P53 Mutation Rate Research Articles

Abstract 556: Oncogenic properties and response to HDAC inhibitor treatment of H1299 cells expressing GOF p53 mutants

Abstract The tumor suppressor p53 protein is mutated in over 50% of cancers. The most frequent mutations in p53, termed “hotspot” mutations, occur in the DNA binding domain of the p53 protein. These mutations not only remove normal p53 function but also convey additional oncogenic properties greater than loss of tumor suppressor activities associated with loss of wild-type (WT) p53 activities. With newly acquired oncogenic properties, mutations in the DNA binding domain of p53 are classified as gain of function (GOF) mutations. GOF p53 mutants can be subdivided into conformational mutations (e.g. R175H, H193Y) involving residues necessary for maintenance of structure and contact mutations (e.g. R248Q, R273H) involving resides necessary for DNA interaction. Cancer cells bearing a GOF p53 mutation increase metastasis with application of histone deacetylase inhibitors (HDACi). This uncharacterized pro-oncogenic GOF p53 property is of clinical relevance as HDACi are FDA approved for hematological cancers but have failed in clinical trials for solid tumors. To explore this hypothesis, metastatic capacity and proliferation rates of common GOF p53 mutations were assessed with and without HDACi treatment and in the presence or absence of specific inhibitors of target GOF p53. The p53-null, H1299 non-small cell lung carcinoma line was transfected with GOF p53 mutants and WT p53 using a tetracycline inducible expression system. Inducible expression of stable subclones was varified by immunofluorescence and western blot. Proliferation rate was measured by incorporation of EdU and metastatic capacity was assessed by measuring MMP activity and migration assays with and without HDACi treatment. Specific GOF p53 mutations possess a subset of the oncogenic properties of the entire GOF p53 class. Data suggest that the GOF p53 mutants display differing rates of proliferation with the R175H mutation exhibiting the highest rate. Application of NSC319726 significantly decreases the proliferation rate and appears to successfully refold the R175H mutant to a WT conformation. The R248Q mutation exhibited morphological change suggestive of the epithelial to mesenchymal transition associated with increased metastatic capacity. Preliminary data suggest that HDACi affect among these cell lines in conjunction with NSC 319726 and SCH 529074 treatment. In conclusion, HDACi treatment may be contraindicated while treatment with HDACi may have great benefit in tumors which are WT or null for p53 protein expression. Citation Format: Brianna Flores, Elizabeth E. Hull. Oncogenic properties and response to HDAC inhibitor treatment of H1299 cells expressing GOF p53 mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 556.

P53 mutation status is a primary determinant of placenta-specific protein 1 expression in serous ovarian cancers.

Placenta-specific protein 1 (PLAC1) expression is co-opted in numerous human cancers. As a consequence of PLAC1 expression, tumor cells exhibit enhanced proliferation and invasiveness. This characteristic is associated with increased aggressiveness and worse patient outcomes. Recently, the presence of the tumor suppressor p53 was shown in vitro to inhibit PLAC1 transcription by compromising the P1, or distal/cancer, promoter. We sought to determine if this phenomenon occurs in primary patient tumors as well. Furthermore, we wanted to know if p53 mutation influenced PLAC1 expression as compared with wild-type. We chose to study serous ovarian tumors as they are well known to have a high rate of p53 mutation. We report herein that the phenomenon of PLAC1 transcription repression does occur in serous ovarian carcinomas but only when TP53 is wild-type. We find that mutant or absent p53 protein de-represses PLAC1 transcription. We further propose that the inability of mutant p53 to repress PLAC1 transcription is due to the fact that the altered TP53 protein is unable to occupy a putative p53 binding site in the PLAC1 P1 promoter thus allowing transcription to occur. Finally, we show that PLAC1 transcript number is significantly negatively correlated with patient survival in our samples. Thus, we suggest that characterizing tumors for TP53 mutation status, p53 protein status and PLAC1 transcription could be used to predict likely prognosis and inform treatment options in patients diagnosed with serous ovarian cancer.

Expression of CD10 in cancer-associated fibroblasts and its effect on initiation and progression of colorectal carcinoma

