TP53 Gene Mutation Status Research Articles

Abstract 4644: A cell-line based TP53 expression pattern control panel with interobserver variability and next-generation sequencing analysis

Abstract Introduction: TP53 gene mutation status is known to be manifested through distinct p53 immunohistochemical staining patterns; diffuse, wild-type and null. This stratified staining pattern is routinely utilized in subtyping ovarian and endometrial cancer subtypes and distinguishing low- and high-grade serous carcinoma. Experimental Procedure: Three ovarian cancer cell lines were used in the construction of an immunohistochemical control panel slide that highlights each respective TP53 expression pattern. Cell lines were expanded, harvested and resuspended in agarose gel and subsequently sectioned onto a microscope slide. Procured tissue specimens were submitted along with either standard control tissue or the TP53 cell line control panel to pathologists of varying experience. Next-generation sequencing analysis was performed on cases in which there was discordance in expression pattern interpretation. Results: Interobserver variability was assessed on 44 samples between five raters of varying experience. The results were similar with the use of the TP53 expression panel (Fleiss kappa= 0.6 vs 0.53). Next-generation sequencing analysis demonstrated low-frequency variant mutations in cases in which there was reviewer discordance. Conclusion: This study suggests the addition of a TP53 cell line expression pattern control panel could potentially increase diagnostic accuracy for ovarian and endometrial cancer subtypes and distinguishing ovarian low-grade and high-grade serous carcinoma. The cell line control has refined quality assurance potential for antibody optimization.This cell line control panel has the potential to be utilized in routine immunohistochemical staining with difficult to interpret p53 expression patterns directed to submission for next-generation sequencing. Citation Format: Yaling Zhou, Sorana Raiciulescu, Viqar Syed, Patrick Malafronte, Justin Wells, Mica Grantham, Michael Allgaeuer, Heping Gong, Joel Moncur, Deyin Xing, John D. Andersen. A cell-line based TP53 expression pattern control panel with interobserver variability and next-generation sequencing analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4644. doi:10.1158/1538-7445.AM2017-4644

Abstract DPOC-002: A NOVEL CELL LINE BASED IMMUNOHISTOCHEMICAL TP53 EXPRESSION PATTERN CONTROL PANEL

Abstract BACKGROUND: TP53 gene mutation status is known to be manifested through distinct p53 immunohistochemical staining patterns; diffuse, “wild-type” and null. This stratified staining pattern is commonly utilized in subtyping ovarian and endometrial cancer subtypes as well as distinguishing between ovarian low- and high-grade serous carcinoma. EXPERIMENTAL PROCEDURES: Three ovarian cancer cell lines consistently demonstrating either diffuse, wild-type or null p53 staining were utilized in the construction of an immunohistochemical control panel slide that highlights each TP53 expression pattern. Cell lines were expanded, harvested and resuspended in agarose gel and subsequently sectioned onto a microscope slide (Figure 1A). Procured tissue specimens were submitted along with either standard control tissue or the TP53 cell line control panel to three pathologists of varying experience. RESULTS: Interobserver variability was demonstrated to be similar with the use of the TP53 expression panel (Fleiss kappa= 0.79 vs 0.76) and there was a net decrease in categorization errors. The TP53 cell line expression pattern control panel sections demonstrated consistent clean and easily interpretable diffuse, wild-type and null p53 staining (Figure 1B). CONCLUSIONS: We developed a novel cell line-based TP53 expression pattern control panel with refined quality assurance potential that would act, as a minimum, a model for antibody optimization. This study suggests the addition of a cell line TP53 control panel could potentially increase the accuracy of identification for ovarian and endometrial cancer subtypes as well as ovarian low grade versus high-grade serous carcinoma distinction. This control panel has the potential to be utilized in other disease states where TP53 expression pattern assists in diagnosis. Citation Format: Viqar Syed PhD, Imran Hussain, BSc, Jeptha Johnson MD, Patrick Malafronte MD, Justin Wells MD, Yaling Zhou PhD, Deyin Xing MD PhD, John D. Andersen DO. A NOVEL CELL LINE BASED IMMUNOHISTOCHEMICAL TP53 EXPRESSION PATTERN CONTROL PANEL [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr DPOC-002.

Inhibition of ATR potentiates the cytotoxic effect of gemcitabine on pancreatic cancer cells through enhancement of DNA damage and abrogation of ribonucleotide reductase induction by gemcitabine.

