TP53 Mutation Carriers Research Articles

International survey of awareness of genetic risk in the clinical sarcoma community.

Integration of clinical genetics into oncology is variable. Sarcomas have a strong genetic component, with up to 1/30 patients carrying germline TP53 mutations. This study aimed to define genetic risk awareness among sarcoma physicians. Outcomes were attitudes toward genetic testing, level of cancer risk and awareness of risk reduction measures. An online survey was administered to members of the Connective Tissue Oncology Society and the Australasian Sarcoma Study Group. Sarcoma physicians (N = 124) from 21 countries participated, 40% of whom favored TP53 mutation testing in children regardless of family history, increasing to ∼83% for all age groups if a family history was present and ∼85% if multiple primary cancers were present. However, 33% were not aware that risk reduction strategies might identify some cancers at a more curable stage in carriers. Clinical genetics is not yet standard of care for multidisciplinary management of sarcoma. Awareness of genetic risk is important among sarcoma physicians. Attitudes among the sarcoma community were generally positive, but education on genetic risk in sarcoma patients and collaboration with clinical genetics services might improve quality of care. Sarcoma physicians need routine access to clinical genetics services so that potential germline TP53 mutation carriers are recognized.

Abstract PD7-03: Characterization of Li-Fraumeni syndrome diagnosed using a 25-gene hereditary cancer panel

Abstract Background: Clinical diagnostic criteria for Li-Fraumeni syndrome (LFS) have evolved with increased utilization of TP53 germline testing and subsequent improved understanding of the diversity of the associated cancer phenotypes. However, data on LFS still suffer from ascertainment bias as patients are typically selected to undergo TP53 testing based on the presence of hallmark features of LFS. Analyzing TP53 mutation carriers identified from multi-gene panel testing, for which the diagnosis of LFS may not have been suspected or was included in a longer differential diagnosis, affords an opportunity to characterize additional TP53 carriers who might not otherwise have been ascertained. Methods: Patients with a deleterious or suspected deleterious germline TP53 mutation were identified from 80,748 consecutive cases that underwent a 25-gene hereditary cancer panel test between September 2013 and March 2015 at a commercial diagnostic laboratory. Patient clinical data were obtained by healthcare provider report on test requisition forms. Each TP53 mutation carrier was evaluated to determine whether the National Comprehensive Cancer Network's (NCCN) guidelines were met for TP53 testing. Results: Eighty-one TP53 mutation carriers were identified and had a total of 115 cancers (0.1% overall prevalence). Among the 76 carriers with at least one cancer, the average age at first diagnosis was 42 years (range 11-76 years) and 24% were first diagnosed older than age 50 years. The most common first cancers were of the breast (n=45), ovary (n=9), and gastrointestinal tract (n=8). Fifty-two of the 75 (69%) women had breast cancer, 44% of which were first diagnosed at 35 years or younger, and 21% were first diagnosed at 50 years or older. Only 27 TP53 carriers met NCCN criteria for TP53 testing, 14 of whom only met based on having early-onset breast cancer. An additional 8 did not meet criteria themselves but had a first- or second-degree relative who did. Among the 28 individuals with more than one primary cancer, 21 (75%) developed their second primary at a site for which increased surveillance is recommended in LFS, but only 4 would have met NCCN criteria for TP53 testing at their first cancer diagnosis. The most common second cancers were of the breast (n=16), gastrointestinal tract (n=4), or kidney (n=2) and occurred an average of 11 years after the first cancer (range 0-36 years). Conclusion: In this analysis, a large proportion of carriers would not have been identified as TP53 testing candidates based on NCCN guidelines. Our data are consistent with other studies demonstrating high second primary cancer risks in LFS, and highlight the value of multigene panel testing in identifying individuals who may be candidates for increased surveillance and/or cancer risk-reducing management options. Citation Format: Rich T, Lotito M, Kidd J, Saam J, Lancaster J. Characterization of Li-Fraumeni syndrome diagnosed using a 25-gene hereditary cancer panel. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD7-03.

Higher Percentages of Ring Sideroblasts and SF3B1 Mutations in Patients with AML Correlate with Mutations in TP53 and Adverse Cytogenetics, but Have No Independent Impact on Outcome

