TP53 Protein Research Articles

Having Pancreatic Cancer with Tumoral Loss of ATM and Normal TP53 Protein Expression Is Associated with a Poorer Prognosis

To determine how often loss of ataxia-telangiectasia-mutated (ATM) protein expression occurs in primary pancreatic ductal adenocarcinomas and to determine its prognostic significance. The expression of ATM and TP53 was determined by immunohistochemistry in 397 surgically resected pancreatic ductal adenocarcinomas (Hopkins; Johns Hopkins Medical Institutions, Baltimore, MD), a second set of 159 cases (Emory; Emory University Hospital, Atlanta, GA), and 21 cancers after neoadjuvant chemoradiotherapy. Expression was correlated with the clinicopathologic parameters, including survival. Tumoral ATM loss was observed in one cancer known to have biallelic inactivation of ATM and 50 of the first 396 (12.8%) cases, significantly more often in patients with a family history of pancreatic cancer (12/49; 24.5%) than in those without (38/347; 11.0%; P = 0.019). In the Hopkins series, ATM loss was associated with a significantly decreased overall survival in patients whose cancers had normal TP53 expression (P = 0.019) and was a significant independent predictor of decreased overall survival (P = 0.014). Seventeen (10.7%) of 159 Emory cases had tumoral ATM loss and tumoral ATM loss/normal TP53 was associated with poorer overall survival (P = 0.1). Multivariate analysis of the combined Hopkins/Emory cases found that tumoral ATM loss/normal TP53 was an independent predictor of decreased overall survival [HR = 2.61; confidence interval (CI), 1.27-5.37; P = 0.009]. Of 21 cancers examined after neoadjuvant chemoradiotherapy, one had tumoral loss of ATM; it had no histologic evidence of tumor response. Tumoral loss of ATM protein was detected more often in patients with a family history of pancreatic cancer than in those without. Patients whose pancreatic cancers had loss of ATM but normal TP53 had worse overall survival after pancreatic resection.

BCL2, BCL6, IGH, TP53, and MYC protein expression and gene rearrangements as prognostic markers in diffuse large B-cell lymphoma: a study of 44 Turkish patients

The purpose of this study was to determine the frequency of BCL2, BCL6, IGH, TP53, and MYC protein expression and rearrangements of the respective genes in diffuse large B-cell lymphoma (DLBCL) patients and to assess their prognostic values. Samples from 44 patients with DLBCL were evaluated using fluorescence in situ hybridization and immunohistochemical analyses. BCL6 was the most rearranged gene (63.6%), followed by MYC (31.8%), TP53 (22.7%), and BCL2 (18.2%). Multiple rearrangements were detected in 40.9% of the cases. BCL6 was the most expressed protein (78.6%), followed by TP53 (69.04%), BCL2 (59.5%) and MYC (14.3%). Expression of multiple proteins was detected in 67.4% of the cases. BCL2 (P = .003) expression had a significant negative influence on overall survival,whereas BCL6 (P = .014) expression had a significant positive influence. Our results with a different pattern of gene rearrangements and associated protein overexpression indicate the molecular genetic complexity of DLBCLs, which reflects the morphologic, biologic, and clinical heterogeneity of these lymphomas.

TP53Promoter Methylation in Primary Glioblastoma: Relationship withTP53mRNA and Protein Expression and Mutation Status

Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The frequency of TP53 promoter methylation was found in 21.4%. To the best of our knowledge, this is the first report showing such high frequency of TP53 promoter methylation in primary GB. There was no relation between TP53 promoter methylation and TP53 mRNA level (p=0.5722) and between TP53 promoter methylation and TP53 protein expression (p=0.2045). No significant associations were found between TP53 mRNA expression and mutation of TP53 gene (p=0.9076). However, significant association between TP53 mutation and TP53 protein expression was found (p=0.0016). Our data suggest that in primary GB TP53 promoter methylation does not play a role in silencing of TP53 transcriptional and protein expression and is probably regulated by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway.