Pancreatic cancer is a highly malignant disease with a dismal prognosis. Gemcitabine (GEM)-based chemotherapy is the first-line treatment for patients with advanced disease, although its efficacy is very limited, mainly due to drug resistance. Ataxia telangiectasia and Rad3-related (ATR) plays a critical role in the DNA damage response (DDR) which has been implicated in GEM resistance. Thus, targeting ATR represents a promising approach to enhance GEM antitumor activity. In the present study, we tested the antitumor activity of AZ20, a novel ATR-selective inhibitor, alone or combined with GEM in 5pancreatic cancer cell lines. AZ20 treatment of the pancreatic cancer cell lines resulted in growth inhibition, with IC50 values ranging from 0.84 to 2.4µM, but limited cell death. As expected, treatment of pancreatic cancer cell lines with AZ20 caused decreased phosphorylation of CHK1 (S-345). However, this was accompanied by DNA damage and SandG2/M cell cycle arrest, independent of TP53 gene mutational status. Importantly, combination of AZ20 with GEM resulted in synergistic inhibition of cell growth and cooperative induction of cell death in the pancreatic cancer cell lines. AZ20 significantly increased GEM-induced DNA damage and almost completely abrogated GEM-induced expression of the M2 subunit of ribonucleotide reductase. These findings suggest that inhibition of ATR is a promising strategy to enhance the antitumor activity of GEM for treating pancreatic cancer.

PIN1 in hepatocellular carcinoma is associated with TP53 gene status.

Phosphorylation of proteins on serine/threonine residues that precede proline (pSer/Thr-Pro) is specifically catalyzed by the peptidyl-prolyl cis-trans isomerase PIN1. PIN1-mediated prolyl-isomerization induces cell cycle arrest and growth inhibition through the regulation of target proteins, including TP53. We examined whether PIN1 acts in a different manner according to TP53 gene status in hepatocellular carcinoma (HCC). We investigated the expression of PIN1 and TP53 proteins in 119HCC tissue samples. We also analyzed PIN1 expression in combination with TP53 gene mutation and its correlation with the clinical outcome. In addition, we used synthetic small interfering RNA to silence PIN1 gene expression in TP53 wild-type and TP53 mutant HCC cell lines, and then evaluated cell proliferation, migration and invasion. Expression of PIN1 was strongly associated with expression of TP53 protein or TP53 mutation of HCC samples. PIN1 and TP53 expression in TP53 mutant HCC cell lines was higher than that in TP53 wild-type HCC cell lines. Silencing of PIN1 in HLE cells containing mutant TP53 significantly decreased cell proliferation, migration and invasion. In contrast to PIN1 silencing in HLE cells, PIN1 silencing in HepG2 cells containing functional wild-type TP53 resulted in enhanced tumor cell proliferation. HCC patients bearing PIN1 expression with wild-type TP53 were predicted to demonstrate favorable relapse-free survival. Our results suggest that PIN1 plays a role in cancer cell proliferation, migration and invasion in a different manner according to the TP53 gene mutation status in HCC. In particular, interaction of PIN1 with mutant TP53 can act as a tumor promoter and increase its oncogenic activities in HCC.

Anaplastic histology Wilms' tumors registered to the Japan Wilms' Tumor Study Group are less aggressive than that in the National Wilms' Tumor Study 5.

To evaluate the clinical features and treatment results of anaplastic histology (AH) Wilms' tumor (WT) patients registered in the Japan Wilms' Tumor Study (JWiTS) group to elucidate the clinical characteristics of AH in the Japanese population. Of 344 WT patients who were enrolled in JWiTS between 1995 and 2013, 17 had AH. Treatment using the JWiTS protocols was similar to the fifth National Wilms' Tumor Study 5 (NWTS-5) protocols. Clinical characteristics and mutation status of TP53 gene were evaluated and compared with those in NWST-5 study. AH incidences in JWiTS were 4.9%, lower than that in NWTS-5. Seven tumors had focal AH and 10 had diffuse AH. Clinical stages of AH patients were stage I in seven, stage II in three, stage III in five, stage IV in one and unknown in one. Four-year event-free survival and overall survival rates were 90.9 and 86.7%, respectively. Two patients with diffuse AH and none with focal AH had TP53 mutation. Japanese patients presented with higher incidence, earlier stages and may have better outcomes than American patients, indicating a possible biological heterogeneity of AH WT. Further analysis is necessary to elucidate the different characteristic of AH WT between Japanese and American populations.

