Nuclear P53 Staining Research Articles

Somatostatin receptor expression related to TP53 and RB1 alterations in pancreatic and extrapancreatic neuroendocrine neoplasms with a Ki67-index above 20%

Somatostatin receptor 2A expression is a feature of well-differentiated neuroendocrine neoplasms and is important for their diagnosis and therapy. Little is known about somatostatin receptor 2A expression in poorly differentiated neuroendocrine neoplasms in relation to TP53 and RB1 status and how these features may contribute to the separation of well from poorly differentiated neuroendocrine neoplasms with a proliferation index above 20%. This study investigates the expression of somatostatin receptors, p53 and Rb1, and TP53 alterations in pancreatic and extrapancreatic well and poorly differentiated neuroendocrine neoplasms (Ki67-index >20%). Thirty-seven poorly differentiated neuroendocrine neoplasms of pancreatic (n=12) and extrapancreatic origin (n=25) as well as 10 well-differentiated neuroendocrine neoplasms of the pancreas (n=9) and rectum (n=1) with a Ki67-index >20% were immunostained for synaptophysin, chromogranin A, Ki67, CD56, p53, Rb1, ATRX, DAXX, progesterone receptor, somatostatin receptor 2A, somatostatin receptor 5, and cytokeratin 20, and sequenced for TP53, exons 5–9. Somatostatin receptor 2A was positive in 6/37 of poorly differentiated and in 8/10 of well-differentiated neuroendocrine neoplasms. One well-differentiated and two poorly differentiated neuroendocrine neoplasms expressed somatostatin receptor 5. Abnormal nuclear p53 and Rb1 staining was found in 29/37 and 22/37 poorly differentiated neuroendocrine neoplasms, respectively, whereas all well-differentiated neuroendocrine neoplasms showed normal p53 and Rb1 expression. TP53 gene alterations were restricted to poorly differentiated neuroendocrine neoplasms (24/34) and correlated well with p53 expression. All cases were progesterone receptor negative. Somatostatin receptor 2A expression is not limited to well-differentiated neuroendocrine neoplasms but also occurs in 16% of poorly differentiated neuroendocrine neoplasms from various sites. Most poorly differentiated neuroendocrine neoplasms are characterized by TP53 alterations and Rb1 loss, usually in the absence of somatostatin receptor 2A expression. In the pancreas, these criteria contribute to separate well-differentiated neuroendocrine neoplasms with a Ki67-index above 20% from poorly differentiated neuroendocrine neoplasms.

Social isolation induces autophagy in the mouse mammary gland: link to increased mammary cancer risk.

Social isolation is a strong predictor of early all-cause mortality and consistently increases breast cancer risk in both women and animal models. Because social isolation increases body weight, we compared its effects to those caused by a consumption of obesity-inducing diet (OID) in C57BL/6 mice. Social isolation and OID impaired insulin and glucose sensitivity. In socially isolated, OID-fed mice (I-OID), insulin resistance was linked to reduced Pparg expression and increased neuropeptide Y levels, but in group-housed OID fed mice (G-OID), it was linked to increased leptin and reduced adiponectin levels, indicating that the pathways leading to insulin resistance are different. Carcinogen-induced mammary tumorigenesis was significantly higher in I-OID mice than in the other groups, but cancer risk was also increased in socially isolated, control diet-fed mice (I-C) and G-OID mice compared with that in controls. Unfolded protein response (UPR) signaling (GRP78; IRE1) was upregulated in the mammary glands of OID-fed mice, but not in control diet-fed, socially isolated I-C mice. In contrast, expression of BECLIN1, ATG7 and LC3II were increased, and p62 was downregulated by social isolation, indicating increased autophagy. In the mammary glands of socially isolated mice, but not in G-OID mice, mRNA expressions of p53 and the p53-regulated autophagy inducer Dram1 were upregulated, and nuclear p53 staining was strong. Our findings further indicated that autophagy and tumorigenesis were not increased in Atg7(+/-) mice kept in social isolation and fed OID. Thus, social isolation may increase breast cancer risk by inducing autophagy, independent of changes in body weight.

