Germline P53 Research Articles

Abstract B07: Gain of function activities of p53R245W in a conditional knock-in mouse model

Abstract Missense mutations of the p53 tumor suppressor gene are present in more than 50% of human tumors. Emerging evidence suggests that p53 missense mutations in human cancer patients are associated with the poor prognosis and drug resistance due to their gain-of-function (GOF) properties, rather than just the loss of normal p53 function. Interestingly, these missense mutations have been shown to contribute to tumor progression by different mechanisms: interactions with Smad and SREBPs, modulation of EGFR/integrin signaling, and inhibition of p63 and p73 tumor suppressors. Combined, these studies suggest that the GOF of p53 missense mutations may be dependent on cell contexts and mutation types. Of particular interest, the p53R248W mutation is one of the most frequent missense mutation among human cancer patients; yet to date a mouse model that faithfully mimics this mutation does not exist. Additionally, the majority of human cancers evolve from somatic inactivation of p53 and current mouse models do not mimic somatic mutation of p53. Thus, we generated a conditional p53WMR245/+ knock-in mouse model, which normally expresses wild type p53, but produces p53R245W by Cre-mediated deletion of wild type p53 sequences. Germ line mutation of p53R245W/+ can rescue the early embryonic lethality of Mdm2 and Mdm4 null alleles like p53 null. Preliminary analyses of p53R245W/+ cohorts indicate differences in tumor spectrum in comparison to p53+/- and p53R172H/+ mice. Somatic mutation of p53R245W has also been performed to examine germ line versus somatic differences in tumor development. This study will help us understand the in vivo contributions of different p53 missense mutations to tumorigenesis, as well as the genetic alterations that occur during tumor evolution initiated by both germ line and somatic p53 mutations. Citation Format: Shunbin Xiong, Yun Zhang, Guillermina Lozano. Gain of function activities of p53R245W in a conditional knock-in mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B07.

The Evolution of Tumors in Mice and Humans with Germline p53 Mutations.

Mice with a homozygous p53 gene deletion develop thymic lymphomas by 9 wk of age. Using the sequence of the rearranged T-cell receptor gene from each clone of cells in the thymus, one can determine the number of independent transformation events. These tumors are oligoclonal, occurring at a frequency of 0.13-0.8 new cancer clones per day. By 20 wk only a few clones are detected, indicating competition among transformed cell clones. DNA sequencing of these tumors demonstrates a point mutation frequency of one per megabase and many genes that are consistently amplified or deleted in independent tumors. The tumors begin with an inherited p53 gene deletion. Next is a PTEN mutation in a stem cell or progenitor cell, before the rearrangement of the T-cell receptor. After that, the T-cell clone selects gene amplifications in cyclin D and cdk-6, and in Ikaros in the Notch pathway. Humans heterozygous for the p53 mutant gene in the germline (Li-Fraumeni syndrome) develop cancers at an early age. The penetrance of heterozygous p53 mutations is ∼93% of individuals developing tumors over their lives. At older ages the remaining 7% of this Li-Fraumeni population actually have a lower risk of developing tumors than the population at large with wild-type p53 genes.

Targeted p16(Ink4a) epimutation causes tumorigenesis and reduces survival in mice.

Cancer has long been viewed as a genetic disease; however, epigenetic silencing as the result of aberrant promoter DNA methylation is frequently associated with cancer development, suggesting an epigenetic component to the disease. Nonetheless, it has remained unclear whether an epimutation (an aberrant change in epigenetic regulation) can induce tumorigenesis. Here, we exploited a functionally validated cis-acting regulatory element and devised a strategy to induce developmentally regulated genomic targeting of DNA methylation. We used this system to target DNA methylation within the p16(Ink4a) promoter in mice in vivo. Engineered p16(Ink4a) promoter hypermethylation led to transcriptional suppression in somatic tissues during aging and increased the incidence of spontaneous cancers in these mice. Further, mice carrying a germline p16(Ink4a) mutation in one allele and a somatic epimutation in the other had accelerated tumor onset and substantially shortened tumor-free survival. Taken together, these results provide direct functional evidence that p16(Ink4a) epimutation drives tumor formation and malignant progression and validate a targeted methylation approach to epigenetic engineering.