Associations between Cigarette Smoking, Hormone Therapy, and Folate Intake with Incident Colorectal Cancer by TP53 Protein Expression Level in a Population-Based Cohort of Older Women

Cigarette smoking (CS), hormone therapy (HT), and folate intake (FI) are each thought to influence colorectal cancer risk, but the underlying molecular mechanisms remain incompletely defined. The TP53 (p53) protein, encoded by the TP53 tumor-suppressor gene that is commonly mutated in colorectal cancer, can be readily assessed to differentiate biologically distinct colorectal cancer subtypes. In this prospective cohort study, we examined CS-, HT-, and FI-associated colorectal cancer risks by TP53 protein expression level among Iowa Women's Health Study (IWHS) participants. The IWHS recruited 41,836 randomly selected Iowa women, ages 55 to 69 years, with a valid driver's license at study entry in 1986. Self-reported exposure variables were assessed at baseline. Incident colorectal cancer cases were ascertained by annual linkage with the Iowa Cancer Registry. Archived, paraffin-embedded tissue specimens were collected and evaluated for TP53 protein expression by immunohistochemistry. Multivariate Cox regression models were fit to estimate relative risks (RR) and 95% confidence intervals (CI) for associations between CS, HT, or FI and TP53-defined colorectal cancer subtypes. Informative environmental exposure and protein expression data were available for 492 incident colorectal cancer cases: 222 (45.1%) TP53 negative, 72 (14.6%) TP53 low, and 198 (40.2%) TP53 high. Longer duration (>5 years) of HT was inversely associated with TP53 high colorectal cancers (RR, 0.50; 95% CI, 0.27-0.94). No other statistically significant associations were observed. These data support possible heterogeneous effects from HT on TP53-related pathways of colorectal carcinogenesis in older women.

TP53 mutations in colorectal cancer from Tunisia: relationships with site of tumor origin, microsatellite instability and KRAS mutations

Loss of TP53 function through gene mutation is a critical event in the development and progression of colorectal cancer (CRC). Here we examined 51 primary CRC tumors from Tunisia for mutations in TP53 exons 4-9 using PCR-direct sequencing. TP53 status and mutation site/type were than correlated with nuclear protein accumulation, familial and clinicopathologic variables and data on KRAS mutations and microsatellite instability (MSI-H). The TP53 mutation analysis was possible in the tumor of 47 patients and a deleterious somatic mutation has been detected in 59.6% of the patients (28/47) including 20 (71.4%) missense mutations, 7 nonsense mutations (25%) and 1 (3.6%) frameshift mutation. 89.3% (25/28) of the detected mutations were in exons 5-8, whereas 10.7% (3/28) were in exon 4. Among the 27 non frameshift mutations, 89% (24/27) were transitions and 11% (3/27) were transversions. 64.3% (18/27) of the altered amino acids corresponded to arginine. 74% (20/27) were G>C to A>T transitions, and more than half (14/27) occur at hotspots codons with CpG sites. TP53 mutations correlated closely with TP53 accumulation (p = 0.0090) and inversely with MSI phenotype (p = 0.0658). A KRAS somatic mutation was identified in 25% (7/28) of the TP53 mutated tumors. All these mutations were G>A transitions in codon 12 and all the tumors with combined alterations but one were distally located and MSS. In conclusion, frequency and types of TP53 mutations and correlations with TP53 protein accumulation, and MSI were as reported for non-Tunisian patients. However, no significant associations have been detected between TP53 mutations and clinicopathological data in Tunisian patients as previously reported.

The expression of TP53 mRNA splice variants relates to progression of cancer in human colorectal cancer

Background The carcinogenesis of colon and rectum is caused by accumulation of genetic alternation or mutation. Recent works have shown significant biomarkers that include loss of hetero-zygosity, gene mutations or gene methylation of genomic DNA. Furthermore, it is also reported that the functional analysis of mRNAs and miRNAs are significance for clarification of cancer progress. In this study, we examined the expression of TP53 mutation, TP53 mRNA splice variants, TP53 protein and TP53-related miRNA in human colorectal cancer, and evaluated significance as the clinicopathological biomarker. Design Tumors were collected from 30 patients diagnosed with primary advanced colorectal cancer. Both genomic DNA and total RNA were extracted with TRIzol reagent, and TP53 mutation was analyzed by direct sequence method. TP53 mRNA splice variants were analyzed by RT-PCR, TP53-related miRNA were analyzed by qRT-PCR, and TP53 protein was detected by immunohisto-chemistry. Statistical analysis was carried out by SPSS21. Results TP53 alfa showed significantly low expression in TP53 mutation cancer. TP53 beta showed significantly low expression in TP53 mutation cancer. TP53 gamma showed significantly high expression in TP53 mutation cancer, and it also showed low expression in pTMN stage I + II as compared to pTMN stage III + IV.