PD-L1 expression correlation with TP53 gene mutation status in lung cancer but not in colorectal cancer.

11557Background: Expression of PD-L1 is, in general, associated with response to immunotherapy. However, it is believed that additional intrinsic factors play a role in determining the potential of...

An Amplicon-Targeted Ultra-Deep Sequencing Approach Reveals the Presence at the Onset of Multiple Myeloma and the Selection over Time of TP53 Sub-Clonal Variants, Which Adversely Influence Patients' Overall Survival

INTRODUCTION In newly diagnosed Multiple Myeloma (MM) patients (pts), Copy Number (CN) losses of chromosome 17p13, carrying the TP53 tumor-suppressor gene, are strong predictors of poor outcomes. On the contrary, the prognostic relevance of TP53 mutations at the onset of the disease is less clear, due to the very limited frequency of clonal lesions, as revealed by Sanger sequencing. To address this poorly investigated issue, we used an ultra-deep sequencing (UDS) approach to characterize the TP53 structural architecture in both newly diagnosed and relapsed MM pts and to assess the prognostic role and evolution over time of small TP53 mutated sub-clones.SAMPLES AND METHODS A cohort of 99 newly diagnosed MM pts treated up-front with bortezomib-based regimens and autologous stem cell transplantation, was included in this molecular study. In 29 cases, samples were collected both at diagnosis and at relapse(s). DNA was obtained from CD138+ highly purified plasma cells. TP53 gene mutational status was analysed by using an amplicon-targeted UDS approach (GSJ, 454 Roche Life Sciences). In order to discriminate between low frequency sub-clonal TP53 variants and sequencing errors, sequencing raw data were filtered according to cut-off values based on different ranges of sequences' coverage depth. Additional filters were also applied, based on both quality and biological cut-offs, to obtain a final confident list of variants. Analysis of CN alterations (CNAs) was performed by SNPs array and results analysed with ChAS software.RESULTS With a median coverage of 1386X, a list of 129 correctly called TP53 variants (either missense, or nonsense or splice ones), including 20 INDELs, was detected. Only deleterious and N/A variants (according to SIFT classification) were included in the list. Most newly diagnosed MM pts (55%) carried at least one TP53 sub-clonal variant (on average 1.08 variants per pts), with 45/99 (45%) carrying non-mutated TP53. Pts carrying TP53 sub-clonal variants bared also TP53 CN hemizygous losses (20%), CKS1B gains (56%) and cdkn2c losses (14%). According to TP53 sub-clonal mutational load, pts were stratified in two sub-groups, including 28 pts with ≥2 (high load) and 71 with <2 variants (low load), respectively. Eleven out of 129 variants were recurrent (RVs), as being detected in at least 3% of pts, with Variants Allele Frequencies (VAFs) ranging from 0.24 to 70.1% (median 0,53%); RVs were observed in 29 pts.The clinical impact of the TP53 sub-clonal mutational load, as well as of variants recurrence, was evaluated in 90/99 MM (median follow up = 70 months). Results of statistical analysis are summarized in Table 1. Pts carrying either high TP53 sub-clonal mutational load or RVs had significantly shorter OS and OS after relapse, as compared to the others, while no difference between these two groups was seen regarding PFS and TTP. Multivariate analysis showed that high TP53 mutational load, as well as the presence of TP53 RVs, both resulted independent factors adversely affecting OS and OS after relapse (Table 2). Of note, none of the detected genomic aberrations significantly influenced the response to front-line induction therapy.The distribution of both TP53 sub-clonal variants and genomic CNAs was overall modified in samples collected at relapse(s): 90% of relapsed pts carried at least one sub-clonal variant (on average 1.63 variants per pts) with 3/29 (10%) relapsed MM carrying non-mutated TP53. Moreover, 5 different sub-clonal lesions proved a linear increment of both TP53 VAFs (from 29.4% to 54.6%; from 7.8% to 12.4%; from 0.5% to 4.3%) and TP53 CN loss smooth signal (from 7% to 89% and from 50% to 100%), as evaluated in longitudinally collected samples.CONCLUSIONS The UDS analysis of TP53 coding sequence in newly diagnosed MM highlighted for the first time a high rate of variants, recurring with a wide range of frequencies among samples. The increased number of TP53 sub-clonal variants per pts in samples collected at relapse(s), compared to that seen at the onset of the disease, suggests a sub-clonal dynamics over time. This finding might explain the adverse impact of high TP53 sub-clonal mutational load and TP53 RVs on OS, due to a shorter OS after relapse.Acknowledgments: Roche Diagnostics for applicationsupport in the realization of this project. [Display omitted] [Display omitted] DisclosuresZamagni:Celgene Corporation: Honoraria, Speakers Bureau; Janssen Pharmaceuticals: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Ariad: Consultancy; AMGEN: Consultancy; ROCHE: Consultancy; Pfizer: Consultancy; MSD: Consultancy. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

