Oncology/endocrine
PIK3CA Mutation Status in Japanese Esophageal Squamous Cell Carcinoma

https://doi.org/10.1016/j.jss.2007.03.044Get rights and content

Background

A somatic mutation of the PIK3CA (phosphatidylinositol 3-kinase catalytic subunit) gene has been found in human cancer patients. However, this mutation has not yet been extensively studied in esophageal squamous cell carcinomas.

Materials and methods

We analyzed a mutation of the PIK3CA gene in 88 Japanese cases of esophageal squamous cell carcinomas that had all undergone surgery at the Department of Surgery II, Nagoya City University Medical School, between 1996 and 2003. The TE and KYSE series of cell lines are human esophageal cancer cell lines. Two PIK3CA mutation hot spots (exon 9 and exon 20) were analyzed by a real time polymerase chain reaction (PCR)-based assay and the data were confirmed by direct sequencing. We performed a cell proliferation assay to determine the effects of a PI3K inhibitor LY294002.

Result

In exon 9, a somatic mutation was found in two patients (2.2%) and in two cell lines. The mutations included three E545K (G1633A) mutations and one E545Q (G1633C) mutation. However, in exon 20, no mutation was observed in our esophageal cancer patients. PI3K inhibitor (LY294002) inhibited the growth of an esophageal cancer cell line with a PIK3CA mutation (E545K) in vitro.

Conclusions

We found LY294002 to reduce the proliferation of the esophageal cancer cell line in vitro. Importantly, a cell line with a PIK3CA gene mutation was more susceptible to a PI3K inhibition than those without any such mutation. Further functional analyses of the PIK3CA mutations are warranted to determine whether or not they may be potentially useful targets of therapy for esophageal cancer.

Introduction

It is now well established that cancer is a genetic disease and that somatic mutation of the oncogenes or tumor suppressor genes are the initiator of the carcinogenic process [1]. The phosphatidylinositol 3-kinase signaling pathway has recently been suggested to play a pivotal role in the oncogenesis of human cancers [1]. Recently, high frequencies of somatic mutations in the PIK3CA gene have been reported in several cancer types, including cancer of the colon, brain, stomach, breast, and ovaries [2, 3, 4, 5, 6, 7]. More than 75% of these mutations are clustered in the helical (exon 9) and kinase domains (exon 20) of the PIK3CA gene [2]. Mutations in the three mutation hotspots in PIK3CA (i.e., E542K, E545K, and H1047R) have been shown to elevate the lipid kinase activity and thereby leading to the activation of the downstream Akt signaling pathway [2, 8]. In a recent report, a PIK3CA mutation was identified in 4 of 35 esophageal squamous cell carcinomas and in 3 of 50 adenocarcinomas [9].

For these known mutations, real time polymerase chain reaction followed by melting curve analysis, using hybridization probes, has been demonstrated to be a highly sensitive, rapid, and an efficient approach to mutation detection [10, 11, 12]. To determine the PIK3CA mutation status in Japanese esophageal cancer for screening and diagnostic purposes, we used this PCR-based screening method [13]. The PIK3CA gene was then sequenced to confirm the results of such screening.

Section snippets

Patients

The study groups included 88 esophageal cancer patients who had undergone surgery at the Department of Surgery II, Nagoya City University Medical School between 1996 and 2003. All patients gave their written informed consent. The esophageal tumors were classified according to the general rules for clinical and pathological record of esophageal cancer in Japan. All tumor samples were immediately frozen and stored at −80°C until assayed.

The clinical and pathological characteristics of the 88

Mutational Analysis of PIK3CA Exon 9 and Exon 20 in Esophageal Cancer

For the PCR-based mutation analysis of the exon 9 of PIK3CA, the anchor probe was matched for the wild type sequence. As shown in Fig. 1A, the wild type PCR product showed a melting curve with a single peak at 66°C, whereas the heterozygous products (mutant) showed an additional peak at 59°C. Among the 88 esophageal cancer patients, three patients had an additional peak (in two, the mutation was confirmed by sequencing; see below). Using the probe for exon 20, the homozygous wild type PCR

Discussion

In the experiments reported in this paper, we found that 2.2% (2/88) of the esophageal cancer patients and 7.4% (2/27) of the esophageal cancer cell lines harbored a somatic mutation in the exon 9 of the PIK3CA gene (TABLE 1, TABLE 2, Fig. 2). No exon 20 mutation was found. We specifically examined exon 9 and 20 of PIK3CA genes because a previous study on a large number of colon cancers reported that four-fifths of the observed mutations were clustered in these two exons [2]. The mutation

Acknowledgments

The authors thank Ms. Shinobu Makino for her excellent technical assistance. This work was supported by the Grant-in-aid for Science Research (No. 19790949) from the Ministry of Education, Culture, Sports, Science and Technology.

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