Loss Of P16 Expression Research Articles

Loss of p16INK4A Expression and Homozygous CDKN2A Deletion Are Associated with Worse Outcome and Younger Age in Thymic Carcinomas

IntroductionThymic carcinomas are aggressive tumors. Biomarkers and alternative treatment modalities are needed. We studied the expression of p16 and cytogenetic abnormalities of cyclin-dependent kinase inhibitor 2A gene (CDKN2A) and correlated findings with clinical features and outcome in a large cohort of thymic carcinomas. MethodsThymic carcinomas (1963–2013) were stained with p16. Fluorescence in situ hybridization was utilized to assess for the presence of CDKN2A gene (at 9p21). Statistical analysis was performed. ResultsA total of 27 patients (including 15 men) with thymic carcinoma at a median age of 51.7 years at time of surgery and available follow-up information were included. Loss of p16 expression was found in 12 of 26 cases (46.2%) and was associated with worse recurrence and metastasis–free survival (p = 0.01), which in multivariate analysis was independent of resection status (p = 0.007) and T stage (p = 0.01). Four of 22 tumors (18.2%) showed homozygous CDKN2A deletion that was correlated with loss of p16 expression (p=0.02). In tumors of the squamous cell carcinoma subtype, loss of p16 expression was associated with worse recurrence and metastasis–free survival (p = 0.006) and overall survival (p = 0.0009). Patients with thymic squamous cell carcinomas lacking p16 expression were younger (p = 0.006). Although similar trends for younger age were noted in patients with thymic squamous cell carcinomas with homozygous CDKN2A deletion, the small number of such cases (n = 2) did not allow for statistical analysis. ConclusionsLoss of p16 expression and homozygous deletion of CDKN2A are promising prognostic biomarkers in thymic carcinoma. On the basis of our findings, a subset of thymic carcinomas have the potential to respond to CDK4/6 inhibitors; however, further functional studies are needed.

PUMA decreases the growth of prostate cancer PC-3 cells independent of p53.

PUMA (p53 upregulated modulator of apoptosis), a member of the B-cell lymphoma 2 (Bcl-2) protein family, is a pro-apoptotic protein. PUMA expression is modulated by the tumor suppressor p53. PUMA has a role in rapid cell death via p53-dependent and -independent mechanisms. To evaluate whether p53 is required for PUMA-mediated apoptosis in prostate cancer cells, p53 protein was silenced in human prostate cancer PC-3 cells by using p53 small interfering RNA (siRNA). The interference efficiency of p53 on RNA and protein levels was detected by reverse transcription-quantitative polymerase chain reaction and western blotting. Cell proliferation and p21 expression were subsequently examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and western blot analysis, respectively. p53-silenced or control PC-3 cells were transfected with pCEP4-(hemagglutinin)-PUMA plasmid, or non-carrier plasmid. Enzyme-linked immunosorbent assay was used to determine cell apoptosis by measuring histone release and caspase-3 activation, and MTT assay was used to measure cell viability. In addition, the expression of pro-apoptosis protein Bax and anti-apoptosis protein Bcl-2 were evaluated. The results of the present study revealed that p53 siRNA significantly suppressed p53 RNA and protein expression in PC-3 cells. Deficiency of p53 increased the cell growth rate and decreased p21 expression. However, PUMA overexpression remained able to induce apoptosis in p53-silenced and control cells by increasing Bax expression and decreasing Bcl-2 expression, leading to the activation of caspase-3. These results suggest that PUMA may mediate apoptosis of prostate cancer PC-3 cells, potentially independently of p53. Furthermore, PUMA gene treatment to induce cancer cell apoptosis may be more efficient compared with p53-dependent apoptosis, where loss of p53 expression or function may lead to limited efficacy of PUMA expression. Therefore, the present study proposes the significant hypothesis that increasing PUMA expression may be an effective approach for the treatment of prostate cancer, regardless of p53 status.

Activation of EVI1 transcription by the LEF1/β-catenin complex with p53-alteration in myeloid blast crisis of chronic myeloid leukemia

The presence of a BCR-ABL1 fusion gene is necessary for the pathogenesis of chronic myeloid leukemia (CML) through t(9;22)(q34;q11) translocation. Imatinib, an ABL tyrosine kinase inhibitor, is dramatically effective in CML patients; however, 30% of CML patients will need further treatment due to progression of CML to blast crisis (BC). Aberrant high expression of ecotropic viral integration site 1 (EVI1) is frequently observed in CML during myeloid-BC as a potent driver with a CML stem cell signature; however, the precise molecular mechanism of EVI1 transcriptional regulation during CML progression is poorly defined. Here, we demonstrate the transcriptional activity of EVI1 is dependent on activation of lymphoid enhancer-binding factor 1 (LEF1)/β-catenin complex by BCR-ABL with loss of p53 function during CML-BC. The activation of β-catenin is partly dependent on BCR-ABL expression through enhanced GSK3β phosphorylation, and EVI1 expression is directly enhanced by the LEF1/β-catenin complex bound to the EVI1 promoter region. Moreover, the loss of p53 expression is inversely correlated with high expression of EVI1 in CML leukemia cells with an aggressive phase of CML, and a portion of the activation mechanism of EVI1 expression is dependent on β-catenin activation through GSK3β phosphorylation by loss of p53. Therefore, we found that the EVI1 activation in CML-BC is dependent on LEF1/β-catenin activation by BCR-ABL expression with loss of p53 function, representing a novel selective therapeutic approach targeting myeloid blast crisis progression.