Objective: To study CD10 expression in cancer-associated fibroblasts (CAF) and related effect on colorectal cancer initiation and progression. Methods: A total of 226 surgical removed colorectal cancer specimens were collected with patient follow-up data. A panel of immunohistochemical markers(CD10, Ki-67, p53, cyclin D1 and β-catenin) were evaluated in carcinomas, adjacent adenomas and paired normal colorectal mucosa with correlation of clinicopathological parameters. Results: CD10 expression was not detected in the normal colorectal mucosa by immunohistochemistry. The percentages of CD10 expression in CAF were 80.1% (181/226) in carcinoma tissue and 58.4% (132/226) in adjacent adenomas, respectively. SMA was positive and desmin was negative. The proliferation index of Ki-67 was 84.1%(190/226) in tumor tissue, 63.7%(144/226)in adenoma zone, and 7.1% (16/226) in the normal mucosa. The rate of p53 mutation was 50.4% (114/226)in tumor tissue, 53.1%(120/226)in adenoma and 8.8%(20/226)in the normal mucosa. The expression rate of cyclin D1 was 83.6%(189/226) in tumor tissue, 48.7%(110/226)in adenoma zone, but negative in the normal mucosa. For normal tissue, the percentages of β-catenin expression was 53.1%(120/226), 95.6%(216/226) and 10.6%(24/226) in the cell membrane, cytoplasm and nucleus, respectively.The β-catenin expression in cell membrane, cytoplasm and nucleus was 17.7%(40/226), 100.0% (226/226) and 49.1% (111/226), respectively, in the tumor cells. The expression of Ki-67, p53, cyclin D1 and β-catenin in the tumor cells was positively associated with CD10 in CAF (P<0.05). The CD10 expression was different in patients with different gender, age and differentiation degree (P<0.05), whereas the depth of invasion, lymph node metastasis and tumor emboli did not relate to it. Overall survival rates in CD10 negative group were higher than that in CD10 positive group. Conclusions: CD10 is only expressed in CAF. Significant correlation is found between CAF with high CD10 expression and neighboring tumor cells with p53, Ki-67 and cyclin D1 expression and nuclear β-catenin expression. CD10-positive CAF and p53, β-catenin, cyclin D1 and Ki-67-labelled colorectal cancer cells together with nuclear β-catenin expression may provide helps for diagnosis of colorectal carcinoma from an angle of tumor interstitial microenvironment. CAF with CD10 expression may promote the initiation and progression of colon cancer cells by activating Wnt signaling pathway.

Clinicopathological and molecular characteristics of Ku 70/80 expression in Nigerian breast cancer and its potential therapeutic implications

Ku 70/80 is a regulator of the Non-Homologous End Joining (NHEJ) roles in clinicopathological features, and has prognostic significance in breast cancer (BC) in Caucasian populations. However, its significance in the Nigerian BC population, which is characterized by a higher rate of the triple-negative and basal phenotype, p53 mutation rate and BRCA1 deficiency, still needs to be investigated. We hypothesize that Ku70/80 expression shows adverse expression in Nigerian BC and, furthermore, that it is likely to have a therapeutic implication for Black BC management.This study investigated the biological, clinicopathological and prognostic significance of Ku 70/80 expression in a BC cohort from a Nigerian population. Ku 70/80 expression was determined in 188 well-characterized formalin-fixed, paraffin-embedded (FFPE) BC samples using tissue microarray and immunohistochemistry. Ku 70/80 expression was correlated with clinicopathological, molecular and prognostic characteristics of patients.Ku 70/80 was expressed in 113 (60.1%) tumors, and was positively associated with metastatic disease, triple-negative and basal phenotype, BRCA1 down regulators (MTA-1 and ID4), p-cadherin, PI3KCA and p53 expression. It inversely correlated with BRCA1, BRCA2, BARD1 and p27. Ku 70/80 was predictive of breast cancer-specific survival in multivariate analysis, but not of disease-free interval.This study demonstrated that Ku 70/80 expression is associated with triple negativity and down-regulation of the homologous recombination pathway of DNA repair. Therefore, the development of novel drugs to target KU70/80 may improve the patients’ outcome in the treatment of Black BC.

Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells

O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked β-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma.

Abstract 4178: Pigs as a new weapon against cancer: Modeling solid tumors in porcine

Abstract Widely-used genetically modified mice have revolutionized the field of cancer research by providing reliable in vivo systems with similar, if not identical, molecular alterations. Furthermore, Cre/Lox technology has afforded greater tissue specificity, with many of the developing cancer phenotypes recapitulating those observed in humans. However, despite these strengths, there are a number of notable limitations when comparing mice to humans including small sample sizes, dramatic differences in physiology, and often dissimilar drug responses. Pig models would alleviate these shortcomings if they were to have a repertoire of gene signatures observed in human cancer, like those engineered in mice. Therefore, to achieve this goal, we have engineered a pig model with the Cre-responsive transgene encoding KRASG12D and TP53R167H. Hence, tissue-specific targeted Cre would generate simultaneous expression of mutant KRAS and p53 alleles to induce cellular changes leading to cancer. Pig pancreases were injected with adenovirus containing a cre expression vector (adenoCre) in an attempt to generate a model of pancreatic cancer. Initial injections of adenoCre into the parenchymal body of the pancreas led to the development of acinar-ductal metaplasia (ADM), though the clear predominant histotype was leiomyosarcoma immediately adjacent to the pancreas. Pancreatic ADM was characterized by concomitant localization of amylase (acinar cell marker) and CK19 (ductal cell marker) in the same pancreatic acinus, with an occasional few cells expressing both cell markers. These lesions were further characterized and exhibited increased proliferation via PCNA staining, as well the development of surrounding desmoplastic stroma (enhanced trichrome, vimentin, and aSMA staining; a marker of pancreatic stellate cells) with a robust inflammatory component (strong CD11b and CAE staining). Based on the lack of pancreas specificity, the experiment was repeated by injecting adenoCre into the main pancreatic duct. This 10-month-old pig appears relatively healthy after, which was expected considering this was a fully adult pig prior to manipulation. We will fully access this pig in 2 months to determine if strict pancreatic duct injection of adenoCre induces ADM, neoplastic disease, and/or ductal adenocarcinoma. Based on these encouraging findings, we anticipate having a robust model of pancreatic cancer in the pig, and plan to extend this study to generate models of other solid tumors with high rates of KRAS and p53 mutations. These porcine models may significantly impact preclinical studies, as diseases induced in pigs are likely more physiologically relevant to those in humans, with a potentially similar pharmacokinetic profile following drug treatment. Indeed, due to the size of the organs and volume of blood, samples can be more readily shared among multiple groups of investigators facilitating greater collaboration and expanding future research. Citation Format: Andrew Diaz, Daniel Principe, Brian DeCant, Paul J. Grippo, Laurie Rund, Lawrence Schook. Pigs as a new weapon against cancer: Modeling solid tumors in porcine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4178.

