Purpose of review: We used two examples of genes, TP53 and EGFR, which are somatically altered by intragenic mutations in common cancer types to illustrate how somatic mutations have followed very different routes to clinical applications.
Recent findings: TP53 somatic mutations are frequent in many cancers. Their prognostic and predictive values are currently assessed in several clinical trials and TP53 gene therapy is in use in China. Mutations in EGFR have been proved to be predictive of response to tyrosine kinase inhibitors, allowing for the licensing of gefitinib in lung adenocarcinomas carrying a mutated EGFR gene.
Summary: With the accumulation of knowledge on the predictive and prognostic value of somatic mutations, and with recent advances in large-scale sequencing techniques and reduction in cost of sequencing, sequencing several genes in human tumors is on the verge of becoming routine clinical practice.
aInternational Agency for Research on Cancer, Group of Molecular Carcinogenesis, Lyon, France
bDepartment of Cellular Pathology, Medical School, University Hospital Birmingham Foundation Trust, Birmingham, UK
Correspondence to Magali Olivier, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon Cedex 8, France Tel: +33 472 738 532; fax: +33 472 738 322; e-mail: firstname.lastname@example.org
Cancer growth involves the sequential accumulation of genetic alterations in genes controlling cell proliferation, lifespan, responses to stress, relationships with neighbors, and gene homeostasis. Among these alterations, intragenic mutations play an important role in activating oncogenes or inactivating tumor suppressor genes. These mutations may be associated with specific types of cancers or may be common to several types of cancers. The most frequently mutated gene in human tumors is the TP53 tumor suppressor gene. TP53 encodes the p53 protein, which mediates antiproliferative mechanisms in response to various forms of cellular stresses, in particular DNA damage . As most conventional chemotherapies act by inducing DNA damage, the predictive value of TP53 mutations has been intensely investigated over the last 15 years, without reaching clinical practice due to inconsistent results . On the contrary, treatments targeting specific genes or pathways have flourished in recent years, revolutionizing clinical practice. These treatments are highly selective and effective in selective types of cancers that carry specific genetic alterations. For example, drugs have been licensed only in lung adenocarcinomas carrying a mutated EGFR gene . Although these treatments are targeted, the presence of the target is not associated with a 100% response. As for other types of treatment, de-novo or secondary treatment resistance is a major issue and finding additional markers that can predict treatment efficacy is an intense field of research.
Here, we review recent data on the prognostic and predictive value of intragenic somatic mutations in TP53 and EGFR genes. These genes constitute two interesting examples of the use of somatic mutations as predictive or prognostic markers, which have followed very different routes to clinical applications.
Prognostic and predictive values of TP53 mutations: lost in translation or on the rise again?
Mutations in the TP53 gene are found in almost every type of cancer, with prevalence ranging from 5 to 50%, depending on the tumor type . These mutations result in loss of p53 functions, promoting cell growth under conditions that suppress the proliferation of normal cells and impairing the antiproliferative activities of DNA-damaging drugs. The majority of mutations are missense mutations producing mutant proteins that accumulate in the nucleus of tumor cells and can be detected by immunohistochemistry (IHC). Because of the initial findings that mutations in TP53 could be detected by IHC, massive translational efforts have been made using IHC to assess the prognostic and predictive value of TP53 mutations in various types of cancers. Although some studies have shown an association between p53 positive immunostaining and poor outcomes, several studies have produced conflicting results and expectations on the use of p53 as a useful clinical biomarker faded . In fact, a significant number of mutations are small insertions or deletions that produce truncated proteins that stain negative by IHC. IHC is, thus, a poor surrogate marker for assessing TP53 gene status, as it produces a high rate of false positive (overexpression of p53 wild-type protein is frequent) and false negative cases (a significant number of truncating mutations that stain negative by IHC).
Studies that have used gene sequencing to assess TP53 status have produced more consistent results, at least for some types of cancers such as breast, head and neck squamous cell carcinoma (HNSCC), and leukemia, in which the presence of a TP53 mutation is associated with poor outcomes (Table 1). However, the example of breast cancer shows that the use of TP53 status as a prognostic or predictive marker is not straightforward. Although TP53 mutations have been associated with poor outcomes in most studies [5–9], two recent studies reported an association with good response to treatment. In Bertheau et al. , patients were treated with a dose-dense epirubicin–cyclophosphamide regimen that targets highly proliferating tumors, a hallmark of TP53 mutated tumors. In a small study that included only triple-negative breast cancers treated with cisplatin in the neoadjuvant setting, nonsense and frameshift TP53 mutations were associated with good response to treatment [11•]. In other types of cancer such as brain and pancreas, mutations were also found to be associated with both poor and good prognosis, depending on the study (Table 1). These results show that the type of tissue and the type of treatment may be important determinants of the prognostic and predictive value of TP53 mutations. This may be explained by the biology of p53, which is activated through different pathways according to treatment type, causing different posttranslational modifications on p53 and resulting in different responses ranging from senescence, cell-cycle arrest to apoptosis . The p53 response is also dependent on the cellular context. The clinical value of TP53 mutations should, thus, be evaluated in homogeneous sets of tumors and in the context of controlled treatment regimens to determine in which context TP53 mutation status may be a useful clinical marker.