Promoter methylation status of MGMT, hMSH2, and hMLH1 and its relationship to corresponding protein expression and TP53 mutations in human esophageal squamous cell carcinoma

To determine the relevance of O-6-methylguanine-DNA methyltransferase (MGMT), human mutS homolog 2 (hMSH2), and human mutL homolog 1 (hMLH1) in TP53 mutations in esophageal squamous cell carcinoma, we employed methylation-sensitive high-resolution melting technology and methylation-specific polymerase chain reaction (PCR) to analyze promoter hypermethylation of MGMT, hMSH2, and hMLH1, respectively, in 51 paired tumors and their adjacent normal tissues. The protein expression of the three proteins was also evaluated by Western blot analysis, and the PCR products of TP53, from exon 5 to exon 8, were directly sequenced to measure the mutation spectrum. Esophageal tumor tissues embraced statistically higher MGMT and hMSH2 promoter methylation level than normal tissue. The promoter methylation status of MGMT and hMSH2 corresponds positively with the protein expression level of MGMT and hMSH2. However, such relevance was not found for hMLH1. Furthermore, TP53 mutation status was well associated with MGMT and hMSH2 promoter methylation status, indicating that silencing of the two genes could lead to TP53 mutation in ESCC.

Association of the p53 codon 72 polymorphism with clinicopathological characteristics of colorectal cancer through mRNA analysis

TP53 represents a suitable candidate for a colorectal cancer susceptibility locus. The polymorphism in the p53 72nd codon involves a proline to arginine substitution, leading to changes in gene transcription activity, interaction with other proteins and modulation of apoptosis. Studies evaluating the association between this polymorphism and colorectal cancer (CRC) have shown inconsistent results, and none have evaluated the mRNA status of TP53. The aim of the present study was to evaluate the association between this SNP expression at the mRNA level in CRC samples and patient clinicopathological variables and prognosis, p53 protein expression and TP53 mutation. This is the first report to describe the mRNA expression of p53 codon 72 alleles in CRC. We evaluated 101 non-related patients with CRC treated at the A.C. Camargo Cancer Center in Brazil. RNA was isolated from frozen tumor tissues using a trizol-based protocol. The polymorphism was detected using RT-PCR followed by Sanger sequencing. Associations were analyzed using Pearson's Chi-square or Fisher's exact tests, logistic regression and Cox. This polymorphism was significantly associated with clinicopathological variables related to increased tumor aggressiveness. The expression of Arg72 (OR, 3.83; CI 1.02-14.35; P=0.046) and the TNM stage (OR, 7.15; CI 1.45-35.29; P=0.016) were found to be independent predictors for recurrence. These data suggest that the mRNA expression of the Pro72 allele is associated with less favorable tumor features. The allele frequency of the p53 Pro72 was 0.26. The analysis of mRNA is important to determine the specific contribution of the allele expressed. These results suggest that this polymorphism may play a role in CRC.

Proliferating CLL Cells Express Abundant But Transcriptionally Compromised TP53 Protein

Current models of CLL pathogenesis invoke specialized microenvironments within the lymph nodes and bone marrow that harbor proliferating cells. Such proliferating CLL cells are more resistant to current immuno-chemotherapeutic regimens than cells in the peripheral blood and are thought to be the cause of resistant disease. Various models have been used to recapitulate these CLL proliferation centers in vitro, including stimulating cells with CD154 and IL4. We studied 40 patients (6 with p53 mutations/deletions) using this system and observed that >50% of CLL cells undergo proliferation after 72 hours of stimulation, as assessed by Ki67 staining. Unexpectedly, under these conditions we also observed a 30-40 fold induction of TP53 protein in all cases of CLL analyzed, irrespective of TP53 mutational status. Nearly all cells showed increased TP53 protein expression (Figure 1), suggesting that such high-level TP53 protein expression did not hinder cell proliferation. Given that induction of wild-type TP53 protein usually induces cell cycle arrest if not apoptosis, we examined for transcriptional up-regulation of TP53 target genes using a combination of qRT-PCR, RNA arrays and RNA-Seq approaches. 4 out of 12 cases showed induction of some TP53 target genes, but overall there was no consistent pattern of transcriptional up-regulation of target genes, suggesting that the induced TP53 is transcriptionally compromised in CLL cells following CD154/IL4 stimulation. In contrast, DNA damage induced by doxorubicin in CD154/IL4 stimulated cells induced wild type TP53 protein to even higher levels, resulting in TP53 target gene up-regulation and apoptosis, as expected. CD154/IL4 stimulation also induced a 10-fold elevation in ROS levels in all cases. This resulted in significant oxidative DNA damage, as measured by a modified comet assay, which could explain the induction of TP53 in proliferating cells. qRT-PCR and RNA-Seq experiments failed to show a significant increase in TP53 mRNA levels, indicating that elevation of TP53 protein levels was occurring post-transcriptionally. Increased phosphorylation of TP53 at S15 was seen in all cases, which may account for the observed increased protein stability through dissociation from MDM2. All TP53 mRNA isoforms expressed retained transcriptional activation and DNA binding domains. In view of these results, we propose a model whereby oxidative stress induced by proliferation in CLL triggers TP53 protein expression. TP53 becomes phosphorylated but, for reasons that remain unclear, is unable to transactivate its target genes normally and induce cell-cycle arrest. Apoptosis could be suppressed by high-level expression of anti-apoptotic BCL2 proteins. However, TP53 remains able to trigger a full apoptotic response after further DNA damage and a higher threshold of protein levels is reached. Reactivation of the full transcriptional activities of wild-type TP53 in proliferating CLL cells may provide a new therapeutic approach. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Detection of TP53 dysfunction in chronic lymphocytic leukemia by an in vitro functional assay based on TP53 activation by the non-genotoxic drug Nutlin-3: a proposal for clinical application

