Annals of Surgical Oncology Cite Track
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10.1245/ASO.2006.08.006
Annals of Surgical Oncology 13:961-966 (2006)
© 2006 Society of Surgical Oncology
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Original Article

PIK3CA and TFRC Located in 3q Are New Prognostic Factors in Esophageal Squamous Cell Carcinoma

Shinsuke Wada, MD, Tsuyoshi Noguchi, MD, PhD, Shinsuke Takeno, MD, PhD and Katsunobu Kawahara, MD, PhD

Department of Oncological Science (Surgery 2), Oita University Faculty of Medicine, Idaigaoka 1-1, Hasamam-machi, Oita, 879-5593, Japan

Correspondence: Address correspondence and reprint requests to: Shinsuke Wada, MD; E-mail: hasamada2000{at}yahoo.co.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 STATISTICAL ANALYSIS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Amplification of the chromosome 3q seems to occur frequently in esophageal squamous cell carcinoma (ESCC). This study analyzed the clinical effect of messenger RNA (mRNA) expression for PIK3CA (the gene that encodes phosphatidylinositol-3 kinase catalytic {alpha}-polypeptide) and TFRC (the gene that encodes the transferrin receptor), which map within chromosome 3q in ESCC.

Methods: Formalin-fixed, paraffin-embedded ESCC tissues were examined. Total RNAs were extracted, and reverse transcription products were subjected to polymerase chain reaction amplification of ß-actin, PIK3CA, and TFRC.

Results: Expression of ß-actin mRNA was detected in 67 (55.8%) of 120 samples, with PIK3CA mRNA expression in 22 (32.8%) of these 67 samples and TFRC mRNA expression in 15 (22.4%) of the 67 samples. PIK3CA mRNA expression correlated with regional lymph node metastasis (P = .04). TFRC mRNA expression correlated with distant metastasis (P = .04). Patients with positive results for either PIK3CA or TFRC mRNA displayed a significantly worse prognosis than patients with negative results (PIK3CA, P = .045; TFRC, P = .009). TFRC mRNA expression represented an independent prognostic factor in multivariate analysis (P = .0233), but PIK3CA did not (P = .7585).

Conclusions: PIK3CA and TFRC mRNA represent prognostic factors in patients with ESCC. TFRC mRNA offers an independent prognostic factor, and expression may have clinically important implications.

Key Words: Esophageal squamous cell carcinoma • PIK3CA • TFRC • Formalin fixed, paraffin embedded


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 STATISTICAL ANALYSIS
 RESULTS
 DISCUSSION
 REFERENCES
 
Esophageal squamous cell carcinoma (ESCC) remains one of most malignant tumors in the world. Despite various therapies tried for ESCC, including surgery, chemotherapy, radiotherapy, and combinations thereof, prognosis remains unfavorable, with ≤40% 5-year survival.1,2 Numerous studies have attempted to clarify the underlying molecular mechanisms and determine the biological behaviors of ESCC.

Gene amplification represents one of the essential mechanisms in oncogene activation. The recurrent DNA copy number increases in tumors revealed by karyotypic abnormalities, such as double minute or homogeneously staining regions, have been considered to offer clues to areas that may harbor putative oncogenes. Comparative genomic hybridization (CGH) provides a molecular method that enables the analysis of each chromosome of tumor cells for genetic amplification and deletions.3,4 Various chromosomal amplicons have recently been identified in ESCC by CGH analysis.

Genomic alterations involving chromosome 3 seem to be one of the most frequent events in squamous cell carcinoma, and amplification of the 3q26–29 region seems particularly frequent in ESCC.5–9 However, the specific gene targets within these amplicons remain largely unknown. PIK3CA encodes the catalytic subunit of PI3K{alpha}, which is required for numerous physiological processes, including differentiation, cell adhesion, and protein synthesis.10 TFRC encodes the transferrin receptor that is associated with neoplastic cellular proliferation.11 To clarify the significance of the amplification of chromosome 3q26–29, this study analyzed the clinical effect in ESCC of messenger RNA (mRNA) expression for PIK3CA and TFRC, which map within the region of 3q26–29.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
STATISTICAL ANALYSIS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Samples
Samples were obtained from 120 patients with ESCC who had undergone radical esophagectomy without prior anticancer treatment in the Second Department of Surgery at Oita University between 1995 and 2000. They were classified according to the tumor-node-metastasis classification system of the International Union Against Cancer.12

Tissue Preparation and RNA Extraction From Tissues
Formalin-fixed, paraffin-embedded specimens were cut in 10-µm-thick sections. Six serial sections were made, and one was stained with hematoxylin and eosin. Cancer tissue was collected by scraping the remaining five sections with a razor blade, with reference to the hematoxylin and eosin–stained section. Total RNA was extracted from tissue samples by using a modification of the method described by Tachibana et al.13 Briefly, samples were deparaffinized with xylene and rehydrated in 100% ethanol. The resulting tissue pellet was dried and resuspended in 50 µL of RNA-extracting mixture (PicoPure RNA Isolation Kit; Arcturus Bioscience Inc., Mountain View, CA). RNA was extracted according to the instructions of the manufacturer. RNA concentrations were measured with a spectrophotometer.