This has been done in some recent studies, which have used tumors from patients enrolled in clinical trials. Di Leo et al.  evaluated the predictive value of TP53 mutations in a small series of advanced breast cancers randomly treated with doxorubicin (an anthracycline) or docetaxel (a taxane) in the context of a phase III clinical trial. Although the series was too small to reach statistical significance, it was found that TP53 mutations may compromise the efficacy of doxorubicin but not of docetaxel. In another series of advanced/noninflammatory breast cancer patients randomly assigned to an anthracycline-based or a taxane-based regimen, TP53 mutations were significantly associated with anthracycline resistance (P < 0.05) . Interestingly, mutations in the CHEK2 gene, which is upstream of p53 in the DNA-damage response pathway, were also associated with anthracycline resistance. These results confirmed earlier studies and suggest that investigating p53 functional pathways may improve prediction of treatment response. Finally, another phase III trial aimed at assessing the predictive value of TP53 mutations for taxane versus nontaxane primary chemotherapy of advanced/inflammatory breast cancers showed that TP53 status was not a predictive but a prognostic marker [15•]. All these studies concur on the predictive value of TP53 status for anthracyclines and the absence of predictive value for taxanes in breast cancer.
If past translational efforts have focused on determining the prognostic or predictive value of TP53 mutations, recent efforts have aimed at developing therapeutic strategies that exploit TP53 status in tumors. Strategies that target mutant p53 have been developed to restore normal p53 functions in tumor cells. They include molecules that re-activate mutant p53 suppressive functions (PRIMA, RITA, scFv), drugs that target specific point mutations to restore wild-type-like structure (Phikan059 targeting R220C), peptides that interact specifically and strongly with several p53 mutants and block their nonspecific transactivation capacities, inhibiting their gain of function properties [16•,17]. Although these molecules are in different stages of clinical development, another strategy that has finally reached clinical practice is gene therapy. Gendicine has been approved in China to treat HNSCC in combination with radiation therapy. Indeed, TP53 mutations are found in more than half of HNSCC tumors and have been associated with poor prognosis in most studies (Table 1). This is in agreement with the fact that radiation therapy is the most common form of treatment for HNSCC and acts mainly through p53. In the USA, Advexin (Introgen Therapeutics, Austin, Texas, USA), a similar gene therapy, has shown efficacy in several clinical trials . In particular, in a phase III clinical trial, Advexin was shown to work best in cells that express wild-type p53 or do not express p53 due to the presence of a truncating mutation [19•]. TP53 status should, thus, be assessed by both IHC and gene sequencing to predict gene therapy efficacy.
After 25 years of research, TP53 has, thus, finally made it to the clinic through gene therapy. Further translational efforts are needed to validate other strategies, but for all these strategies, screening TP53 intragenic mutations will be necessary.
Predictive value of EGFR mutations and tyrosine kinase inhibitor treatments in lung cancers
EGFR intragenic mutations have recently found their clinical utility, as they were found to confer sensitivity to tyrosine kinase inhibitors (TKIs) in lung adenocarcinomas. Receptor tyrosine kinases (RTKs) such as EGFR are deregulated in a variety of human solid tumors, resulting in the upregulation of downstream signalling cascades that control cell proliferation, survival, and motility, and this upregulation is a major force that drives tumor development. Targeting these pathways to stop cancer progression has led to the development of several new molecules that are used in clinical practice or tested in clinical trials [20•].
Gefitinib and erlotinib are small molecules that specifically target the tyrosine kinase domain of EGFR, inhibiting its kinase activity. In lung adenocarcinomas, several gain of function mutations located at specific residues in the tyrosine kinase domain (exons 18–21) of EGFR have been described that result in the constitutive activation of EGFR and sensitization to TKIs (Fig. 1). Two main types of mutation, inframe deletions in exon 19, and a substitution within exon 21 (L858R), represent nearly 90% of all mutations. However, other mutations do occur at varying incidence. Some have been shown to convey resistance to anti-EGFR small molecules, like the missense mutation T790M in exon 20, which can be a primary or a secondary mutation detected in patients who initially responded well but started progressing under treatment .
