Background
Esophageal cancer is one of the most aggressive types of cancer. In contrast to the predominance of adenocarcinoma in western countries, esophageal squamous cell carcinoma (ESCC) is mostly prevalent in eastern Asia, including Japan and China. Epidemiologic studies have established that cigarette smoking and alcohol consumption are strong risk factors for developing ESCC [
1]. However, only small number of studies have investigated the prognostic effect of smoking and the association between the molecular characteristics and smoking status in esophageal cancer.
Despite the development of multimodality therapies, including surgical treatment with two- to three-field lymph node dissection [
2], adjuvant radiotherapy, chemotherapy [
3], and chemoradiotherapy [
4], long-term outcome is still unfavorable, even in patients who undergo complete resection of their carcinomas [
5].
To improve treatment outcome in patients with esophageal cancer, novel strategies have been developed, especially those that are molecularly targeted. Information on molecular characteristics may have novel therapeutic potential against esophageal cancer. Furthermore, their prognostic or predictive value is extremely useful not only for risk-adapted therapeutic strategies, but also for stratifying patients into future clinical trials for molecular-targeting drugs. For clinical use, practical and reliable genotyping procedures with a considerable number of samples are required. Advances in mutation testing for molecular-targeting drugs, including next-generation sequencing (NGS), have led to the increased use of formalin-fixed paraffin-embedded (FFPE) tissue. Although molecular profiling obtained from a validated comprehensive genomic assay is necessary, there is concern regarding sequencing quality or accuracy when using the DNA extracted from FFPE. We previously demonstrated that the combination strategy of quantitative PCR (qPCR)-based DNA quality assessment and uracil DNA glycosylase (UDG) pretreatment improved the accuracy of amplicon-based massively parallel sequencing (MPS) implemented with damaged DNA from FFPE [
6].
The goal of this study was to evaluate the profiles of genetic alterations in esophageal cancer and to assess the effect of molecular characteristics on clinical outcome. To this end, we extensively analyzed gene expression and mutations obtained by automated quantitative fluorescent immunohistochemistry (AQUA) and MPS using archived FFPE samples from 135 esophageal cancer patients who underwent surgical resection, and correlated these results with the clinicopathological features, such as smoking status and survival outcomes.
Discussion
In this study, we determined the genetic profiles of 126 Japanese esophageal cancer patients by NGS and AQUA using DNA from FFPE samples. Our cohort was non-biased consecutive cases, which consisted mostly of ESCC, but also of those with non-ESCC histology. Amplicon-based MPS identified mutations in TP53, PIK3CA, APC, ERBB4, and FBXW7 as the most frequent gene alterations. We further examined the prognostic effect of these gene mutations, and found that the PIK3CA mutation, as well as the clinical T stage were independent prognostic factors. Importantly, patients with the PIK3CA mutations had significantly better survival than those with the wild-type PIK3CA. To the best of our knowledge, the present report is the first to investigate the prognostic significance of PIK3CA mutations based on NGS data in esophageal cancer.
One of the goals in this study was to characterize the smoking status in esophageal cancer. However, there was no association between clinicopathological features or prognosis and smoking status. Furthermore, our molecular analysis by NGS suggested there were no significant differences in the mutation spectrum and the pattern of base substitutions between smokers and non-smokers, unlike that of non-small-cell lung cancer patients who underwent surgery [
9]. These results are consistent with previous exome sequencing in ESCC from China [
10], and support the hypothesis that smoking might contribute to tumorigenesis of esophageal cancer through distinct mechanisms similar to those in other smoking-related cancers.
PIK3CA, which encodes the p110a catalytic subunit of the phosphoinositide 3-kinase (PI3K) [
11,
12], is an oncogene in various cancers, and its mutation or amplification and subsequent activation of the PI3K/AKT signaling pathway regulates cell proliferation, growth, survival, apoptosis, and glucose metabolism [
13]. The frequency of PIK3CA mutations has been reported to range from 1.5 to 22.9% in ESCC [
10,
14‐
23], as well as in Barrett’s esophagus [
24]. In our study, 13.5% of cases were identified as having a PIK3CA mutation or amplification, all of which presented squamous cell carcinoma histology. Therefore, PIK3CA serves as a potential therapeutic target in ESCC. Hotspot mutations of PIK3CA in exon 9 and exon 20 are known to be oncogenic in various tumor types, including esophageal, colorectal, brain, and gastric cancers [
25]. PIK3CA mutations were not significantly associated with any clinicopathological characteristics, except for the APC and BRAF genotype as discussed below, which was consistent with the results of other studies in Korea, China, and Japan [
17,
19,
26].
