TP53 Mutations in Human Cancers: Origins, Consequences, and Clinical Use

Somatic mutations in the TP53 gene are one of the most frequent alterations in human cancers, and germline mutations are the underlying cause of Li-Fraumeni syndrome, which predisposes to a wide spectrum of early-onset cancers. Most mutations are single-base substitutions distributed throughout the coding sequence. Their diverse types and positions may inform on the nature of mutagenic mechanisms involved in cancer etiology. TP53 mutations are also potential prognostic and predictive markers, as well as targets for pharmacological intervention. All mutations found in human cancers are compiled in the IARC TP53 Database (http://www-p53.iarc.fr/). A human TP53 knockin mouse model (Hupki mouse) provides an experimental model to study mutagenesis in the context of a human TP53 sequence. Here, we summarize current knowledge on TP53 gene variations observed in human cancers and populations, and current clinical applications derived from this knowledge.Genetic variations in the tumor suppressor gene TP53 (OMIM #191117) contribute to human cancers in different ways. First, somatic mutations are frequent in most cancers (Hollstein et al. 1991). The antiproliferative role of p53 protein in response to various stresses and during physiological processes such as senescence makes it a primary target for inactivation in cancer (Levine 1997). The main modes of TP53 inactivation are single-base substitution and loss of alleles, with inactivation by viral or cellular proteins playing a major role in specific cancers (Tommasino et al. 2003). Second, inheritance of a TP53 mutation causes predisposition to early-onset cancers including breast carcinomas, sarcomas, brain tumors, and adrenal cortical carcinomas, defining the Li-Fraumeni (LFS) and Li-Fraumeni-like (LFL) syndromes (Li et al. 1988; Olivier et al. 2003). Third, TP53 is highly polymorphic in coding and noncoding regions and some of these polymorphisms have been shown to increase cancer susceptibility and to modify cancer phenotypes in TP53 mutation carriers (Whibley et al. 2009).Whereas tumor suppressors are commonly inactivated by frameshift or nonsense mutations, most TP53 mutations are missense and cause single amino-acid changes at many different positions. Mutations are thus diverse in their type, sequence context, position, and structural impact, making it possible to identify mutation patterns in relation with cancer type and etiology. The occurrence of special mutation patterns may inform on the nature of the mutagens that have caused them, making TP53 an interesting gene to analyze in the realm of molecular epidemiology.Data on mutation prevalence in human cancer can be conveniently accessed through the IARC TP53 database (http://www-p53.iarc.fr/), a resource that compiles all TP53 gene variations reported in human cancers with annotations on tumor phenotype, patient characteristics, and structural and functional impact of mutations (Petitjean et al. 2007b). Recently, it has become possible to confront these observations with experimental data generated in a novel mouse model, the HupKi mouse, that contains a human TP53 sequence at the mouse TP53 locus and recapitulates the effects of environmental mutagens in a human sequence context (Luo et al. 2001). In this article, we review the current knowledge on the origin, causes, and consequences of TP53 variations and mutations in cancer and we discuss their significance as biomarkers in epidemiology and in the clinics.     

Mutational spectra of human cancer

The purpose of this review is to summarize the evidence that can be used to reconstruct the etiology of human cancers from mutations found in tumors. Mutational spectra of the tumor suppressor gene p53 (TP53) are tumor specific. In several cases, these mutational spectra can be linked to exogenous carcinogens, most notably for sunlight-associated skin cancers, tobacco-associated lung cancers, and aristolochic acid-related urothelial tumors. In the TP53 gene, methylated CpG dinucleotides are sequences selectively targeted by endogenous and exogenous mutagenic processes. Recent high-throughput sequencing efforts analyzing a large number of genes in cancer genomes have so far, for the most part, produced mutational spectra similar to those in TP53 but have unveiled a previously unrecognized common G to C transversion mutation signature at GpA dinucleotides in breast cancers and several other cancers. Unraveling the origin of these G to C mutations will be of importance for understanding cancer etiology.     

