Yasuyuki Fukai

Genetic Alterations in Esophageal Cancer

Esophageal cancer is a common malignancy with a striking variation in geographical distribution; a reflection of exposure to specific environmental factors, which are still poorly defined. We discuss the recent progress made in the investigation of the molecular biology of esophageal cancer, addressing the topics of genetic alterations, methylation, overexpression of molecules thought to cause malignant transformation, carcinogenesis, invasion, and metastasis. We review six aspects of the research literature on esophageal cancer: epidemiology and etiology, epidermal growth factor receptor and related growth factor receptors, cell cycle regulatory proteins, transforming growth factor-β/Smad proteins, mismatch repair genes, and other genes. This article provides a conceptual basis for evaluating studies on the molecular mechanism of esophageal carcinogenesis and for devising therapeutic and preventive strategies based on molecular biology. We hope that in the near future, the clinical outcome of patients with esophageal carcinoma will be improved by a better understanding of the basic mechanisms of carcinogenesis.

Increased expression of c-Ski as a co-repressor in transforming growth factor-β signaling correlates with progression of esophageal squamous cell carcinoma

Transforming growth factor‐β (TGF‐β) regulates cell growth inhibition, and inactivation of the TGF‐β signaling pathway contributes to tumor development. In our previous study, altered expression of TGF‐β, TGF‐β‐specific receptors and Smad4 was shown to correlate with tumor progression in esophageal squamous cell carcinoma (SCC). These components, however, were maintained normally in some patients with esophageal SCC. In our study, the mechanism by which aggressive esophageal SCC maintains these components was investigated, with particular emphasis on the participation of c‐Ski and SnoN as transcriptional co‐repressors in TGF‐β signaling. Immunohistochemistry for c‐Ski and SnoN was carried out on surgical specimens obtained from 80 patients with esophageal SCC. The expression of c‐Ski and SnoN was also studied in 6 established cell lines derived from esophageal SCC and compared to an immortalized human esophageal cell line by Western blotting. High levels of expression of c‐Ski, detected immunohistologically, were found to correlate with depth of invasion (p = 0.0080) and pathologic stage (p = 0.0447). There was, however, no significant correlation between expression of SnoN and clinicopathologic characteristics. A significant correlation between c‐Ski and TGF‐β expression was observed. Moreover, in patients with TGF‐β negative expression, the survival rates of patients with c‐Ski positive expression were significantly lower than those of patients with c‐Ski negative expression (p = 0.0486). c‐Ski was expressed at a high level in 5 of 6 cell lines derived from esophageal SCC compared to immortalized esophageal keratinocytes. Furthermore, the cyclin‐dependent kinase (CDK) inhibitor, p21 that was up‐regulated by TGF‐β signaling was expressed at a low level in the 5 cell lines. The expression of c‐Ski protein as a transcriptional co‐repressor in TGF‐β signaling seems to be correlated with tumor progression of esophageal SCC.

Reduced expression of transforming growth factor-β receptors is an unfavorable prognostic factor in human esophageal squamous cell carcinoma

Transforming growth factor‐β (TGF‐β) inhibits epithelial cell proliferation. Inactivation of the TGF‐β signaling pathway is thought to play a role in tumorigenesis. Our purpose was to clarify the correlation between TGF‐β receptors or TGF‐β1 expression and the clinicopathologic characteristics of patients with esophageal squamous cell carcinoma (SCC). Immunohistochemical staining for TGF‐β type I receptor (TGF‐βR‐I), TGF‐βR‐II and TGF‐β1 was performed on surgical specimens obtained from 80 patients with esophageal SCC. Preoperative plasma TGF‐β1 levels were measured and correlated with pathologic features and clinical outcomes. Expression of TGF‐βR‐I and TGF‐βR‐II was reduced in 43 (53.8%) and 23 (28.8%) specimens, respectively. TGF‐β1 was overexpressed in 29 (36.3%). Reduced expression of TGF‐βR‐I and TGF‐βR‐II showed a significant association with depth of invasion (p = 0.0015 and p = 0.0012), lymph node metastasis (p = 0.0309 and p = 0.0059) and pathologic stage (p = 0.0103 and p = 0.0401). Overexpression of TGF‐β1 had a significant association with depth of invasion only (p = 0.0335). Reduced expression of TGF‐βR‐I and TGF‐βR‐II was correlated with cancer‐specific survival (p = 0.0324 and p = 0.0243). The mean preoperative plasma TGF‐β1 level was 10.5 ± 0.8 ng/ml in patients with esophageal carcinoma and was significantly higher compared to healthy controls (p < 0.01). We demonstrate that reduced expression of TGF‐β receptors in esophageal SCC appears to be correlated with depth of invasion, lymph node metastasis, pathologic stage and poor prognosis. TGF‐β receptor expression may play a key role in the progression of this cancer.