Rational Manual and Automated Scoring Thresholds for the Immunohistochemical Detection of TP53 Missense Mutations in Human Breast Carcinomas

Missense mutations in TP53 are common in human breast cancer, have been associated with worse prognosis, and may predict therapy effect. TP53 missense mutations are associated with aberrant accumulation of p53 protein in tumor cell nuclei. Previous studies have used relatively arbitrary cutoffs to characterize breast tumors as positive for p53 staining by immunohistochemical assays. This study aimed to objectively determine optimal thresholds for p53 positivity by manual and automated scoring methods using whole tissue sections from the Carolina Breast Cancer Study. p53-immunostained slides were available for 564 breast tumors previously assayed for TP53 mutations. Average nuclear p53 staining intensity was manually scored as negative, borderline, weak, moderate, or strong and percentage of positive tumor cells was estimated. Automated p53 signal intensity was measured using the Aperio nuclear v9 algorithm combined with the Genie histology pattern recognition tool and tuned to achieve optimal nuclear segmentation. Receiver operating characteristic curve analysis was performed to determine optimal cutoffs for average staining intensity and percent cells positive to distinguish between tumors with and without a missense mutation. Receiver operating characteristic curve analysis demonstrated a threshold of moderate average nuclear staining intensity as a good surrogate for TP53 missense mutations in both manual (area under the curve=0.87) and automated (area under the curve=0.84) scoring systems. Both manual and automated immunohistochemical scoring methods predicted missense mutations in breast carcinomas with high accuracy. Validation of the automated intensity scoring threshold suggests a role for such algorithms in detecting TP53 missense mutations in high throughput studies.

Loss of p27 expression is associated with MEN1 gene mutations in sporadic parathyroid adenomas.

MEN1 is the main gene responsible for tumorigenesis of syndromic and sporadic primary hyperparathyroidism (PHPT). Germline mutations of the CDKN1B/p27Kip gene have been associated with multiple endocrine tumors in rats and humans. To evaluate the involvement of the CDKN1B gene and its relationship with MEN1 in sporadic PHPT, we carried out sequencing and loss of heterozygosity analyses of the CDKN1B gene in 147 sporadic parathyroid adenomas. p27 immunohistochemistry and genetic screening of the MEN1 gene were performed in 50 cases. Three germline CDKN1B variants (c.-80C>T, c.-29_-26delAGAG, c.397C>A) were identified in 3/147 patients. Reduction of CDKN1B gene transcription rate was demonstrated in vitro for the novel c.-80C>T and the c.-29_-26delAGAG variants. Loss of p27 expression was detected in the tumor carrying the c.-29_-26delAGAG variant. Two tumors carrying the CDKN1B variants also harbored a MEN1 mutation. Fifty-four percent of 50 CDKN1B mutation-negative tumors had a reduction of p27 nuclear staining. Somatic MEN1 mutations, identified in 15/50 samples, significantly segregated in tumors negative for nuclear and cytoplasmic p27 staining. The germline nature of the CDKN1B mutations suggests that they might predispose to PHPT. The lack of somatic CDKN1B mutations in our samples points to a rare involvement in parathyroid adenomas, despite the frequent loss of nuclear p27 expression. MEN1 biallelic inactivation seems to be directly related to down-regulation of p27 expression through the inhibition of CDKN1B gene transcription.

Abstract B05: Assessment of TP53 mutation status in primary high-grade serous ovarian cancer and cell line models: Comparison between immunohistochemistry and next-generation sequencing.