The prognostic relevance of PIK3CA mutations has been investigated in various solid tumors, and PIK3CA mutations are generally associated with an unfavorable prognosis in patients with colorectal [
27‐
30] or lung cancer [
31]. On the other hand, studies on breast and ovarian cancer demonstrated that the patients with the PIK3CA mutation showed a trend towards a favorable prognosis [
32‐
34]. These reports suggest that PIK3CA mutations might behave differently according to tumor type. Our multivariate analysis revealed that PIK3CA gene mutations were associated with a favorable prognosis among Japanese patients with curatively resected esophageal cancer, mainly ESCC, suggesting that the PIK3CA gene mutational status may be a prognostic biomarker for Japanese esophageal cancer patients. This finding supports other studies in Chinese and Japanese ESCC patients [
16,
19,
22]. We further separately analyzed the survival in patients with PIK3CA mutations in coding exon 9 and 20. Median OS in patients with exon 9 mutation was not reached, and that in patients with exon 20 mutation was 2902 days (95% CI 693 days–not reached). That is, both patients with exon 9 and 20 mutation had better OS than patients with wild-type PIK3CA. These findings suggest that both exon 9 and 20 mutation might be favorable prognostic factors. However, due to limited sample size for each type of PIK3CA mutation (6 patients in exon 9, 7 patients in exon 20, and 1 in exon 7), it is hard to differentially conclude the significance of each mutation as a prognostic marker. Taken together, the prognostic effect of the PIK3CA mutation in ESCC has been controversial, despite a number of investigations dating from the 2010s in Asia (Table
4).
Table 4
Prognostic significance of PIK3CA alterations in esophageal cancer
| 80 | ESCC | Exon 9 and 20 mutations | 11.5 | N.S. | NA | Primary& Metastatic sites FFPE | Mass-spectrometry based assay |
Shigaki et al. (2013) [ 19] | 219 | ESCC | Exon 9 and 20 mutations | 21 | Favorable OS | 0.35 (0.10–0.90) | Surgically resected FFPE | Pyrosequencing |
| 96 | ESCC | Exon 9 and 20 mutations | 12.5 | Trend towards favorable OS | NA | Surgically resected FFPE | Mutant enriched PCR method |
| 406 | ESCC | Exon 9 mutations | 7.4 | N.S. | 1.256 (0.748–2.108) | Surgically resected FFPE | Direct sequencing |
| 534 | ESCC | Amplification | 10.5 | Trend towards unfavorable OS | 1.21 (0.83–1.77) | Surgically resected FFPE | Fluorescent in situ hybridization |
388 | Exon 9 and 20 mutations | 1.5 | N.S. | NA | Direct sequencing |
| 210 | ESCC | Exon 9 mutations | 22.9 | Favorable OS | NA | Surgically resected FFPE | Pyrosequencing |
Current study | 126 | Mostly ESCC | Exon 9 and 20 mutations | 11.1 | Favorable OS | 0.34 (0.12–0.96) | Surgically resected FFPE | MPS with TSACP |
Amplification | 2.4 | N.S. | 2.66 (0.64–11.05) |
The possible reasons for the different results might be different patient cohorts, sample sizes, methods used to assess PIK3CA mutations, or ethnicity. First, the patient cohort in Maeng et al. had metastatic ESCC, which differed from those studies using surgically resected primary sites [
17]. Next, we used amplicon-based MPS, which is a NGS technology and increasingly being used for mutational analysis of tumors for both clinical and research applications. NGS facilitates multi-gene mutational profiling with only nanograms of DNA and has better sensitivity than traditional sequencing platforms, such as direct sequencing [
35,
36]. Indeed, the limited sensitivity of direct sequencing may result in an apparent the low frequency of PIK3CA mutations [
21,
23]. Although allele-specific mutation testing, including pyrosequencing and mass-spectrometry based assays, was shown to be more sensitive than regular direct sequencing, its potential disadvantage is the ability to assess only limited hotspot regions in given genes [
37]. Therefore, different sequencing methodologies may have an effect on the frequency of PIK3CA mutations, leading to different prognostic values. Furthermore, although FFPE samples are the most practically available material when performing mutation testing, one of the pitfalls of using FFPE samples is DNA fragmentation [
38] and artificial C: G > T: A single nucleotide variants because of deamination of cytosine to uracil. Therefore, DNA quality assessment is essential in mutation testing, especially in highly sensitive sequencing methods. We previously demonstrated that UDG pretreatment is efficacious for excluding nonspecific single nucleotide variants in amplicon-based MPS that occur if poor-quality DNA from FFPE samples was used. This may be a reason why the frequency of the PIK3CA mutation in our study was not higher than previous allele-specific mutation testing. Although the data on molecular profiling in this study was obtained from a validated comprehensive genomic assay, one of the limitations of this study is that our findings were not validated by other methods. Furthermore, because the sample size for each type of mutation was small, our results should be further validated in some independent cohorts in the future.