Loss of p16 expression and copy number changes of CDKN2A in a spectrum of spitzoid melanocytic lesions

Spitzoid melanocytic lesions, including Spitz nevi (benign), spitzoid melanoma (malignant), and borderline atypical Spitz tumors (ASTs), frequently present challenges for accurate diagnosis and prognosis. Evaluation for loss of the tumor suppressor p16, encoded by CDKN2A gene on chromosome 9p21.3, has been proposed to be useful for evaluation of spitzoid melanocytic lesions. However, reports on the utility of p16 immunohistochemistry for spitzoid lesions have been conflicting, and few studies have directly compared p16 immunohistochemistry with fluorescence in situ hybridization (FISH) for CDKN2A genomic status. We analyzed a spectrum of benign (n=24), borderline (n=27), and malignant (n=19) spitzoid lesions for p16 protein expression by immunohistochemistry and CDKN2A copy number by FISH. Immunohistochemistry was evaluated by 2 scoring methods: H score and 2-tiered score (positive or negative for p16 loss). By immunohistochemistry, loss of p16 expression was not observed in Spitz nevi (0/24) but was seen in ASTs (7/27; 26%) and spitzoid melanomas (3/19; 16%). By H score, p16 expression was significantly higher in Spitz nevi relative to ASTs or spitzoid melanomas. Similarly, copy number aberrations of CDKN2A by FISH were absent in Spitz nevi but were found in 2 (9.5%) of 21 ASTs and 4 (33%) of 12 spitzoid melanomas. Our findings from this large cohort suggest that p16 aberrations are highly specific for borderline and malignant spitzoid neoplasms relative to Spitz nevi. Similar to ASTs, p16 loss in spitzoid melanomas may occur in the presence or absence of genomic CDKN2A loss.

Immunohistochemical staining for p16 and BRAFV600E is useful to distinguish between sporadic and hereditary (Lynch syndrome-related) microsatellite instable colorectal carcinomas.

DNA mismatch repair (MMR) protein analysis by immunohistochemistry (IHC) can identify colorectal cancer (CRC) with microsatellite instability (MSI). As MLH1-deficient CRC can be hereditary or sporadic, markers to distinguish between them are needed. MLH1 promoter methylation assay is the reference method; however, sometimes, it is challenging on formalin-fixed paraffin-embedded tissue samples. We assessed by IHC the expression of BRAFV600E, p16, MGMT, and CDX2 in 55 MLH1-deficient MSI CRC samples (of which 8 had a germline MLH1 mutation) to determine whether this panel differentiates between sporadic and hereditary CRCs. We also analyzed MLH1 promoter methylation by methylation-specific PCR and pyrosequencing and BRAF status by genotyping. None of the hereditary CRCs showed MLH1 methylation, BRAF mutation, BRAFV600E-positive immunostaining, or loss of p16 expression. We detected MLH1 promoter methylation in 67% and a BRAF mutation in 42% of CRC, all showing MLH1 promoter methylation. BRAFV600E IHC and BRAF genotyping gave concordant results in all but two samples. Loss of expression of p16 was found in 30% of CRC with methylation of the MLH1 promoter, but its expression was retained in all non-methylated and part of MLH1-methylated tumors (100% specificity, 30% sensitivity). CDX2 and MGMT expression was not associated with MLH1 status. Thus, BRAFV600E and p16 IHC may help in differentiating sporadic from hereditary MLH1-deficient CRC with MSI. Specifically, p16 IHC might be used as a surrogate marker for MLH1 promoter methylation, because all p16-negative CRCs displayed MLH1 methylation, whereas hereditary CRCs were all p16-positive.

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.

Loss of p53 expression is accompanied by upregulation of beta-catenin in meningiomas: a concomitant reciprocal expression.