Pretreatment evaluation of combined HIF‐1α, p53 and p21 expression is a useful and sensitive indicator of response to radiation and chemotherapy in esophageal cancer

Tumor hypoxia has been known to be associated with resistance to radiation and chemotherapy (CRT). Hypoxia‐inducible factor‐1α (HIF‐1α), a transcription factor induced by hypoxic condition, plays a major role in the pleiotropic response observed under hypoxic conditions. It encodes proteins that play key roles in critical development and physiologic processes, including angiogenesis, glucose transport and erythropoiesis. On the other hand, cell cycle‐ and apoptosis‐control genes p53 and p21 may play major roles in the tumor response to cytotoxic agents such as radiation and chemotherapy. Previous reports have suggested that the regulation of p53 and p21 is HIF‐1‐dependent. Our aim was to evaluate the expression of the HIF‐1α, p53 and p21 proteins by immunohistochemistry in biopsy specimens of esophageal squamous cell carcinoma, which were obtained endoscopically from 65 patients before CRT, and then determine whether the levels of expression of these proteins predicted the clinical effectiveness of CRT in individual cancers. Also, to assess the relationship between expression of these proteins and cell death and cellular proliferation activity, we evaluated Ki67 expression and the apoptosis index (TUNEL). HIF‐1α expression in esophageal cancer was significantly and negatively related to the response to CRT, independently of p53 and p21 expression. Interestingly, 44.4% (12/27) of the HIF‐1α‐negative group showed a complete response to therapy. There was no significant correlation between the expression of HIF‐1α, p53 and p21 and proliferation and apoptosis. HIF‐1α overexpression may predict resistance to CRT and may be a helpful guide in choosing between therapeutic strategies, such as intensive combined modality therapy vs. palliative therapy. Combined immunohistochemical evaluation of HIF‐1α, p53 and p21 protein expression at the pretreatment biopsy is a very useful and powerful indicator of sensitivity to CRT in human esophageal cancer. Our data also indicate the importance of having a clear grasp of the degree of hypoxia (HIF‐1α) of a tumor, rather than its cellular character (proliferation and apoptosis), to indicate the likely impact of CRT.

Plasma Level of Transforming Growth Factor β1 Measured from the Azygos Vein Predicts Prognosis in Patients with Esophageal Cancer

Purpose: Transforming growth factor (TGF)-β regulates cell growth inhibition. When tumor cells lose their sensitivity to TGF-β growth inhibition, the excess TGF-β that results may act on tumor cells to facilitate tumor development. Previously, we have shown that an elevated systemic TGF-β1 level is not related to tumor progression in esophageal cancer (Y. Fukai et al., Int J Cancer 2003;104:161–6). We considered that systemic inflammation or chronic disease, in addition to the tumor, may influence the plasma TGF-β level. Therefore, we examined the hypothesis that the plasma TGF-β level measured from the azygos vein would independently predict tumor progression and prognosis in patients with esophageal cancer. Experimental Design: Fifty-seven plasma samples were obtained intraoperatively from the azygos vein in patients with esophageal cancer. ELISA was used to quantify the plasma TGF-β1 levels, which were correlated with pathological features and patient survival. Results: The mean plasma TGF-β1 level measured from the azygos vein of esophageal cancer patients was 5.09 ± 0.48 ng/ml (mean ± SE). The survival rates of patients with a high TGF-β1 level (defined as a level above the 4.6 ng/ml level of normal controls) in the azygos vein were significantly lower than those of patients with a low TGF-β1 level (P = 0.0317). Moreover, the TGF-β1 level in the azygos vein was an independent prognostic factor for overall survival (P = 0.0474). Conclusions: The level of plasma TGF-β1 measured from the azygos vein is an independent predictor in patients with esophageal cancer and may reflect tumor progression more specifically because the azygos vein is responsible for venous return from the esophagus.