It is expected that PIK3CA mutations would imply poor clinical outcome, because the presence of oncogene activation leads to aggressive tumor behavior. However, this was not true. One possible hypothesis to explain the paradoxical result may be a negative feedback mechanism through which the PI3K/AKT pathway is inactivated in PIK3CA mutant tumors. In wild-type PIK3CA tumors, on the contrary, the PI3K/AKT pathway may be activated by several factors, such as EGFR and HER2, independent of PIK3CA mutation. Indeed, the relationship between PIK3CA mutation and p-AKT expression has been different among tumor types [
19,
34]. Otherwise, wild-type PIK3CA tumors may require alternative molecular alterations to the PI3K/AKT pathway to acquire more aggressive phenotypes. However, all of them have not been proven yet, and our AQUA system did not include p-AKT expression. Therefore, such compensatory mechanisms need to be further elucidated in the future.
To further characterize the PIK3CA mutation and wild-type, we also investigated the clinicopathological characteristics of esophageal cancer patients with respect to PIK3CA status. PIK3CA mutations were significantly associated with the APC mutation. The type of APC gene alteration detected in this study was different from that reported to occur frequently in upper gastrointestinal cancers, such as at codons 1450, 1462–1465, and 1554–1556 [
39]. The most frequently noted mutations in our cohort were nonsense mutations (11/17; 64.7%), which resulted in truncated APC proteins. APC frameshift deletions in the codon 1556 hot spot, 1301, and 1384 detected in this study also lead to loss of APC function. The coexistence of APC alterations with PIK3CA mutation may be partially explained by a previous study using a mouse model with PIK3CA mutation, which demonstrated that the PIK3CA mutation alone was insufficient to initiate intestinal tumorigenesis in intestinal cancers. Thus, loss of APC activity may synergistically act with active PI3K, resulting in tumorigenesis [
40,
41].
As compared to PIK3CA alterations, the mutations involved in the RAS–RAF pathway were rare. Previous analysis by a mass-spectrometry based assays revealed that only one of 80 patients harbored an oncogenic BRAFV600E mutation [
17]. In our series, no BRAFV600E mutation was detected. Instead, there were nine cases with BRAF mutations at codons 598 (
n = 3), 469 (
n = 2), and 444 (
n = 2). We also found statistically significant coexistence of BRAF mutations and PIK3CA mutations. However, the biological relevance of the coexistence of these mutations remains unclear.
Furthermore, Zhang et al. successfully established and characterized patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models, and found that PDECX models with PIK3CA mutation had no significant response to cytotoxic agents. This result suggests that PIK3CA mutation is also involved in sensitivity to chemotherapy, and may provide an information for the treatment of ESCC patients in the future [
42].
Recent treatment strategies for advanced esophageal cancer have shifted to neoadjuvant treatment, such as chemotherapy and chemoradiotherapy [
3,
4]. The limitation of this study is that some of the surgically resected specimens may have been modified by preoperative therapy. Therefore, we also separately analyzed the survival for a cohort with preoperative treatment (
n = 65) from that without preoperative therapy (
n = 61). However, PIK3CA mutational status was not significantly associated with survival, probably because of the small sample size for PIK3CA mutations in each cohort. Furthermore, protein expression measured by AQUA may be modified by preoperative treatment. That could be a main reason why any gene expression was not significantly associated with prognosis in our study. Indeed, for instance, the prognostic effect of EGFR protein expression was proved by immunohistochemistry using surgically resected tumor tissue in patients with ESCC who underwent surgery alone or surgery and postoperative radiotherapy [
43].