Crosstalk between Wnt and p53 signalling pathways in cancer has long been suggested. Therefore in this study we have investigated the involvement of these pathways in meningiomas by analysing their main effector molecules, beta-catenin and p53. Cellular expression of p53 and beta-catenin proteins and genetic changes in TP53 were analysed by immunohistochemistry, PCR/RFLP and direct sequencing of TP53 exon 4. All the findings were analysed statistically. Our analysis showed that 47.5% of the 59 meningiomas demonstrated loss of expression of p53 protein. Moderate and strong p53 expression in the nuclei was observed in 8.5% and 6.8% of meningiomas respectively. Gross deletion of TP53 gene was observed in one meningioma, but nucleotide alterations were observed in 35.7% of meningiomas. In contrast, beta-catenin, the main Wnt signalling molecule, was upregulated in 71.2%, while strong expression was observed in 28.8% of meningiomas. The concomitant expressions of p53 and beta-catenin were investigated in the same patients. In the analysed meningiomas, the levels of the two proteins were significantly negatively correlated (P=0.002). This indicates that meningiomas with lost p53 upregulate beta-catenin and activate Wnt signalling. Besides showing the reciprocal relationship between proteins, we also showed that the expression of p53 was significantly (P=0.021) associated with higher meningioma grades (II and III), while beta-catenin upregulation was not associated with malignancy grades. Additionally, women exhibited significantly higher values of p53 loss when compared to males (P=0.005). Our findings provide novel information about p53 involvement in meningeal brain tumours and reveal the complex relationship between Wnt and p53 signalling, they suggest an important role for beta-catenin in these tumours.

P21 in chronic and acute liver injury.

p21 historically has been considered a tumor suppressor since first studies showed that p21−/− mice display spontaneous tumor formation after 16 months and additionally these mice are more sensitive to chemically induced carcinogenesis [1,2]. On the contrary, recently a potential function as an oncogene has been described for p21. For instance mice deficient for p53 spontaneously develop multiple tumors and an additional deletion of p21 lead to a significant reduction of thymic lymphomas [3]. This argues that the complete spectrum of p21 function during tumorigenesis is not clearly identified. The role of p21 has been further studied in the NEMOΔhepa mice model. The NF-κB pathway regulator NEMO (also known as IKKγ) has been shown to control chronic inflammation and hepatocarcinogenesis in mice. The hepatocyte specific deletion of NEMO (NEMOΔhepa), is of clinical interest as these animals develop a cascade of events which resemble the spectrum of human chronic liver disease, which leads from chronic hepatitis to liver cirrhosis and growth of hepatocellular carcinomas (HCC). Additionally, a recent study using human HCC tissue found a downregulation of NEMO in tumor tissue, further supporting the translational relevance of the NEMOΔhepa mice model [4]. The deletion of NEMO in hepatocytes triggers increased p21 expression [5,6]. In order to study the relevance of p21 overexpression for disease progression of NEMOΔhepa livers, double knockout (NEMOΔhepa/p21−/−) mice carrying a hepatocyte specific deletion for NEMO and an additional constitutional deletion for p21 were generated. Although p21 is a cell cycle inhibitor its deletion had no impact on cell proliferation in 8 week-old NEMOΔhepa/p21−/− livers compared to NEMOΔhepa livers. This result was unexpected since p21 binds to CcnE/cdk2 and CcnA/cdk2 complexes thereby preventing progression from G1- to S-phase. Very likely the loss of p21 expression is compensated by other cell cycle inhibitors such as p-p27 and p18. Despite the unchanged cell cycle activity in p21 deficient NEMOΔhepa livers, the cell cycle regulator CcnA2 and CcnE2 were overexpressed. However, recent studies discovered that ectopic overexpression of CcnA or CcnE in mouse embryonic fibroblast (MEFs) lead to an increase in DNA double strand breakage [7]. Therefore the enhanced liver injury caused by exacerbation of DNA damage in p21-deficient NEMOΔhepa mice could be explained by elevated CcnA2 and CcnE2 expression. The DNA double strand breakage was quantified by pH2AX Immunofluorescence staining. p21 is not only protective against DNA damage in the chronic liver injury model as challenging double mutant NEMOΔhepa/p21−/− mice with Lipopolysaccharide (LPS) enhanced DNA damage massively compared to NEMOΔhepa mice. After LPS administration NEMOΔhepa mice suffer from severe liver injury which is reflected in the increased alanine aminotransferase (ALT) and aspartataminotransferase (AST) serum values and apoptotic cells in the liver of these mice. However, in NEMOΔhepa/p21−/− mice transaminases and cell death were significantly enhanced. Finally, this enhanced liver injury in the double knockout animals resulted in a higher lethality of this mice after LPS administration. The observed hypersensitivity against LPS due to the lack of p21 is mediated via the Tumor Necrosis Factor (TNF), since NEMOΔhepa/p21−/− mice which carry in addition a deletion for the TNF receptor 1 (NEMOΔhepa/p21−/−/TNF-R1−/−), showed a strong attenuation of the DNA damage and cell death. The protective role of p21 in carcinogenesis was the first time visible in 26 week old knockout animals. Here, the double knockout mice (NEMOΔhepa/p21−/−) showed enhanced hepatocyte proliferation as revealed by Ki67 staining. This resulted consequently into a higher liver weight/body weight ratio but more interestingly p21- deficient NEMOΔhepa livers showed more frequently small tumors in comparison to NEMOΔhepa livers. Finally, a significantly increased number of HCCs were found in 52 week-old NEMOΔhepa/p21−/− animals, meaning that the loss of p21 expression caused exacerbation of hepatocarcinogenesis. Analysing the livers of these mice revealed that only the number of nodules was increased, whereas the sizes of the tumors were not significantly enlarged. This suggests that the loss of p21 overexpression in NEMOΔhepa animals has more impact on tumor initiation than on tumor progression. Beside hepatocarcinogenesis p21 had an additional protective role in cholestasis. Livers of 52 week old NEMOΔhepa/p21−/− animals display yellow inclusions and serum values for alkaline phosphatase, direct and total bilirubin confirmed the cholestatic phenotype. These cholestatic serum markers were significantly lower in NEMOΔhepa mice. Taken together, the enhanced p21 expression in NEMOΔhepa animals has a protective function in this model, as p21 protects against DNA damage, acceleration of hepatocarcinogenesis and cholestasis. Since liver disease progression is reduced in the presence of p21 expression, p21 has been shown to act as a tumor suppressor in the NEMOΔhepa model.