Expression of p53 protein related to smoking and alcoholic beverage drinking habits in patients with esophageal cancers

In esophageal squamous cell carcinoma (SCC), we used immunohistochemical analysis to further elucidate the correlation of p53 protein expression with clinicopathological factors, as well as with risk factors, such as tobacco smoking, alcohol consumption and a family history of cancer, using odds ratios (ORs). The expression of p53 protein was demonstrated in 55.1% of 89 esophageal SCC cases examined by immunohistochemistry. The expression of p53 protein did not correlate with gender, age, histological grading, lymph node metastasis, or TNM stage. The prevalence of p53 expression was significantly higher in patients with multiple primary esophageal cancers (P<0.05). p53 expression did not correlate with prognosis in univariate survival analysis. The esophageal SCC in either smokers or alcohol users was 4.67-5.83 times more likely to express p53 protein, while the likelihood of p53 expression in patients who use both tobacco and alcohol was more than 14.0 times. However, a significant association was not found between p53 expression and a family history of cancer, this having an OR as low as 1.85. The expression of p53 protein did not correlate with clinicopathological factors and prognosis in univariate and multivariate survival analyses. In contrast, tobacco smoking and alcohol consumption were shown to be strongly associated with p53 mutations in esophageal carcinogenesis.

Surgical Treatment for Esophageal Cancer

Esophageal cancer is one of the most difficult malignancies to cure. The prognosis remains unsatisfactory despite significant advances in surgical techniques and perioperative management. The optimal treatment strategy for localized esophageal cancer has not yet been established. Surgical resection remains the mainstay of treatment for esophageal cancer, and curative resection is the most important surgery. Extended esophagectomy with three-field lymphadenectomy provides the highest quality of tumor clearance and prolongation of patient survival. There has been intense effort in developing novel strategies to treat patients with resectable esophageal cancer. Various combined-modality approaches have been attempted to improve treatment outcomes. Definitive chemoradiotherapy has an impact on long-term survival in patients with resectable esophageal cancer. Accordingly, there are three main combined-modality approaches: esophagectomy with adjuvant chemotherapy or chemoradiotherapy; primary definitive chemoradiotherapy with or without salvage esophagectomy, and preoperative chemoradiotherapy followed by planned esophagectomy. Recently, owing to the remarkable advances in optical technology, minimally invasive esophagectomy using endoscopic instruments has been introduced into esophageal cancer surgery. This article reviews recent changes in the treatment of esophageal cancer surgery, and considers the role of esophagectomy.

Correlation between Laminin-5 γ2 Chain and Epidermal Growth Factor Receptor Expression in Esophageal Squamous Cell Carcinomas