IntroductionRing sideroblasts (RS) are common findings in myeloid malignancies as MDS or MDS/MPN, especially in RARS-T, and are associated with SF3B1 mutations. However, the incidence of both RS and SF3B1 mutations have not yet been assessed properly in acute myeloid leukemia (AML).AimDetermine the frequency of RS and SF3B1 mutations in 1857 patients with de novo and therapy-related AML (t-AML). Define their impact on survival and association with other frequently mutated genes, as well as with cytogenetic abnormalities.Patients and MethodsFrom a total of 1857 AML patients (excluding those with cytogenetically-defined entities according to WHO), bone marrow assessment revealed 473 (25%) with RS ≥ 1% and thereof 183 (10% of all) with ≥15 RS. Sequencing or melting curve analysis were performed in a subcohort of 340/473 patients for the detection of mutations in: SF3B1, ASXL1, DNMT3A, FLT3- TKD, IDH1 R132, IDH2 R140, IDH2 R172, KRAS, NPM1, NRAS, RUNX1, TET2, FLT3 -ITD and MLL -PTD. These 340 cases were subject to the study. Out of these 340, 141 cases (42%) had RS ≥ 15% and the remaining 199/340 (59%) had RS ≥1 to <15%. The cohort consisted of 303 (89%) de novo AML (FAB: M0 n=18, M1 n=67, M2 n=165, M4 n=30, M5 n=3, M6 n=20) and 37 (11%) t-AML. 148 were female and 192 were male, with median age 74 years, range: 20-93 years. Chromosome banding analysis (assisted by FISH if needed) was performed in all 340 cases.ResultsThe percentage of bone marrow blasts correlated inversely with the percentage of RS present (r 0.213, p<0.001). 136 (40%) patients had a normal karyotype. Intermediate cytogenetics according to MRC criteria were found in 193 (57%), and adverse in 147 (43%). Patients with RS ≥15% more frequently had adverse cytogenetics in comparison to those with RS between 1-14% (54% vs 36%, p=0.001).The frequencies of gene mutations were as follows: TP53 103/331 (31%), RUNX1 84/315 (26%), DNMT3A 86/337 (25%), TET2 68/330 (20%), ASXL1 58/334 (17%), IDH2 R140 53/338 (15%), NPM1 43/340 (12%), SF3B1 34/334 (10%), FLT3 -ITD 33/340 (10%), NRAS 29/340 (8%), IDH1 R132 21/339 (6%), MLL -PTD 22/337 (6%), FLT3 -TKD 18/333 (5%), KRAS codon 12 13/299 (4%), IDH2 R172 13/338 (3%) and KRAS codon 61 3/299 (1%). Moreover, in total 30 variants in 28 patients were identified in DNMT3A, RUNX1, TET2 and TP53, which according to current knowledge cannot be assigned to mutations or SNPs yet.Patients with ≥15% more frequently had TP53 mutations (mut) (44% vs 22%, p<0.001) and less frequently IDH2 R140 mut (11% vs 19%, p=0.094) and MLL -PTD (2% vs 6%, p=0.006). Accordingly, patients with TP53 mutations had higher percentages of RS as compared to those without (28% vs 16%, p<0.001) and patients with IDH2 R140 mut and MLL -PTD, respectively, had lower percentages of RS as compared to those without (15% vs 21%, p=0.043 and 11% vs 21%, p=0.025, respectively). Furthermore, patients with mutations in the following genes had fewer RS than patients with the respective wild-types: ASXL1 (15% vs 21%, p=0.040), FLT3 -ITD (14% vs 21%, p= 0.049), IDH2 R140 (15% vs 21%, p=0.043), MLL -PTD (11% vs 21%, p=0.025), NPM1 (13% vs 21%, p=0.018) and KRAS codon 61 (3% vs 20%, p<0.001). Conversely, patients with mutated SF3B1 had more RS than patients with wild type (27% vs 19%, p=0.054). However, the number of RS did not translate into an increase in the mutational burden of SF3B1.Given the limited degree of overlap between mutations of the four most frequently mutated genes, we hierarchically separated the patients into 5 groups: TP53 mut, ASXL1 mut, NPM1 mut, SF3B1 mut and patients without any of these mutations. Interestingly, the percentage of RS was very similar in the two groups, TP53 mut and SF3B1 mut, and significantly higher as compared to all other groups (TP53 mut/SF3B 1mut: 28% vs ASXL1 mut/NPM1 mut/others 14%, p<0.001).The number of RS did not have an impact on the overall survival (OS) and event free survival (EFS) of patients.Conclusion1. Ring sideroblasts ≥ 15% are present in 10% of de novo and t-AML.2. The blast count correlates inversely with the number of RS.3. Patients with ≥ 15% RS more frequently carry TP53 mutations and adverse cytogenetics.4. Although patients with ≥ 15% RS have worse molecular (TP53 mut) and cytogenetic features, there is no statistically significant impact on survival when compared to patients with <15% RS.5. Patients with mutated ASXL1, FLT3 -ITD, IDH2 R140, MLL -PTD and NPM1 have less RS than wild type patients while those with TP53 or SF3B1 mutations have higher RS. [Display omitted] DisclosuresMartin-Cabrera:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Mutational analysis by next generation sequencing of gastric type dysplasia occurring in hyperplastic polyps of the stomach: Mutations in gastric hyperplastic polyps

