Concordance between PIK3CA mutations in endoscopic biopsy and surgically resected specimens of esophageal squamous cell carcinoma

Squamous cell carcinoma is the predominant histological subtype of esophageal cancer in Asia, whereas adenocarcinomas predominate in the United Kingdom, some other Western European countries, and the United States [1]. To date, multidisciplinary treatment approaches for esophageal cancer with different histological subtypes including surgery, chemotherapy, and radiation have been employed; however, the prognosis of these patients remains poor [2, 3]. For patients with metastatic or recurrent esophageal squamous cell carcinoma (ESCC), the available agents are quite limited. A combination of platinum agents and fluorouracil derivatives is commonly used as first-line chemotherapy, and taxanes are options for second-line chemotherapy [1, 4]. In addition, no molecular-target therapies have been established for the treatment of ESCC. Therefore, there is an unmet medical need for ESCC treatment, particularly for patients who are in good physical condition but who are refractory or intolerant to standard therapies.

The phosphoinositide 3-kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) pathway plays a pivotal role in cancer cell proliferation, and mutations in the PIK3CA gene are commonly found in various cancers regardless of histological subtypes [5]. More than 80% of PIK3CA mutations occur in two major regions: the helical domain (exon 9), and the kinase domain (exon 20); moreover, three mutations (E542K, E545K, and H1047R) have been regarded as hotspot mutations [6]. In a phase 1 trial evaluating an mTOR inhibitor, a case with advanced ESCC exhibited a partial response, although the PIK3CA mutation status was unknown [7]. In addition, PIK3CA mutations have been suggested to be a potential predictive biomarker for PI3K–Akt–mTOR inhibitors in a review of early phase clinical trials for the testing of such agents in various solid cancers [8]. In this report, a case with squamous cell head and neck carcinoma, which is genetically similar to ESCC, harboring a PIK3CA mutation (H1047R) demonstrated a partial response to a PI3K–Akt–mTOR inhibitor. The frequency of PIK3CA mutations in ESCC has been reported to range from 2.2 to 21% [9–16], whereas mutations in genes in the RAS–RAF pathway are very rare [15, 17, 18]. Accordingly, PIK3CA mutations may be a potential target molecule in ESCC treatment.

Previous studies investigating the frequency of PIK3CA mutations in ESCC used available clinical samples obtained from either surgically resected specimens or biopsy specimens [9–16]. Clarifying whether PIK3CA mutations from biopsy specimens can be detected in corresponding surgically resected specimens is important for the future clinical development of ESCC treatment. Therefore, the present study examined the frequency of PIK3CA mutations and the concordance between PIK3CA mutations detected in endoscopic biopsy specimens and those detected in corresponding surgically resected specimens in patients with ESCC.

To the best of our knowledge, this is the first report to investigate the concordance of the PIK3CA mutation status between endoscopic biopsy and surgically resected specimens using FFPE tissues from patients with ESCC. In the present study, we demonstrated that, although not frequent, a certain proportion of patients with ESCC harbored PIK3CA mutations, and the mutation statuses of the two types of specimens were highly concordant.

Among the five hotspot mutations assessed in the present study, E542K, E545K and E546K are located in exon 9, corresponding to the helical domain, and H1047R and H1047L are located in exon 20, corresponding to the kinase domain. The mutations in both of these domains increase the kinase activity of PI3K and activate the PI3K–Akt–mTOR pathway, resulting in the activation of cell signaling and the promotion of cell growth and invasion [23, 24]. The present study showed no significant association between the PIK3CA mutation status and the clinicopathological characteristics of the ESCC cases, suggesting that an examination of the clinicopathological factors prior to genetic analysis might not be capable of predicting the presence of a PIK3CA mutation. In other words, ESCC patients with a PIK3CA mutation constitute a subgroup only in terms of the applicability of a PI3K inhibitor, since emerging evidence suggests that patients with PIK3CA-mutated cancer might benefit from treatment with PI3K inhibitors [8, 25].