Reverse Transcriptase-Polymerase Chain Reaction
Reverse transcriptase (RT)-polymerase chain reaction (PCR) was performed at 42°C for 60 minutes by using 100 ng of RNA, random primer (25 pmol/µL), and ReverTra Ace (Toyobo, Osaka, Japan) in a 20-µL reaction mixture. Subsequently, 2 µL of each RT product was subjected to PCR amplification by using a ReverTra-Plus (Toyobo) in accordance with the instructions of the manufacturer. PCR protocols were as follows: initial denaturation at 94°C for 2 minutes, followed by 40 cycles at 98°C for 10 seconds; annealing at 60°C for 30 seconds for ß-actin, at 50°C for 30 seconds for PIK3CA, and at 52°C for 30 seconds for TFRC; and extraction at 72°C for 2 minutes. The RT-PCR product was spread by using 2% agarose gel electrophoresis in Tris-borate-ethylene diamine tetra-acetic acid buffer and visualized by using ethidium bromide staining. RT-PCR product was detected as a 100-base pair (bp) fragment for ß-actin, a 99-bp fragment for PIK3CA, and a 105-bp fragment for TFRC (Fig. 1Go). Primers used for PCR are listed in Table 1Go. Each RT-PCR was performed twice per sample. Samples were judged as positive only when each fragment was clearly detected in both assays.


Figure 1
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  		FIG. 1. Detection of ß-actin, PIK3CA, and TFRC messenger RNA (mRNA) in formalin-fixed, paraffin-embedded esophageal squamous cell carcinoma sections. Expression of ß-actin mRNA was detected in all cases. Expression of PIK3CA and TFRC mRNA was detected in three (samples 1–3) and three (samples 1, 2, and 4) of five samples, respectively. bp, base pair.

 

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  		TABLE 1. Primer used
 
Clinical Follow-Up
After discharge, all patients underwent follow-up every 1 to 3 months with physical examinations, routine blood tests, and serum tumor marker tests and every 3 to 6 months by using computed tomography. Follow-up data were obtained for all patients.


   STATISTICAL ANALYSIS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 STATISTICAL ANALYSIS
RESULTS
 DISCUSSION
 REFERENCES
 
Correlations between PIK3CA or TFRC expression and clinicopathologic factors were analyzed by using the Mann-Whitney U-test, Fisher’s exact probably test, or the {chi}2 test. Disease-specific survival after surgery was examined by using Kaplan-Meier methods, and survival characteristics were compared by using the log-rank test. A Cox proportional hazards regression model was used for multivariate analysis. Values of P < .05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 STATISTICAL ANALYSIS
 RESULTS
DISCUSSION
 REFERENCES
 
Amplification of ß-Actin mRNA From Tissues
To confirm extraction of mRNA, preliminary experiments were conducted to amplify ß-actin mRNA from sections. The band of ß-actin mRNA was clearly detectable in 67 (55.8%) of 120 ESCC samples. Analysis for expression of PIK3CA and TFRC was therefore confined to these 67 samples. Details of the 67 patients are listed in Table 2Go.


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  		TABLE 2. Background of 67 patients
 
Relationship Between PIK3CA mRNA Expression and Clinicopathologic Findings
Positive results for PIK3CA mRNA were identified in 22 (32.8%) of the 67 patients. Relationships between PIK3CA mRNA expression in ESCC samples and clinicopathologic factors were examined (Table 3Go). For the 67 ESCC samples studied, only regional lymph node metastasis (pN) displayed significant correlations with PIK3CA mRNA expression (P = .04). Although no significant relationship was observed, lymphatic invasion was common in patients with positive results for PIK3CA mRNA (P = .07). Survival analysis revealed that patients with positive results for PIK3CA mRNA displayed significantly unfavorable outcomes (34.6 ± 4.6 months) compared to patients with negative results for PIK3CA mRNA (43.8 ± 2.6 months) in univariate analysis (Fig. 2Go).


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  		TABLE 3. Clinicopathologic implications of PIK3CA mRNA expression
 

Figure 2
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  		FIG. 2. Disease-specific survival after surgery for patients with esophageal squamous cell carcinoma. Patients with positive results for PIK3CA messenger RNA (mRNA; black line) displayed more unfavorable outcomes than patients with negative results (gray line; P = .045).

 
Relationship Between TFRC mRNA Expression and Clinicopathologic Findings
Positive results for TFRC mRNA were identified in 15 (22.4%) of the 67 patients. Relationships between TFRC mRNA expression and clinicopathologic factors were examined (Table 4Go). For the 67 ESCC samples studied, distant metastasis (pM; P = .04) correlated significantly with TFRC mRNA expression. Patients with positive results for TFRC mRNA displayed a significantly worse prognosis (31.0 ± 5.9 months) than patients with negative results for TFRC mRNA (43.2 ± 2.4 months) according to univariate analysis (Fig. 3Go).