BackgroundTP53 defects, i.e. 17p13 deletion and/or nucleotide mutations, associate with short survival and chemorefractoriness in chronic lymphocytic leukemia (CLL). In this context, since direct sequencing of the TP53 gene does not evaluate TP53 functionality, a functional assessment of TP53 pathway may be of interest to identify high risk CLL. By taking advantage of a training cohort of 100 CLL and a validation cohort of 40 CLL with different patterns of TP53 mutation/deletion by FISH and sequencing, we propose an in-vitro assay in which the modulation of TP53 protein and CDKN1A mRNA were investigated upon 24-hour exposure of CLL cells to Nutlin-3.MethodsThe functional assay was set-up on cell lines recapitulating all TP53 genotypes (EHEB, TP53wt/wt; RAJI, TP53mut/wt; MEC-1 and MAVER1, TP53mut/del; HL-60, TP53del/del) and evaluated in two multi-institutional cohorts, purposely enriched in CLL bearing TP53 disruption: a training cohort of 100 cases and a validation cohort of 40 cases, both characterized by FISH and TP53 direct sequencing. Cells were exposed to 10 μM Nutlin-3 for 24 hours; TP53 accumulation was evaluated by Western blotting; TP53 transcriptional activity was determined by quantitative realtime PCR (qRT-PCR) of the TP53 target gene CDKN1A.ResultsAccording to TP53 protein modulation, in the training cohort we identified: i) 63 cases (51 TP53wt/wt, 12 TP53del/wt) with absence of basal TP53 and induction after treatment (normal pattern); ii) 18 cases (3 TP53mut/wt, 15 TP53mut/del) with high basal TP53 without increase after treatment (mutant pattern); iii) 19 cases (5 TP53mut/wt; 3 TP53mut/del; 11 TP53wt/wt) with basal TP53 that increases upon treatment (intermediate pattern). Evaluation of CDKN1A mRNA levels upon Nutlin-3 exposure showed that the 26 TP53 mutated (TP53mut/del or TP53mut/wt) cases had lower induction levels than the majority (57/63) of cases with normal pattern, and 10/12 cases with intermediate pattern without evidence of TP53 derangement by FISH and sequencing. These results were confirmed in the independent validation cohort of 40 cases (13 TP53wt/wt, 3 TP53del/wt, 12 TP53mut/del, 12 TP53mut/wt).ConclusionsThe proposed functional assay may integrate the conventional analyses for the identification of TP53 dysregulated CLL.