Abstract Mutations in TP53 have been shown to occur in around 96% of high-grade serous ovarian cancer (HGSOC). Different TP53 mutations can lead to accumulation of mutant p53 in the cell that support gain of function effects, or loss or abrogation of both wild-type and mutant p53. Success of new therapeutic strategies for HGSOC that target mutant p53 will likely depend on detailed knowledge of the type of TP53 mutation present and its influence on mutant p53 levels in the cell. The aim of this study was to identify the spectrum of TP53 mutations in a cohort of primary HGSOCs and compare this with p53 immunostaining patterns. In addition, we sought to identify TP53 mutations in the previously uncharacterised HGSOC cell line models OV167 and OV202, as well as the clear cell adenocarcinoma line OV207. DNA from fresh frozen tumors and cell line samples were processed for next generation sequencing using The Fluidigm Access TP53 Array System run on the Fluidigm Biomark HD™ Real-Time PCR fluidics system (Fluidigm Corporation). Amplicon libraries were pooled and analyses performed in a single massively parallel sequencing run using the MiSeq Sequencing System (Illumina Inc.). Unaligned BAM files were analyzed within the Broad Institute's Genome Analysis Toolkit (GATK) best practice pipeline. The ANNOVAR software tool was used to generate an annotated variants list. Variants were further filtered based on their frequency in the 1000 Genome Database and predicted SIFT scores. Mutations were visualized using the Broad Institute's Integrative Genomics Viewer. All samples in which a mutation was identified underwent Sanger sequencing using primers recommended in the International Agency for Research on Cancer TP53 database. Immunohistochemistry for p53 was performed on formalin fixed paraffin embedded tissue sections using a commercially available mouse monoclonal anti-human antibody (Protein Clone DO-7, cat. #M7001, Dako) on an automated staining platform (Leica BOND-III™ autostainer). The percentage of cells showing positive nuclear staining for p53 was reported. A mutation in TP53 was identified in 94% (68 of 72) of HGSOC. The majority of mutations identified (62%, 42 of 68) were missense, with all except one of these mutations located in the p53 DNA binding domain. Overall, 82% (56 of 68) of mutations occurred within the DNA binding domain. Frameshift, stop or splice mutations (38%, 26 of 68) were identified in exons or flanking intronic sequence between exons 4 – 10. The presence or location of a TP53 mutation did not influence overall survival. Sanger sequencing was unable to reliably detect variants at an allele frequency less than 25%. TP53 mutations were identified in all cell lines at a mutant allele frequency ≥99%. Based on percent of nuclei positive for p53, tumors were immunohistochemically graded as High, Intermediate or Low for p53. All missense mutations scored High for expression of the p53 protein. Considerable variability was observed in p53 staining levels in samples containing stop, frameshift or splice mutations, with samples in all of High, Intermediate and Low categories. Furthermore, a single sample shown to be wild-type for TP53 was graded as High for p53, raising the possibility that p53 had been activated in this tumor via an alternative mechanism. The level of p53 did not affect overall survival. Taken together, these results suggest that caution needs to be exercised when using p53 immunohistochemistry as a surrogate marker for either presence or type of TP53 mutation. A combination of next generation sequencing and p53 immunohistochemistry is required to robustly define both mutation type and effect on mutant p53 levels in the cell. Citation Format: Alexander J. Cole, Trisha Dwight, Ying Zhu, Anthony J. Gill, Kristie-Ann Dickson, Adele Clarkson, Gregory B. Gard, Jayne Maidens, Susan Valmadre, Roderick J. Clifton-Bligh, Deborah J. Marsh. Assessment of TP53 mutation status in primary high-grade serous ovarian cancer and cell line models: Comparison between immunohistochemistry and next-generation sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B05.

Evaluation of microvessel density and p53 expression in pancreatic adenocarcinoma

OBJECTIVE:To evaluate the prognostic significance of microvessel density and p53 expression in pancreatic cancer.METHODS:Between 2008 and 2012, 49 patients with pancreatic adenocarcinoma underwent resection with curative intention. The resected specimens were immunohistochemically stained with anti-p53 and anti-CD34 antibodies. Microvessel density was assessed by counting vessels within ten areas of each tumoral section a highpower microscope.RESULTS:The microvessel density ranged from 21.2 to 54.2 vessels/mm2. Positive nuclear staining for p53 was found in 20 patients (40.6%). The overall median survival rate after resection was 24.1 months and there were no differences in survival rates related to microvessel density or p53 positivity. Microvessel density was associated with tumor diameter greater than 3.0 cm and with R0 resection failure.CONCLUSIONS:Microvessel density was associated with R1 resection and with larger tumors. p53 expression was not correlated with intratumoral microvessel density in pancreatic adenocarcinoma.

Pituitary Adenoma and the Chemokine Network: A Systemic View.