The high overall concordance rate (98.3%) between endoscopic biopsy and surgically resected specimens observed in this study suggests that PIK3CA mutations are homogeneously distributed in the primary tumor in most cases. Although the intratumoral heterogeneity of the PIK3CA mutation status has not yet been investigated for ESCC, the results of our study are in line with the homogeneous distribution of the PIK3CA mutation status in the primary tumor observed in colorectal and breast cancers [26, 27]. In colorectal cancer, the concordance rate of the KRAS mutation status between endoscopic biopsy and surgically resected specimens is high [28], similar to that of the PIK3CA mutation status in ESCC demonstrated in the present study, and endoscopic biopsy specimens are used for the molecular analysis of KRAS mutations to evaluate the clinical indications for anti-epidermal growth factor receptor antibody therapy. These findings suggest that FFPE clinical samples obtained from endoscopic biopsies are applicable to the identification of PIK3CA mutations in ESCC. In contrast, the discordant mutation status between endoscopic biopsy and surgically resected specimens that was observed in three cases may be attributable to intratumoral heterogeneity. Because endoscopic biopsy specimens represent a limited and superficial sampling of the primary tumor, intratumoral heterogeneity can be an obstacle to establishing a precise biomarker diagnosis. Obtaining multiple endoscopic biopsy samples from primary tumors may improve the likelihood of detecting a mutation and may minimize potential mutational discordances.

The PIK3CA mutation frequency of 7.2% observed in this study is based on Luminex (xMAP) technology targeting five hotspot mutations in the PIK3CA gene with a detection limit of 5% and is compatible with the COSMIC database published by the Sanger institute (9.5%), and with those of the previous studies ranging from 2.2 to 21% [9–16]. The variation in mutation frequency among these studies is thought to be mainly attributable to differences in the methods used to detect PIK3CA mutations as well as differences in the patient cohorts, with factors such as disease stage, prior therapy, and ethnicity playing major roles. The frequency of PIK3CA mutations has been reported to be 2.2%–7.7% using direct sequencing, 11.5%–21% using pyrosequencing or other high-sensitivity methods, and 4.5%–9.0% using NGS [9–12, 14–16, 29, 30]. The results of the present study were likely influenced by the sensitivity of the mutation testing. Collecting a sufficient number of cancer cells and excluding non-cancerous cells from biopsy specimens is difficult using manual microdissection for DNA extraction because the original size of the biopsy specimens is considerably smaller than that of the surgically resected specimens. One strategy is to use a detection method with a higher sensitivity, thereby reducing the risk of missing a relatively small fraction of cancer cells carrying a PIK3CA mutation. However, the relationship between the proportion of cancer cells with a PIK3CA mutation and biological differences has not yet been reported, and the relationship between the proportion of cancer cells with a PIK3CA mutation and differences in the response to PI3K inhibitors for any cancer type, including ESCC, remains unknown. Importantly, the present study revealed a case in which a PIK3CA mutation was observed in a biopsy specimen, but the wild-type was observed in the surgically resected specimen. This event suggests the existence of another problem in the detection of PIK3CA mutations: that is, heterogeneity. In addition to the sensitivity of mutation testing, tumor heterogeneity is also likely to influence the detection sensitivity. Since the efficacy of agents inhibiting the PI3K–Akt–mTOR pathway has been demonstrated clinically, further investigation of the optimal detection method and its detection limit is needed to ensure that patients who might benefit from such treatment are accurately identified.

Recently, phase 1, phase 2, and phase 3 clinical trials examining a number of PI3K inhibitors have begun for patients with various types of cancer [31, 32]. Among these agents, the clinical efficacy of buparlisib (BKM120), a pan-PI3K inhibitor, has been demonstrated in breast cancer patients in a phase 3 trial, and the presence of a PIK3CA mutation was shown to predict a response to this agent [25]. Several phase 2 trials examining buparlisib in ESCC patients are also currently ongoing (registration ID: NCT01806649, UMIN000011217). Additional biomarker studies performed during these clinical trials may reveal whether the PIK3CA mutation can be used as a biomarker to predict the efficacy of PI3K inhibitors in patients with ESCC.

Although heterogeneity in the PIK3CA mutation status between primary tumors and corresponding lymph nodes or distant organ metastases is reportedly rare for colorectal and breast cancers, limited information is available with regard to ESCC. For the development of therapies targeting the PIK3CA mutation in patients with ESCC, the potential for heterogeneity between primary tumors and metastases must be further investigated.

The detection of PIK3CA mutations could be used to define a subset of patients who may be potential candidates for treatment with inhibitors of the PI3K–Akt–mTOR pathway. The PIK3CA mutation status was highly concordant between endoscopic biopsy and surgically resected specimens in patients with ESCC, suggesting that endoscopic biopsy specimens are clinically applicable for the detection of PIK3CA mutations.