A small organ takes center stage: selected topics in fallopian tube pathology.

In this paper we consider a number of non-neoplastic and neoplastic lesions of the fallopian tube. Emphasis has been placed on diagnostically difficult entities, some of which result in misdiagnosis and consequent alteration of treatment, including "pseudocarcinomas" that represent a florid epithelial response to acute and/or chronic salpingitis. Endometriosis-related lesions may cause infertility, or undergo malignant transformation to a Mullerian carcinoma, most frequently endometrioid and clear cell types. Pregnancy-related tubal lesions include the easily misdiagnosed metaplastic papillary tumor as well as several manifestations of ectopic pregnancy. Covered briefly are familial conditions such as the Peutz-Jeghers syndrome and its association with tubal mucinous metaplasia, clear cell papillary cystadenoma associated with von Hippel-Lindau syndrome, and the Li Fraumeni syndrome's germline p53 mutation and its association with distal tubal p53 signatures. Miscellaneous tumors discussed include the common adenomatoid tumor and the uncommon female adnexal tumor of probable Wolffian origin. Important issues including the updated staging of fallopian tube carcinomas, and the histopathologic variants of endometrioid carcinomas and their sometimes unusual patterns that engender the potential for confusion with other tumors are briefly noted. The final section covers the relatively recent and novel concept of the fallopian tube as the predominant site of origin of ovarian and peritoneal carcinomas. Discussed are the histologic, immunohistochemical, and molecular biologic evidence that support the tubal fimbria as the site of serous tubal intraepithelial carcinoma, possibly the immediate precursor to high-grade ovarian and peritoneal serous carcinoma.

Identification of new p53 target microRNAs by bioinformatics and functional analysis

BackgroundThe tumor suppressor p53 is a sequence-specific transcription factor that regulates an extensive network of coding genes, long non-coding RNAs and microRNAs, that establish intricate gene regulatory circuits influencing many cellular responses beyond the prototypical control of cell cycle, apoptosis and DNA repair.MethodsUsing bioinformatic approaches, we identified an additional group of candidate microRNAs (miRs) under direct p53 transcriptional control. To validate p53 family-mediated responsiveness of the newly predicted target miRs we first evaluated the potential for wild type p53, p63β and p73β to transactivate from p53 response elements (REs) mapped in the miR promoters, using an established yeast-based assay.ResultsThe REs found in miR-10b, -23b, -106a, -151a, -191, -198, -202, -221, -320, -1204, -1206 promoters were responsive to p53 and 8 of them were also responsive to p63β or p73β. The potential for germline p53 mutations to drive transactivation at selected miR-associated REs was also examined. Chromatin Immuno-Precipitation (ChIP) assays conducted in doxorubicin-treated MCF7 cells and HCT116 p53+/+ revealed moderate induction of p53 occupancy at the miR-202, -1204, -1206, -10b RE-containing sites, while weak occupancy was observed for the miR-23b-associated RE only in MCF7 cells. RT-qPCR analyses cells showed modest doxorubicin- and/or Nutlin-dependent induction of the levels of mature miR-10b, -23b, -151a in HCT116 p53+/+ and MCF7 cells. The long noncoding RNA PVT1 comprising miR-1204 and −1206 was weakly induced only in HCT116 p53+/+ cells, but the mature miRs were not detected. miR-202 expression was not influenced by p53-activating stimuli in our cell systems.ConclusionsOur study reveals additional miRs, particularly miR-10b and miR-151a, that could be directly regulated by the p53-family of transcription factors and contribute to the tuning of p53-induced responses.

Abstract C114: Clinical response to a lapatinib-based therapy of a Li-Fraumeni Syndrome patient with a novel HER2-V659E mutation.