Pituitary adenomas (PAs) are adenohypophysial neoplasms, representing ~10–15% of all intracranial tumors, which are found to occur in almost 20% of the general population (1, 2). Although recognized as benign lesions, 20–45% of PAs are invasive and some exhibit clinically aggressive behavior (1, 3). Advancements have allowed the pathological classification of PAs from a histochemical classification (i.e., acidophilic, basophilic, and chromophobic PAs) to an immunohistochemical-based one, which essentially recognizes PAs as lactotrophic, somatotrophic, corticotrophic, gonadotrophic, thyrotrophic, and null cell adenomas (4). Electron microscopy has identified additional subtypes, based on the appearance of specific morphology/arrangement of ubiquitous cytoplasmic constituents (5). It is now accepted that the hypothalamic–anterior pituitary axis integrates a set of stimulatory and inhibitory central and peripheral signals to synthesize and secrete hormones by highly differentiated cell types, namely somatotrophs, gonadotrophs, lactotrophs, thyrotrophs, and corticotrophs. Each of these cell types expresses unique G protein-coupled receptors (GPCRs), which are specific for hypothalamic releasing and inhibiting hormones. These peptides traverse the vascular system that connects the hypothalamus with the anterior pituitary gland and impinge upon the pituitary cells to regulate the synthesis and secretion of anterior pituitary hormones (6). It has been demonstrated that PAs arise from any of these cells as monoclonal neoplasms (7). The functional classification of PAs has also been facilitated by the measurement of circulating trophic and target hormone concentrations (6). Several hallmarks of PAs (i.e., benign nature, slow growth) point to a unique growth behavior distinct from that of other endocrine and non-endocrine malignancies (8). The general failure to proceed to true malignancy with demonstrable extra-cranial metastases remains an intriguing feature not completely investigated yet (8). Atypical and/or aggressive PAs can share some histological features with carcinomas, including atypical morphologic appearances, elevated mitotic index (i.e., Ki-67 labeling index >3%), or extensive nuclear staining for p53 (9). Nonetheless, the endocrine signs due to hormonal hypersecretion or pituitary deficit, pituitary abnormal growth, or invasion represent a greater clinical and therapeutic challenge (8). The subset of PAs with clinically aggressive behavior should be promptly identified, as patients with such tumors might require closer clinical, biochemical, and imaging surveillance or multimodal therapeutic treatments. Despite the paramount importance of identifying specific criteria for tumor aggressiveness, no clinical signs, biochemical biomarkers, or imaging techniques have been universally accepted and no reliable histological biomarker or classification system for characterizing pituitary aggressive neoplasms are available today (10). It has also been suggested that the histological features of “typical” and “atypical” PAs as defined by the World Health Organization (WHO) do not entirely correlate with clinical outcome. In particular, some typical PAs have an aggressive behavior while many atypical PAs lack an aggressive clinical dynamics (1, 7). Additionally, the lack of standardization of the terminology in the literature remains a source of confusion (i.e., the terms “aggressive” and “invasive” are often used synonymously).

Histological criteria for atypical pituitary adenomas - data from the German pituitary adenoma registry suggests modifications.

IntroductionThe term atypical pituitary adenoma (APA) was revised in the 2004 World Health Organization (WHO) classification of pituitary tumors. However, two of the four parameters required for the diagnosis of APAs were formulated rather vaguely (i.e., “extensive” nuclear staining for p53; “elevated” mitotic index). Based on a case-control study using a representative cohort of typical pituitary adenomas and APAs selected from the German Pituitary Tumor Registry, we aimed to obtain reliable cut-off values for both p53 and the mitotic index. In addition, we analyzed the impact of all four individual parameters (invasiveness, Ki67-index, p53, mitotic index) on the selectivity for differentiating both adenoma subtypes.MethodsOf the 308 patients included in the study, 98 were diagnosed as APAs (incidence 2.9 %) and 10 patients suffered from a pituitary carcinoma (incidence 0.2 %). As a control group, we selected 200 group matched patients with typical pituitary adenomas (TPAs). Cut-off values were attained using ROC analysis.ResultsWe determined significant threshold values for p53 (≥2 %; AUC: 0.94) and the mitotic index (≥2 mitosis within 10 high power fields; AUC: 0.89). The most reliable individual marker for differentiating TPAs and APAs was a Ki-67-labeling index ≥ 4 % (AUC: 0.98). Using logistic regression analysis (LRA) we were able to show that all four criteria (Ki-67 (p < 0.001); OR 5.2// p53 (p < 0.001); OR 3.1// mitotic index (p < 0.001); OR 2.1// invasiveness (p < 0.001); OR 8.2)) were significant for the group of APAs. Furthermore, we describe the presence of nucleoli as a new favorable parameter for TPAs (p = 0.008; OR: 0.4; CI95 %: 0.18; 0.77).ConclusionsHere we present a proposed rectification of the current WHO classification of pituitary tumors describing an additional marker for TPA and specific threshold values for p53 and the mitotic index. This will greatly help in the reliable diagnosis of APAs and facilitate further studies to ascertain the prognostic relevance of this categorization.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0229-8) contains supplementary material, which is available to authorized users.

Signet ring cell carcinoma of the non-ampullary duodenum: A case report

We describe a case of signet ring cell carcinoma of the non-ampullary duodenum in an 86-year-old woman. Endoscopic examination revealed a fungating lesion (Borrmann classification; type 2) on the anterior wall of the descending duodenum (second part). Pylorus-preserving pancreaticoduodenectomy and lymphadenectomy were performed. The tumor (pT3 and pN0) was predominantly composed of signet ring cells, characterized by a central, optically clear, globoid droplet of cytoplasmic mucin with an eccentrically placed nucleus. The cells were positive for periodic acid Schiff with diastase and Alcian blue pH 2.5. On immunohistochemical examination, MUC-5AC, MUC-2 and CDX2 were diffusely positive, and MUC-6 was negative in the tumor cells, which consisted of mixed gastric foveolar and intestinal phenotypes. Cytokeratin 7 was focally positive, but cytokeratin 20 was negative. Nuclear staining of p53 was diffusely and weakly positive. Signet ring cell carcinoma occurring in the non-ampullary duodenum might derive from duodenal goblet cells with MUC-5AC expression.