Gastric hyperplastic polyps (GHP) are the most common type of polyps occurring in the stomach. Although GHP are broadly interpreted as benign lesions, they may progress to dysplasia and adenocarcinoma. Objective: In this study, we aimed to identify genomic mutations that characterize and may drive malignant transformation in GHP by using next-generation sequencing. Eight GHP (2 with dysplasia, 1 indefinite for dysplasia and 5 without dysplasia) were studied. Only large polyps (>1cm) with gastric differentiation were included in this study, while adenomatous polyps (intestinal-type) were excluded. Immunohistochemistry for MUC2, MUC5A, MUC6, CDX2, p53, and Ki67 was performed. DNA was extracted from formalin-fixed paraffin-embedded sections and sequenced for the detection of somatic mutations. Multiplex sequencing was done with the TrueSeq Amplicon Cancer Panel in the MiSeq platform. Variant annotation and visualization were performed using NextGENe (SoftGenetics) software. No pathogenic mutations were detected in GHP without dysplasia. TP53 gene mutations were the most common alteration in dysplastic GHP (2 of 2 dysplastic cases). PIK3CA mutation was identified in a GHP with pyloric-type dysplasia, whereas foveolar-type dysplasia carried TP53 mutations. In conclusion, TP53 gene mutations are a common alteration in the early dysplastic stage during malignant transformation of GHP. GHP with dysplasia may show dual differentiation. In our study, pyloric-type dysplasia was associated with a PIK3CA alteration whereas foveolar dysplasia carried TP53 mutations. The identification of carcinoma-associated mutations in large GHP provides additional evidence of their neoplastic potential and emphasizes the need for their complete resection and follow-up.

Abstract 4664: Impact of TP53 mutations, single nucleotide variants and global methylation patterns on pre-menopausal breast cancer risk

Abstract INTRODUCTION: Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome that confers a high lifetime risk of developing malignancies, including pre-menopausal breast cancer. Over 80% of LFS patients harbor germline mutations of the TP53 tumor suppressor gene. OBJECTIVE: We sought to determine if the spectrum of TP53 variants, as well as global methylation methylation patterns, correlates with the presence of TP53 mutations or influences cancer risk in pre-menopausal breast cancer patients. METHODS: Constitutional DNA was obtained from peripheral blood lymphocytes (PBL) of 10 patients with pre-menopausal breast cancer harboring germline TP53 mutations, 10 patients diagnosed with pre-menopausal breast cancer that are non-mutation carriers, 15 TP53 mutation carriers who do not have cancer, and 15 healthy female controls. Genome-wide methylation analysis of PBL-derived DNA was performed using the Illumina 450K array. Sanger sequencing of exons 2-11, and flanking intron regions of the TP53 gene was performed. RESULTS: The most commonly observed single nucleotide polymorphism (SNP) in TP53 across all samples was at c.215C&amp;gt;G (p.Pro72Arg) in exon 4 (rs1042522). We observed a G allele frequency of 66.0% in our study group. Three additional low-frequency variants in TP53 were observed: the exon 4: c.108G&amp;gt;A (p.Pro36Pro) variant was found in 2 patients, exon 6: c.639A&amp;gt;G (p.Arg213Arg) was found in 9 patients and intron 9: c.993+12T&amp;gt;C was found in 4 patients. Methylation patterns were not associated with a specific TP53 mutation or variant; however, distinct methylation patterns were observed in TP53 mutation carriers when compared to either TP53 wild-type healthy individuals or patients with early-onset breast cancer. DISCUSSION: This study represents an epigenome-wide analysis and targeted genetic survey of germline TP53 in early-onset, pre-menoupausal breast cancer. As a result of the mutation frequency observed, germline mutations in TP53 may need to be included in the genetic work-up of patients with early-onset breast cancer. Parallel studies of genome-wide methylation demonstrate these patterns may serve as an independent biomarker of early-onset breast cancer risk. Note: This abstract was not presented at the meeting. Citation Format: Nardin Samuel, Mathieu Lemire, Ana Novokmet, Thomas J. Hudson, David Malkin. Impact of TP53 mutations, single nucleotide variants and global methylation patterns on pre-menopausal breast cancer risk. [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 4664. doi:10.1158/1538-7445.AM2015-4664

Abstract 2749: Cumulative cancer risk in the NCI Li-Fraumeni Syndrome Cohort

Abstract Background: Li-Fraumeni Syndrome (LFS) is a rare hereditary cancer predisposition syndrome characterized by the early onset of a wide range of cancers and a very high lifetime cumulative cancer risk. Despite significant progress in understanding LFS, uncertainty remains regarding the cancer incidence rates and cancer spectrum for TP53 mutation carriers. In addition, the cancer incidence for members of families meeting the clinical diagnostic criteria of LFS or Li-Fraumeni like (LFL) syndrome but in which no TP53 mutation has been identified (TP53- families) has not been established. Methods: The NCI's LFS Study (NCT01443468) is a natural history study that opened to accrual in August 2011. Inclusion criteria include LFS or LFL syndrome; a germline TP53 mutation; or a personal history of choroid plexus carcinoma, adrenocortical carcinoma (ACC), or ≥3 primary cancers. Participants complete a detailed family history and individual information questionnaires. Confirmation of all reported cancer diagnoses is pursued. Results: Of the 122 families enrolled to date, 95 families have a germline TP53 mutation (TP53+ families) and 27 are TP53-families. TP53+ families: 243 mutation carriers from 95 mutation-positive families were included in the cancer risk estimates. The cumulative risk to first cancer diagnosis for the entire group is 43% by age 30 and close to 100 by age 70, with males having higher risk in childhood adolescence and females having higher risk starting in the 20s, mostly due to breast cancer. When examined by cancer type, the cumulative risk to age 70 is 35% for breast cancer, 16% for soft tissue sarcoma, 10% for brain cancer, 7% for bone cancer, 2% for leukemia, 2% for ACC, and 26% for others. Overall survival is approximately 80% by age 50. TP53- families: The underlying etiology contributing to cancer risk in the TP53- families is not known at this time; therefore, for the estimation of cancer risk among these families, we included all bloodline members belonging to the lineage contributing to the eligibility criteria. Among the 522 individuals from the 27 TP53- families, the cumulative risk to first cancer diagnosis is 7% by age 30 and 45% by age 70. The lifetime cumulative risk is higher for breast, brain, lung cancer, and melanoma compared with other cancer types. Overall survival is approximately 90% by age 50. Conclusion: We report the cumulative cancer incidence and overall survival among TP53 mutation carriers in TP53+ families as well as among members of TP53- families. Identification of additional causative gene(s) in TP53- families will help refine the cancer risk of family members. We are evaluating the risks of second primary cancers, and whether the rate is dependent on the type of the first cancer in all families. Citation Format: Phuong L. Mai, Philip S. Rosenberg, June A. Peter, Rosamma DeCastro, Payal P. Khincha, Jennifer T. Loud, Renee Bremer, Sharon A. Savage. Cumulative cancer risk in the NCI Li-Fraumeni Syndrome Cohort. [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 2749. doi:10.1158/1538-7445.AM2015-2749