Abstract 3761: The MDM2 inhibitor AMG 232 causes tumor regression and potentiates the anti-tumor activity of MEK inhibition and DNA-damaging cytotoxic agents in preclinical models of acute myeloid leukemia

Abstract AMG 232 is a potent inhibitor of the MDM2-p53 interaction and is a promising clinical candidate for treating tumors, in particular those harboring wild-type p53. AML represents a compelling indication for AMG 232 given the low rate of p53 mutation, frequent MDM2 overexpression, and unmet medical need. We evaluated the effect of AMG 232 treatment on AML tumors in vitro and in vivo, elucidated the mechanism of anti-tumor efficacy, and tested the effect of combining AMG 232 with targeted agents and chemotherapeutics. Combinations were identified based on evidence of in vitro synergy from cell based screens, or based on biological rationale and clinical opportunity with standard of care agents. Combinations evaluated included MEK inhibitors, and p53-inducing, DNA-damaging cytotoxics cytarabine, doxorubicin, and decitabine. In vitro assays demonstrated that AMG 232 as a single agent was effective at inducing cell death across a panel of p53 wild-type AML cell lines. The anti-tumor efficacy involved activation of the p53 pathway, robust inhibition of the cell cycle and induction of apoptosis. AMG 232 treatment caused AML tumor regression in vivo which was related to dose- and time-dependent induction of the p53 targets p21 and PUMA. The combination of AMG 232 and MEK inhibition resulted in synergistic tumor cell killing in vitro, and enhanced in vivo anti-tumor activity which was significantly better than either single agent. Combinations of AMG 232 with chemotherapies which induce DNA damage resulted in synergistic in vitro cell killing, and superior anti-tumor efficacy in vivo with increased induction of p53 signaling in tumors. These data support a clinical strategy for evaluating AMG 232 as a monotherapy and in combination with targeted and cytotoxic agents to treat AML patients. Citation Format: Jude R. Canon, Tao Osgood, Anne Y. Saiki, Jonathan D. Oliner. The MDM2 inhibitor AMG 232 causes tumor regression and potentiates the anti-tumor activity of MEK inhibition and DNA-damaging cytotoxic agents in preclinical models of acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3761.

Differences of malignant proliferation and infiltrating growth related molecular changes in different pathological types of glioma

Objective To investigate the distribution of the cell cycle alteration and cell migration as well as infiltrating growth process and related genetic changes in each grade and each molecular subtype of gliomas. Methods A total of 300 glioma specimens(WHO grade Ⅱ-Ⅳ)in Chinese Glioma Genome Atlas(CGGA)were collected. Its tissue RNA and DNA were extract respectively. Sanger sequencing was used to detect the mutation status of PTEN, p53, and TERT promoter. The expression levels of MET and matrix metalloproteinase 9(MMP-9)were derived from whole-genome RNA microarray expression profile. The distribution differences of the mutations of PTEN, p53, TERT promoter and high expression of MET and MMP-9 were compared in each level and each molecular subtypes of glioma. Results (1)In WHO grade Ⅱ, Ⅲ and Ⅳ, the mutation frequencies of PTEN were 2.8%(2/72), 5.0%(1/20), and 23.0%(20/87), respectively; the mutation rates of p53 were 28.0%(21/75), 10.0%(2/20), and 20.7%(18/87), respectively; the mutation rates of TERT promoter were 30.2%(26/86), 47.1%(8/17), and 46.8%(29/62), respectively; the incidences of MET high expression were 28.9%(35/121), 33.3%(17/51), and 37.5%(48/128), respectively; and the high expression rates of MMP-9 were 3.3%(4/121), 39.2%(20/51), and 59.4%(76/128), respectively. There were significant differences in each grade of gliomas between the mutation rate of PTEN and the high expression rate of MMP-9(all P<0.01).(2)The mutation rates of PTEN in the proneural type, neural type, classical type and molecular type of mesenchymal glioma were 1.9%(1/52), 12.5%(4/32), 15.0%(3/20), and 20.0%(15/75), respectively; the mutation rates of p53 were 34.0%(18/53), 6.1%(2/33), 40.0%(8/20), and 17.1%(13/76), respectively; the mutation rates of TERT promoter were 37.5%(21/56), 34.5%(10/29), 31.3%(5/16), and 45.1%(23/51), respectively; the MET high expression rates were 25.6%(21/82), 34.5%(19/55), 28.9%(11/38), and 40.0%(38/95), respectively; the MMP-9 high expression rates were 9.8%(8/82), 1.8%(1/55), 44.7%(17/38), and 68.4%(65/95), respectively. There were significant differences in the mutation rates of PTEN and p53, and the MMP-9 high expression rates among each molecular subtype of glioma(all P<0.05). Conclusions The mutation rate of PTEN and the high expression rates of MMP-9 were associated with the malignant degree of gliomas. The differences of the mutation rates of PTEN and p53, and the high expression rates of MMP-9 in each molecular subtype of gliomas suggested that they could represent the molecular pathologic features of the specific molecular subtypes of gliomas. Key words: Glioma; Pathology; Molecular subtype; Malignant proliferation; Infiltrating growth