Gaining insights into relevance across cancers based on mutation features of TP53 gene

The tumor suppressor gene TP53, one of the most frequently mutated genes, is recognized as the guardian of genome and can provide a significant barrier to neoplastic transformation and tumor progression. Traditional theory believes that TP53 mutations are equal among cancer types. However, to date, no study has explored the TP53 mutation profile from a holistic and systematic standpoint to discovery its relevance and feature with cancers. Mutation signature, an unbiased approach to identify the mutational processes, can be a potent indicator for exploring mutation-driven tumor occurrence and progression. In this research, several features such as hotspots, mutability and mutation signature of somatic TP53 mutations derived from 18 types of cancer tissues from cBioPortal were analyzed and manifested the organizational preference among cancers. Mutation signatures found in almost all cancer types were Signature 6 related to mismatch repair deficiency, and Signature 1 that reflects the natural decomposition of 5-methylcytosine into thymine associated with aging. Meanwhile, several signatures of TP53 mutations displayed tissue-selective. Mutations enriched in bladder, skin, lung cancer were associated with signatures of APOBEC activity (Signature 2 and 13), alkylating agents (Signature 11), and tobacco smoke (Signature 4), respectively. Moreover, Signature 4 and 29 associated with tobacco smoking or chewing found in lung, sarcoma, esophageal, and head and neck cancer may be related to their smoking history. In addition, several digestive cancers, including colorectal, stomach, pancreatic and esophageal cancers, showed the high correlation in context and mutation signature profiles. Our study suggests that the tissue-selective activity of mutational processes would reflect the tissue-specific enrichment of TP53 mutations and provides a new perspective to understand the relevance of diverse diseases based on the spectrum of TP53 mutations.     

Application of multiplex PCR with histopathologic features for detection of familial breast cancer in formalin-fixed, paraffin-embedded histologic specimens

Breast cancer is the most common malignancy among females in the world. Age and familial history are the major risk factors for the development of this disease in Iran. Mutations of BRCA1 and BRCA2 genes are associated with a greatly increased risk for development of familial breast cancer. Frequency of BRCA mutations was identified in familial breast cancers (FBC) and non-familial breast cancers (NFBC) by molecular genetics, morphological and Immunohistochemical methods. Thirty forth formalin-fixed, paraffin-embedded breast tissue tumors were analyzed from 16 patients with FBC and 18 patients with NFBC. Three 5382insC mutations detected by multiplex PCR in 16 familial breast cancers. Immunohistochemical method was used to detect estrogen receptor (ER) and progesterona receptor (PR) and TP53. Comparison of ER, PR and TP53 exhibited high difference (P < 0.0001) in familial breast cancers and non-familial breast cancers. Our results demonstrated that 5382insC mutation, ER, PR, TP53, mitotic activity, polymorphism, necrosis and tubules can serve as the major risk factors for the development of FBC.     

Polymorphisms in insulin-related genes predispose to specific KRAS2 and TP53 mutations in colon cancer

Risk factors for colon cancer may not only influence the overall risk of cancer but also the risk for specific types of mutations. We evaluated the effect of polymorphisms in four insulin-related genes (G972R in IRS1, G1057D in IRS2, a CA repeat in IGFI and an A/C polymorphism at -202 of IGFBP3) on the risk of microsatellite instability and KRAS2 and TP53 mutations in a population-based set of 1788 cases of colon cancer and 1981 controls. The GR/RR IRS1 genotypes were associated with an increased risk of colon cancers with the KRAS2 G12D mutation (OR 2.3, 95% CI 1.5, 3.5 versus controls, OR 1.7, 95% CI 1.1, 2.6 versus KRAS2 wild type), the "no 192" IGFI genotype increased the risk of the KRAS2 G13D mutation (OR 2.3, 95% CI 1.2, 4.2 versus controls, OR 2.1, 95% CI 1.1, 4.0 versus wild type), and the DD IRS2 genotype increased the risk of the G12V KRAS2 mutation (OR 1.8, 95% CI 0.9, 3.5 versus controls, OR 2.0, 95% CI 1.0, 4.0 versus wild type). Polymorphisms in IRS1 and IGF1 were also associated with an approximately two-fold increased risk of specific TP53 mutations relative to controls without cancer. We conclude that polymorphisms in some insulin-related genes are associated with an increased risk of colon cancer with specific KRAS2 and TP53 mutations, implying a link between these genetic changes and specific mutational pathways in carcinogenesis.     