A randomized phase III trial, the IRESSA Pan-Asia Study (IPASS), has been instrumental in demonstrating the predictive value of EGFR mutations and has led to the licensing of gefitinib as first-line monotherapy for EGFR mutated lung adenocarcinomas in Europe. This study showed that EGFR mutations were a strong predictor of better outcome with gefitinib and that gefitinib was superior to carboplatin–paclitaxel as first-line treatment in EGFR-mutated lung adenocarcinomas [22•]. A recent pooled analysis of 54 studies evaluating clinical outcomes in Asian and non-Asian nonsmall cell lung cancer (NSCLC) patients with EGFR mutations confirmed the better outcome obtained with TKIs as compared with chemotherapy [23•]. Progression-free survival was 13.2 months with erlotinib (365 patients), 9.8 months with gefitinib (1069 patients) compared with 5.9 months (375 patients) with chemotherapy. Another pooled analysis of five phase II clinical trials from the USA and Europe evaluated the impact of EGFR and KRAS mutations in 223 NSCLC patients treated with first-line erlotinib or gefitinib [24•]. It was found that EGFR mutation status was a better predictor of outcome than clinical predictors (race, sex, smoking status, and tumor histology). Patients with exon 19 deletions had a longer survival (overall and progression free) compared with patients with the L858R (in exon 21) mutation, although the two mutation types were associated with similar response rates. In patients with sensitizing EGFR mutations, there were no statistically significant differences between erlotinib and gefitinib treatments. KRAS mutations were not associated with survival in this study, although one patient with both an EGFR exon 19 deletion and a KRAS mutation achieved stable disease with erlotinib. The better outcome associated with exon 19 deletions compared with the L858R mutation was also confirmed in a large multicentric study from Spain that included 217 patients treated with erlotinib [25•].
Overall, a large amount of data on the predictive value of the most frequent EGFR mutations is now available in NSCLC. The next challenge will be to assess the clinical significance of rare mutations and of mutations in EGFR and related pathways.
Mutation screening in clinical practice
Mutation screening should be seen as a supplementary test to histology diagnosis that is guided by histological type and availability of the drugs for prescription. For EGFR, mutation rates vary according to histological type, ethnicity of patient and smoking history, with highest frequencies in adenocarcinomas from nonsmoking women of Asian origin . Indeed, mutation rates are over 60% in Asia and between 10 and 15% in white patients. Some may, thus, recommend EGFR testing in these clinically defined groups of patients. However, mutations have also been detected in rare cases of squamous cell carcinomas or in smokers, and EGFR genotype was shown to be more effective than clinical characteristics at selecting appropriate patients for first-line therapy with a TKI [24•]. Because no consensus exists on the criteria for selecting patients to be tested, the decision to request EGFR mutation testing should be made by the treating physician.
The challenge for the molecular pathologist is then to complete the mutation tests within a few working days following the histological diagnosis. Several techniques may be used to screen for specific mutations or entire gene sequence, including direct sequencing, real-time PCR, pyrosequencing, DNA fragment analysis, denaturing high-performance liquid chromatography (DHPLC), kit-based mutation screen, among others. Furthermore, testing has often to be performed in small cytology or formalin-fixed-paraffin embedded histology specimens containing relatively poorly preserved DNA. As it is not feasible to obtain extra fragments of tissue dedicated to molecular pathology in routine practice, the pathologists ought to select for each test the most appropriate method depending on the type of sample, their own experience, the equipment in place, and the cost per test. Internal and external quality control schemes and regular audits will, thus, be essential to guarantee good practice.
Clinical interpretation of the molecular results can also be a challenge. Clinical data on the panel of mutations that can be seen in tumors are still limited and the clinical significance of certain rare mutations is not known. The systematic collection in well maintained databases of standardized molecular and clinical data from routine pathology will be essential to allow for pooled analysis of these data and to understand the role of rare mutations as well as of the combination of multiple mutations.
The search for genes that are altered in cancer and drive tumor development has identified more than 400 genes that carry somatic mutations in human cancers. These genes are all candidates for targeted therapies or as predictive/prognostic markers. Large-scale sequencing efforts in well designed trials will, thus, be required to identify sets of clinically significant mutations, in the hope that the mutation outfit of each patient tumor will allow for the selection of the most appropriate treatment. On the basis of the TP53 and EGFR experiences, reaching this stage may not be trivial. It will be important to collect tissues on most patients in clinical trials to allow for prospective–retrospective studies on biomarkers, as unforeseen new markers may emerge during the course of clinical trials. Another important aspect that should not be overlooked is to develop databases that systematically collect standardized information on clinical outcomes associated with specific genotypes, as they will be instrumental in achieving this goal.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 126–127).
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