Clinicopathological Characteristics of Mucinous Breast Cancer: A Retrospective Analysis of a 10-Year Study.

BackgroundMucinous breast carcinoma (MC) is a special type of breast cancer that presents with a large amount of extracellular mucin. MC comprises approximately 4% of all invasive breast cancers. This type of tumor has a better prognosis and higher incidence in peri- and post-menopausal patients. Pathologically, there are two main subtypes of MC: pure and mixed. In this study, we describe 10 years of experience with MC at the Zhejiang Cancer Hospital in China, specifically, clinical data, histological findings and immunohistochemical features.MethodsWe identified MC patients who were diagnosed as operable and completed clinical treatment from January 2001 to January 2011. The clinicopathological data included the age at diagnosis, tumor size, TNM stage, presence and number of lymph node (LN) metastases, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) status and p53 expression. If the tumor was defined as mixed mucinous carcinoma (MMC), IHC was performed on a non-mucinous part, such as invasive ductal and lobular cancer. We evaluated the clinical characteristics of all MC patients using chi-square, one-way ANOVA and LSD tests. We also studied the correlations between all of the clinical parameters and LN metastasis in a binary logistic regression analysis. We used ten consecutive years of data that were collected at Zhejiang Cancer Hospital.ResultsWe identified 48 cases of pure mucinous carcinoma (PMC) and 77 cases of MMC. The 48 PMC cases consisted of 38 PMC-A and 10 PMC-B subtypes. The MMCs were divided into two groups, those with partial mixed mucinous breast carcinoma (pMMC, 58 cases) and those with main mixed mucinous breast carcinoma (mMMC, 19 cases). pMMC was defined by tumors with less than 50% mucinous components, while mMMC was defined by tumors where the mucinous component accounted for 50% to 90% of the tumor. No significant differences in the clinicopathological characteristics were noted between the patients with PMC-A and those with PMC-B. The tumor size was larger in the mMMC than PMC cases (44.84 mm vs. 30.06 mm, p = 0.021). The number of positive LN metastases was greater in pMMC than PMC patients (p = 0.024). The clinical stages were significantly different among the three groups, with the pMMC group having more stage III-IV patients than the other two groups (p = 0.005). The incidence of LN metastasis was also higher in the pMMC cases (pMMC vs. mMMC and PMC, 50% vs. 31.58% and 18.75%, p = 0.003). The PMC patients had much lower p53 expression than the other two groups (PMC vs. pMMC and mMMC, 27.08% vs. 55.17% and 57.89%, p = 0.007). The tumor size (>30mm), p53 expression and less proportion of the mucinous component are associated with risk of LN metastasis.ConclusionBased on the results of this study, we conclude that the tumor size, status of LN metastasis, clinical stage, and p53 mutation rate may differ between MMC and PMC patients. The tumor size (>30mm), p53 mutation and less proportion of the mucinous component should be considered risk factors of LN metastasis in MC patients.

Circulating tumor DNA(ctDNA) testing to detect identical gene mutation rates of KRAS, NRAS, PIK3CA and P53 compared to tissue-based testing and reflects treatment efficacy in patients with colorectal cancer.

e23066Background: Molecular profiling for CRC commonly utilizes tissue, however, there are two major limitations. It is hard to overcome the complications of tumor heterogeneity and inconvenient to...