Occurrence of Neuroblastoma among TP53 p.R337H Carriers

The high incidence of adrenocortical tumors and choroid plexus carcinoma in children from South and Southeastern regions of Brazil is associated with the germline p.R337H mutation of TP53 gene. The concomitant occurrence of neuroblastoma and adrenocortical tumors in pediatric patients harboring the p.R337H mutation at our institution prompted us to investigate the putative association between p.R337H and pediatric neuroblastoma. Genomic DNA samples from 83 neuroblastoma patients referred to a single institution during the period of 2000–2014 were screened for the p.R337H mutation. Available samples from carriers were investigated for both nuclear p53 accumulation and loss of heterozigosity in tumor. Clinical data were obtained from medical records in order to assess the impact of 337H allele on manifestation of the disease. Seven out 83 neuroblastoma patients (8.4%) were carriers of the TP53 p.R337H mutation in our cohort. Immunohistochemical analysis of p.R337H-positive tumors revealed nuclear p53 accumulation. Loss of heterozigosity was not found among available samples. The presence of 337H allele was associated with increased proportion of stage I tumors. Our data indicate that in addition to adrenocortical tumors, choroid plexus carcinoma, breast cancer and osteosarcoma, genetic counseling and clinical surveillance should consider neuroblastoma as a potential neoplasia affecting p.R337H carriers.     

Application of multiplex PCR with histopathologic features for detection of familial breast cancer in formalin-fixed,paraffin-embedded histological specimens

Breast cancer is the most common malignancy among females in the world. In Iran, age and family history are the major risk factors for the development of this disease. Mutations of BRCA1 and BRCA2 genes are associated with a greatly increased risk for familial breast cancer. The frequency of BRCA mutations was identified in familial breast cancers (FBCs) and nonfamilial breast cancers (NFBCs) by molecular genetics, and morphological and immunohistochemical methods. Thirty-four formalin-fixed, paraffin-embedded breast tissue tumors were analyzed from 16 patients with FBCs and 18 patients with NFBCs. Three 5382insC mutations were detected by multiplex PCR in 16 FBCs. The immunohistochemical method was used to detect estrogen receptors (ER), progesterona receptors (PR), and TP53. Comparison of ER, PR, and TP53 exhibited a high difference (P < 0.0001) in FBCs and NFBCs. Our results demonstrated that 5382insC mutation, ER, PR, TP53, mitotic activity, polymorphism, necrosis and tubules can serve as the major risk factors for FBC. The text was submitted by the authors in English.     

TP53 and mutations in human cancer

TP53 is the most frequently mutated gene in human cancer, with a predominance of missense mutations scattered over 200 codons. In many cancers, specific mutation patterns can be identified, which are shaped by site-specific mutagenesis and by biological selection. In tobacco-related cancers (lung, head and neck), organ-specific patterns are observed, with many mutations compatible with the ones experimentally induced by tobacco carcinogens. In several other cancers, such as squamous cell carcinoma of the oesophagus or hepatocellular carcinoma (HCC), mutation patterns show geographic variations between regions of high and low incidence, suggesting a role for region-specific risk factors. HCC from high-incidence regions showing also a high prevalence of a specific Ser-249 TP53 mutation is one of the most striking examples of a mutagen fingerprint. All such assessments are useful to generate clues on the mutagenic mechanisms involved in human cancer. Moreover, it has been shown that DNA retrieved from plasma can be successfully used for detection of TP53 mutations, which gives hope for earlier more accurate detection of human cancers.     

Molecular Characterization of an Intact p53 Pathway Subtype in High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) is the most aggressive histological type of epithelial ovarian cancer, which is characterized by a high frequency of somatic TP53 mutations. We performed exome analyses of tumors and matched normal tissues of 34 Japanese patients with HGSOC and observed a substantial number of patients without TP53 mutation (24%, 8/34). Combined with the results of copy number variation analyses, we subdivided the 34 patients with HGSOC into subtypes designated ST1 and ST2. ST1 showed intact p53 pathway and was characterized by fewer somatic mutations and copy number alterations. In contrast, the p53 pathway was impaired in ST2, which is characterized by abundant somatic mutations and copy number alterations. Gene expression profiles combined with analyses using the Gene Ontology resource indicate the involvement of specific biological processes (mitosis and DNA helicase) that are relevant to genomic stability and cancer etiology. In particular we demonstrate the presence of a novel subtype of patients with HGSOC that is characterized by an intact p53 pathway, with limited genomic alterations and specific gene expression profiles.     