Abstract POSTER-TECH-1101: Characterization of genomic landscapes of BRCA1 and BRCA2 implicated ovarian cancer specimens from a founder french canadian population

Abstract Although molecular genetic profiling of ovarian cancers harboring germline BRCA1/BRCA2 mutations suggest that pathways in common with sporadic cases are involved in the pathogenesis of the disease, overall survival differs between BRCA1, BRCA2 and sporadic disease. To further dissect molecular pathways involved, which could account for differences in pathogenesis of the disease, we have investigated genomic landscapes in the BRCA1 and BRCA2 mutated ovarian cancers. Chromosomal anomalies were assessed in 28 specimens with BRCA1 (n=15), BRCA2 (n=12) or BRCA1 and BRCA2 (n=1) mutations using high-density Illumina SNP arrays. The majority (22/28) are serous adenocarcinomas while the remaining samples were either endometroid (2/28) or mixed adenocarcinomas (4/28). The cases harbor BRCA1/BRCA2 mutations from a French Canadian population, which exhibit strong founder effects. Allelic imbalance, copy number differences, intrachromosomal breaks and homozygous deletions were inferred visually using the Genome Viewer module of the BeadStudio software followed by ASCAT analysis. A statistical analysis was used to directly compare genotypes of BRCA1 versus BRCA2 positive samples. TP53 gene mutation status was also assessed. All samples were found to harbor a somatic TP53 mutation comprised of missense (19/28), nonsense (2/28), frame-shift (4/28) and (3/28) splice mutations. The results were compared to independently derived data generated previously from our group, which was largely comprised of sporadic cases (Wojnarowicz et al 2012), and to the genomic data from the Cancer Genome Atlas project (TCGA). The genomic landscapes of BRCA1 and BRCA2 mutated cancers overlap those reported in previous studies. However, there were significant differences in the genomic landscapes involving chromosome 6q between BRCA1 and BRCA2 mutation-positive cancers. This genomic distinction between BRCA1 and BRCA2 ovarian cancer samples may point to a region containing genes important in the etiology or progression of hereditary cancer. Citation Format: Eman AlShehri, Moria Belanger, Suzanna Arcand, Kathleen Klein Oros, Celia Greenwood, Patricia N. Tonin. Characterization of genomic landscapes of BRCA1 and BRCA2 implicated ovarian cancer specimens from a founder french canadian population [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1101.

Abstract 4248: A long tail of sub-clonal TP53 mutations emerged by ultra-deep sequencing of newly diagnosed multiple myeloma (MM)