MYC, FBXW7 and TP53 copy number variation and expression in Gastric Cancer

BackgroundMYC deregulation is a common event in gastric carcinogenesis, usually as a consequence of gene amplification, chromosomal translocations, or posttranslational mechanisms. FBXW7 is a p53-controlled tumor-suppressor that plays a role in the regulation of cell cycle exit and reentry via MYC degradation.MethodsWe evaluated MYC, FBXW7, and TP53 copy number, mRNA levels, and protein expression in gastric cancer and paired non-neoplastic specimens from 33 patients and also in gastric adenocarcinoma cell lines. We also determined the invasion potential of the gastric cancer cell lines.ResultsMYC amplification was observed in 51.5% of gastric tumor samples. Deletion of one copy of FBXW7 and TP53 was observed in 45.5% and 21.2% of gastric tumors, respectively. MYC mRNA expression was significantly higher in tumors than in non-neoplastic samples. FBXW7 and TP53 mRNA expression was markedly lower in tumors than in paired non-neoplastic specimens. Moreover, deregulated MYC and FBXW7 mRNA expression was associated with the presence of lymph node metastasis and tumor stage III-IV. Additionally, MYC immunostaining was more frequently observed in intestinal-type than diffuse-type gastric cancers and was associated with MYC mRNA expression. In vitro studies showed that increased MYC and reduced FBXW7 expression is associated with a more invasive phenotype in gastric cancer cell lines. This result encouraged us to investigate the activity of the gelatinases MMP-2 and MMP-9 in both cell lines. Both gelatinases are synthesized predominantly by stromal cells rather than cancer cells, and it has been proposed that both contribute to cancer progression. We observed a significant increase in MMP-9 activity in ACP02 compared with ACP03 cells. These results confirmed that ACP02 cells have greater invasion capability than ACP03 cells.ConclusionIn conclusion, FBXW7 and MYC mRNA may play a role in aggressive biologic behavior of gastric cancer cells and may be a useful indicator of poor prognosis. Furthermore, MYC is a candidate target for new therapies against gastric cancer.

Carcinoma Ex Pleomorphic Adenoma, with Particular Emphasis on Early Lesions

Carcinoma ex pleomorphic adenoma (CXPA) is a broad category of carcinomas of the salivary glands which includes at least 2 clinically relevant categories; one is referred here as early CXPA (ECXPA), the other as widely invasive CXPA. The former includes several histological patterns ranging from non-invasive/in situ/intraductal/intratubular, early invasive/extratubular/intracapsular and extracapsular (up to 6 mm). The latter includes any CXPA with invasion of >6 mm. The clinical behaviour of ECXPA is not aggressive and tends to overlap that of a pleomorphic adenoma (PA) which makes the histological report of carcinoma contradictory. These early malignant changes in PA are known since the 1970s but it has been the use of immunohistochemical and molecular genetic analysis for HER-2 and TP53 gene in the last decade that has clarified the genuine malignant nature of the cells. HER-2 and TP53 gene and protein are involved in the early stages of malignant transformation of PA. Moreover the immunohistochemical over-expression HER-2, p53 protein and Mib-1 proliferation marker may be useful markers to identify malignant areas in PA.

Overexpression of RBBP6, Alone or Combined with Mutant TP53, Is Predictive of Poor Prognosis in Colon Cancer

Retinoblastoma binding protein 6 (RBBP6) plays an important role in chaperone-mediated ubiquitination and interacts with TP53 in carcinogenesis. However, the clinicopathologic significance of RBBP6 expression in colon cancer is unknown; in particular, the prognostic value of RBBP6 combined with TP53 expression has not been explored. Therefore, quantitative real-time PCR and western blot analyses were performed to detect RBBP6 expression in colon cancer tissues. RBBP6 and TP53 expression were assessed by immunohistochemistry in a tissue microarray format, in which the primary colon cancer tissue was paired with noncancerous tissue. Tissue specimens were obtained from 203 patients. We found that RBBP6 was overexpressed in colon tumorous tissues and was significantly associated with clinical stage, depth of tumor invasion, lymph node metastasis (LNM), distant metastasis, and histologic grade. Further studies revealed that a corresponding correlation between RBBP6 overexpression and mutant TP53 was evident in colon cancer (r = 0.450; P<0.001). RBBP6 expression was an independent prognostic factor for overall survival (OS) and disease free survival (DFS). Interestingly, patients with tumors that had both RBBP6 overexpression and mutant TP53 protein accumulation relapsed and died within a significantly short period after surgery (P<0.001). Multivariate analysis showed that patients with LNM and patients with both RBBP6- and TP53-positive tumors had extremely poor OS (HR 6.75; 95% CI 2.63–17.35; P<0.001) and DFS (HR 8.08; 95% CI 2.80–23.30; P<0.001). These clinical findings indicate that the assessment of both RBBP6 and mutant TP53 expression will be helpful in predicting colon cancer prognosis.