Abstract Genomic characterization of recurrent breast and lung tumors developed over the course of 10 years in a 29-year-old patient with a germline p53 mutation (Li-Fraumeni Syndrome) identified oncogenic alterations in the HER2 and EGFR genes across all tumors, including HER2 amplifications, an EGFR-exon 20 insertion, and the first-in-human HER2-V659E mutation showing a phenotypic convergent evolution towards HER2 and EGFR alterations. Following the identification of HER2-activating events in the most recent lung carcinoma and in circulating tumor cells, we treated the reminiscent metastatic lesions with a lapatinib-based therapy. A clinical response both symptomatic and radiologic was achieved. HER2-V659E sensitivity to lapatinib was confirmed in the laboratory. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C114. Citation Format: Violeta Serra, Ana Vivancos, Xose Puente, Enriqueta Felip, Daniel Silberschmidt, Ginevra Caratù, Josep-Lluis Parra, Leticia De Mattos-Arruda, Judit Grueso, Javier Hernández-Losa, Joaquín Arribas, Ludmila Prudkin, Paolo Nuciforo, Maurizio Scaltriti, Joan Seoane, José Baselga. Clinical response to a lapatinib-based therapy of a Li-Fraumeni Syndrome patient with a novel HER2-V659E mutation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C114.

Two co-existing germline mutations P53 V157D and PMS2 R20Q promote tumorigenesis in a familial cancer syndrome

Germline mutations are responsible for familial cancer syndromes which account for approximately 5–10% of all types of cancers. These mutations mainly occur at tumor suppressor genes or genome stability genes, such as DNA repair genes. Here we have identified a cancer predisposition family, in which eight members were inflicted with a wide spectrum of cancer including one diagnosed with lung cancer at 22years old. Sequencing analysis of tumor samples as well as histologically normal specimens identified two germline mutations co-existing in the familial cancer syndrome, the mutation of tumor suppressor gene P53 V157D and mismatch repair gene PMS2 R20Q. We further demonstrate that P53 V157D and/or PMS2 R20Q mutant promotes lung cancer cell proliferation. These two mutants are capable of promoting colony formation in soft agar as well as tumor formation in transgenic drosophila system. Collectively, these data have uncovered the important role of co-existing germline P53 and PMS2 mutations in the familial cancer syndrome development.

Mutant p53 Prolongs NF-κB Activation and Promotes Chronic Inflammation and Inflammation-Associated Colorectal Cancer

The tumor suppressor p53 is frequently mutated in human cancer. Common mutant p53 (mutp53) isoforms can actively promote cancer through gain-of-function (GOF) mechanisms. We report that mutp53 prolongs TNF-α-induced NF-κB activation in cultured cells and intestinal organoid cultures. Remarkably, when exposed to dextran sulfate sodium, mice harboring a germline p53 mutation develop severe chronic inflammation and persistent tissue damage, and are highly prone to inflammation-associated colon cancer. This mutp53 GOF is manifested by rapid onset of flat dysplastic lesions that progress to invasive carcinoma with mutp53 accumulation and augmented NF-κB activation, faithfully recapitulating features frequently observed in human colitis-associated colorectal cancer (CAC). These findings might explain the early appearance of p53 mutations in human CAC.

Novel p53 splicing site mutation in Li‐Fraumeni‐like syndrome with osteosarcoma

We describe a 15-year-old girl with a novel germline p53 splice site mutation who developed an osteosarcoma. She received several cycles of chemotherapy with complete resection of the primary tumor without amputation, and has maintained remission for 18 months. Li-Fraumeni-like syndrome was suspected based on familial history. Sequence analysis revealed the presence of a novel germline p53 gene mutation resulting in a G to A transition at position +1 at the donor splice site of intron 6, creating a 6 amino acid insertion. This case provides interesting insight into the phenotype-genotype correlation in LFL syndrome with a TP53 splicing mutation.

