PIK3CA mutations are frequent in esophageal squamous cell carcinoma associated with chagasic megaesophagus and are associated with a worse patient outcome

Esophageal cancer is the eighth most frequent type of cancer in the world and the sixth most lethal, occurring mainly in developing countries such as Brazil [1]. The most frequent histological subtype is esophageal squamous cell carcinoma (ESCC), accounting for 90% of cases, especially in high-risk areas [2]. The main risk factors for ESCC are alcohol consumption, tobacco (mainly in association) and hot-beverage consumption [3]. It is also reported that chronic diseases, such as the chagasic megaesophagus, can be associated with ESCC development [4].

Chagasic megaesophagus is the late manifestation of Chagas’ disease (caused by the protozoan Trypanosoma cruzi) [5]. Direct infection of Trypanosoma cruzi will lead to destruction of the intramural myenteric neurons in the esophagus, causing inflammation and production of neurotoxins. This will result in uncoordinated contractions and reduction of peristalsis of the organ, altering the functioning of the lower esophageal sphincter and progressive dilation of the esophagus (megaesophagus) [6]. In Brazil, one of the endemic regions of Chagas’ disease, approximately 4 million people are infected with the parasite and about 6–7% of these patients will develop chagasic megaesophagus [5]. Patients affected with this lesion are more likely to develop ESCC (3–10%) when compared to the general population [4].

The carcinogenic mechanisms of ESCC development in the context of chagasic megaesophagus have been little explored. Recently, our group showed the high frequency (13/32, 40.6%) of TP53 mutations in ESCC associated with chagasic megaesophagus [7]. Moreover, we also reported the presence of microsatellite instability (MSI) in a small fraction (1/19, 5.3%) of cases [8]. However, many other genes are known to be involved in ESCC carcinogenesis as demonstrated by the TCGA consortium [9].

One of these genes is the PI3KCA, which encodes the protein phosphatidylinositol 3-kinase (PI3K), that belongs to a family of lipid kinases that encodes the p110α catalytic subunit [10]. PI3K is a quite complex signaling pathway since it regulates cell growth, proliferation, cell motility, the production of new proteins, apoptosis and cell survival [10]. Therefore, its activation will lead to many downstream pathways that regulate several cellular functions, including those involved in the development of cancer [10, 11]. Recurrent PI3KCA oncogenic mutations were identified in several types of tumors, including colorectal, breast, ovary, gastric, and recently in ESSC [12]. The mutations occur mainly in exons 9 (E542K and E545K) and 20 (H1047R) [12]. Recently, it was shown that PIK3CA mutations, namely H1047R, also disrupt cellular genetic stability, increasing the frequency of chromosomal errors and leading to tetraploidy [13]. Importantly, therapeutic strategies targeting the PIK3/Akt signaling pathway have been developed and could constitute effective treatment options for patients harboring PI3KCA mutations [14].

Among the several risk factors for the development of ESCC, the chagasic megaesophagus (late complication of Chagas’ disease) has been a minor etiological factor and little explored [4]. Nevertheless, Chagas’ disease is still an important public health problem, particularly in Latin-America, where approximately 20 million people are infected with Chagas’ disease and approximately 6–7% of these people will develop chagasic megaesophagus [5, 16].

In the present study, we investigated the frequency of PIK3CA mutations in regions of hotspot (exons 9 and 20) in patients with chagasic megaesophagus associated with esophageal squamous cell carcinoma (CM/ESCC) and compared with patients with esophageal squamous cell carcinoma without chagasic megaesophagus (ESCC) and patients with chagasic megaesophagus without esophageal squamous cell carcinoma (CM). We observed that patients in the CM/ESCC group had a higher frequency of mutations (5/23, 21.7%) followed by patients in the ESCC group (4/38, 10.5%), and in the CM group (1/28, 3.6%). This is the first report of PIK3CA mutation in ESCC that developed in the context of chagasic megaesophagus and the significant frequency of mutations (~ 22%) suggest that PIK3CA plays an important role in the carcinogenesis of CM/ESCC patients. Moreover, the presence of PIK3CA mutation in a benign lesion further supports the putative role of chagasic megaesophagus as an ESCC-related condition. The frequency of mutations identified in our study is in line with that reported in the literature for ESCC patients, with frequencies varying from 2.2 to 32.8% (Table 6) [9, 17‐33]. This variation can be due to several factors, such as type of tissue (frozen vs FFPE), distinct methodologies for mutation screening and distinct ethnic groups of patients. (Table 6).

The PIK3CA gene is often mutated in several tumors types and most of its mutations occur in hotspot regions, such as E542K and E545A located in the helical domain (exon 9), and H1047R and H1047L in the kinase domain (exon 20) [11]. These mutations lead to the activation of the PIK3 pathway and have a great potential in oncogenic activities [11]. Interestingly, most of these mutations (E545A, H1047R and H1047L) occurred in patients in the CM/ESCC group and only one (E545A) in one patient in the ESCC group. We also identified other previously described important mutations (Table 3), the D549H mutation observed in the CM/ESCC group was reported in vulva and hepatocellular cancer [34]; R524K mutation found in the ESCC group was reported in colorectal cancer [35]; and the R555K mutation was reported in ovary cancer [36]. Interestingly, it is important to note that we identified three mutations in exon 20 that have not yet been reported (A1027D and K1030R in CM/ESCC group; T1053K in CM group). All these mutations occurred in patients with chagasic megaesophagus whose mutational profile of PIK3CA was never reported.

Importantly, we observed that CM/ESCC patients harboring PIK3CA mutations were associated with lower overall survival, suggesting its role as a prognostic biomarker in this group of patients. Interestingly, the results of our analyzes of the survival of the mutated patients differ from those reported by others studies, especially in regions of some risk such as Asia, in which patients with ESCC with mutations of the PIK3CA gene had a favorable overall survival compared to patients wild-type [37].

Notably, inhibitors of the PIK3-Akt-mTOR pathway have been developed as cancer target therapy alternatives, and patients harboring PIK3CA gene mutations could be potential candidates for such therapeutic approach [14]. Interestingly, phase I and II clinical trials using pan-PIK3CA agents (PIK3-class I), such as buparlisib (BKM120), an oral agent that affects α, β, γ and δ isoforms of PI3K [38], showed efficacy in several solid tumors, including head and neck cancer [39]. Copanlisib (BAY80–6946), an intravenous agent that affects α and δ isoforms of PI3K, also showed promising results in non-Hodgkin’s lymphomas [40]; as well as pictilisib (GDC-0941), an oral agent that affects γ and δ isoforms of PI3K, where a good response was reported in breast, colorectal, ovarian and non-small cell lung cancer [41]. Therefore, we can hypothesize that a subset of ESCC and CM/ESCC patients with PIK3CA mutations may benefit from these targeted-therapies and consequently improve their dismal survival.