O‐GlcNAc Regulates p53 in Ovarian Cancer

O‐GlcNAcylation is a dynamic post‐translational modification (PTM) that consists of an addition of a single N‐acetylglucosamine sugar to serine and threonine residues in nuclear, cytoplasmic and mitochondrial proteins. The enzyme O‐linked β‐N‐acetylglucosamine transferase (OGT) adds the modification, while enzyme O‐GlcNAcase (OGA) removes the modification. O‐GlcNAc regulates enzymatic activity, localization, stability and protein‐protein interactions on a wide range of proteins such as transcription factors, structural proteins and kinases. In cancer, when comparing O‐GlcNAc levels in normal and tumor cells, tumor samples present higher O‐GlcNAcylation in vitro and in vivo. This aberrant O‐GlcNAc pattern contributes to changes in important signaling pathways and could contribute to the progression of the disease. Interestingly, the tumor suppressor p53 is modified by O‐GlcNAc. The transcription factor p53 regulates diverse cellular processes like cell survival and the cell cycle. Most solid tumors contain mutations in p53 leading to increased degradation or loss of p53 function, which promotes malignant tumor growth. Ovarian cancer has one of the highest frequencies of p53 mutation rates. The goal of this work is to understand the relationship between O‐GlcNAcylation and p53 function in ovarian cancer. First, O‐GlcNAcylation, OGT and OGA protein and mRNA levels were measured in a series of human ovarian cancer cell lines and no correlation was found between p53 genetic status and O‐GlcNAcylation. Overexpression or silencing of p53 in a p53 wild‐type cell line (A2780) had little effect on O‐GlcNAcylation. On the other hand, pharmacological inhibition of OGA with the inhibitor Thiamet‐G (TMG) leads to an increase in nuclear localization of p53 and increased mRNA levels of p53‐target genes (p21 and Bax). Overexpression of OGA or OGT leads to increased p53 protein but not mRNA levels and a concurrent increase in p53‐targets (Mdm‐2, p21 and Bax) expression. Also, p53 acetylation on lysine 382, a transcriptional activating modification, is higher in OGA/OGT overexpressing cells. In conclusion, O‐GlcNAcylation regulates p53 function through stabilization and activation.Support or Funding InformationSupport and funding: Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Science Without Borders Program, NIH NIDDK R01 DK 100595.

Relationships between p53 mutation, HPV status and outcome in oropharyngeal squamous cell carcinoma

Background and purposeThis study aimed to examine the rate and type of p53 mutation in oropharyngeal cancer (OSCC). Relationships were sought between human papillomavirus (HPV) status and p53 mutation. The role of p53 mutation as a prognostic factor independent of HPV status and as a modifier of the effect of HPV on outcomes was also examined. MethodsThe HPV status of 202 cases was determined by HPV DNA by RT-PCR and p16 immunohistochemistry. P53 mutation in exon 5–8 was determined by pyrosequencing. Findings were correlated with known clinicopathological factors and outcomes. Results48% of the cases were HPV positive and they were significantly less likely to have a p53 mutation than HPV-negative OSCCs (25.8% vs 46.7%, p=0.0021). Mutation was most common in exon 5. Among patients with HPV-positive OSCC, there was no significant difference in p53 mutation by smoking status (22.2% for never smokers and 30.8% for current or ex-smokers). Patients with p53 mutant OSCC had significantly worse overall survival (p=0.01). There was no statistical evidence that p53 mutation modified the effect of HPV status on outcomes. In the multivariate analysis, positive HPV status remained the strongest predictor of outcomes. p53 mutation status was not a significant predictor of outcome after adjusting for age, gender, T stage, N stage and HPV status. ConclusionsIn summary, HPV-positive OSCC are less likely to have mutant p53 than HPV-negative OSCC. Our study did not show any evidence that p53 mutation could modify the effect of HPV status on outcomes.

The presence of the intron 3 16 bp duplication polymorphism of p53 (rs17878362) in breast cancer is associated with a low Δ40p53:p53 ratio and better outcome.

Breast cancer is the most common female cancer, but it has relatively low rates of p53 mutations, suggesting other mechanisms are responsible for p53 inactivation. We have shown that the p53 isoform, Δ40p53, is highly expressed in breast cancer, where it may contribute to p53 inactivation. Δ40p53 can be produced by alternative splicing of p53 in intron 2 and this is regulated by the formation of G-quadruplex structures in p53 intron 3, from which the nucleotides forming these structures overlap with a common polymorphism, rs17878362. rs17878362 alters p53 splicing to decrease fully spliced p53 messenger RNA (mRNA) in vitro following ionizing radiation and this in turn alters Δ40p53:p53. Hence, the presence of rs17878362 may be important in regulating Δ40p53:p53 in breast cancer. This study aimed to determine if rs17878362 was associated with altered Δ40p53 and p53 expression and outcome in breast cancer. We sequenced p53 in breast tumours from 139 patients and compared this with Δ40p53 and p53 mRNA expression. We found that the ratio of Δ40p53:p53 was significantly lower in tumours homozygous for the polymorphic A2 allele compared with those who were wild-type (A1/A1). Furthermore, there was a lower proportion of breast cancers carrying the A2 allele from patients who subsequently developed metastasis compared with those that did not. Finally, we show that patients whose tumours carried the polymorphic A2 allele had significantly better disease-free survival. These results show that rs17878362 is associated with a low Δ40p53:p53 ratio in breast cancer and that this is associated with better outcome.