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  		TABLE 4. Clinicopathologic implications of TFRC mRNA expression
 

Figure 3
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  		FIG. 3. Disease-specific survival after surgery for patients with esophageal squamous cell carcinoma. Patients with positive results for TFRC messenger RNA (mRNA; black line) displayed more unfavorable outcomes than patients with negative results (gray line; P = .009).

 
Multivariate Analysis
Multivariate analysis of prognostic factors including pT, pN, and pM and expression of PIK3CA mRNA and TFRC mRNA suggested that pM (P = .0467) and TFRC mRNA expression (P = .0223) represented independent prognostic factors, but that PIK3CA mRNA expression did not (P = .7585; Table 5Go).


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  		TABLE 5. Multivariate analysis by Cox proportional hazards regression model
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 STATISTICAL ANALYSIS
 RESULTS
 DISCUSSION
REFERENCES
 
Although formalin-fixed samples are known to offer poor materials for molecular biological applications, several reports have demonstrated the feasibility of extracting mRNA from formalin-fixed, paraffin-embedded tissues.13–20 Large numbers of formalin-fixed, paraffin-embedded tissue samples have been collected throughout the years and stored for verification of preliminary diagnoses in various diseases. The wide availability of these tissues leads to the possibility of further retrospective investigations of diseases. These tissues thus represent a valuable resource for molecular biological investigations. Studies of gene expression can be performed with several methods, including Northern blot, micro-array, in situ hybridization, and RT-PCR. Messenger RNA in a cell offers a snapshot of the real-time activity of the genome, depicting what genes are being expressed and to what extent.

To evaluate the quality of extracted RNA, expression of ß-actin mRNA was investigated, and this revealed ß-actin mRNA expression in 67 of the 120 samples. This imperfection may result from fix-ation conditions. Fixation time in formalin is often variable in tissue processing and has been shown to affect the detection of target RNA sequences. Formalin fixation for 24 to 48 hours is reportedly optimal.21,22 More uniform fixation conditions may allow more efficient RNA extraction.

A common amplification target encompassing chromosome region 3q has been identified by CGH in various types of carcinoma.5,23 Amplification of 3q26–29 has been reported in ESCC.5–9 Racz et al.24 noted that the amplified region contains genes for ribosomal protein L22 (RPL22), butyrylcholinesterase (BCHE), glucose transfer 2 (SKC2A2), thrombopoietin (THPO), PIK3CA, and TFRC. PIK3CA and TFRC caught our attention because both genes are related to cell proliferation.10,11

PIK3CA encodes the p110{alpha} catalytic subunit of phosphatidylinositol-3 kinase and has recently been suggested to play a critical role as an oncogene in ovarian carcinoma and squamous cell carcinoma of the oral tongue.25,26 PIK3CA was found as a major phosphotyrosyl protein subsequent to growth factor stimulation and oncogenic transformation,27 and it is involved in several cell-signaling pathways, including those of epidermal growth factor, platelet-derived growth factor, and insulin-like growth factor.28,29 This study found that mRNA expression of PIK3CA was correlated with pN status and a worsened prognosis of ESCC. Woenckhaus et al.30 reported that PIK3CA expression was associated with tumor progression of head and neck squamous cell carcinoma. The observed association between PIK3CA mRNA expression and pN status suggests that PIK3CA may also play an important role in tumor progression in ESCC, influencing disease-specific survival in these patients.

TFRC encodes the transferrin receptor, which plays an important role in controlling cell growth through iron uptake. Transferrin receptor expression occurs in various neoplastic tissues, including lung, breast, and other carcinomas; some sarcomas; leukemia; and malignant lymphoma.31,32 In non–small-cell lung cancer, expression of the transferrin receptor is reportedly a prognostic indicator.33,34 Kondo et al.33 reported significant correlations between transferrin receptor expression in lung cancer cells and the mitotic index. Some authors reported significant correlations between various indices of proliferation and tumor progression and survival in ESCC.35 The present findings lend further credence to these reports. In multivariate analysis including pT, pN, pM, PIK3CA mRNA expression, and TFRC mRNA expression, expression of TFRC mRNA was an independent prognostic factor. This result presumably reflects the effect of cell proliferation on biological behaviors in ESCC.

This study has some limitations. Expression levels of mRNA were investigated only with qualitative analyses. Further quantitative analyses, such as real-time PCR, would allow more precise outcomes.

The results suggest that PIK3CA and TFRC, both of which are located on 3q, are associated with tumor progression and prognosis in patients with ESCC who undergo radical esophagectomy. In particular, TFRC offers an independent prognostic factor and might be an indicator of malignant potential in patients with ESCC. Expression of TFRC mRNA in ESCC may have clinically important implications and might be an important target for novel chemotherapeutic or gene-therapeutic approaches to ESCC.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Yuzo Uchida, Professor Emeritus of Oita Medical University, for advice and helpful comments during the study.

Received for publication August 2, 2005. Accepted for publication December 22, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 METHODS
 STATISTICAL ANALYSIS
 RESULTS
 DISCUSSION
 REFERENCES
 

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