Abstract 2762: LFSpro: A risk assessment tool to estimate TP53 mutation status in families with Li-Fraumeni syndrome

Abstract Li-Fraumeni syndrome (LFS) is a rare disorder and characterized by autosomal dominant mutation inheritance with frequent occurrence of multiple cancer types including soft tissue/bone sarcoma, breast cancer, leukemia, brain tumors and many others. A majority of LFS is caused by germ-line mutations in p53 tumor suppressor gene. Given the diverse cancer spectrum and incidence in Li-Fraumeni syndrome (LFS) and limitations of the clinical criteria, accurate identification of candidates for prospective TP53 mutation testing has been difficult. A more efficient prediction tool is needed for TP53 mutation carrier identification, which, followed by appropriate management and screening, may ultimately decrease mortality. To this date, we developed LFSpro, which is built on a Mendelian model and estimates TP53 mutation probability through the Elston-Stewart algorithm. With independent validation data from 765 families (19,530 individuals), we demonstrated the accurate prediction of TP53 mutation status using LFSpro. Family history of cancer evolves, and LFSpro is sensitive to mutation carriers in families newly presenting in high-risk clinics and in families followed for years. It is more broadly applicable than the current clinical criteria and therefore may improve clinical management for families with LFS. Citation Format: Gang Peng, Jasmina Bojadzieva, Mandy L. Ballinger, David M. Thomas, Louise Strong, Wenyi Wang. LFSpro: A risk assessment tool to estimate TP53 mutation status in families with Li-Fraumeni syndrome. [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 2762. doi:10.1158/1538-7445.AM2015-2762

Surveillance recommendations for patients with germline TP53 mutations.

Li-Fraumeni syndrome is associated with germline TP53 mutations and carriers have a high lifetime risk of cancer, the most common being sarcoma, breast cancer, brain tumors, adrenocortical carcinoma and leukemia. Germline TP53 mutation carriers are increasingly being identified as more genomic sequencing is performed in both clinical and research settings. There is a pressing clinical need for effective cancer risk management approaches in this group. Current clinical surveillance guidelines mainly focus on breast and bowel cancer risk with little consideration for the other cancers common to the syndrome. Imaging technologies are such that the utilization of whole-body MRI imaging for surveillance is viable. Globally, several research groups have included whole-body MRI along with other diagnostic measures in formulating surveillance protocols for TP53 mutation carriers. Early reports suggest a survival benefit. Surveillance protocols for TP53 mutation carriers have the potential to improve outcomes in individuals and families. Further research is needed to guide the development of an effective and comprehensive surveillance schedule.

Genotype Versus Phenotype: The Yin and Yang of Germline TP53 Mutations in Li-Fraumeni Syndrome

Genotype Versus Phenotype: The Yin and Yang of Germline <i>TP53</i> Mutations in Li-Fraumeni Syndrome

APR-246 overcomes resistance to cisplatin and doxorubicin in ovarian cancer cells.

Two main causes of platinum resistance are mutation in the tumor suppressor gene TP53 and drug-induced increase in intracellular glutathione concentration. Mutations in TP53 occur in about 50% of human tumors. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53 and induces apoptosis. APR-246 is a prodrug that is converted to the active compound methylene quinuclidinone (MQ), a Michael acceptor that binds to cysteine residues in mutant p53 and restores its wild-type conformation. Here, we show that MQ also binds to cysteine in glutathione, thus decreasing intracellular free glutathione concentration. We also show that treatment with APR-246 completely restores the cisplatin and doxorubicin sensitivity to p53-mutant drug-resistant ovarian cancer cells. We propose that this unique ability of APR-246/MQ to bind to cysteines in both mutant p53 and glutathione has a key role in the resensitization as well as in the outstanding synergistic effects observed with APR-246 in combination with platinum compounds in ovarian cancer cell lines and primary cancer cells. However, MQ binding to cysteines in other targets, for example, thioredoxin reductase, may contribute as well. Strong synergy was also observed with the DNA-damaging drugs doxorubicin and gemcitabine, while additive effects were found with the taxane docetaxel. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with platinum-based therapy in patients with p53-mutant recurrent high-grade serous (HGS) ovarian cancer. More than 96% of these patients carry TP53 mutations. Combined treatment with APR-246 and platinum or other DNA-damaging drugs could allow dramatically improved therapy of a wide range of therapy refractory p53 mutant tumors.