Interrelationships Between Ki67, HER2/neu, p53, ER, and PR Status and Their Associations With Tumor Grade and Lymph Node Involvement in Breast Carcinoma Subtypes: Retrospective-Observational Analytical Study.

Various predictive and prognostic factors could affect breast carcinoma behavior, but to date no definitive correlation has been established between them and breast carcinoma subtypes. The present study was conducted to examine the interrelationships of these predictive and prognostic factors as well as their effects on breast carcinoma subtypes.The archives of all patients with breast carcinoma (from 2008 to 2014) were studied. Patients’ data were extracted using a checklist that included age, histology type, size and grade of tumor, lymph node involvement, estrogen receptor (ER) and progesterone receptor (PR) status, along with the overexpression of human epidermal growth factor receptor (HER2/neu) and the rate of Ki67 and p53 mutations. All data were analyzed by SPSS-17 software with χ2 and Fisher exact tests, as well as the least significant difference pairwise comparison test.A total of 566 patients’ records were included in this study. The mean age of patients was 50 ± 12.9 with an age range of 17 to 98 years. A meaningful correlation was found between age and the type of tumor (P = 0.001). Infiltrating lobular carcinoma had a higher ER positivity between groups (85.7%), whereas noninvasive carcinomas had a higher PR positivity (67%). In addition, a meaningful correlation was detected between the type and grade of tumor (P = 0.001). No meaningful relationship was observed between the type of tumor and HER2/neu overexpression and number of lymph nodes involved. Between the groups, medullary carcinoma had the highest Ki67 index (P = 0.007). Meaningful correlation was found between the grade of tumor and lymph node involvement (P = 0.005) and also with HER2/neu overexpression (P = 0.002). Higher grades had greater positivity in Ki67 index and p53 mutation rates (P = 0.002, P = 0.01). HER2/neu positive tumors had a higher Ki67 index (P = 0.03).Higher Ki67 index tumors showed more HER2/neu overexpression, larger size, and more lymph node involvement compared with other types and maybe considered aggressive. Moreover, in young patients with breast carcinoma, the rates of Ki67 with the overexpression of HER2/neu and p53 mutations are higher, and it shows a more aggressive behavior than other tumors assessed in this age group.

Determination of HER2 and p53 Mutations by Sequence Analysis Method and EGFR/Chromosome 7 Gene Status by Fluorescence in Situ Hybridization for the Predilection of Targeted Therapy Modalities in Immunohistochemically Triple Negative Breast Carcinomas in Turkish Population

Triple negative breast cancer (TNBC), an agressive subtype accounts nearly 15 % of all breast carcinomas. Conventional chemotherapy is the only treatment modality thus new, effective targeted therapy methods have been investigated. Epidermal growth factor receptor (EGFR) inhibitors give hope according to the recent studies results. Also therapeutic agents have been tried against aberrant p53 signal activity as TNBC show high p53 mutation rates. Our aim was to detect the incidence of mutations/amplifications identified in TNBC in our population. Here we used sequence analysis to detect HER2 (exon 18-23), p53 (exon 5-8) mutations; fluorescence in situ hybridization (FISH) method to analyse EGFR/chromosome 7 centromere gene status in 82 immunohistochemically TNBC. Basaloid phenotype was identified in 49 (59.8 %) patients. EGFR amplification was noted in 5 cases (6.1 %). All EGFR amplified cases showed EGFR overexpression by immunohistochemistry (IHC). p53 mutations were identified in 33 (40.2 %) cases. Almost 60 % of the basal like breast cancer cases showed p53 mutation. Only one case showed HER2 mutation (exon 20:g.36830_3). Our results showed that gene amplification is not the unique mechanism in EGFR overexpression. IHC might be used in the decision of anti-EGFR therapy in routine practice. p53 mutation rate was lower than the rates reported in the literature probably due to ethnic differences and low sensitivity of sanger sequences in general mutation screening. We also established the rarity of HER2 mutation in TNBC. In conclusion EGFR and p53 are the major targets in TNBC also for our population.

Effect of FHIT loss and p53 mutation on HPV-infected lung carcinoma development.