Abstract TP53 is rarely reported as being affected either by deletions or mutations in MM, even if chr17p13 copy number (CN) loss defines a samples subgroup with a particularly poor prognosis. Here we aim at retrospectively analyzing by Next Generation Sequencing (NGS) the TP53 gene inactivation in newly diagnosed MM, assessing the mutational events’ frequency and their clinical impact. A cohort of 92 MM, receiving up-front velcade (vel)-based regimens, followed by autologous stem cell transplantation, was included in the study. TP53 gene mutational status was analysed by amplicon-targeted deep NGS approach. An upcoming NGS data analysis software was employed to detect vars (var) and to compare them to IARC database. p53 activity was evaluated by immunoblot assays. Ultra-deep TP53 coding sequence analysis (median depth: 1060) highlighted the presence of a median of 1.8 vars per samples in 73/92 (79%) MM: a total of 131 nucleotide substitutions emerged, with vars allele frequencies (VAF) ranging from 1 to 99% (median 1.3%). TP53 vars, were defined either neutral or deleterious mutations, according to their predicted effect at amino acid level. 36 cases carried 42 deleterious vars (VAF = 1.2%), 67% of which were predicted as non-functional at protein level. 9 samples carried 21 neutral vars (VAF = 1.1%), mainly affecting the DNA binding domain. 28 samples carried 68 SIFT unclassified vars, among which several polymorphisms were counted (VAF = 94.6%). SNPs arrays were performed on 83/92 samples: 14/83 (17%) carried a TP53 CN loss on chr17p13.1. The incidence of TP53 hemizygous deletion was higher among cases carrying deleterious mutations, as compared to cases carrying either neutral or unclassified mutations (25%, 12,5% and 12%, respectively). 2/17 non-mutated samples carried TP53 hemizygous deletion, as well. Interestingly, an Rb1 tumour suppressor gene CN loss on chr13q14.2 significantly characterized samples carrying either mutated (deleterious var) or deleted TP53 (p = 0.006). By immunoblot assay we showed the absence of phosphorilation both in 3 TP53 deleted cases and in 3 MM carrying deleterious vars; on the contrary p-p53 was observed in 4 non-mutated cases. Clinical correlations were performed on 81 MM showing that the presence of either TP53 hemizygous deletions or at least one TP53 deleterious var was more likely associated to the response to vel-based induction therapy (frequencies of ≤partial response: 35% and 64% among samples carrying or not impaired TP53, respectively; p = 0.05). Conversely, the frequency of progression events was slightly higher among MM carrying impaired TP53 (69% vs. 50%). In conclusion, the TP53 coding sequence analysis by ultra-deep NGS of a cohort of newly diagnosed MM highlighted an unexpected, still un-explored, high rate of TP53 vars. The impact of TP53 damage on MM disease course has to be confirmed in randomized clinical trial. Supported by Roche Diagnostics and FP7 NGS-PTL project Citation Format: Marina Martello, Giovanni Martinelli, Angela Flores Dico, Barbara Santacroce, Enrica Borsi, Mauro Procacci, Torsten Haferlach, Flavio Mignone, Igor Saggese, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Anna Maria Brioli, Serena Rocchi, Annalisa Pezzi, Michele Cavo, Carolina Terragna. A long tail of sub-clonal TP53 mutations emerged by ultra-deep sequencing of newly diagnosed multiple myeloma (MM). [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 4248. doi:10.1158/1538-7445.AM2015-4248

A Long Tail of Sub-Clonal TP53 Mutations Emerged By Ultra-Deep Sequencing of Newly Diagnosed Multiple Myeloma (MM)