A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

Congenital reduction in nephron number (renal hypoplasia) is a predisposing factor for chronic kidney disease and hypertension. Despite identification of specific genes and pathways in nephrogenesis, determinants of final nephron endowment are poorly understood. Here, we report that mice with germ-line p53 deletion (p53−/−) manifest renal hypoplasia; the phenotype can be recapitulated by conditional deletion of p53 from renal progenitors in the cap mesenchyme (CMp53−/−). Mice or humans with germ-line heterozygous mutations in Pax2 exhibit renal hypoplasia. Since both transcription factors are developmentally expressed in the metanephros, we tested the hypothesis that p53 and Pax2 cooperate in nephrogenesis. In this study, we provide evidence for the presence of genetic epistasis between p53 and Pax2: a) p53−/− and CMp53−/−embryos express lower Pax2 mRNA and protein in nephron progenitors than their wild-type littermates; b) ChIP-Seq identified peaks of p53 occupancy in chromatin regions of the Pax2 promoter and gene in embryonic kidneys; c) p53 binding to Pax2 gene is significantly more enriched in Pax2 -expressing than non-expressing metanephric mesenchyme cells; d) in transient transfection assays, Pax2 promoter activity is stimulated by wild-type p53 and inhibited by a dominant negative mutant p53; e) p53 knockdown in cultured metanephric mesenchyme cells down-regulates endogenous Pax2 expression; f) reduction of p53 gene dosage worsens the renal hypoplasia in Pax2 +/− mice. Bioinformatics identified a set of developmental renal genes likely to be co-regulated by p53 and Pax2. We propose that the cross-talk between p53 and Pax2 provides a transcriptional platform that promotes nephrogenesis, thus contributing to nephron endowment.

Genetic Testing by Cancer Site

Gastric cancer is a global public health concern, ranking as the fourth leading cause of cancer mortality, with a 5-year survival of only 20%. Approximately 10% of gastric cancers appear to have a familial predisposition, and about half of these can be attributed to hereditary germline mutations. We review the genetic syndromes and current standards for genetic counseling, testing, and medical management for screening and treatment of gastric cancer. Recently, germline mutations in the E-cadherin/CDH1 gene have been identified in families with an autosomal dominant inherited predisposition to gastric cancer of the diffuse type. The cumulative lifetime risk of developing gastric cancer in CDH1 mutation carriers is up to 80%, and women from these families also have an increased risk for developing lobular breast cancer. Prophylactic gastrectomies are recommended in unaffected CDH1 mutation carriers, because screening endoscopic examinations and blind biopsies have proven inadequate for surveillance. In addition to this syndrome, gastric cancer risk is elevated in Lynch syndrome associated with germline mutations in DNA mismatch repair genes and microsatellite instability, in hereditary breast and ovarian cancer syndrome due to germline BRCA1 and BRCA2 mutations, in familial adenomatous polyposis caused by germline APC mutations, in Li-Fraumeni syndrome due to germline p53 mutations, in Peutz-Jeghers syndrome associated with germline STK11 mutations, and in juvenile polyposis syndrome associated with germline mutations in the SMAD4 and BMPR1A genes. Guidelines for genetic testing, counseling, and management of individuals with hereditary diffuse gastric cancer are suggested. A raised awareness among the physician and genetic counseling communities regarding these syndromes may allow for increased detection and prevention of gastric cancers in these high-risk individuals.

Potential genotype-specific single nucleotide polymorphism interaction of common variation in p53 and its negative regulator mdm2 in cholangiocarcinoma susceptibility

Aberrant cell cycle control and apoptosis deregulation are involved in biliary carcinogenesis. The tumor suppressor gene p53 and its key negative regulator murine double minute 2 (mdm2) cooperate in modulating these basic cell functions and germline p53 alteration promotes cholangiocarcinoma (CCA) formation in animal models. The potential association between common functional genetic variation in p53 (SNP72 G/C) and mdm2 (SNP309 T/G) and susceptibility to bile duct cancer, however, has not been studied. p53/SNP72 G/C (rs1042522) and mdm2/SNP309 T/G (rs2279744) were genotyped in 182 Caucasian CCA patients and 350 controls using TaqMan assays. Allelic and genotypic differences, including exploratory data analyses (according to gender, tumor localization, early onset and genotypic interactions) were compared in contingency tables using the χ(2) and Fisher's exact tests. The overall comparison of allele and genotype frequencies yielded no significant association between either SNP and CCA susceptibility. Similarly, gender- and localization-specific analyses did not reveal deviations in allelic or genotypic distributions. In carriers of the low-apoptotic p53 genotype CC, the mdm2 SNP309 T allele conferred borderline significant CCA risk [P=0.049; odds ratio (OR), 4.36; 95% CI, 0.92-20.77]. Power analysis confirmed adequate statistical power to exclude major SNP effects (each >97% for OR 1.7). Collectively, the results we obtained from the largest European CCA cohort do not support the hypothesis of a prominent role of common p53 and mdm2 variation in the genetic susceptibility to bile duct cancer. However, epistatic effects may modulate genetic CCA risk in individual subsets.