Abstract PD6-01: Cooperation of clinically-relevant genomic alterations transforms mammary progenitor cells to metaplastic breast cancer

Abstract Background: We have previously identified the MAPK phosphatase dual specificity phosphatase 4 (Dusp4) as frequently down-regulated in triple-negative and basal-like breast cancer (TNBC and BLBC, respectively). DUSP4 is deregulated in BLBCs by heterozygous and homozygous loss, as well as by promoter CpG methylation. In TNBC and BLBC, DUSP4 deregulation frequently co-occurs with MYC amplification and mutations in TP53. In addition, loss of DUSP4 promotes activation of ETS-1 and cJUN, transcription factors downstream of Ras/MAPK and JNK, while imparting resistance to chemotherapy and promoting tumor-initiating cells, suggesting DUSP4 is a tumor suppressor. Thus, we hypothesized that loss of Dusp4 in the mammary epithelium, together with MYC amplification and mutations in Trp53, would promote tumorigenesis. Methods: We generated transgenic mice with a conditional floxed Dusp4 allele (Dusp4FLOX) and isolated mammary epithelial progenitor cells. The resulting cells were transduced with adenovirus to deliver either Cre recombinase to excise the Dusp4 locus (Dusp4NULL) or GFP control. CRISPR-mediated mutagenesis (or non-targeting CRISPR control) was used to disrupt exon 7 or 8 of Trp53 causing relevant p53 mutations and MYC was overexpressed (or LACZ control). Isogenic cell lines were assessed for tumorigenicity, ploidy, sensitivity to chemotherapy, proliferation, and cell signaling. Results: The isolated parental (Dusp4FLOX) mammary cell line maintained proliferation through >50 passages without senescing and was highly cytokeratin (Ck) 5/14+ and moderately Ck 8/18+, suggestive of a bipotent progenitor-like lineage. As expected, p53 disruption, regardless of Dusp4 status, resulted in tetraploidy and loss of p21 expression. With concurrent Dusp4, p53 and MYC alterations, cells became increasingly resistant to chemotherapy and targeted agents, as measured with proliferation assays. In order to determine whether cells formed tumors in vivo, we implanted isogenic cell lines subcutaneously into nude mice. We found that only Dusp4NULL cells, together with p53 disruption and MYC overexpression (DPM) generated aggressive tumors (forming and ulcerating within 1-2 weeks), while the Dusp4FLOX counterparts (p53 disruption and MYC overexpression; PM) did not. Syngeneic transplantation revealed that DPM cells could also form tumors in an immune competent setting. Cells with Dusp4 loss and p53 mutation were also able to develop tumors without MYC amplification, but at a much slower rate. Tumors derived from the DPM cells were primarily metaplastic or basal-like in nature, including intra-tumor heterogeneity (pockets of Ck5 and pockets of Ck8, with regions of Ck8+ keratin pearls). Conclusions: We have developed a genetically relevant mouse model to study BLBC in immune competent mice for unique opportunities to understand the pathogenesis and therapy response in BLBC, including immunotherapy. These results support the hypothesis that DUSP4 loss promotes tumorigenesis in BLBC. An understanding of the mechanism whereby Dusp4 loss contributes to this process in vivo should provide a rational for novel therapeutic strategies to treat BLBCs with DUSP4 loss. Citation Format: Hicks M, Owens P, Moore P, Giltnane J, Estrada M, Sanders M, Cook R, Arteaga C, Balko J. Cooperation of clinically-relevant genomic alterations transforms mammary progenitor cells to metaplastic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD6-01.

