using these markers to optimize immunohistochemical classification, agreement between immunohistochemical and morphological classification was only moderate (kappa = 0.53). In pancreatobiliary differentiated tumours, MUC1 and/or MUC4 expression was an independent prognostic factor (hazard ratio 2.02, 95% confidence interval 1.02, 3.98) when adjusting for nodal involvement, vessel involvement and tumour size. In intestinally differentiated tumours, none of the markers was significantly associated with prognosis. CONCLUSIONS: Agreement between immunohistochemical and morphological classification of pancreatic head adenocarcinomas is moderate. In pancreatobiliary adenocarcinomas, MUC1 and/or MUC4 expression indicates a particularly poor prognosis.

Nondysplastic Ulcerative Colitis Has High Levels of the Homologous Recombination Repair Protein NUCKS1 and Low Levels of the DNA Damage Marker Gamma-H2AX.

Background: The colon and rectum are continuously exposed to oxidative stress that generates reactive oxygen species, which are a major cause of DNA double-strand breaks (DSB). Furthermore, chronic inflammatory diseases such as ulcerative colitis (UC) are characterized by an excess of reactive nitrogen species that can also lead to DNA double-strand breakage and genomic instability. We investigated the expression of the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) protein in UC and sporadic colorectal cancer (CRC) due to its involvement in both DNA double-strand break repair and inflammatory signaling. Methods: NUCKS1 expression and expression of the DNA double-strand break marker gamma-H2AX (γH2AX) were assessed in formalin-fixed, paraffin-embedded UC and CRC patient biopsies using peroxidase immunohistochemistry. Expression levels for both proteins were evaluated together with previously published expression-level data for hTERT and TP53 proteins in the same material. Results: Nondysplastic UC lesions had 10-fold lower γH2AX expression and approximately 4-fold higher NUCKS1 expression compared with sporadic CRC, indicating minimal DNA DSB damage and heightened DNA DSB repair in these lesions, respectively. NUCKS1 expression in UC tended to decrease with increasing grades of dysplasia, whereas γH2AX, hTERT, and TP53 expression tended to increase with increasing grades of dysplasia. The highest γH2AX expression was seen in sporadic CRC, indicating considerable DNA DSB damage, whereas the highest NUCKS1 expression and hTERT expression were seen in nondysplastic UC. Conclusions: Overall, our data suggest that NUCKS1 may be involved in DNA DSB repair and/or inflammatory signaling in UC, but a more thorough investigation of both pathways in UC is warranted.

Subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 assessed by immunohistochemistry.

The spindle checkpoint, the primary mechanism to ensure that two daughter cells receive the same amount of DNA, is compromised in many malignant tumors and has been implicated as a contributor to aneuploidy and carcinogenesis. The extent of expression and subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 varies considerably in different immunohistochemical (IHC) reports from archival tumor tissues. Given the conflicting reports in the literature about the localization of these proteins, we examined the subcellular localization of Aurora kinase A, Mad2, and BUBR1 in normal and cancerous human tissues by IHC. In normal tissues, Aurora A was mainly localized to the nucleus when monoclonal or purified polyclonal antibodies were used, and Mad2 was localized to the nucleus, whereas BUBR1 was localized to the cytoplasm. In malignant tissues, Aurora A showed additional staining in the cytoplasm in the majority of tumors analyzed. Furthermore, BUBR1 was also localized to both the nucleus and cytoplasm in a significant fraction of tumors. Subcellular localization of Mad2 was similar in normal and malignant tissues. Thus, the validity of some earlier IHC studies of Aurora A, Mad2, and BUBR1 should be reconsidered, indicating that high-quality antibodies and a high-alkaline antigen-retrieval technique are required to achieve optimal results. We conclude that the subcellular localizations of these spindle proteins are different, although they have overlapping biological functions, and that Aurora A and BUBR1 undergo a shift in the subcellular localization during malignant transformation.

TP53/p53 alterations and Aurora A expression in progressor and non-progressor colectomies from patients with longstanding ulcerative colitis.

Aneuploidy is a common feature in the colonic mucosa of patients suffering from the inflammatory bowel disease ulcerative colitis (UC) and often precedes the development of dysplasia and cancer. Aneuploidy is assumed to be caused by missegregation of chromosomes during mitosis, often due to a faulty spindle assembly checkpoint. p53 is a tumour suppressor protein known to regulate the spindle assembly checkpoint and is frequently mutated in aneuploid cells. Aurora A is a presumed oncoprotein, also involved in regulation of the spindle assembly checkpoint. In the present study, we examined the mutational frequency of TP53 and the protein levels of p53 in a set of 20 progressor and 10 non-progressor colectomies from patients suffering from longstanding UC. In addition, we re-examined previously published immunohistochemical data on Aurora A expression using the same material. Levels of Aurora A were re-examined with regard to DNA ploidy status and dysplasia within the progressors, as well as in relation to p53 accumulation and TP53 mutational status. We detected p53 accumulation only within the progressor colectomies, where it could be followed back 14 years prior to the colectomies, in pre-colectomy biopsies. TP53 mutations were detected in both progressors and non-progressors. Expression levels of Aurora A were similar in the progressors and non-progressors. Within the group of progressors however, low levels of Aurora A were associated with areas of DNA aneuploidy, as well as with increasing degrees of dysplasia. Our results indicate that alterations in p53 may be an early biomarker of a progressor colon, and that p53 is accumulated early in UC-related carcinogenesis. Furthermore, a decreased Aurora A expression is associated with the development of DNA aneuploidy, as well as with dysplasia in UC progressors.