Lymphocytes of Patients with Alzheimer’s Disease Display Different DNA Damage Repair Kinetics and Expression Profiles of DNA Repair and Stress Response Genes

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, characterized by loss of memory and cognitive capacity. Given the limitations to analyze brain cells, it is important to study whether peripheral lymphocytes can provide biological markers for AD, an interesting approach, once they represent the overall condition of the organism. To that extent, we sought to find whether lymphocytes of AD patients present DNA damage and repair kinetics different from those found in elderly matched controls (EC group) under in vitro treatment with hydrogen peroxide. We found that AD patient cells indeed showed an altered DNA repair kinetics (comet assay). Real-time quantitative analysis of genes associated with DNA stress response also showed that FANCG and CDKN1A are upregulated in AD, while MTH1 is downregulated, compared with the control group. In contrast, the expression of ATM, ATR and FEN1 genes does not seem to differ between these groups. Interestingly, TP53 protein expression was increased in AD patients. Therefore, we found that kinetics of the stress response in the DNA were significantly different in AD patients, supporting the hypothesis that repair pathways may be compromised in AD and that peripheral lymphocytes can reveal this condition.

Comparison of Modules of Wild Type and Mutant Huntingtin and TP53 Protein Interaction Networks: Implications in Biological Processes and Functions

Disease-causing mutations usually change the interacting partners of mutant proteins. In this article, we propose that the biological consequences of mutation are directly related to the alteration of corresponding protein protein interaction networks (PPIN). Mutation of Huntingtin (HTT) which causes Huntington's disease (HD) and mutations to TP53 which is associated with different cancers are studied as two example cases. We construct the PPIN of wild type and mutant proteins separately and identify the structural modules of each of the networks. The functional role of these modules are then assessed by Gene Ontology (GO) enrichment analysis for biological processes (BPs). We find that a large number of significantly enriched () GO terms in mutant PPIN were absent in the wild type PPIN indicating the gain of BPs due to mutation. Similarly some of the GO terms enriched in wild type PPIN cease to exist in the modules of mutant PPIN, representing the loss. GO terms common in modules of mutant and wild type networks indicate both loss and gain of BPs. We further assign relevant biological function(s) to each module by classifying the enriched GO terms associated with it. It turns out that most of these biological functions in HTT networks are already known to be altered in HD and those of TP53 networks are altered in cancers. We argue that gain of BPs, and the corresponding biological functions, are due to new interacting partners acquired by mutant proteins. The methodology we adopt here could be applied to genetic diseases where mutations alter the ability of the protein to interact with other proteins.

Small bowel adenocarcinoma phenotyping and prognostic factors.

3585 Background: SBA is a rare tumor with poor prognosis. Data regarding SBA molecular alterations are lacking. Methods: We searched for several candidate oncogenic alterations and characterised the immunophenotype according to the primary tumour location in pts with SBA (all stages; ampullary tumours excluded) treated in 11 centres from 1996 to 2008. Tissue microarrays were constructed from tumour samples, and DNA was extracted from formalin-fixed, paraffin-embedded samples. HER2, β-catenin, TP53, and mismatch repair (MMR) protein expression was assessed by immunohistochemistry. A molecular analysis assessed microsatellite instability, KRAS mutation and BRAFV600E mutation. Results: We obtained samples from 63 SBA pts (median age, 58 years; tumour stage: I-II, n=19 (30%); III, n=22 (35%); IV, n=20 (32%); locally advanced, n=2 (3%)). HER2 overexpression (3+) was observed in 2/62 (3%) pts. Overexpression of TP53 was observed in 26/62 (42%) pts. Abnormal expression of β-catenin was observed in 12/62 (19%) pts. MMR deficiency (dMMR) was observed in 14/61 (23%) pts, consistent with Lynch syndrome in 7/14 (50%) pts. All of the dMMR tumours were in duodenum or jejunum. Only one of dMMR tumour was stage IV. A KRAS mutation was observed in 21/49 (43%) pts. BRAFV600E mutation was observed in only 1/40 pt. Median overall survival (OS) was 36.6 months (95% confidence interval [CI], 26.9-72.2). In univariate analysis, stage I-II (p&lt;0.001) and dMMR phenotype (p=0.02) were significantly associated with longer OS. In multivariate analysis, only disease stage independently predicted longer OS (p&lt;0.001). For stage IV pts, median OS was 17.9 months (CI, 12.6-36.6). For stage I-III pts median recurrence-free survival (RFS) was 26.7 months (CI: 14.6-42.4). A trend for a better RFS was observed if tumour was dMMR HR: 0.39 (CI, 0.10-1.44], p=0.15. Conclusions: This large study suggests that molecular alterations in SBA are closer to those in colorectal cancer (CRC) than those in gastric cancer, with low levels of HER 2 overexpression and high rates of KRAS mutations. The seemingly higher rate of dMMR than in CRC may be explained by the higher frequency of Lynch syndrome in SBA in pts. A trend for good prognosis was associated with dMMR.