Cyclin kinase inhibitor p27 is downregulated in conjunctival melanoma

PurposeAs loss of cyclin dependent kinase inhibitors p27 and p21 have been identified in cutaneous melanoma and associated with a poorer prognosis, we decided to assess the expression of p27 and p21 in benign and malignant conjunctival melanocytic proliferations.MethodsThe expression of p27 and p21 was assessed by immunohistochemistry in 35 conjunctival naevi and 32 conjunctival melanomas. Statistical analysis was performed with JUMP 8.0 software. Immunohistochemistry was evaluated independently by two observers.ResultsThere was a concordance in 94.02% and 92.54% of the cases in the evaluation of the expression of p27 and p21, respectively. Discrepant cases were simultaneously reviewed to achieve complete agreement.There were 13 subepithelial nevi and 22 compound nevi. There were 14 females and 21 males with a mean age was 36.9 ± 3.6 yo (SEM). Nuclear expression of p27 was found in all the nevi. p21 was identified in only 4 nevi (11.4%).The melanoma group was composed of 18 females and 14 males with a mean age of 65.9 ± 3.38 yo (SEM). p27 and p21 were respectively lost in 18 cases (56.25%) and in 24 cases (75%). There was a significant downregulation of p27 in conjunctival melanoma compared to benign conjunctival nevi (p > 0.0001).In the melanomas, there was a correlation between loss of p27 expression and depth of invasion (p = 0.0141) as well as non‐bulbar tumor localization (p = 0.0279). Although the proliferation index was more elevated in melanomas with p27 loss, this was not significant.ConclusionsThe downregulation of p21 in benign melanocytic proliferations identified in the skin appears also to occur in the conjunctiva. Our results also demonstrate in vivo a significant downregulation of p27 in conjunctival melanoma. Restauration of p27 function in conjunctival melanoma might represent a potential therapeutical option for this tumor.

Loss of p27Kip¹ promotes metaplasia in the pancreas via the regulation of Sox9 expression.

p27Kip1 (p27) is a negative regulator of proliferation and a tumor suppressor via the inhibition of cyclin-CDK activity in the nucleus. p27 is also involved in the regulation of other cellular processes, including transcription by acting as a transcriptional co-repressor. Loss of p27 expression is frequently observed in pancreatic adenocarcinomas in human and is associated with decreased patient survival. Similarly, in a mouse model of K-Ras-driven pancreatic cancer, loss of p27 accelerates tumor development and shortens survival, suggesting an important role for p27 in pancreatic tumorigenesis. Here, we sought to determine how p27 might contribute to early events leading to tumor development in the pancreas. We found that K-Ras activation in the pancreas causes p27 mislocalization at pre-neoplastic stages. Moreover, loss of p27 or expression of a mutant p27 that does not bind cyclin-CDKs causes the mislocalization of several acinar polarity markers associated with metaplasia and induces the nuclear expression of Sox9 and Pdx1 two transcription factors involved in acinar-to-ductal metaplasia. Finally, we found that p27 directly represses transcription of Sox9, but not that of Pdx1. Thus, our results suggest that K-Ras activation, the earliest known event in pancreatic carcinogenesis, may cause loss of nuclear p27 expression which results in derepression of Sox9, triggering reprogramming of acinar cells and metaplasia.

Abstract 3293: Molecular evidence for a parent of origin effect in pediatric insulinomas

Abstract Insulinomas are well-differentiated pancreatic endocrine tumors that cause abnormal beta cell proliferation and hyperinsulinimic hypoglycemia. With an incidence of 2-4 cases per million per year, insulinomas are rare in adults and even less common in children. Since congenital focal hyperinsulinism, another developmental lesion, is associated with paternal isodisomy for the imprinted 11p15 locus, we sought to characterize the role of this region and of mutations in MEN1 on 11q13 in pediatric insulinoma cases. We identified 13 pediatric insulinoma cases, which is the largest pediatric series reported, and performed immunohistochemical analysis for the maternally expressed tumor suppressor marker p57 located in 11p15. Using blood and tumor DNA, we tested 9 candidate genes associated with congenital hyperinsulinism including ABCC8, KCNJ11 and the MEN1 tumor suppressor. We performed methylation analysis of imprint control regions (ICR) that included the paternally expressed IGF2 (ICR1) and maternally expressed CDKN1C (ICR2) located on 11p15 and genome-wide SNP array analysis with tumor DNA. MEN1 mutations were found in 4 cases and suspected in one (MEN1 group); all 5 tumors stained negative for p57 and were hypermethylated at ICR1 and hypomethylated at ICR2 indicating loss of the maternal allele. SNP arrays confirmed loss of heterozygosity (LOH) of entire chromosome 11 in 4 cases. Four out of the 6 remaining non-MEN1 cases also stained negative for p57; in confirmation, 2 of the 4 cases carried mosaic copy number abnormality of chromosome 11. SNP array data demonstrated extensive genome-wide aneuploidy in MEN1 and non-MEN1 cases; however interstitial changes were not present in any of the cases. Notably, MEN1 cases carried only entire chromosome changes, while non-MEN1 cases carried whole arm changes in addition whole chromosome changes. In conclusion, MEN1 mutations are more common in pediatric insulinomas than in adult insulinoma cases (40% in this study vs. 8% in adults). Compared to adult cases, our pediatric insulinoma cases had smaller sized tumors and a shorter median duration of symptoms, suggesting that these tumors are rapidly growing and that the most common chromosome gains and losses very likely represent early events in insulinoma development. Mechanistically, our data suggest that loss of p57 expression, in conjunction with loss of either MEN1 and/or additional imprinted genes on 11p, play a role in the pathogenesis of most insulinomas. In cases with a germ-line MEN1 mutation, we identified a previously unreported paternal parent-of-origin effect that is unique to pediatric insulinomas but not to other MEN1 endocrine tumors (e.g., prolactinoma, parathyroid adenoma). Since a congenital focal hyperinsulinism has a similar parent of origin effect this suggests a broader role for the imprinted locus, 11p15, in not just the pathogenesis of pediatric insulinomas but also in beta-cell replication. Citation Format: Arupa Ganguly, Tricia Bhatti, Karthik Ganapathy, Laura Conlin, Courtney MacMullen, Susan Becker, Eduardo Ruchelli, Charles Stanley. Molecular evidence for a parent of origin effect in pediatric insulinomas. [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 3293. doi:10.1158/1538-7445.AM2015-3293