Introduction. The role of TP53 tumor-suppressor gene in mediating cellular basic tumor suppressive mechanisms is crucial: its utmost importance is underscored by a 50% mutational rate among most human cancers. In MM, TP53 is rarely reported as being affected either by deletions or mutations, even if chromosome (chr) 17p13 copy number (CN) loss defines a samples subgroup with a particularly poor prognosis. Here we aim at retrospectively analyzing by Next Generation Sequencing (NGS) the TP53gene inactivation in newly diagnosed MM, assessing the mutational events’ frequency and their clinical impact.Samples and methods. A cohort of 92 MM, receiving up-front velcade (vel)-based regimens, followed by autologous stem cell transplantation, was included in this molecular study. Response to therapy was evaluated according to the IMWG criteria. DNA was obtained from CD138+ highly purified plasma cells and profiled by SNPs array (Affymetrix). TP53gene mutational status was analysed employing an amplicon-targeted deep NGS approach (GSJ, 454 Life Sciences). A novel upcoming NGS data analysis software was employed to detect variants from raw sequencing data and to compare them to IARC database. As a validation, 30 randomly chosen samples have been re-sequenced. p53 activity was evaluated by p-p53 and p53 immunoblot assays.Results. Ultra-deep TP53coding sequence analysis (median depth: 1060) highlighted the presence of a median of 1.8 variants per samples in 73/92 (79%) MM: a total of 131 nucleotide substitutions emerged, with variants allele frequencies (VAF) ranging from 1 to 99% (median 1.3%). The polymorphism c.215C>G (validated as SNP1042522) was the most prevalent detected variant, recurring in ~40% of the analysed samples.TP53variants were assessed according to the SIFT classification, in order to identify either neutral or deleterious mutations, on the basis of their predicted effect at amino acid level. 36 cases carried 42 deleterious variants (VAF=1.2%): they were mostly missense substitutions affecting mainly the DNA binding domain; most deleterious variants (67%) were predicted as non-functional at protein level. 9 samples carried 21 neutral variants (VAF=1.1%), which were mostly missense mutations, again mainly affecting the DNA binding domain. Finally, 28 samples carried 68 SIFT unclassified variants – among which several polymorphisms were counted – (VAF=94.6%). Of these, 29 were silent substitutions affecting the DNA-binding domain, 32 were missense variants affecting the SH3-like/Proline- rich domain.To assess the genomic background of deep-sequenced MM, SNPs arrays were performed on 83/92 samples: 14/83 (17%) carried a TP53 CN loss (1N) on chr17p13.1. The incidence of TP53 hemizygous deletion was higher among cases carrying deleterious mutations, as compared to cases carrying either neutral or unclassified mutations (25%, 12,5% and 12%, respectively). 2/17 non-mutated samples carried TP53 hemizygous deletion, as well. Interestingly, an Rb1 tumour suppressor gene CN loss on chr13q14.2 significantly characterized samples carrying either mutated (deleterious variant) or deleted TP53 (p=0.006).The activation state of p53 was evaluated by p-p53 immunoblotting assay, showing the absence of phosphorilation either in 3 TP53 deleted cases, or in 3 cases carrying deleterious variants; on the contrary p-p53 was observed in 4 non-mutated cases.Finally, clinical correlations were performed on 81 MM, for whom clinical data were available, showing that the presence of either TP53 hemizygous deletions or at least one TP53 deleterious variant was more likely associated to the response to vel-based induction therapy (frequencies of ≤partial response were 35% and 64% among samples carrying or not impaired TP53, respectively; p=0.05). Conversely, the frequency of progression events was slightly higher among MM carrying impaired TP53 (69% vs. 50%).Conclusions. The analysis by ultra-deep next generation sequencing of TP53 coding sequence in a cohort of newly diagnosed MM highlighted an unexpected, still un-explored, high rate of TP53 variants, recurring with a wide range of frequencies among samples. The impact of TP53 damage on MM disease course has to be confirmed in randomized clinical trial.Acknowledgments: Roche Diagnostics for supporting us in the realization of this project; ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Cavo:Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy; Roche: Honoraria.

Carcinoma Cuniculatum of the Esophagus and Tongue: Report of Two Cases, Including TP53 Mutational Analysis

Carcinoma cuniculatum (CC) is a rare variant of extremely well differentiated squamous cell carcinoma. We present the clinicopathological features of two cases of CC; one lingual and one esophageal case with a molecular genetic study regarding the TP53 gene mutational status. Case 1 was a 62year old male with enlarging chronic ulcer in the tongue. Case 2 was a 77year old male with progressive dysphagia and odynophagia. Both patients were treated surgically. Both tumors showed deeply invaginating, keratin-filled, burrowing crypts lined by very well differentiated squamous epithelium. The esophageal tumor showed varying degrees of reactive nuclear atypia largely limited to the areas with dense intratumoral infiltration of neutrophils. No mutation of TP53 was identified in the esophageal case. Cytologic atypia limited to areas of significant acute inflammation may occur in CC and should, in the absence of aggressive stromal invasion, not preclude a diagnosis of CC.

Association between hTERT rs2736100 polymorphism and sensitivity to anti-cancer agents

Background: The rs2736100 single nucleotide polymorphism (SNP) is located in the intron 2 of human telomerase reverse transcriptase (hTERT) gene. Recent genome-wide association studies (GWAS) have consistently supported the strong association between this SNP and risk for multiple cancers. Given the important role of the hTERT gene and this SNP in cancer biology, we hypothesize that rs2736100 may also confer susceptibility to anti-cancer drug sensitivity. In this study we aim to investigate the correlation between the rs2736100 genotype and the responsiveness to anti-cancer agents in the NCI-60 cancer cell panel.Methods and Materials: The hTERT rs2736100 was genotyped in the NCI-60 cancer cell lines. The relative telomere length (RTL) of each cell line was quantified using real-time PCR. The genotype was then correlated with publically available drug sensitivity data of two agents with telomerase-inhibition activity: Geldanamycin (HSP90 inhibitor) and RHPS4/BRACO19 (G-quadruplex stabilizer) as well as additional 110 commonly used agents with established mechanism of action. The association between rs2736100 and mutation status of TP53 gene was also tested.Results: The C allele of the SNP was significantly correlated with increased sensitivity to RHPS4/BRACO19 with an additive effect (r = −0.35, p = 0.009) but not with Geldanamycin. The same allele was also significantly associated with sensitivity to antimitotic agents compared to other agents (p = 0.003). The highest correlation was observed between the SNP and paclitaxel (r = −0.36, p = 0.005). The telomere length was neither associated with rs2736100 nor with sensitivity to anti-cancer agents. The C allele of rs2736100 was significantly associated with increased mutation rate in TP53 gene (p = 0.004).Conclusion: Our data suggested that the cancer risk allele of hTERT rs2736100 polymorphism may also affect the cancer cell response to both TERT inhibitor and anti-mitotic agents, which might be attributed to the elevated telomerase-independent activity of hTERT, as well as the increased risk for TP53 gene mutagenesis conferred by the polymorphism. Detailed mechanisms need to be further investigated.