OP201: EGFR amplification in saliva of oral cancer patients: A pilot study

Objectives We aim to examine EGFR, CCND1, TP53 and P16 expression in oral squamous cell carcinoma (OSCC) and pre-and post-operative saliva samples from the same patients. Materials and Methods 28 OSCC samples were used to assess EGFR, CCND1, TP53 and P16 protein expression in OSCC and paired adjacent normal mucosa, by immunohistochemistry (IHC), and EGFR amplification, by fluorescent in situ hybridization (FISH). Examination for TP53 mutation(s) on exons 5–8 by sequencing, HPV status using the Roche linear array, and EGFR copy number by quantitative real-time PCR was also performed. Saliva from 14 healthy controls and 27 oral cancer patients were analyzed for genetic changes in OSCC, and associated with clinicopathological data. Results EGFR protein over-expression was commonly found in OSCC and was associated with perineural invasion (p = 0.04) and advanced tumor grade (p = 0.03). EGFR copy number gains in OSCC were significantly associated with T3–T4 category (p = 0.04) and perineural invasion (p = 0.02). EGFR copy number was increased in OSCC and not significantly different from pre-operative saliva; EGFR copy number in saliva from healthy controls was significantly different from pre-operative saliva (p = 0.02). Conclusions Analysis of EGFR amplification in saliva could become useful for monitoring patients for recurrence or the appearance of a second primary tumor.

Differential effects on lung cancer cell proliferation by agonists of glucocorticoid and PPARα receptors

Glucocorticoids (GCs) are well-known anti-inflammatory compounds, but they also inhibit cell proliferation depending on cell type. Similarly, peroxisome proliferator-activated receptors (PPARα, PPARδ, and PPARγ) also possess anti-proliferation properties beyond their canonical roles as metabolic mediators. In the present study, we investigated the potential additive or synergistic inhibitory effects on cancer cell proliferation by simultaneous application of fenofibrate and budesonide, agonists for PPARα and glucocorticoid receptor, respectively. We observed differential effects on cell proliferation in A549 and SK-MES-1 lung cancer cells by budesonide and fenofibrate. Fenofibrate inhibited cell proliferation in both TP53 wild type and deficient lung cancer cells. The anti-proliferation effect of budesonide in TP53 wild type A549 cells was abolished in SK-MES-1 cells that do not have wild type TP53 protein. An additive effect against cell proliferation by budesonide and fenofibrate combination was observed only in TP53 wild type A549 cancer cells. Analysis of cell cycle distribution and cyclin profile indicated that the inhibition of cell proliferation was associated with G1 cell cycle arrest. The suppression of NF-κB activity and ERK signaling may contribute to the inhibition of cell proliferation by budesonide and or fenofibrate. The additive inhibitory effect on cell proliferation by budesonide and fenofibrate combination suggests that the same or greater therapeutic effect could be achieved with reduced dosage and side effects when the two compounds are applied simultaneously.

Abstract 1063: Examining early events in carcinogenesis by profiling premalignant lesions in the airways of patients with lung squamous cell carcinomas.

