pISSN : 2093-582X, eISSN : 2093-5641 J Gastric Cancer 2016;16(2):85-92 http://dx.doi.org/10.5230/jgc.2016.16.2.85
Clinicopathological Significance of Elevated PIK3CA Expression in Gastric Cancer Si-Hyong Jang, Kyung-Ju Kim, Mee-Hye Oh, Ji-Hye Lee, Hyun Ju Lee, Hyun Deuk Cho, Sun Wook Han1, Myoung Won Son1, and Moon Soo Lee1 Departments of Pathology and 1General Surgery, Soonchunhyang University Cheonan Hospital, Soonchunhyang University College of Medicine, Cheonan, Korea
Purpose: PIK3CA is often mutated in a variety of malignancies, including colon, gastric, ovary, breast, and brain tumors. We investigated PIK3CA expression in gastric cancer and explored the relationships between the PIK3CA expression level and clinicopathological features as well as survival of the patients. Materials and Methods: We examined PIK3CA expression in a tissue microarray of 178 gastric adenocarcinomas by immunohistochemistry and reviewed patients’ medical records. Results: In our study, 112 of the 178 gastric cancer patients displayed positive PIK3CA expression. Overexpression of PIK3CA was correlated with low grade differentiation (P=0.001), frequent lymphatic invasion (P=0.032), and high T stage (P=0.040). Patients with positive PIK3CA staining were more likely to display worse overall survival rate than those with negative PIK3CA staining, as determined by Kaplan-Meier survival analysis with log-rank test (P=0.047) and a univariate analysis using the Cox proportional hazard model (hazard ratio=1.832, P=0.051). Conclusions: Elevated PIK3CA expression was significantly correlated with tumor invasiveness, tumor phenotypes, and poor patient survival. Key Words: PIK3CA; Stomach neoplasms; Immunohistochemistry
half of gastric cancer patients are not cured.2 Both environmental factors and genetic/epigenetic alterations are thought to be asso-
Gastric cancer is the fifth most common malignant tumor
ciated with tumor development and progression, but the precise
and the third leading cause of cancer-related death worldwide.
pathogenesis has not been determined.3 Therefore, the discovery
Although incidence of gastric cancer has decreased in recent
of gastric cancer-specific markers that are clinically applicable
years, it remains a major global health problem, with a particu-
for early cancer detection and targeted therapeutics is crucial.
larly high prevalence rate in Asia. Improvements in surgical
The phosphatidylinositol 3-kinase (PI3K) signaling pathway
and chemotherapeutic interventions have led to lower gastric
is pivotal to drive the extracellular growth signaling during tu-
cancer morbidity and mortality rates; however, approximately
morigenesis, progression, and drug resistance of breast, colorectal, endometrial, and gastric cancers.3-7 During malignant trans-
Correspondence to: Moon Soo Lee Department of General Surgery, Soonchunhyang University Cheonan Hospital, 31 Suncheonhyang 6-gil, Dongnam-gu, Cheonan 31151, Korea Tel: +82-41-570-3628, Fax: +82-41-571-0129 E-mail: [email protected]
Received December 21, 2015 Revised March 30, 2016 Accepted April 14, 2016
formation, various genetic alterations may occur in any of the PI3K pathway components, such as the receptor tyrosine kinase genes, EGFR, HER2, KIT, PTEN, PIK3CA, and AKT. PI3K is a heterodimer comprising of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit that possess an enzymatic activity. To date, four isoforms of PI3K catalytic subunit (α, β, γ, and δ) have
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyrights © 2016 by The Korean Gastric Cancer Association
86 Jang SH, et al.
been identified. Among these, the gene encoding the α-isoform
reviewed. Patients who received neoadjuvant chemotherapy and/
catalytic subunit (PIK3CA, p110α) was found to be frequently mutated or amplified, leading to the acquisition of oncogenic
or had other critical medical conditions were excluded in this study. Cases with no tissue samples available were also excluded.
properties or increased tumor aggressiveness. PIK3CA mutation and amplification in gastric cancer have also been described; in particular, the amplification of PIK3CA is frequently observed in gastric adenocarcinoma.10,11
2. Tissue microarray construction Tissue microarrays (TMA) were prepared as previously described.15 H&E-stained slides were evaluated under light mi-
PIK3CA mutation and amplification in gastric cancer can act
croscopy to define representative and non-necrotic areas in the
as a driver mutation, and they are associated with carcinogenesis
tumors. Single tissue cores (2 mm in diameter) were obtained
In gastric cancer, dysregulation of the
from paraffin blocks (donor block) and transferred to a recipient
PI3K-Akt pathway due to PIK3CA mutation and inactivation of
block using a trephine apparatus (Superbiochips Laboratories,
the phosphatase and tensin homolog (PTEN) has been identified
Seoul, Korea). Cores of non-tumorous gastric mucosa tissue
and adverse prognosis.