Novel TP53 gene mutation and correlation with p53 immunohistochemistry in a mixed epithelial carcinoma of the endometrium

► We examine the correlation between p53 immunohistochemistry and TP53 gene mutation status in a mixed epithelial endometrial carcinoma. ► We describe a novel R306* (c.916C>T) mutation in exon 8 of the TP53 gene. ► We propose that the distinction between Type I and Type II endometrial carcinomas may be more fluid than previously believed.

Profile of p53 expression in bladder and oral tumours: effects of in vitro manipulations of p53 on the behaviour of established human tumour cell lines

In this investigation the profile of p53 and epidermal growth factor receptor (EGFR) expression in tumour tissue biopsies of transitional cell carcinoma of bladder (TCC) and of oral-pharyngeal carcinoma (OP) were compared using an immunocytochemical staining method. In addition, various techniques including sodium dodecyl sulphate-polyacrylamide gel elecrophoresis (SDS-PAGE), colorimetric assay and gene transfection were used to investigate the influence of p53 on the behaviour of human tumour cell lines in vitro. The results showed that: (a) p53 was detectable in more than 45% of cases in both tumour types, although the profile and intensity of expression differed. (b) Concomitant strong expression of EGFR and p53 for TCC and OP was 21% and 38% ( P>0.05%), respectively. (c) Treatment of tumour cells by either gamma radiation or by cisplatin resulted in the induction of p53 independent of the origin of the tumour. (d) Susceptibility of two cell lines, one with and one without constitutive expression of p53 showed that the expressing cells were more sensitive to gamma radiation (the percentage inhibition at 250 cGy was 57% versus −15%, P<0.01), and also cisplatin (the percentage inhibition at 1 μg/ml was 71.0±6.0 versus 2.6±7.0, P<0.001). (e) Transfection of wild-type TP53 gene into a bladder tumour cell line resulted in a rapid cell apoptosis (by as much as 90%) whereas cells receiving mutated TP53 survived. A similar frequency of TP53 mutation in TCCs and OPs was observed. In addition, the pattern of p53 expression within the squamous type of TCC was similar to that in OPs. If the data from the in vitro studies could be translated into an in vivo setting, one could envisage a situation where the introduction of wild-type TP53 gene by gene transfection into tumour cells (independent of their TP53 gene mutational status), would prove to be beneficial. If the cellular TP53 gene is mutated, then an introduction of the normal TP53 gene would induce cells to undergo apoptosis. Alternatively, if TP53 is wild-type, then the increased levels of p53 expression would enable the cells to become more susceptible to DNA damaging treatments such as cisplatin or gamma radiation.

TP53 gene mutation status in pretreatment biopsies of oesophageal adenocarcinoma has no prognostic value

Identification of markers which help to predict response to treatment and overall survival at the time of diagnosis would assist in the management of patients with oesophageal adenocarcinoma. In the present study we investigated the prognostic significance of mutations to the TP53 tumour suppressor gene in a large, consecutive series of oesophageal adenocarcinomas. The incidence of TP53 mutation determined by molecular analysis of endoscopic biopsy specimens was 36% (49/135). No statistically significant difference was observed in patient survival according to the TP53 status of the tumour biopsy. The median survival time for patients with mutation was 12±1 months compared with 14±2 months for patients with TP53. These results demonstrate that mutation of the TP53 gene is not a useful predictive marker for patient survival in oesophageal adenocarcinoma.