High-risk human papillomavirus (HPV)16/18 infection in the development of lung cancer has previously been identified, and fragile histidine triad (FHIT) loss and p53 mutation are frequently observed in the disease. However, the association between these factors has not been well studied. The present study aimed to further investigate the significance of HPV infection, FHIT loss and p53 mutations in the development of lung cancer and their possible associations. DNA was extracted from paraffin-embedded specimens from 88 cases of squamous cell carcinoma (SCC), 56 of adenocarcinoma (AC), 36 of small cell lung carcinoma (SCLC) and 110 non-cancer control cases of lung neoplasms. The prevalence of HPV infection was determined by polymerase chain reaction analysis, and FHIT loss and p53 mutations were detected by immunohistochemistry. The χ2, Fisher's exact and Pearson correlation tests were applied for statistical analysis. The results of the present study demonstrated that HPVL1 (the major capsid protein of HPV), HPV16 and HPV18 infection were more prevalent in the lung cancer samples compared with the non-cancer controls (all P<0.001). FHIT loss occurred more frequently in the lung cancer samples (44.44%) compared with the non-cancer controls (7.25%) (P<0.001). FHIT loss in the HPVL1-positive group was significantly increased compared with the HPVL1-negative group in the lung cancer cases and the non-cancer controls (P<0.05). In the lung cancer cases, the p53 mutation rates in the HPVL1- and HPV16/18-positive groups were significantly increased compared with the HPVL1- and HPV16/18-negative groups (P<0.05). In the 180 lung cancer cases, the coexistence rate of FHIT loss and a history of smoking was 38.33% (69/180; Pearson contingency coefficient of r=0.318; P<0.001). FHIT loss and p53 mutation exhibited a synergistic effect on HPV-associated lung cancer (Pearson contingency coefficient r=0.357, P<0.001). The present study demonstrated that FHIT loss may be important in the occurrence of lung cancer, particularly in lung SCCs. FHIT loss may therefore be used as an early indicator for lung cancer, particularly for patients with a history of smoking. HPV infection in lung tumorigenesis may, at least in part, be mediated through FHIT loss. FHIT loss and p53 mutation may coordinate together in the development of HPV-associated lung cancer, and accelerate the occurrence and development of lung cancer.

Abstract P3-06-17: Multiplatform molecular profiling of BC reveals significant differences in actionable targets from matched female breast carcinomas

Abstract Introduction Male breast cancer (MBC) is rare, comprising ∼1% of all breast cancers. While clinically characterized as being similar to postmenopausal ER+ BC, MBC has been much less characterized molecularly than female BC. Methods 60 male (ages 37-84) and 5000 female (ages 27-98) breast cancer samples were evaluated for common gene mutations (Sanger or Illumina TruSeq), protein expression (immunohistochemistry), microarray, and/or amplification/rearrangement (CISH or FISH). The samples were analyzed for patterns within the MBC cohort and similarities/differences compared to the female (FBC) subtypes (TNBC, non-TNBC, HER2+, and ER+) evaluated at Caris Life Sciences. Results Within the MBC cohort, approximately 23% were negative for ER, PR, and HER2 (TNBC); of those 18% were also negative for AR; 72% were ER+; 51% were both ER+ and PR+. The incidence of high ER and PR protein expression was greater (72% vs. 56%, 54% vs. 40%) but incidence of HER2 overexpression (IHC, 3+) and amplification (FISH, HER2/CEP17 ratio higher than 2) was lower (8.8% vs. 11%, 5% vs. 14.9%) when compared to FBC overall. The rate of EGFR amplification (measured as ≥ 4 copies in 40% or more tumor cells by FISH) was not different from FBC (15%), while the percentage of MBC pts with AR protein expression (70%) was most similar to ER, PR positive FBC patients. Other biomarkers: the rate of ERCC1 overexpression was lower in MBC when compared to FBC (36% vs. 49), the rate of PTEN loss was lower (36% vs. 61%), and the rates of MGMT, TLE3, and RRM1 overexpression were higher (73% vs. 64%, 70% vs. 53%, and 47% vs. 30%, respectively). In the 14 MBC cases evaluated by NGS, no PTEN gene mutations were identified, although PIK3CA gene mutations were seen at a similar rate (41%) as in the &amp;gt;50yo ER+ FBC (37%), and TP53 gene mutations (21%) were seen slightly less frequently than in the &amp;gt;50yo ER+ FBC (27%). Comparison of the TN MBC to the ER+ MBC cohort identified differences in the mTOR pathway (PTEN loss of 13% vs. 28% and PIK3CA mutation rate of 13% vs. 50%, respectively), in P53 mutation rates (33% vs. 0%), and in AR protein expression (33% vs. 82% overexpression), TLE3 (14% vs. 83% overexpression), and ERCC1 (100% vs. 77% low). Conclusions The gene mutation, amplification, and protein expression profiles in MBC patients, including HER2 protein expression/amplification, AR and TLE3 protein expression and PIK3CA gene mutation, may inform standard and investigational therapeutic options for this rare cancer population. Citation Format: Sherri Z Millis, Joanne Xiu, Zoran Gatalica, Joyce O'Shaughnessy. Multiplatform molecular profiling of BC reveals significant differences in actionable targets from matched female breast carcinomas [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-17.