Reduced hTERT protein levels are associated with DNA aneuploidy in the colonic mucosa of patients suffering from longstanding ulcerative colitis.

Longstanding ulcerative colitis (UC) is a disease of chronic inflammation of the colon. It is associated with the development of colorectal cancer through a multistep process including increasing degrees of dysplasia and DNA-ploidy changes. However, not all UC patients will develop these characteristics even during lifelong disease, and patients may therefore be divided into progressors who develop dysplasia or cancer, and non-progressors who do not exhibit such changes. In the present study, the amount of hTERT, the catalytic subunit of the enzyme telomerase, was estimated by using peroxidase immunohistochemistry (IHC) in a set of progressor and non-progressor UC colectomies. The protein levels in the colonic mucosa of the progressors and non­progressors were compared, and further comparisons between different categories of dysplastic development and to DNA-ploidy status within the progressors were made. Levels of hTERT were elevated in the colonic mucosa of the progressors and non-progressors when compared to non-UC control samples, but no difference was observed between the hTERT levels in the mucosa of progressors and non-progressors. The levels of hTERT associated with levels of Ki67 to a significant degree within the non-progressors. hTERT expression in lesions with DNA-aneuploidy were decreased as compared to diploid lesions, when stratified for different classes of colonic morphology. Our results indicate an association between hTERT protein expression and aneuploidy in UC-progressor colons, and also a possible protective mechanism in the association between hTERT and Ki67, against development of malignant features within the mucosa of a UC-colon.

Aneuploidy is associated with TP53 expression but not with BRCA1 or TERT expression in sporadic colorectal cancer.

BACKGROUND: Defective expression of genes involved in mitotic chromosome segregation (e.g. AURKA, BUB1B), DNA damage response (e.g. TP53, BRCA1), and telomere function (e.g. TERT) may play a role in the development of tumor aneuploidy. MATERIALS AND METHODS: The levels of TP53, BRCA1 and TERT were assessed in 55 sporadic colorectal tumors and 37 normal mucosas using tissue microarrays and immunohistochemical detection, and their associations with DNA aneuploidy, levels of mitotic spindle proteins AURKA, AURKB, MAD2L1 and BUB1B and clinicopathological parameters were investigated. RESULTS: DNA aneuploidy was associated only with TP53 alterations. BRCA1 expression in tumors was significantly correlated with individual mitotic spindle protein expressions, and TERT and MAD2L1 expressions were moderately correlated in the tumor group, suggesting a putative role for TERT in MAD2L1 regulation. CONCLUSION: Loss of TP53 function appears to be involved in the development of aneuploidy, but not in the deregulation of mitotic spindle protein function.

Identification of human papillomavirus in keratoacanthomas.

BACKGROUND: Keratoacanthomas are benign, clinically distinct skin tumors that may infiltrate and show cellular atypia. A viral etiology has been suggested, and the aim was to search for human papillomavirus (HPV) in keratoacanthomas. METHODS: From 21 immunosuppressed organ transplant recipients and 11 non-immunosuppressed patients, 72 fresh biopsies with diagnosis of keratoacanthomas were analyzed. For detection of cutaneous and genital HPV DNA, single-tube nested "hanging droplet" polymerase chain reaction (PCR) and another PCR (GP5+ and 6+) were used, respectively. RESULTS: Among 21 immunosuppressed patients, 71% (15/21) harbored HPV DNA at least in one sample. Of the keratoacanthoma lesions, 55% (33/60) were HPV DNA positive. Fourteen samples from eight immunosuppressed patients contained HPV types 5, 9, 10, 14, 19, 20, 21, 38, 49, 80, putative HPV types as HPVvs20-4, HPVvs75, and HPVvs92 and FA16.1, FA23.2, FA37, FA75, and FA81. Among 11 non-immunosuppressed patients, 36% (4/11) harbored HPV DNA at least in one sample, and 33% (4/12) of their keratoacanthomas were HPV DNA positive. In total, HPV DNA was detected in 51% (37/72) of the keratoacanthomas. CONCLUSIONS: By the use of PCR, cutaneous HPV DNA was detected in 51% (37/72) of the keratoacanthomas. No predominating HPV type or genital HPV type was identified. The role of HPV in keratoacanthomas remains thus elusive.