Sa1926 SOX2 and p53 Protein Expression Predicts Response to Preoperative Chemoradiotherapy in Patients With Esophageal Adenocarcinoma

Objective Preoperative chemoradiotherapy has recently become common practice in treatment of esophageal cancer with a gain in 5-year survival of 10-15%. However, a significant proportion of patients do not respond well and experiencing unnecessary severe side-effects. Accurate risk-stratification of patients using informative biomarkers before therapy may help to avoid unnecessary morbidity due to ineffective treatment. The aim of this study was to investigate the correlation between the expression of SOX2 and P53 in pre-treatment tumor biopsies and grade of pathological tumor response in resected specimen of patients with esophageal adenocarcinoma (EAC) treated with neoadjuvant chemoradiotherapy (nCRT). Methods All EAC patients who received nCRT according to the CROSS regimen followed by esophagectomy, between January 2003 and July 2011 at the Erasmus University Medical Center, were included. SOX2 and P53 protein expression was visualized by immunohistochemistry on all pre-treatment tumor biopsies and scored independently by two investigators who were blinded for clinical outcome. Aberrant expression was defined as negative expression of SOX2 and overexpression or complete loss of P53 expression. The overall Tumor Regression Grade (TRG) was evaluated using the modified Mandard scoring system. Patients with TRG 1 or TRG 2 were classified as major responders (ie, 10% of tumor cells remaining). Results In total 77 patients were included. Forty (53%) patients had a major pathological response (TRG 1-2) and 37 (47%) a minor response (TRG 3-4). In pre-treatment biopsies aberrant SOX2 and P53 expression was seen in 40% (31/77) and 83% (64/77), respectively. A major response was significantly associated with an aberrant SOX2 expression (OR 3.9, 95% CI: 1.5 10.2, p=0.005) and aberrant p53 expression (OR 4.5, 95% CI: 1.15 18.2, p=0.031). Aberrant expression of both biomarkers increased the probability of a major response in the individual patient (OR of 5.6; 95% CI: 2.1 14.9, p= 0.001), with a sensitivity of 68%, specificity of 73% and a positive predictive value of 73%. Conclusion SOX2 and P53 expression in the pre-treatment biopsies predict response to nCRT in patients with EAC. These biomarkers might help to identify patients who are likely to benefit most from this multimodality treatment.

Sa1927 Significant Genetic Variability Associated With the Evolution of Gastric Cancer Precursor Lesions in a Spanish Population

Objective Preoperative chemoradiotherapy has recently become common practice in treatment of esophageal cancer with a gain in 5-year survival of 10-15%. However, a significant proportion of patients do not respond well and experiencing unnecessary severe side-effects. Accurate risk-stratification of patients using informative biomarkers before therapy may help to avoid unnecessary morbidity due to ineffective treatment. The aim of this study was to investigate the correlation between the expression of SOX2 and P53 in pre-treatment tumor biopsies and grade of pathological tumor response in resected specimen of patients with esophageal adenocarcinoma (EAC) treated with neoadjuvant chemoradiotherapy (nCRT). Methods All EAC patients who received nCRT according to the CROSS regimen followed by esophagectomy, between January 2003 and July 2011 at the Erasmus University Medical Center, were included. SOX2 and P53 protein expression was visualized by immunohistochemistry on all pre-treatment tumor biopsies and scored independently by two investigators who were blinded for clinical outcome. Aberrant expression was defined as negative expression of SOX2 and overexpression or complete loss of P53 expression. The overall Tumor Regression Grade (TRG) was evaluated using the modified Mandard scoring system. Patients with TRG 1 or TRG 2 were classified as major responders (ie, 10% of tumor cells remaining). Results In total 77 patients were included. Forty (53%) patients had a major pathological response (TRG 1-2) and 37 (47%) a minor response (TRG 3-4). In pre-treatment biopsies aberrant SOX2 and P53 expression was seen in 40% (31/77) and 83% (64/77), respectively. A major response was significantly associated with an aberrant SOX2 expression (OR 3.9, 95% CI: 1.5 10.2, p=0.005) and aberrant p53 expression (OR 4.5, 95% CI: 1.15 18.2, p=0.031). Aberrant expression of both biomarkers increased the probability of a major response in the individual patient (OR of 5.6; 95% CI: 2.1 14.9, p= 0.001), with a sensitivity of 68%, specificity of 73% and a positive predictive value of 73%. Conclusion SOX2 and P53 expression in the pre-treatment biopsies predict response to nCRT in patients with EAC. These biomarkers might help to identify patients who are likely to benefit most from this multimodality treatment.