Abstract 1447: Investigating susceptibility to sporadic osteosarcoma by genome-wide copy number analysis

Abstract Constitutional copy-number variants (CNVs) frequently overlap genomic regions that contain genes associated with cancer. In addition, copy number alterations (CNAs) in tumors often coincide with sites of CNVs and may arise through progression of CNVs. Osteosarcoma (OS), the most common human bone malignancy, can arise in familial or sporadic forms. While germline mutations of tumor suppressor genes have been implicated in a minority of OS cases, little is known about genetic changes that contribute to the etiology of sporadic OS. Using high-resolution SNP/CNV arrays, we observed a higher global germline CNV frequency in patients with sporadic OS compared to healthy controls. Given the early age of onset and the extensive constitutional CNVs, we hypothesize that these genomic alterations are linked to susceptibility to OS. Here we investigate whether specific constitutional CNVs are related to susceptibility to sporadic OS and whether OS tumor CNAs arise via progression of constitutional CNVs in these patients. Blood derived DNA samples from patients with sporadic OS with wild-type germline p53 sequence and gene dosage were selected for analysis on Affymetrix GW6.0 arrays with paired pre-therapy tumor biopsies. Initial results from a discovery set of samples (n=43 pairs) reveal a copy number loss at 1q43, in a region that overlaps several genes that have a role in bone formation, microtubules and cytoskeletal organization. This loss was present in 43% of the blood-derived DNA samples and 44% of the tumor-derived samples, whereas this variant was noted in only 1.2% of controls. Furthermore, the germline 1q43 deletion correlates with poor survival of the OS patients (p=0.04, Long Rank). Analysis of an independent validation set (n=32 pairs) is currently underway to confirm the initial finding and further characterize specific constitutional CNVs among sporadic OS patients. The findings of this study will open new avenues for the discovery of novel loci implicated in susceptibility to OS with potential for improved diagnostic and prognostic options as well as further insight into disease pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1447. doi:1538-7445.AM2012-1447

Functional studies of a novel Germline p53 splicing mutation identified in a patient with Li–Fraumeni‐Like syndrome

Most p53 mutations identified in Li-Fraumeni syndrome (LFS) are missense mutations; splicing mutations have rarely been reported. A novel splicing p53 mutation was identified in a patient with Li-Fraumeni-like syndrome (LFL). Usually, p53 missense mutants identified in LFS and cancer cells function as dominant negative mutations interfering with wild-type p53 function. However, the mechanism by which p53 haploinsufficiency causes carcinogenesis is not well characterized. In this study, we describe a novel splicing mutation that results in the loss-of-function of p53. These findings suggest a linkage between the loss-of-function type p53 mutation and a LFL phenotype.

MDM2 309 polymorphism predicts outcome in platinum-treated locally advanced head and neck cancer

Chemo-radiotherapy (CRT) with cisplatin-based regimens is curative in a subset of patients with locally advanced (stage III and IV) squamous carcinomas of the head and neck (LAHNSCC), but causes considerable toxicity. To seek predictive biomarkers, we analysed single nucleotide polymorphisms (SNPs) in the p53 and MDM2 genes in LAHNSCC patients treated with cisplatin-based CRT. We analysed germ-line p53 72 Arg/Pro (R/P) and MDM2 309 SNPs and somatic p53 mutational status in 140 LAHNSCC and determined their utility as predictive biomarkers. In cases with wild-type p53, overall survival (OS) was longest in 72RR (median OS=60.8 months) and less favourable in 72PP (median OS=6.7 months, p<0.0001). OS in individuals with 72RP was intermediate between 72RR and 72PP, while in patients with missense p53 mutations, median OS did not reach statistical significance. Median OS was significantly shorter in patients with MDM2 309 SNP genotypes GG or GT, compared to TT (15 vs. 86 months; p<0.0001). The predictive effect of the G allele was maintained independent of age, gender, stage, primary site, nodal status, performance status, EGFR grade, HPV status, p53 mutation and p53 SNP (HR for death 3.241; 95% CI 1.90-5.52, p<0.001). The predictive utility of the MDM2 germ-line 309 SNP, which can be easily determined from peripheral blood, implies that it may be of value in the objective selection of patients for radical CRT. In contrast, the predictive utility of the 72 Arg/Pro SNP in p53 requires mutational analysis of p53, limiting its routine clinical use.