Identification of a Comprehensive Spectrum of Genetic Factors for Hereditary Breast Cancer in a Chinese Population by Next-Generation Sequencing

The genetic etiology of hereditary breast cancer has not been fully elucidated. Although germline mutations of high-penetrance genes such as BRCA1/2 are implicated in development of hereditary breast cancers, at least half of all breast cancer families are not linked to these genes. To identify a comprehensive spectrum of genetic factors for hereditary breast cancer in a Chinese population, we performed an analysis of germline mutations in 2,165 coding exons of 152 genes associated with hereditary cancer using next-generation sequencing (NGS) in 99 breast cancer patients from families of cancer patients regardless of cancer types. Forty-two deleterious germline mutations were identified in 21 genes of 34 patients, including 18 (18.2%) BRCA1 or BRCA2 mutations, 3 (3%) TP53 mutations, 5 (5.1%) DNA mismatch repair gene mutations, 1 (1%) CDH1 mutation, 6 (6.1%) Fanconi anemia pathway gene mutations, and 9 (9.1%) mutations in other genes. Of seven patients who carried mutations in more than one gene, 4 were BRCA1/2 mutation carriers, and their average onset age was much younger than patients with only BRCA1/2 mutations. Almost all identified high-penetrance gene mutations in those families fulfill the typical phenotypes of hereditary cancer syndromes listed in the National Comprehensive Cancer Network (NCCN) guidelines, except two TP53 and three mismatch repair gene mutations. Furthermore, functional studies of MSH3 germline mutations confirmed the association between MSH3 mutation and tumorigenesis, and segregation analysis suggested antagonism between BRCA1 and MSH3. We also identified a lot of low-penetrance gene mutations. Although the clinical significance of those newly identified low-penetrance gene mutations has not been fully appreciated yet, these new findings do provide valuable epidemiological information for the future studies. Together, these findings highlight the importance of genetic testing based on NCCN guidelines and a multi-gene analysis using NGS may be a supplement to traditional genetic counseling.     

Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors

P53 is one of the most important tumor suppressor proteins in human cancers. Mutations in the TP53 gene are common features of malignant tumors and normally correlate to a more aggressive disease. In breast cancer, these gene alterations are present in approximately 20% of cases and are characteristically of missense type. In the present work we describe TP53 mutations in breast cancer biopsies and investigate whether wild and mutant p53 participate in protein aggregates formation in these breast cancer cases. We analyzed 88 biopsies from patients residing in the metropolitan area of Rio de Janeiro, and performed TP53 mutation screening using direct sequencing of exons 5-10. Seventeen mutations were detected, 12 of them were of missense type, 2 nonsenses, 2 deletions and 1 insertion. The presence of TP53 mutation was highly statistically associated to tumor aggressiveness of IDC cases, indicated here by Elston Grade III (p<0.0001). Paraffin embedded breast cancer tissues were analyzed for the presence of p53 aggregates through immunofluorescence co-localization assay, using anti-aggregate primary antibody A11, and anti-p53. Our results show that mutant p53 co-localizes with amyloid-like protein aggregates, depending on mutation type, suggesting that mutant p53 may form aggregates in breast cancer cells, in vivo.     

p53 and disease: when the guardian angel fails

The p53 tumor suppressor gene (TP53) is mutated more often in human cancers than any other gene yet reported. Of importance, it is mutated frequently in the common human malignancies of the breast and colorectum and also, but less frequently, in other significant human cancers such as glioblastomas. There is also one inherited cancer predisposing syndrome called Li-Fraumeni that is caused by TP53 mutations. In this review, we discuss the significance of p53 mutations in some of the above tumors with a view to outlining how p53 contributes to malignant progression. We also discuss the usefulness of TP53 status as a prognostic marker and its role as a predictor of response to therapy. Finally, we outline some evidence that abnormalities in p53 function contribute to the etiology of other non-neoplastic diseases.     