Abstract P4-12-06: Results of next-generation sequencing panels in a large community-based hereditary cancer risk program

Abstract Background: Next-generation sequencing (NGS) allows for broader germline genetic testing for hereditary cancers. Since the Supreme Court decision of AMP v. Myriad on June 13, 2013, hereditary cancer multi-gene panels can now include BRCA1 and BRCA2, making these panels an option for first-tier testing. However, questions have been raised about the clinical utility and implications of extended panels for medical management given the inclusion of unknown to moderate penetrant genes. Methods: We reviewed all patients who underwent multi-gene panel testing from July 1, 2013 through May 23, 2014. The indications for testing included personal and/or family history of breast or ovarian cancer. The panels were ordered in a single genetic counseling clinic within a large community-based cancer center. Results: A total of 136 patients underwent panel testing via a single commercial laboratory. We identified 12 (8.8%) patients who were positive for a pathogenic or likely pathogenic mutation in a cancer susceptibility gene; 4 had prior negative BRCA1 and BRCA2 sequencing and deletion/duplication testing. These positive results included 4 BRCA2 mutations, 2 TP53 mutations, 1 CDH1 mutation, 2 ATM mutations, and 1 patient each with a CHEK2, NBN, or PALB2 mutation. Of the patients found to have a positive test result, 100% met the National Comprehensive Cancer Network (NCCN) guidelines for Hereditary Breast and Ovarian Cancer (HBOC) genetic testing. The CDH1 mutation carrier did not meet NCCN guidelines for hereditary diffuse gastric cancer testing and only one of the TP53 mutation carriers met NCCN guidelines for Li-Fraumeni syndrome. Within our cohort (136), 21 (15.4%) patients had a total of 25 variants of uncertain significance (VUS) and 103 (75.7%) patients had negative test results. Conclusion: Testing through NGS panels identified 7/12 (58%) patients with a mutation which led to changes in current medical management and 3/7 (43%) had a mutation in a gene other than BRCA1 or BRCA2. Our findings suggest that there is clinical utility of NGS panels for use in this patient population despite the inclusion of unknown to moderate penetrant genes and a higher rate of VUS than single gene testing. Citation Format: J L Blum, C A Garby, A E Simmons, S A Walker, L E Panos. Results of next-generation sequencing panels in a large community-based hereditary cancer risk program [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 P4-12-06.

Germline TP53 mutational spectrum in French Canadians with breast cancer.

BackgroundSpecific germline mutations in the hereditary breast-ovarian cancer susceptibility (HBC/HBOC) genes, BRCA1, BRCA2 and PALB2, have been shown to recur in French Canadians of Quebec, Canada, and this has been attributed to common ancestors. Germline TP53 mutation carriers are known to segregate in Li-Fraumeni syndrome families, which feature young age of onset breast cancer. We have reported rare TP53 mutation carriers in French Canadian HBC families, though none recurred possibly due to the limited number of cancer families investigated. Here we describe TP53 germline mutations found in French Canadian cancer families provided from hereditary cancer clinics; investigate 37 new BRCA1 and BRCA2 mutation-negative HBC/HBOC families for the TP53 mutations; and assess the frequency of TP53 mutations in a 1235 French Canadian breast cancer cases not selected for family history of cancer.MethodsTP53 mutation-positive pedigrees from French Canadian cancer families were provided from local hereditary cancer clinics. Bidirectional Sanger sequencing of all protein encoding exons of TP53 was performed using peripheral blood lymphocyte DNA from breast/ovarian cancer probands from 37 HBC/HBOC families of French Canadian descent. Targeted bidirectional Sanger sequencing assay of regions containing the identified TP53 mutations was performed on 1235 French Canadian breast cancer cases not selected for family history cancer.ResultsFive new TP53 mutations were identified in six pedigrees from hereditary cancer clinics. No deleterious mutations were identified in cancer probands from 37 HBC/HBOC families. A targeted mutation screen of the 1235 breast cancer cases identified a c.844C>T [p.Arg282Trp] mutation carrier. This mutation was also found among the six mutation-positive cancer families provided by the local hereditary cancer clinics. The targeted screen also uncovered a new TP53 mutation, c.685T>C [p.Cys229Arg] that was found in two breast cancer cases. All TP53 mutation carriers were among the 656 women with breast cancer diagnosed less than 50 years of age.ConclusionsIn all six new TP53 mutations were identified in French Canadians, where two each occurred in independently ascertained cases/families. Although all newly identified breast cancer mutation carriers reported a family history of cancer, none were consistent with features of Li-Fraumeni syndrome families.

(18)F-FDG PET-CT for Surveillance of Brazilian Patients with Li-Fraumeni Syndrome.