An identical, complex TP53 mutation arising independently in two unrelated families with diverse cancer profiles: the complexity of interpreting cancer risk in carriers

Most inherited TP53 mutations have been identified in individuals with a family cancer predisposition syndrome, in which the activity of p53 mutants is severely reduced. However, germline p53 mutants in children with ‘sporadic' adrenocortical or choroid plexus tumors exhibit a wide range of functional activity. Here, we demonstrate the occurrence of a complex germline TP53 mutation in two unrelated families with different cancer phenotypes, neither fulfilling the classic criteria for Li-Fraumeni syndrome. The TP53 mutation consists of a duplication of 7 bp in exon 4, resulting in a frame shift and premature stop signal. Haplotype analysis indicated that the mutation arose independently in the two families. Analysis of the DNA secondary structure predicts the TP53 mutation occurred within a hairpin loop. Additional germline complex mutations occurring within the same region of exon 4 have been identified in the IARC database. Our findings suggest that certain TP53 regions are prone to intrinsic genetic alterations, possibly through defects in DNA replication or repair. Further, carriers of the same TP53 mutation can have diverse cancer profiles, illustrating the complexity of genetic counseling and risk prediction.

Inherited Hematologic and Oncologic Syndromes

Inherited Hematologic and Oncologic Syndromes

Pleomorphic carcinoma of the lung arising in a patient with Li-Fraumeni syndrome: Report of a case

We herein report the case of a patient with Li-Fraumeni syndrome (LFS) who developed lung pleomorphic carcinoma. A 28-year-old female patient with a family history of early-onset malignancies was diagnosed with lung carcinoma and treated by surgical resection. Histological examination revealed a heterogeneous tumor with epithelial and mesenchymal components. The final pathological diagnosis was pulmonary pleomorphic carcinoma. In this patient, a constitutional mutation at codon 213 in exon 6 of the p53 gene was identified in the peripheral lymphocytes and the resected tumor, and LFS was suspected. This mutation causes a nonsense mutation (Arg-to-Stop codon) that has been shown to attenuate p53 function. This is the first report of pulmonary pleomorphic carcinoma developing in an LFS patient, and may suggest a relationship between germline p53 mutation and carcinogenesis in pulmonary pleomorphic carcinoma.

Abstract 5446: The activity and pharmacokinetics of capecitabine in a mouse model (K8484) of pancreatic adenocarcinoma