Predictive Biomarker Profiling of > 6000 Breast Cancer Patients Shows Heterogeneity in TNBC, With Treatment Implications

BackgroundTriple-negative breast cancer (TNBC) is an aggressive disease without established targeted treatment options for patients with metastatic disease. This study was undertaken to evaluate potentially actionable biomarkers in a large cohort of TNBC and compare them with non-TNBCs. Materials and MethodsWe evaluated 6341 (2111 TNBC and 4230 non-TNBC) breast cancer samples at a central laboratory for biomarkers of potential drug response across multiple platforms, including gene sequencing, protein expression, and gene copy number. ResultsTNBC expresses androgen receptor (AR) in a significantly (P < .05) lower percentage of cases (17%) than hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-positive breast carcinomas (59% and 79%, respectively), and gene comutations were differentially associated with AR-positive versus AR-negative cases. Higher AR expression levels in TNBC predicted for lower Ki-67 levels. Seventy percent of TNBC harbored a phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), v-akt murine thymoma viral oncogene homolog 1 (AKT1), or phosophatase and tensin homolog (PTEN) aberration. TNBC patients had a significantly lower PIK3CA mutation rate (13%) than all other subtypes (P < .05) and a higher tumor protein p53 (TP53) mutation rate (64%) than the estrogen receptor (ER)-positive cases (approximately 30%; P < .05). Topoisomerase 2 (TOP2A) amplification was observed in 1.3% of TNBC and in 1.6% of HER2-negative, HR-positive cancers; in contrast, HER2-positive, HR-negative or HR-positive cancers exhibited TOP2A amplification in 19% and 40% of cases, respectively (P <.05). ConclusionMulti-platform molecular profiling identifies subgroups of TNBC with different biomarker profiles, suggesting numerous potentially targetable alterations in TNBC. TNBC is further characterized by different gene mutations and proliferative activity relative to AR expression, highlighting a need for comprehensive pathologic examination with potential to develop different, individualized treatment options.

A novel cell-permeable RDP-p53 fusion protein for specific inhibition on the growth of cancerous neural cells

Objective: There is 25–35% mutation rate of p53 in cancerous neural cells and this rate reaches 70–76% in glioma cell line. Complement of wild-type p53 has become a potential strategy for protein therapy of cancerous neural cells. Here we investigated the feasibility of a novel RDP-p53 fusion protein for anti-proliferation of cancerous neural cell and the possible mechanism, which would provide an effective approach for targeted delivery of p53 protein to treat cancerous neural cells.Methods: The RDP-p53 fusion proteins are expressed in Escherichia coli, and they are labeled with FITC and rhodamine B by chemical modification. The fluorescence-labeled proteins are added to human hepatocellular carcinoma cells (HepG-2) and human neuroblastoma cells (SH-SY5Y) in order to investigate the possibility of RDP enhancing the cell uptake efficiency into neural cells as a cell-permeable carrier. The inhibitory effect of RDP-p53 on SH-SY5Y and human glioma cells (U251) was evaluated by MTT assay. Moreover, the anti-proliferation mechanism of RDP-p53 was determined by Apoptosis and Necrosis Assay Kit and flow cytometric analysis.Results: The results showed that RDP-p53 could enter SH-SY5Y cells with high efficiency and selectively inhibit the growth of cancerous neural cells, including SH-SY5Y and U251. Also, cell apoptosis pathway and cell-cycle arrest at the G2/M phase were associated with the inhibition mechanism of RDP-p53 according to the data of flow cytometric analysis.Conclusions: RDP-p53 could be a novel antitumor candidate for targeting treatment of cancerous neural cells.

Gene Mutation Analysis in Non-Small Cell Lung Cancer Patients using Bronchoalveolar Lavage Fluid and Tumor Tissue as Diagnostic Markers

Non-small cell lung cancer (NSCLC) is one of the main causes of cancer death in the world. Early detection of NSCLC can improve its outcome. The aim of this study was to identify the mutations of the KRAS and p53 genes in bronchoalveoar lavage (BAL) fluid for the early detection of peripheral NSCLC. We examined the DNA obtained from the tumor, nearby normal lung tissue, and matched BAL fluid for mutations in the KRAS and p53 genes; the material was obtained from 48 patients with peripheral NSCLC, and was analyzed by PCR-single strand conformation polymorphism and DNA sequencing. BAL fluids from 26 patients with benign lung disease were used as controls. Positive rates of KRAS and p53 mutations were distributed as follows: in NSCLC tissue, 52% and 58%; in BAL fluid of NSCLC patients, 38% and 44%; in normal lung tissue, 6% and 4%; and in BAL fluid of patients with benign lung disease, 8% and 4%. The combined detection of both KRAS and p53 mutations yielded a sensitivity of 66% for the diagnosis of peripheral NSCLC, which is markedly higher than that of cytology plus histology by first bronchoscopy (38%, p=0.008). In each patient with the 2 gene mutations in BAL fluid, mutation type and location were the same as those of the primary tumor. Our study indicates that the detection of the KRAS and p53 mutations in BAL fluids could be a helpful addition to cytology and histology examination for the diagnosis of peripheral NSCLC.