Purpose: To evaluate the effectiveness of 18F-Fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) for detecting early cancer in carriers of germline TP53 mutation, the genetic defect underlying Li-Fraumeni and related syndromes, which predisposes to many forms of cancer throughout life.Patients and methods: A total of 30 adult patients from six families with germline TP53 mutations were recruited. These patients did not have a diagnosis of cancer in the 24 months preceding the study. Anomalous concentrations from whole-body 18F-FDG-PET/CT were assessed by two independent experts. Suspicious lesions were excised and subjected to pathological examination.Results: A total of 6/30 patients showed abnormal 18F-FDG-concentration. Confirmation studies revealed three cases of cancer, including one lung cancer, one ovarian cancer, and one disseminated breast cancer. Three patients had non-malignant lesions (one Bartholin’s cyst and two cases of reactive lymph nodes).Conclusion: 18F-FDG-PET/CT is effective in detecting cancer in subjects who are asymptomatic according to current screening guidelines. These results further suggest that 18F-FDG-PET/CT is an appropriate method for surveillance of cancer risk in TP53 mutation carriers.

Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression.

Genomic instability (IN) is a common feature of many human cancers. The TP53 tumour suppressor gene is mutated in approximately half of human cancers. Here, we show that BRCA1 and RAD17 genes, whose derived proteins play a pivotal role in DNA damage repair, are transcriptional targets of gain-of-function mutant p53 proteins. Indeed, high levels of mutp53 protein facilitate DNA damage accumulation and severely impair BRCA1 and RAD17 expression in proliferating cancer cells. The recruitment of mutp53/E2F4 complex onto specific regions of BRCA1 and RAD17 promoters leads to the inhibition of their expression. BRCA1 and RAD17 mRNA expression is reduced in HNSCC patients carrying TP53 mutations when compared to those bearing wt-p53 gene. Furthermore, the analysis of gene expression databases for breast cancer patients reveals that low expression of DNA repair genes correlates significantly with reduced relapse free survival of patients carrying TP53 gene mutations. Collectively, these findings highlight the direct involvement of transcriptionally active gain of function mutant p53 proteins in genomic instability through the impairment of DNA repair mechanisms.

The breast cancer immunophenotype of TP53-p.R337H carriers is different from that observed among other pathogenic TP53 mutation carriers.

Germline TP53 mutations are associated with Li-Fraumeni syndrome, an autosomal dominant disorder characterized by a predisposition to multiple early-onset cancers including breast cancer (BC), the most prevalent tumor among women. The majority of germline TP53 mutations are clustered within the DNA-binding domain of the gene, disrupting the structure and function of the protein. A specific germline mutation in the tetramerization domain of p53, p.R337H, was reported at a high frequency in Southern and Southeastern Brazil. This mutation appears to result in a more subtle defect in the protein, which becomes functionally deficient only under particular conditions. Recent studies show that the BC phenotype in TP53 mutation carriers is often HER2 positive (63-83%). Considering that the immunophenotype of BC among p.R337H carriers has not been reported, we reviewed immunohistochemistry data of 66 p.R337H carriers in comparison with 12 patients with other non-functional TP53 germline mutation. Although 75% of carriers of these mutations showed significant HER2 overexpression (3+), corroborating previous studies, only 22.7% of p.R337H patients had BC overexpressing HER2. These results reinforce the notion that different germline mutations in TP53 may predispose to BC via different mechanisms.

A functional variant in miR-605 modifies the age of onset in Li-Fraumeni syndrome

Li-Fraumeni syndrome (LFS) is a rare cancer predisposition syndrome, typically associated with germline mutations in the TP53 gene. Despite the high penetrance of TP53 mutations, LFS patients display striking phenotypic differences, suggesting the presence of secondary risk loci. To date, all genetic modifiers in LFS have been shown to map to either TP53 or its principal negative regulator, MDM2. Given this strong association, we set out to investigate the contribution of miR-605, a recently described microRNA (miRNA) regulator of the p53-MDM2 loop. We hypothesized that, if functional, the miR-605 gene and its variant (rs2043556) could impact the cancer risk profile of TP53 mutation carriers. Consistent with this proposition, the variant G-allele of miR-605 was associated with a 10-year acceleration in the mean age of LFS tumor onset (P = 0.04) and caused a 2.6-fold reduction in the processing levels of its host miRNA (P < 0.05). We also demonstrate that miR-605 overexpression leads to a decrease in cell proliferation, clonogenicity, and migration in two rhabdomyosarcoma cell lines carrying hotspot TP53 mutations. Together, our results implicate miR-605 as a novel modifier gene of the LFS phenotype and a promising therapeutic target in TP53 mutant cancers.