Genetic Polymorphism and Expression of CXCR4 in Breast Cancer.

CXCR4 genetic polymorphisms, as well as their expression level, have been associated with cancer development and prognosis. The present study aimed to investigate the influence of CXCR4 rs2228014 polymorphism on its mRNA and protein expression in breast cancer samples. It was observed that patients presented higher CXCR4 mRNA relative expression (5.7-fold) than normal mammary gland, but this expression was not correlated with patients clinicopathological features (nuclear grade, nodal status, ER status, PR status, p53 staining, Ki67 index, and HER-2 status). Moreover, CXCR4 mRNA relative expression also did not differ regarding the presence or absence of T allele (p = 0.301). In the immunohistochemical assay, no difference was observed for CXCR4 cytoplasmic protein staining in relation to different genotypes (p = 0.757); however, high cytoplasmic CXCR4 staining was verified in invasive breast carcinoma (p < 0.01). All in all, the results from present study indicated that rs2228014 genetic variant does not alter CXCR4 mRNA or protein expression. However, this receptor was more expressed in tumor compared to normal tissue, in both RNA and protein levels, suggesting its promising applicability in the general context of mammary carcinogenesis.

Overexpression of p53 in the endometrial gland in postmenopausal women.

The p53 signature, which (although morphologically unremarkable) displays diffuse and strong p53 nuclear staining, has been proposed to be a precursor of serous endometrial intraepithelial carcinoma. We examined the overexpression of p53 in postmenopausal endometrial glands. Postmenopausal endometrial tissues of 82 women with benign disease, including 10 hormone users, were evaluated in this study. Tissues with endometrial hyperplasia and/or polyps were excluded based on a histopathologic review. Expressions of estrogen receptor-α, Ki-67, and p53 were immunohistochemically examined. Apoptotic cells were identified using a terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Overexpression of p53 was categorized as moderate to strong in more than 50% of glandular cell nuclei. Focal glandular overexpression of p53 was observed in 1 (9%) of 10 and in 8 (11%) of 72 postmenopausal endometrial tissue specimens in women with and women without a history of hormone use, respectively. Among nonhormone users, the median Ki-67 and apoptotic indices in the postmenopausal endometrial glands of women with and women without overexpression of p53 were 16% and 6% (P = 0.007) and 1% and 1% (P = 0.345), respectively. All postmenopausal endometrial glands were positive for estrogen receptor-α, regardless of the overexpression of p53. The postmenopausal endometrial glands of estrogen users exhibited significantly higher Ki-67 and apoptotic indices than those of nonestrogen users (P = 0.001 and P < 0.001, respectively). Overexpression of p53 may be responsible for the high proliferative activity of postmenopausal endometrial glandular cells associated with conditions of low apoptotic cell death.

Abstract 311: Autophagic pathways and activity in breast and pancreatic cancer

Abstract Autophagy is induced as a homeostatic process by energy shortage and cellular stress but can also be influenced by drugs. Poor autophagic function contributes to carcinogenesis but autophagy also promotes survival of tumours. We performed immunohistochemistry for LC3, pAMP kinase, p62, p53 on 15 samples from breast cancer and 14 from pancreatic cancer. P53 mutations were detected using CDGE. Autophagy was active (LC3 puncta in ≥ 30% of cells) in 5/15 breast carcinomas and 7/14 pancreatic carcinomas. This was associated with energy shortage (strong pAMP kinase expression) in 4/5 in breast samples and 5/7 in those from pancreas, often clearly colocalized when expression was variable. In contrast, activation of AMP kinase was not associated with autophagy in 4/8 breast and 2/7 pancreatic cancers. In 2/5 breast samples and 2/7 from pancreas impaired autophagic flux was suggested by absent cytoplasmic LC3 staining and strong expression of p62. Nuclear expression of p53 was seen in 10/15 breast cancer samples and of those 3 were mutated, 11/14 pancreas samples showed nuclear staining for p53, none mutated. Three out of 7 non-mutated p53-protein-positive breast cancers had low autophagy. Autophagy was thus often associated with energy shortage, can clearly also be induced by other pathways, but p53 appears not to be involved. Lack of autophagy in the presence of energy shortage suggests defects in induction. There is, therefore, considerable variability in autophagic pathways and activity in cancer and this will have to be taken into account when considering therapeutic intervention in the autophagic process. Citation Format: Mar Egilsson, Ulfur Thoroddsen, Jon Gunnlaugur Jonasson, Margret Helga Ogmundsdottir, Helga M. Ögmundsdottir. Autophagic pathways and activity in breast and pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 311. doi:10.1158/1538-7445.AM2014-311

P27 expression in pleomorphic salivary gland adenoma.