Clinical outcome and expression of mutant P53, P16, and Smad4 in lung adenocarcinoma: a prospective study.

BackgroundWhole-exome sequencing has shown that lung adenocarcinoma (LAC) can be driven by mutant genes, including TP53, P16, and Smad4. The aim of this study was to clarify protein alterations of P53, P16, and Smad4 and to explore their correlations between the protein alterations and clinical outcome.MethodsWe investigated associations among P53 mutant (P53Mut) expression, and P16 and Smad4 loss-of-expression, with clinical outcome in 120 LAC patients who underwent curative resection, using immunohistochemical (IHC) methods.ResultsOf the 120 patients, 76 (63.3%) expressed P53Mut protein, whereas 54 (45.0%) loss of P16 expressed and 75 (62.5%) loss of Smad4 expressed. P53Mut expression was associated with tumor size (P = 0.041) and pathological stage (P = 0.025). Loss of P16 expression was associated with lymph node metastasis (P = 0.001) and pathological stage (P < 0.001). Loss of Smad4 expression was associated with tumor size (P = 0.033), lymph node metastasis (P = 0.014), pathological stage (P = 0.017), and tumor differentiation (P = 0.022). Kaplan-Meier survival analysis showed that tumor size (P = 0.031), lymph node metastasis (P < 0.001), pathological stage (P < 0.001), P53Mut protein expression (P = 0.038), and loss of p16 or Smad4 expression (P < 0.001) were significantly associated with shorter overall survival(OS), whereas multivariate analysis indicated that lymph node metastasis (P = 0.014) and loss of p16 or Smad4 expression (P < 0.001) were independent prognostic factors. Analysis of protein combinations showed patients with more alterations had poorer survival (P < 0.001). Spearman correlation analysis showed that loss of Smad4 expression inversely correlated with expression of P53Mut (r = −0.196, P = 0.032) and positively with lost P16 expression (r =0.182, P = 0.047).ConclusionsThe findings indicate that IHC status of P53Mut, P16, and Smad4 may predict patient outcomes in LAC.

Periosteal chondrosarcoma: a histopathological and molecular analysis of a rare chondrosarcoma subtype.

Periosteal chondrosarcoma is a rare, malignant cartilage-forming neoplasm originating from the periosteal surface of bone. We collected 38 cases from the archives of the Netherlands Committee on Bone Tumours, with the aim of studying histological features and evaluating the involvement of isocitrate dehydrogenase 1 (IDH1), EXT, Wnt/β-catenin, the pRB pathway (CDK4 and p16), and the TP53 pathway (p53 and MDM2). Histology showed a moderately cellular matrix with mucoid-myxoid changes and, in 42% of cases, formation of a neocortex. Occasional intramedullary extension (26%) and subsequent host bone entrapment (40%) were seen. Histological grading revealed grade 1 (53%) and grade 2 (45%). The EXT1 protein was normally expressed, and mutations in IDH1 were observed in only 15% of cases. pRb signalling was deregulated by loss of p16 expression in 50% of cases, and Wnt signalling was lost in 89%. No alterations were found in CDK4, p53, or MDM2. We report the first large histological and molecular study on periosteal chondrosarcoma showing that histopathological examination and molecular aberrations do not predict prognosis. Although the mutation frequency of IDH1 was low, we confirm the supposed relationship with central chondrosarcoma. Moreover, we identify loss of canonical Wnt signalling and deregulation of pRb signalling as possible events contributing to its histogenesis.

Differential expression of p63 in hydropic and molar gestations.

To observe the differential expression of p63 in hydropic and molar gestation. Cross-sectional analytical study. Department of Pathology, Basic Medical Sciences Institute, Jinnah Postgraduate and Medical Centre, Karachi, from January 2006 to June 2013. Ninety placental biopsies including 30 cases each of hydropic abortions, partial hydatidiform mole and complete hydatidiform mole were analyzed for morphological features and results of immunohistochemical staining. Results were described as frequency. Significance was determined using test of proportions with significance at p < 0.05. Out of 30 cases of hydropic abortion, 6 were negative, 15 were weak, 4 were moderate and 5 showed strong degree of intensity for p63. Out of 30 cases of partial hydatidiform mole, 3 were negative, 2 showed weak, 4 showed moderate and 21 cases showed strong degree of intensity for p63. All 30 cases of complete hydatidiform mole strongly stained for p63. The intensity of staining of p63 was stronger in cases of molar pregnancy as compared to hydropic abortion. There was loss of p63 expression in cytotrophoblastic cells in all abortions. In limited resources settings, where facilities for PCR/FISH and DNA ploidy analysis is not available, the authors advocate p63 in routine clinical practice to provide the most refined diagnosis of hydatidiform moles.