Next Generation Sequencing (NGS) of TP53 Gene Allows to Identify a Very High Risk Group of Philadelphia Negative Acute Lymphoblastic Leukemia (ALL) Patients

Background and Aim of the StudyFor both childhood and adult Acute Lymphoblastic Leukemia (ALL) patients, clinical risk factors such as age, white cell count, response to steroids, time to complete remission, as well as biologic characteristics such as immunophenotype and cytogenetic at diagnosis are important but not sufficient in predicting clinical outcome. Aberrations of TP53 play a crucial role in the molecular pathogenesis of leukemias and lymphomas in which their presence is associated to disease progression and represents a strong predictor of poor clinical outcome. In childhood ALL, hereditary and acquired TP53 mutations are involved both in the pathogenesis and progression of the disease. In adult ALL, TP53 mutations are frequent in patients negative for recurrent fusion genes and correlate with poor response to induction therapy (Chiaretti S. et al, Haematologica 2013). The aim of this study was to evaluate the impact of TP53 alterations, analyzed by Next Generation Sequencing (NGS), on the outcome of a cohort of T (n= 57) and B (n= 114) precursor, Philadelphia (Ph) negative, adult ALL patients enrolled into the NILG-ALL 09/2000 clinical trial (ClinicalTrials.gov identifier: NCT00358072, Bassan R. et al, Blood 2009) in which molecular minimal residual disease was used to guide post-remissional therapy.Patients and Study designAmong the 171 patients who were investigated for TP53 mutations, 16 proved also positive for t(4;11) and 3 for t(1;19). We analyzed DNA isolated from mononuclear cells obtained from bone marrow or peripheral blood samples containing at least 30% of blasts at diagnosis. The TP53 gene was sequenced using 454 ultra-deep sequencing (Roche Diagnostics) for alterations in exons 4 to 11, following the protocol developed in the IRON-II consortium. The sequencing data were analyzed by the Roche Diagnostics GS Run Browser and GS Amplicon Variant Analyzer software. The probabilities of survival were estimated using the Kaplan Meier method. The log-rank test was used to compare survival probabilities between subgroups of patients.Results and DiscussionThe data obtained by NGS allowed to identify 15 coding mutations detected in the DNA binding domain region (exons 5 to 8). These alterations were observed at diagnosis in 14 patients (8%), (11 B-precursor ALL and 3 T-ALL). In 12 cases these aberrations were single nucleotide changes, in 2 cases we found a duplication (one of 4 and the other of 8 nucleotides) and in one case there was an 11 base pair DNA insertion. Remarkably, all of these DNA alterations led to missense or frame-shift mutations that introduced a premature stop codon. Moreover, they were detected with a wide range of allele burden (from 5% to 97%) pointing out that TP53 mutations can be present at diagnosis in different proportions within the leukemic clones. All patients carrying a TP53 alteration reached complete remission after induction therapy but 13 out of 14 suffered an early relapse. Frequency of relapses was significantly higher in mutated than in wild-type cases (p=0.019). Relapse DNA samples were available in 3 patients and in all of them we detected the same TP53 mutation found at diagnosis, indicating the presence of a stable mutated clone. The univariate analysis enlightens a clear relationship between TP53 mutation with an increasing age (p= 0.0003) but no correlation with other clinical features such as gender, hemoglobin, white blood count, platelets, percentage of blasts and cytogenetics at diagnosis. Moreover, patients with mutated TP53 showed a Disease Free Survival (DFS) and Overall Survival (OS) dramatically shorter than wild-type patients. The 2 years DFS was 43% in the TP53 non-mutated subjects compared to 7% in the mutated (p=0.0007). Similarly, the 2 years OS was of 50% in wild-type patients and of 7% in mutated patients (p=0.0011) (Figure 1).ConclusionsIn adult ALL, response to induction chemotherapy is not different in patients with a wild-type or a TP53 mutated gene, but in these latter cases the leukemia relapse rate is dramatically higher. The frequency of these mutations observed at diagnosis and the poor clinical outcome indicate the need of their identification during the diagnostic work up of adult ALL to guide treatment strategies. The use of a highly sensitive deep sequencing approach is crucial to identify also minor leukemic clones carrying TP53 mutations that may lead to the rapid emergence of a treatment resistant disease. [Display omitted] DisclosuresKohlmann:AstraZeneca: Employment.

TP53 mutational status is a potential marker for risk stratification in Wilms tumour with diffuse anaplasia.

PurposeThe presence of diffuse anaplasia in Wilms tumours (DAWT) is associated with TP53 mutations and poor outcome. As patients receive intensified treatment, we sought to identify whether TP53 mutational status confers additional prognostic information.Patients and MethodsWe studied 40 patients with DAWT with anaplasia in the tissue from which DNA was extracted and analysed for TP53 mutations and 17p loss. The majority of cases were profiled by copy number (n = 32) and gene expression (n = 36) arrays. TP53 mutational status was correlated with patient event-free and overall survival, genomic copy number instability and gene expression profiling.ResultsFrom the 40 cases, 22 (55%) had TP53 mutations (2 detected only after deep-sequencing), 20 of which also had 17p loss (91%); 18 (45%) cases had no detectable mutation but three had 17p loss. Tumours with TP53 mutations and/or 17p loss (n = 25) had an increased risk of recurrence as a first event (p = 0.03, hazard ratio (HR), 3.89; 95% confidence interval (CI), 1.26–16.0) and death (p = 0.04, HR, 4.95; 95% CI, 1.36–31.7) compared to tumours lacking TP53 abnormalities. DAWT carrying TP53 mutations showed increased copy number alterations compared to those with wild-type, suggesting a more unstable genome (p = 0.03). These tumours showed deregulation of genes associated with cell cycle and DNA repair biological processes.ConclusionThis study provides evidence that TP53 mutational analysis improves risk stratification in DAWT. This requires validation in an independent cohort before clinical use as a biomarker.