Aim of the studyThis study aims to characterise alterations in the immunohistochemical expression of p27 in normal tissue of the salivary gland surrounding pleomorphic adenoma and in the tumour cells of pleomorphic adenoma.Material and methodsA retrospective study of data including 120 cases of pleomorphic adenomas (66 female, 54 male) retrieved from the files of the Oral Pathology Departments of Aleppo University, Syria and Al-Farabi Dental and Nursing College. Immunohistochemical expression against p27 was examined in the selected cases. The percentage of p27-positive nuclei was semi-quantitatively assessed by two independent observers and scores were given. The statistical analysis included the use of descriptive statistics and proportional frequencies.ResultsThe results showed that P27 nuclear staining with high staining (moderate to strong positive staining in more than 50% of nuclei, depending on the percentage of nuclei for the cells) was noted in tumour duct cells of pleomorphic adenoma in 90 (75%) cases out of 120, while 30 (25%) cases showed low staining (positive staining in less than 5% of nuclei, depending on the percentage of nuclei for the cells).ConclusionsOur data suggests that p27 might have a minor role in the development of pleomorphic adenoma.

T Antigen Dependent Oncogenic Pathways Are Active in Post-Transplant Tumors Driven By BK Polyoma Virus.

BK Polyoma Virus (BKPyV) transforms primary cells and is homologous to the Merkel Cell Carcinoma virus, but has never been convincingly demonstrated to cause tumors in humans. There have been 18 case reports of urinary tract tumors in solid organ transplant recipients (17 kidney, 1 lung) with diffuse expression of BKPyV’s large T antigen (LTA), partial evidence that BKPyV is an oncovirus. Here we report that known binding partners and downstream effectors of the LTA and Small T Antigen (STA) of BKPyV are active in a series of 10 of these tumors (5 novel). Methods: Formalin fixed tissue was obtained from kidney and bladder tumors with diffuse LTA expression arising in solid organ transplant recipients. Control populations include kidney and bladder tumors arising in transplant recipients without diffuse large T antigen expression, and cases of polyoma virus reactivation and superinfection. Sections were stained for p53, cyclin A, and β-catenin. Results: Tumors with diffuse LTA staining showed universal nuclear staining for p53, 9/10 had high rates of cyclin A nuclear positivity, and 9/10 had at least weak membrane accumulation of β-catenin. Control tumors had variable positivity for p53, with urothelial carcinoma of the bladder showing weak membrane staining for β-catenin and low cyclin A positivity, and renal cell carcinoma showing minimal β-catenin but high levels of cyclin A. Active BKPyV infections stained similarly to tumors withdiffuse LTA, but only in the infected cells. Discussion: Cyclin A is a known target of STA signaling through PP2A. The LTA stabilizes p53 directly and β-catenin indirectly via Rac1. These proteins are involved in distinct pathways of oncogenesis. p53: DNA repair and apoptosis, β-catenin: growth and metastasis, cyclin A: cell cycle. While activation of any single pro-oncogenic pathway is non-specific, tumors with diffuse LTA are generally positive for all 3 proteins. In contrast, control tumors did not stain for the full panel. Staining in tumors and active infections are similar, suggesting that STA and LTA sequences are intact in tumors with BKPyV. This provides additional supporting evidence that BKPyV is an oncovirus in humans. DISCLOSURE:Emerson, L.: Employee, ARUP Laboratories.

Nuclear expression of p53 in mature tumor endothelium of retinoblastoma.

The present study aimed to investigate the p53 expression pattern in tumor cells and in mature tumor vascular endothelium of retinoblastoma. Nuclear p53 accumulation was observed in most of the tumor cells in both the human and orthotopic retinoblastoma animal models using SNUOT-Rb1 and Y79 cells. In the orthotopic animal model, some of the tumor vascular endothelium also demonstrated nuclear p53 immunoreactivity, and the ratio of p53 positivity among the total mature tumor vascular endothelium was slightly higher in the Y79 cell model when compared with the SNUOT-Rb1 cell model. In addition, in the human retinoblastoma specimens, 32.9% of the tumor vascular endothelium showed p53 nuclear staining. In conclusion, some of the mature tumor vascular endothelium in both the human and orthotopic models of retinoblastoma share the same cytogenetic abnormality (an abnormal nuclear accumulation of p53) with retinoblastoma cells.