Abstract Little is known about the mechanisms involved in the initiation and progression of lung cancer, even though lung cancer contributes to the most cancer-related deaths in the world. Lung squamous cell carcinoma (SCC) is the second most common histological subtype, and often arises centrally from a large airway, usually a bronchus. It is believed that squamous lung cancer develops through a series of genetic and epigenetic changes that alter the normal epithelium from squamous metaplasia, to dysplasia, then carcinoma in situ, and finally to invasive SCC. Although there have been studies devoted to discovering the genetic and molecular changes observed in the tumor, few studies have contributed to the investigation of squamous metaplasia or dysplasia. To better understand carcinogenesis in the lung, especially those steps involved in the early and precancerous stages, a precise study of the biology of premalignant lesions is needed. To achieve this, we used laser capture microdissection to specifically isolate normal airway basal cells, premalignant cells, and tumor regions within individual patients with squamous lung cancer. The RNAs obtained were successfully sequenced using Next Generation Sequencing. Using two different statistical models of linear mixed effects and edgeR (empirical analysis of digital gene expression data in R), we identified a list of genes that were differentially expressed in the premalignant lesions and in tumors when compared to the normal basal cells. We were able to validate at RNA and protein levels several of the genes identified to have higher expression in premalignant lesions and tumors than in normal basal cells. We performed a gene network analysis and found that some of these genes are known to be involved in the development of other squamous tumors, cell cycle progression, cell death and survival, and cellular assembly, while some genes are uncharacterized or novel to cancer development. We also discovered that, even though TP53 RNA expression was not significantly different among normal, premalignant, and tumor cells, several proteins that were upregulated in the premalignant lesions were known to target TP53 protein for degradation, cytoplasmic localization, or disruption of its function. This suggests that the disruption of TP53 pathway began at the premalignant stage of carcinogenesis, and persisted through the formation of invasive carcinoma. This is the first comprehensive gene expression profiling of airway premalignant lesions and allows us to study the biology behind these lesions in the setting of carcinogenesis. Citation Format: Aik T. Ooi, Adam Gower, Kelvin Zhang, Jessica Vick, Longsheng Hong, Michael Fishbein, Brian Nagao, W Dean Wallace, David Elashoff, Marc Lenburg, Steven Dubinett, Avrum Spira, Brigitte Gomperts. Examining early events in carcinogenesis by profiling premalignant lesions in the airways of patients with lung squamous cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1063. doi:10.1158/1538-7445.AM2013-1063

Abstract 3063: MicroRNA-605: A novel genetic modifier in Li-Fraumeni Syndrome.

Abstract Background: Li-Fraumeni syndrome (LFS) is a rare autosomal-dominant disorder, typically involving germ-line mutations in the prototypical tumour suppressor gene, TP53. Despite the high penetrance of TP53 mutations, LFS patients display striking differences in the age of disease onset, suggesting the presence of additional genetic modifiers. To date, functional polymorphisms in TP53 (Arg72Pro), MDM2 (SNP 309), as well as DNA copy number variations (CNVs) and telomere attrition, have been associated with cancer risk in LFS. However, it is likely that additional modifiers exist and that a combination of these low-penetrance risk factors act together to predispose TP53 mutation carriers to a spectrum of susceptibilities. Recently, the TP53 signalling network has been linked to a small number of non-coding RNAs, called microRNAs (miRNAs), whose main function is to negatively regulate gene expression. One particular member of this family, called miR-605, has been found to target the MDM2 gene, an important inhibitor of p53 function. Importantly, a common Single Nucleotide Polymorphism (SNP) within miR-605 was also found to modulate gastrointestinal cancer risk. By affecting miR-605 expression, this SNP could impact cancer risk in TP53 mutation carriers. Hypothesis: In this study, we hypothesized that SNP-mediated miR-605 deregulation might modify cancer risk in LFS by eliciting important transcriptional changes in the levels of p53 and/or MDM2. Methods: miR-605 was overexpressed in two TP53 mutant cell lines (RD and Rh30) in order to assess its tumor suppressive functions. MDM2 and TP53 protein expression were analyzed through Western Blotting. Additionally, blood-derived DNA samples from 47 Caucasian TP53 mutation carriers were genotyped for the miR-605 variant by SNP-RFLP. Results: Overexpressing miR-605 in two TP53 mutated cell lines led to a significant attenuation in cell viability as measured by MTS assay. The capacity of these cell lines to form macroscopic colonies was also compromised following transfection by a miR-605 commercial mimic. Moreover, miR-605-transfected cells also displayed higher sensitivity to a wide range of chemotherapeutic drugs. In RD cells, MDM2 downregulation was detected following miR-605 overexpression but TP53 levels remained unchanged. Genotyping our aforementioned study population revealed the G-allele of the miR-605 SNP to be associated with an earlier age of cancer onset (p=0.02). The median age of cancer onset in carriers of the G-allele (n= 15) was 3.75 years compared to 21.5 years in patients with the common homozygous A/A genotype (n= 32). Conclusion: Together, our results identify miR-605 deregulation as a potential cancer modifying event in TP53 mutation carriers. Future work will focus on elucidating the functional consequences of the miR-605 SNP whilst also pursuing a more general understanding of miR-605 tumor suppression. Citation Format: Badr Idsaid, David Malkin. MicroRNA-605: A novel genetic modifier in Li-Fraumeni Syndrome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2013-3063