as a mechanism for trastuzumab resistance. Moreover, aber-
were included and assembled between the tumor cores.
rant increase in PIK3CA immunoreactivity can promote gastric cancer metastasis.9 Here, we investigated PIK3CA expression in
3. Immunohistochemical staining and assessment
human gastric cancer tissue using immunohistochemistry. We evaluated the relationships between PIK3CA expression level
For PIK3CA immunohistochemistry, 4-µm sections were cut from the TMA block and transferred onto coated slides. The
and the conventional clinicopathological parameters, as well as
sections were deparaffinized through incubation in a dry oven at
effects on patient survival. In addition, we sought to determine
60oC for 1 hour and three washes in xylene. Following block-
the use of PIK3CA immunohistochemistry to predict the clinical
age of the endogenous peroxidase activity using 5% hydrogen
outcome of gastric cancer.
peroxide in methanol for 15 minutes at 37oC, the sections were autoclaved in a pH-6.0 epitope retrieval solution for 20 minutes
Materials and Methods
for antigen retrieval. The sections were stained with a 1:75 dilution of rabbit polyclonal anti-PIK3CA antibody (HPA009985;
1. Patients and specimens
Sigma-Aldrich, St. Louis, MO, USA). Following incubation in
For this retrospective study, we enrolled a consecutive series
a humidified chamber at 4oC for 16 hours, a secondary antibody
of 178 gastric cancer patients. Gastric adenocarcinoma was con-
treated using the BOND Polymer Refine Detection kit (Leica
firmed using histology for all patients. All patients underwent
Biosystems, Wetzlar, Germany) was applied. Diaminobenzidine
total or subtotal gastrectomy at the Soonchunhyang University
was used as chromogen for color development.
Cheonan Hospital between July 2006 and December 2012. The
PIK3CA immunostaining was evaluated by two independent
patients had a mean age of 61 years (range, 27 to 81 years). They
pathologists who were blinded to the clinicopathological infor-
were regularly followed every 6 months or yearly after surgery,
mation of the patients. Discrepancy in findings was solved by
and their mean follow-up period was 58 months (range, 0 to 95
reviewing the cases using a multi-head microscope until con-
months). Among 178 patients, 66 patients (37.1%) died and 112
sensus was reached. Cytoplasmic granular staining of PIK3CA
patients (66.9%) remain alive.
was considered as immunoreactivity. Adjacent non-tumorous
All tissue samples obtained in the study were fixed in forma-
mucosa in the tumor core and normal gastric mucosa cores were
lin and embedded in paraffin. Medical records from the initial
used as negative controls. PIK3CA expression was graded based
and follow-up visits, including clinical data, pathology reports,
on the proportion and intensity of staining. The stained cell pro-
and hematoxylin and eosin (H&E)-stained slides were reviewed
portion was scored as: 0 (＜5%); 1 (5% to 25%); 2 (26% to 50%);
to confirm diagnoses. The clinicopathological variables of the
and 3 (＞50%). Intensity of the immunostaining was scored as
patients, including age, sex, tumor size, tumor location, invasion
follows: 0 (negative); 1+ (weak positive); 2+ (moderate posi-
depth, lymph node metastasis, distant metastasis, TNM stage,
tive); and 3+ (strong positive) (Fig. 1). The final score was cal-
differentiation, lymphatic, vascular, and perineural invasions,
culated by multiplying the proportion score with the intensity
Lauren and Ming classifications, and patient survival were also
score. Final scores of ＜4 were considered negative, whereas
87 High PIK3CA Expression in Gastric Cancer
Fig. 1. Representative PIK3CA immunohistochemical staining images of gastric cancers (×400) exhibiting intensity score of 0 (A), 1 (B), 2 (C), or 3 (D).
Fig. 2. (A, B) Normal gastric mucosa, which was used as a negative control, displayed absence of PIK3CA staining (A) or weak PIK3CA staining (B) (×200). (C) Positive PIK3CA staining in tumor glands and negative staining in the adjacent non-tumorous gastric mucosa (×200).
scores of ≥4 were considered positive.16 For negative controls,
4. Statistical analysis
18 normal gastric mucosa cores were included in the TMA
All statistical analyses were performed using the PASW ver.
blocks. PIK3CA immunoreactivity score of these cores ranged
18.0 statistical software (IBM Co., Armonk, NY, USA). The
from 0 to 3. Additionally, non-tumorous mucosa adjacent to the tumor core showed no immunoreactivity or weak PIK3CA-
χ test for linear trends and Fisher’s exact test were utilized to determine the relationships between PIK3CA expression and
positive staining in contrast with the tumor glands (Fig. 2).
clinicopathological features of the patients. Survival curves were
88 Jang SH, et al.
Table 1. Correlations between PIK3CA expression and various clinicopathological features in gastric cancer patients
Gender Male Female Age (yr)