KRAS Mutations in Codon 12 or 13 Are Associated With Worse Prognosis in Pancreatic Ductal Adenocarcinoma

Mutations in the KRAS and P53 genes belong to the most frequently observed genetic alterations in pancreatic ductal adenocarcinoma. The aim of this study was to examine the frequency and prognostic impact of KRAS mutations. In addition, we attempted to define molecular subgroups with distinct biologic behavior by combination of KRAS sequencing data with p53 protein expression data. KRAS mutational analyses were performed in a study group of 153 patients by Sanger sequencing. Immunohistochemistry for p53 was performed on tissue microarrays. KRAS mutations in codon 12 or 13 were found in 68% of cases. Nuclear staining for p53 was detectable in 110 (68%) of 162 evaluable cases. We found no correlation between KRAS mutational status and p53 expression. KRAS mutational status but not p53 immunohistochemistry was an independent prognostic factor in the study group (P = 0.02). In a stratified analysis according to KRAS mutational status, p53 expression failed to define prognostic groups beyond the impact of KRAS mutational status. Our results support the crucial role of KRAS mutational status in pancreatic cancer biology. KRAS mutational status may serve as a prognostic marker. However, its predictive role for targeted therapies remains to be evaluated.

Quantitative Analysis of γ-H2AX and p53 Nuclear Expression Levels in Ovarian and Fallopian Tube Epithelium from Risk-reducing Salpingo-Oophorectomies in BRCA1 and BRCA2 Mutation Carriers

Mutations in BRCA1 and BRCA2 genes confer an increased lifetime risk for breast and ovarian cancer. Increased lifetime ovarian cancer risk among BRCA1/BRCA2 mutation carriers can be substantially decreased by risk-reducing salpingo-oophorectomy (RRSO), which also provides material for molecular research on early pathogenesis of serous ovarian cancer. RRSO studies have suggested fallopian tube as a primary site of serous high-grade ovarian cancer. In this study, the nuclear expression levels of γ-H2AX and p53 using immunohistochemical (IHC) study was quantitatively assessed in ovarian and fallopian tube epithelium derived from RRSOs in 29 BRCA1 and BRCA2 mutation carriers and in 1 patient with a strong family history of breast and ovarian cancer but showing an unknown BRCA status. Both p53 and γ-H2AX nuclear staining levels were significantly higher in BRCA1/2 mutation-positive fallopian tube epithelium compared with the control fallopian tube epithelium (P<0.006 and P=0.011, respectively). Nuclear expression levels of p53 and γ-H2AX were similar between the BRCA1/2 mutation-positive ovarian epithelium and controls. Both γ-H2AX and p53 showed significantly higher nuclear expression levels in BRCA1/2 mutation-positive fallopian tube epithelium compared with BRCA1/2 mutation-positive ovarian epithelium (P<0.0001 and P<0.0001, respectively). BRCA1/2 mutation-positive fallopian tube epithelium showed a positive correlation between the γ-H2AX and p53 nuclear expression levels (Pearson r=0.508, P=0.003). Our results of quantitative nuclear p53 and γ-H2AX expression levels in ovarian and fallopian tube epithelium derived from RRSO in high-risk patients support the previously suggested role of fallopian tube epithelium serving as a possible site of initial serous ovarian carcinogenesis.

The Role of CK20, p53 and p63 in Differentiation of Some Urothelial Lesions of Urinary Bladder, Immunohistochemical Study