Abstract The pharmacokinetics (PK) of the oral fluoropyrimidine capecitabine were investigated in a mouse model of pancreatic cancer. Allografts of a cell line (K8484) derived from a pancreatic adenocarcinoma which occurred in a KrasG12D; p53R172H; Pdx1-Cre (KPC) mouse (S. Hingorani, et al., Cancer Cell 2005) were grown subcutaneously in left and right flanks of syngeneic immunocompetent recipient mice. Mice were dosed with either a single dose or 5 daily doses of capecitabine at 755 mg/kg (2.1 mmol/kg/day) with tumour volumes of approximately 250 mm3. Plasma and tissue homogenates were analysed using an LC-MS/MS assay developed to simultaneously detect capecitabine and its 3 metabolites DFCR, DFUR and 5-FU (S.M.Guichard, et al., J. Chrom. B. 2005). Forty minutes after a single dose the mean plasma concentrations (n=3) were 28 +/- 20 mg/ml capecitabine, 78 +/- 48 mg/ml DFCR, 41 +/- 17 mg/ml DFUR and 0.19 +/- 0.07 mg/ml (1.5 µM) 5-FU, with concentrations of all analytes falling at 2 and 4 hours. In the tumour tissue, mean concentrations at 40 mins were capecitabine: 37 +/- 19 ng/mg tissue, DFCR: 56 +/- 12 ng/mg; DFUR: 20 +/- 8 ng/mg and 5-FU: 3.3 +/- 0.7 ng/mg. At 2 and 4 hours the tumour 5-FU concentrations were 3.0 +/- 1.1 and 1.4 +/- 0.6 ng/mg respectively. In the liver, DFCR concentrations were higher than in the tumour from the same mice but DFUR concentrations were lower and 5-FU was below the limit of quantification (&amp;lt;0.4 ng/mg) in most, consistent with the reported distribution of carboxylesterases, cytidine deaminase and thymidine phosphorylase in tissues (M. Miwa, et al., Eur. J. Cancer 1998). In animals dosed for 5 days, there was no evidence of accumulation of capecitabine or its metabolites in tumour tissue when compared to the single dose. The concentration of tumour 5-FU, ranging from 1.4 to 3.3 ng/mg, is estimated to be equivalent to 11 to 25 µM, more than 10-fold higher than the plasma concentration. The IC50 for 5-FU in K8484 cells, grown in vitro, was 1.4 +/- 0.8 µM suggesting that oral dosing with capecitabine delivers a therapeutically effective dose to the allograft. This was then confirmed in an efficacy study, treating tumour-bearing mice with oral capecitabine for 5 days per week for 3 weeks, which resulted in significant inhibition of tumour growth rate compared to the vehicle treated group, with mean tumour doubling time of 7.5 +/- 3.0 days for capecitabine compared to 3.5 +/- 0.5 days for the vehicle treated group (P &amp;lt; 0.001). Despite identical germline Kras and p53 genotypes, the KPC PDA and KPC allograft tumour types have previously shown differences in gemcitabine sensitivity in vivo, predicted to be due to differences in drug delivery (K. Olive, et al., Science 2009). Therefore, capecitabine PK and efficacy are now being investigated in the autochthonous tumours arising in the KPC model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5446. doi:10.1158/1538-7445.AM2011-5446

Abstract 5351: Prevalence and function of p53 mutations among children with adrenocortical carcinoma (ADCC)

Abstract Adrenocortical carcinoma (ACC) is a rare tumor in children, affecting 0.4 per million children worldwide. Incidence in adults is approximately 5-fold higher and may represent a distinct genetic and biologic entity. Mortality rates among patients with ACC are high and treatment options are limited. Previous studies have suggested that a majority of children who develop ACC possess germline mutations in the p53 tumor suppressor gene. In this study we sought to characterize the prevalence and mutational spectrum of p53 among a large multi-center cohort of children with ACC. We further compared the specific mutations found in these children with mutations found in individuals with the Li-Fraumeni syndrome, a familial cancer predisposition syndrome which is strongly associated with germline p53 mutation. Among 58 independent pedigrees assessed for p53 mutation, 29 (50%) carried a heterozygous mutation in p53. No mutation was identified more than once among this cohort. This is in contrast to families with the LFS, where 6 “hotspot” mutations are disproportionately over-represented, combined accounting for 20% of patients with identified p53 mutations. Missense mutations were noted in 28/29 individuals, with a single intragenic deletion identified in the remaining individual. Mutations were found predominantly within the DNA-binding domain (26/29 (90%)). One mutation was identified in the trans-activation domain and two in the oligomerization domain. In the course of this analysis, we identified several novel p53 mutations among children with ACC. These mutations were functionally characterized in vitro based on the ability of mutant p53 to activate transcription of known p53 target genes including those involved in DNA damage repair, cell-cycle arrest and apoptosis. Dominant-negative activity of these mutations was also assessed. Several of the novel mutations demonstrated either full p53 function or partial loss-of-function. The association of these seemingly milder mutations with adrenal tumorigenesis suggests that adrenal tissues may be more sensitive to partial decrease in p53 function than other LFS-affected tissues and that other predisposing or disease-modifying genetic alterations may exist to propel these cells towards a transformed fate. Further efforts to identify disease modifying loci, as well as pathogenic loci in children with wild-type p53 genes are underway via genome-wide analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5351. doi:10.1158/1538-7445.AM2011-5351