Abstract 1104: The role of DLL4 in cancer immune-surveillance and its pathogenicity in p53-dysregulated environment

Abstract Recent advances in cancer studies reveal that tumorigenesis results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. Li-Fraumeni syndrome (LFS) is a clinically and genetically heterogeneous inherited cancer syndrome characterized by mutation of the TP53 tumor suppressor gene (locus 17p13.1). The p53 transcription factor responds to diverse cellular stresses by regulating target genes that induce apoptosis, cell cycle arrest, DNA repair, and senescence. LFS can provide powerful insights into our understanding of somatic mutations present in sporadic cancers as well as the de-regulation of cell signaling pathways. We will be using LFS cells and mice as models to build on our previous discovery that showed a balanced reciprocal translocation between chromosomes 11q23 and 15q15 in LFS patients' skin fibroblast cells. In our present study, analysis of the breakpoint region reveals that in LFS, breast cancer (MCF7) and neuroblastoma (IMR32) cell lines, there is a dramatically increased activation in the expression of cancer susceptibility candidate 5 gene (CASC5), a part of kinetochore-microtubule attachments associated with cell proliferation. Furthermore, there was an abrogation in the expression of a critical protein, Delta-like ligand-4 (DLL4), in LFS normal skin fibroblasts of TP53 mutation carriers and non-carriers. There was also a down-regulation of DLL4 in unrelated breast cancer and neuroblastoma cell lines by DNA hypermethylation. DLL4 is a ligand for Notch family of receptors with crucial functions in normal development, immune surveillance and tissue homeostasis. TP53 and CTCF (a transcription factor involved in chromatin architecture) have a direct activator effect at the DLL4 promoter. Notch/DLL4 plays a key role in the generation of hematopoietic stem cells during embryonic development and regulates the T cell lineage and subsequent stages of thymopoiesis. WNT2 is also down-regulated in LFS cell lines and abolished in MCF7. Whereas Notch signaling controls cell fate specification in a variety of cell contexts during embryonic and postnatal development, Wnt signaling plays a critical role in cell differentiation and proliferation. Both Notch and Wnt pathways are activated by oncogenic mutations and dysregulated in cancer. The long-term objective of this research is to develop a critical understanding of the mechanisms by which p53 regulates cancer immune-surveillance through cross-talk with the tumor microenvironment so that therapeutic agents can be developed to curtail cancer progression. Citation Format: Zhixing Yao, Zaki A. Sherif. The role of DLL4 in cancer immune-surveillance and its pathogenicity in p53-dysregulated environment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1104. doi:10.1158/1538-7445.AM2014-1104

PIN3 duplication may be partially responsible for TP53 haploinsufficiency.

BackgroundPreviously we have suggested that cancer cells develop a mechanism(s) which allows for either: silencing of the wild-type TP53 transcription, degradation of the wild-type TP53 mRNA, or selective overproduction of the mutated TP53 mRNA, which is the subject of this article. Sequencing of TP53 on the respective cDNA and DNA templates from tumor samples were found to give discordant results. DNA analysis showed a pattern of heterozygous mutations, whereas the analysis of cDNA demonstrated the mutated template only. We hypothesized that different TP53 gene expression levels of each allele may be caused by the polymorphism within intron 3 (PIN3). The aim of this study was to test if one of the polymorphic variants of PIN3 (A1 or A2) in the heterozygotes is associated with a higher TP53 expression, and therefore, responsible for the haploinsufficiency phenomenon.Methods250 tumor samples were tested. To analyze the involvement of PIN3 polymorphic variant (A1 or A2) on TP53 mRNA expression regulation, bacterial subcloning combined with sequencing analyses, dual luciferase reporter assays and bioinformatic analysis were performed.ResultsHaplotype analysis showed the predominance of the mutated template during the cDNA sequencing in all samples showing a heterozygous TP53 mutation and PIN3 heterozygosity. Out of 30 samples (from the total of 250 tested samples) which carried TP53 mutations and had a bias in allelic expression 6 were heterozygous for the A1/A2 polymorphism, and all 6 (p = 0.04) samples carried the mutation within the PIN3 longer allele (A2). Reporter assays revealed higher luciferase activity in cells transfected with the plasmid containing A2 construct than A1 and control. A2/A1 ratio ranged from 1.16 for AD293 cell line (p = 0.019) to 1.59 for SW962 cell line (p = 0.0019). Moreover, bioinformatic analyses showed that PIN3 duplication stabilized secondary DNA structures – G-quadruplexes.ConclusionTP53 alleles are not equivalent for their impact on the regulation of expression of TP53 mRNA. Therefore, in PIN3-heterozygous cases a single TP53 mutation of the longer allele might sufficiently destabilize its function. Secondary DNA structures such as quadruplexes can also play a role in PIN3-dependent TP53 haploinsufficiency.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-669) contains supplementary material, which is available to authorized users.