Background: Differentiation of urothelial hyperplasia, dysplasia and carcinoma in situ (CIS) may pose diagnostic difficulties. We aim to evaluate the role of CK20, p53 and p63 in differentiation of such lesions. Methods: we evaluate these markers in 213 cases of bladder lesions (14 hyperplasia, 7 dysplasia, 5 CIS, 25 noninvasive and 162 invasive urothelial carcinoma) in a retrospective study on sections from formalin-fixed, paraffin-embedded blocks. Cytoplasmic staining considered for CK20 and nuclear staining for p53 and p63. Results: CK20 was expressed in 14 out of 14 hyperplasia (4 strong, 7 moderate, 3 weak), in 7 out of 7 dysplasia (4 strong , 3 moderate), in 5 out of 5 CIS, in 19 out of 25 noninvasive carcinoma; strong in 11 (7 low grade, 4 high grade), moderate in 7 (5 low grade, 2 high grade), and weak in 1 case of low grade, in 136 out of 162 invasive carcinoma; strong in 46 (22 grade II, 24 grade III), moderate in 71 (2 grade I, 58 grade II, 11 grade III), and weak in 19 (2 grade I, 15 grade II, 2 grade III), no expression in 2 cases of grade IV. p53 was expressed in 7 out of 14 hyperplasia (weak in all cases), in 7 out of 7 dysplasia (5 strong, 2 moderate), in 5 out of 5 CIS (strong in all cases), in 20 out of 25 noninvasive carcinoma (8 strong, 9 moderate, 3 weak), in 135 out of 162 invasive carcinoma (34 strong, 73 moderate, 28 weak). p63 was expressed in 14 out of 14 hyperplasia, in 7 out of 7 dysplasia, in 4 out of 5 CIS, in 25 out of 25 noninvasive carcinoma, and in 134 out of 162 invasive carcinoma (28 strong, 63 moderate, 43 weak). CK20 was insignificant in noninvasive and significant in invasive carcinoma, while p53 and p63 were significant in noninvasive and invasive carcinoma. Conclusion: CK20, p53 and p63 are useful in differentiation of different bladder lesions.

Loss of the putative tumor suppressor protein spinophilin is associated with poor prognosis in head and neck cancer

The putative tumor suppressor protein spinophilin has been recently involved in the pathogenesis of lung, liver, and other types of cancer. Previous studies also indicate that a loss of spinophilin in combination with functional impairment of p53 drives tumor progression. To date, no data exist about the role of spinophilin in head and neck squamous cell carcinoma (HNSCC). In the present study, we evaluated spinophilin and p53 expression by immunohistochemistry in 85 patients with nonmetastatic HNSCC. Kaplan-Meier curves and multivariate Cox proportional models were used to define the prognostic relevance of spinophilin for patients with HNSCC. Overall, immunoreactivity for spinophilin was reduced in 40 tumors (47%). Nine cases (10.5%) showed complete loss of spinophilin. Kaplan-Meier curve analysis demonstrated that reduced spinophilin expression is associated with poor overall survival (P = .022). Concomitant analysis of spinophilin and p53 further showed that patients with reduced spinophilin expression and nuclear p53 staining have a significantly decreased overall survival (hazard ratio, 1.96; 95% confidence interval, 1.06-3.61; P = .030). In conclusion, the combination of reduced spinophilin expression and nuclear p53 staining indicates a poor prognosis in HNSCC patients. Based on our results, spinophilin might play a previously unrecognized role in the pathogenesis of HNSCC.

Immunohistochemical analysis of chromogranin A and p53 expressions in ulcerative colitis–associated neoplasia: neuroendocrine differentiation as an early event in the colitis-neoplasia sequence

Pancellular dysplasia involving neuroendocrine cells has been shown to be comparatively rare but crucially implicated in the development of neuroendocrine tumors in ulcerative colitis (UC). We attempted to clarify the prevalence of chromogranin A expression as a marker of neuroendocrine differentiation in UC-associated neoplasia by immunohistochemical analyses of 26 lesions of low-grade dysplasia (LGD), 32 high-grade dysplasias (HGDs) and 27 invasive cancers (INVs), along with p53 expression. We additionally assessed the utility of these proteins for differential diagnosis between LGD and HGD. Chromogranin A was considered positive when immunoreactive cells were more than 5% of neoplastic lesions, and the positivity tended to be higher in HGDs (57.7%) or INVs (46.7%) than LGDs (32.0%). Focal or diffuse nuclear staining for p53 was defined as positive. The positive rate for p53 was also higher in HGDs (59.4%; P = 0.037) or INVs (59.3%) than LGDs (30.8%). A similar trend was found in co-positivity for both proteins (HGDs, 30.7%/INVs, 26.7% versus LGDs, 12.0%). No positivity for both proteins was identified in the non-neoplastic mucosa. The combination of the two proteins improved the sensitivity (66.7%), specificity (80.0%), positive predictive value (72.7%) and negative predictive value (75.0%) for HGD as compared to p53 alone (sensitivity, 57.7%; specificity 68.0%; positive predictive value, 65.2%; negative predictive value, 60.7%). In conclusion, we show here that neuroendocrine differentiation is relatively common and represents an early event in the UC-neoplasia pathway in which p53 and chromogranin A are coordinately up-regulated. Immunohistochemical assessment of their expression might provide a useful adjunct tool for grading dysplasia in UC.