Number of rare germline CNVs and <Emphasis Type="Italic">TP53</Emphasis> mutation types

Background

The Li-Fraumeni syndrome (LFS), an inherited rare cancer predisposition syndrome characterized by a variety of early-onset tumors, is caused by different highly penetrant germline mutations in the TP53 gene; each separate mutation has dissimilar functional and phenotypic effects, which partially clarifies the reported heterogeneity between LFS families. Increases in copy number variation (CNV) have been reported in TP53 mutated individuals, and are also postulated to contribute to LFS phenotypic variability. The Brazilian p.R337H TP53 mutation has particular functional and regulatory properties that differ from most other common LFS TP53 mutations, by conferring a strikingly milder phenotype.

Methods

We compared the CNV profiles of controls, and LFS individuals carrying either p.R337H or DNA binding domain (DBD) TP53 mutations by high resolution array-CGH.

Results

Although we did not find any significant difference in the frequency of CNVs between LFS patients and controls, our data indicated an increased proportion of rare CNVs per genome in patients carrying DBD mutations compared to both controls (p=0.0002***) and p.R337H (0.0156*) mutants.

Conclusions

The larger accumulation of rare CNVs in DBD mutants may contribute to the reported anticipation and severity of the syndrome; likewise the fact that p.R337H individuals do not present the same magnitude of rare CNV accumulation may also explain the maintenance of this mutation at relatively high frequency in some populations.

     

Sex-specific effect of the <Emphasis Type="Italic">TP53</Emphasis> PIN3 polymorphism on cancer risk in a cohort study of <Emphasis Type="Italic">TP53</Emphasis> germline mutation carriers

Germline mutations in the tumor suppressor gene TP53 occur in the majority of families with Li-Fraumeni syndrome, who are at an increased risk for a wide spectrum of early onset cancers. Several genetic polymorphisms in TP53 modify its effect on cancer risk. While some studies indicate that the TP53 PIN3 deletion allele (D) accelerate tumor onset in carriers with TP53 germline mutations, other studies have shown that the TP53 PIN3 insertion allele (I) confers a significantly higher risk of developing cancer than D allele. To further determine the effects of the TP53 PIN3 polymorphism on cancer development among TP53 germline mutations and to evaluate if those are differenence between male and female carriers, we studied a total of 152 germline mutation carriers with available DNA samples that can be used for genotyping. Our results indicate that the TP53 PIN3 polymorphism has a sex-specific effect on the risk of cancer in TP53 mutation carriers, conferring cancer risk in men (P = 0.0041) but not women with DI or II genotypes.     

Ancestry of the Brazilian TP53 c.1010G>A (p.Arg337His,R337H) Founder Mutation: Clues from Haplotyping of Short Tandem Repeats on Chromosome 17p

Rare germline mutations in TP53 (17p13.1) cause a highly penetrant predisposition to a specific spectrum of early cancers, defining the Li-Fraumeni Syndrome (LFS). A germline mutation at codon 337 (p.Arg337His, c1010G>A) is found in about 0.3% of the population of Southern Brazil. This mutation is associated with partially penetrant LFS traits and is found in the germline of patients with early cancers of the LFS spectrum unselected for familial history. To characterize the extended haplotypes carrying the mutation, we have genotyped 9 short tandem repeats on chromosome 17p in 12 trios of Brazilian p.Arg337His carriers. Results confirm that all share a common ancestor haplotype of Caucasian/Portuguese-Iberic origin, distant in about 72–84 generations (2000 years assuming a 25 years intergenerational distance) and thus pre-dating European migration to Brazil. So far, the founder p.Arg337His haplotype has not been detected outside Brazil, with the exception of two residents of Portugal, one of them of Brazilian origin. On the other hand, increased meiotic recombination in p.Arg337His carriers may account for higher than expected haplotype diversity. Further studies comparing haplotypes in populations of Brazil and of other areas of Portuguese migration are needed to understand the historical context of this mutation in Brazil.     