Objectives: Laminin-5 γ2 chain (LN-5 γ2) is an extracellular matrix protein that plays an important role in cell migration and tumor invasion. We evaluated the association of LN-5 γ2 and epidermal growth factor receptor (EGFR) expression in esophageal squamous cell carcinoma (SCC). Methods: LN-5 γ2 and EGFR expression was evaluated in 110 esophageal SCC patients by immunohistochemistry, and was confirmed using esophageal SCC cell lines by Western blot analysis. Results: LN-5 γ2 expression in the invasive front of the tumor was correlated with the depth of invasion (p = 0.0001), lymph node metastasis (p = 0.0011) and pathological stage (p = 0.0001). The strong expression of EGFR was also correlated with lymph node metastasis (p = 0.0456) and the pathological stage (p = 0.0055). In patient survival, LN-5 γ2 positivity and/or strong EGFR expression showed a significantly low survival rate as compared with those with lesser expression of LN-5 γ2 and EGFR. Immunohistochemically, LN-5 γ2 expression was significantly correlated with EGFR expression (p < 0.0001). Western blot analysis also confirmed the correlated expression of LN-5 γ2 and EGFR in SCC cell lines except 2 of the 5 cell lines. Conclusions: This study suggests that coexpression of LN-5 γ2 and EGFR is closely related to the progression and poor prognosis of esophageal SCC.

Prediction of hematogenous recurrence in patients with esophageal carcinoma

OBJECTIVES Despite recent advances in diagnosis and treatment of esophageal carcinoma, the future risk of hematogenous recurrence is still unpredictable. To identify risk factors of hematogenous recurrence in esophageal carcinoma, we used pathological and immunohistochemical analysis to examine relationships among clinical outcomes, clinicopathological features, and E-cadherin expression. METHODS Subjects were 102 patients with thoracic esophageal cancer who had undergone curative esophagectomy without preoperative treatment. We used univariate and multivariate logistic regression analyses to examine the relationship among clinical outcomes, clinicopathological features, and E-cadherin expression. RESULTS There was no significant relationship between E-cadherin expression and clinicopathological features at operation. However, the survival rates of patients with E-cadherin-negative tumors were significantly lower than those of patients with E-cadherin-weak and E-cadherin-positive tumors (P < 0.01). Disease recurrence had occurred in 49 (48.0%), with hematogenous recurrence in 29 (28.4%), of the 102 patients at the time of analysis. Metastasis occurred in liver in 14 patients, lung in 13, bone in 6, and brain in 2. Comparisons of hematogenous recurrences and clinicopathological features by multivariate regression analyses revealed significant associations between hematogenous recurrences; particularly in liver and lung metastasis and negative E-cadherin expression. With regard to the associations between the organ with the recurrence and the number of positive nodes; hematogenous recurrence, equal to or higher than lymphatic recurrence, was more likely to have occurred in patients with high numbers of positive nodes. Interestingly, with regard to the sites of positive nodes, liver metastasis was closely correlated with lymph node metastasis in the mid-thoracic as opposed to the abdominal region. Further, lung metastasis was most likely to occur in patients with cervical lymph node metastasis. CONCLUSIONS Esophageal carcinoma with negative E-cadherin expression tended to be associated with hematogenous recurrence, particularly with liver and lung metastasis. Hematogenous recurrences were significantly associated with high numbers and the site of positive nodes, as well as with lymphatic recurrence.

Nitrotyrosine in esophageal squamous cell carcinoma and relevance to p53 expression

We have examined the expression of nitrotyrosine, a marker of peroxynitrite formation, in 55 esophageal cancers by immunohistochemistry. Nitrotyrosine was detected in 21 of 55 (38.2%) esophageal cancers. Comparison of nitrotyrosine expression and the pathological findings showed that there was a significant association between the expression of nitrotyrosine and each of the depth of tumor invasion (P<0.01), occurrence of metastasis (P<0.05), pathological stage (P<0.01), smoking status (P<0.05) and alcohol intake (P<0.05). The survival rate of patients with nitrotyrosine-negative cancer was significantly higher than that of patients with nitrotyrosine-positive cancer (log-rank test, P<0.01). p53 was detected in 29 of 55 (52.7%) esophageal cancers, however, p53 expression did not correlate with nitrotyrosine expression. In conclusion, nitrotyrosine, a product of nitrogen species, is expressed in esophageal squamous cell carcinoma, which suggests that exogenous risk factors, such as tobacco and alcohol, through NO, are associated with carcinogenesis and progression of esophageal squamous cell carcinoma.