Adaptive Resistance to Immunotherapy Directed Against p53 Can be Overcome by Global Expression of Tumor-Antigens in Dendritic Cells.

Immunotherapy of cancer utilizes dendritic cells (DCs) for antigen presentation and the induction of tumor-specific immune responses. However, the therapeutic induction of anti-tumor immunity is limited by tumor escape mechanisms. In this study, immortalized dendritic D2SC/1 cells were transduced with a mutated version of the p53 tumor suppressor gene, p53M234I, or p53C132F/E168G, which are overexpressed in MethA fibrosarcoma tumor cells. In addition, D2SC/1 cells were fused with MethA tumor cells to generate a vaccine that potentially expresses a large repertoire of tumor-antigens. Cellular vaccines were transplanted onto Balb/c mice and MethA tumor growth and anti-tumor immune responses were examined in vaccinated animals. D2SC/1–p53M234I and D2SC/1–p53C132F/E168G cells induced strong therapeutic and protective MethA tumor immunity upon transplantation in Balb/c mice. However, in a fraction of immunized mice MethA tumor growth resumed after an extended latency period. Analysis of these tumors indicated loss of p53 expression. Mice, pre-treated with fusion hybrids generated from D2SC/1 and MethA tumor cells, suppressed MethA tumor growth and averted adaptive immune escape. Polyclonal B-cell responses directed against various MethA tumor proteins could be detected in the sera of D2SC/1–MethA inoculated mice. Athymic nude mice and Balb/c mice depleted of CD4+ or CD8+ T-cells were not protected against MethA tumor cell growth after immunization with D2SC/1–MethA hybrids. Our results highlight a potential drawback of cancer immunotherapy by demonstrating that the induction of a specific anti-tumor response favors the acquisition of tumor phenotypes promoting immune evasion. In contrast, the application of DC/tumor cell fusion hybrids prevents adaptive immune escape by a T-cell dependent mechanism and provides a simple strategy for personalized anti-cancer treatment without the need of selectively priming the host immune system.

Nuclear iASPP may facilitate prostate cancer progression

One of the major challenges in prostate cancer (PCa) research is the identification of key players that control the progression of primary cancers to invasive and metastatic disease. The majority of metastatic PCa express wild-type p53, whereas loss of p63 expression, a p53 family member, is a common event. Here we identify inhibitor of apoptosis-stimulating protein of p53 (iASPP), a common cellular regulator of p53 and p63, as an important player of PCa progression. Detailed analysis of the prostate epithelium of iASPP transgenic mice, iASPPΔ8/Δ8 mice, revealed that iASPP deficiency resulted in a reduction in the number of p63 expressing basal epithelial cells compared with that seen in wild-type mice. Nuclear and cytoplasmic iASPP expression was greater in PCa samples compared with benign epithelium. Importantly nuclear iASPP associated with p53 accumulation in vitro and in vivo. A pair of isogenic primary and metastatic PCa cell lines revealed that nuclear iASPP is enriched in the highly metastatic PCa cells. Nuclear iASPP is often detected in PCa cells located at the invasive leading edge in vivo. Increased iASPP expression associated with metastatic disease and PCa-specific death in a clinical cohort with long-term follow-up. These results suggest that iASPP function is required to maintain the expression of p63 in normal basal prostate epithelium, and nuclear iASPP may inactivate p53 function and facilitate PCa progression. Thus iASPP expression may act as a predictive marker of PCa progression.

Neoplasms arising in large gastric hyperplastic polyps: endoscopic and pathologic features

Little is known about gastric neoplasms arising from hyperplastic polyps (HPs). To investigate the risk factors associated with neoplasms within HPs and to evaluate the role of alterations of the p16-cyclin D1-pRb pathway in the malignant transformation of HPs. Retrospective, case-control study. Tertiary-care center. Between May 1995 and January 2011, a total of 809 HPs >1 cm were investigated. Associated neoplasms were present in 30 HPs (case group); 30 HPs without neoplasms were selected as a control group. Gastric polypectomy. The risk factors associated with neoplasms within HPs and immunohistochemical expression of p16, cyclin D1, p53, and Ki-67 between case and control groups. Of the 809 HPs, 15 had associated dysplasia, and 15 had carcinoma. Multivariate analysis showed that neoplasm was associated with patient age (odds ratio [OR] 1.159; 95% confidence interval [CI], 1.243-2.044; P < .001), polyp size (OR 1.103; 95% CI, 1.055-1.152; P < .001), and polyp lobulation (OR 4.549; 95% CI, 1.759-11.0766; P < .001) but not with location, multiplicity, intestinal metaplasia, growth pattern, or Helicobacter pylori infection. Loss of p16 expression and high Ki-67 expression were observed in dysplastic areas of HPs compared with the control group (p16 = 14.3% vs 60%; P = .001, Ki-67 = 60.7% vs 36.7%; P < .001). However, no significant differences were found in nondysplastic areas in both groups. Single-center, retrospective study. HPs >1 cm may indicate the presence of neoplasms. Loss of p16 and high Ki-67 expression may be markers of HP-associated dysplasia.