Immunogenomics Analysis Reveals that TP53 Mutations Inhibit Tumor Immunity in Gastric Cancer

Although immunotherapy continues to demonstrate efficacy in a variety of refractory cancers, currently, no any immunotherapeutic strategy is clinically used for gastric cancer (GC) except its microsatellite instable subtype. Thus, it is important to identify molecular biomarkers for predicting the responders to GC immunotherapy. TP53 mutations frequently occur in GC and are associated with unfavorable clinical outcomes in GC. We performed a comprehensive characterization of the associations between TP53 mutations and immune activities in GC based on two large-scale GC cancer genomics data. We compared expression and enrichment levels of 787 immune-related genes and 23 immune gene-sets among TP53-mutated GCs, TP53‐wildtype GCs, and normal tissue, and explored the correlations between p53-mediated pathways and immune activities in GC. Strikingly, almost all analyzed immune gene-sets were significantly downregulated in enrichment levels in TP53-mutated GCs compared to TP53‐wildtype GCs. These less active immune pathways and cell types in TP53-mutated GCs included 15 immune cell types and function, tumor-infiltrating lymphocytes, regulatory T cells, immune checkpoint, cytokine and cytokine receptor, human leukocyte antigen, pro‐inflammatory, and parainflammation. Moreover, we identified a number of p53-mediated pathways and proteins that were significantly associated with immune activities in GC. Furthermore, we demonstrated that the TP53 mutation itself could result in the depressed immune activities in GC and other cancer types. We revealed that chromosomal instability was an important mechanism for the depressed tumor immunity in TP53-mutated cancers. Finally, we showed that immune cell infiltration and immune activities were likely positively associated with survival prognosis in GC. Our findings suggest that p53 may play an important role in activating tumor immunity in GC and other cancer types and that the TP53 mutation status could be useful in stratifying cancer patients responsive to a certain immunotherapy.     

Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing

Single-cell sequencing is a powerful tool for delineating clonal relationship and identifying key driver genes for personalized cancer management. Here we performed single-cell sequencing analysis of a case of colon cancer. Population genetics analyses identified two independent clones in tumor cell population. The major tumor clone harbored APC and TP53 mutations as early oncogenic events, whereas the minor clone contained preponderant CDC27 and PABPC1 mutations. The absence of APC and TP53 mutations in the minor clone supports that these two clones were derived from two cellular origins. Examination of somatic mutation allele frequency spectra of additional 21 whole-tissue exome-sequenced cases revealed the heterogeneity of clonal origins in colon cancer. Next, we identified a mutated gene SLC12A5 that showed a high frequency of mutation at the single-cell level but exhibited low prevalence at the population level. Functional characterization of mutant SLC12A5 revealed its potential oncogenic effect in colon cancer. Our study provides the first exome-wide evidence at single-cell level supporting that colon cancer could be of a biclonal origin, and suggests that low-prevalence mutations in a cohort may also play important protumorigenic roles at the individual level.     

TP53 mutations in circulating tumor DNA in advanced epidermal growth factor receptor-mutant lung adenocarcinoma patients treated with gefitinib

Tumor protein p53 (TP53) is a tumor suppressor gene and TP53 mutations are associated with poor prognosis in non-small cell lung cancer. However, the in-depth classification of TP53 and its relationship with treatment response and prognosis in epidermal growth factor receptor (EGFR)-mutant tumors treated with EGFR tyrosine kinase inhibitors are unclear. Circulating tumor DNA was prospectively collected at baseline in advanced treatment-naïve EGFR-mutant lung adenocarcinoma patients treated with gefitinib in an open-label, single-arm, prospective, multicenter, phase 2 clinical trial (BENEFIT trial) and analyzed using next-generation sequencing. Survival was estimated using the Kaplan–Meier method. Of the 180 enrolled patients, 115 (63.9%) harbored TP53 mutations. The median progression-free survival (PFS) and overall survival (OS) of patients with TP53-wild type tumors were significantly longer than those of patients with TP53-mutant tumors. Mutations in exons 5–8 accounted for 80.9% of TP53 mutations. Mutations in TP53 exons 6 and 7 were significantly associated with inferior PFS and OS compared to wild-type TP53. TP53 mutation also influenced the prognosis of patients with different EGFR mutations. Patients with TP53 and EGFR exon 19 mutations had significantly longer PFS and OS than patients with TP53 and EGFR L858R mutations, and both groups had worse survival than patients with only EGFR mutations. Patients with TP53 mutations, especially in exons 6 and 7, had a lower response rate and shorter PFS and OS when treated with gefitinib. Moreover, TP53 exon 5 mutation divided TP53 mutations in disruptive and non-disruptive types.