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Ann Thorac Surg 1998;66:1745-1750
© 1998 The Society of Thoracic Surgeons

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Original articles: general thoracic

Lewis Y antigen expression and postoperative survival in non–small cell lung cancer

^a Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University Kyoto, Japan

Address reprint requests to Dr Wada, Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University, Shogoinn-kawahara-cho 53, Sakyo-ku, Kyoto 606, Japan
e-mail: (wada{at}chest.kyoto-u.ac.jp)

Presented at the Poster session of the Thirty-fourth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 26–28, 1998.

	Abstract

Top Abstract Introduction Material and methods Results Discussion Acknowledgments References

 
Background. In contrast to other Lewis blood group-related antigens, Lewis Y antigen (Le^Y) has not been fully investigated in non–small cell lung cancer.

Methods. To assess the significance of Le^Y expression, 236 patients with completely resected pathologic stage 1-3a were reviewed with immunohistochemical analysis.

Results. Le^Y expression was positive in 179 patients (75.8%). In poorly differentiated cancer, percentage of Le^Y-positive patients was lower than in moderately to well-differentiated cancer (67.2% versus 81.2%, p = 0.028). Five-year survival rate of Le^Y-positive patients was 78.2%, significantly higher than that of Le^Y-negative patients (59.7%, p = 0.001). Combined with p53 status, differences in survival proved to be marked; 5-year survival rate of patients with positive Le^Y expression and without aberrant p53 expression, was as high as 83.3%, whereas that of patients with negative Le^Y expression and with aberrant p53 expression was only 38.4% (p < 0.001). Multivariate analysis confirmed that Le^Y expression was a significant independent factor to predict better survival.

Conclusions. Le^Y expression is a significant prognostic factor related to grade of cancer differentiation.

	Introduction

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A variety of changes in cell surface carbohydrates occur closely related to malignant transformation. ABH (O) and Lewis blood group-related antigens, which play major roles in alloantigenic systems in human tissues, are carbohydrates carried by two types of backbone structures, type 1 chain containing [Galß1 3GlcNAc] and type 2 chain containing [Galß1 4GlcNAc] [1]. Among them, Lewis A antigen having type 1 chain structure and Lewis X antigen having type 2 chain structure have been well studied as cancer-related carbohydrate antigens [2–5].

On the other hand, the biological significance of Lewis Y antigen (Le^Y, Fuc1 2Galß1 4[Fuc1 3]GlcNAc) having type 2 chain structure, has not been fully investigated yet. Only Le^Y expression in process of malignant transformation of colorectal tissue has been demonstrated in some reports [1, 5–9]. There have been only a few clinical reports on Le^Y expression in colorectal cancer [9], hepatocellular cancer [10], breast cancer [11], and lung cancer [12, 13]; the clinical significance as a cancer-related marker has not been established. In the present study, therefore, significance of Le^Y expression in nonsmall cell lung cancer (NSCLC) was analyzed.

	Material and methods

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A total of 237 consecutive patients with pathologic stage 1–3a NSCLC who underwent complete tumor resection and mediastinal lymph node dissection without chemotherapy nor radiation therapy before operation at the Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University between January 1, 1985 and December 31, 1990, were reviewed. Complete tumor resection was defined when no microscopic cancers were identified either in the resection margins of the tumor or in the highest mediastinal lymph nodes [14]. Pathologic stage was determined histopathologically in accordance with tumor-node-metastasis classification revised in 1986 [15], and pathologic stage of patients treated before 1986 was reevaluated according to the same criteria. Histological type was determined in accordance with the classification by the World Health Organization. One case was excluded from the study due to operation-related death, a total of 236 patients were evaluated in this study. In all patients, the in-patient medical records, chest x-ray films, whole-body computed tomography (CT) films, bone and gallium scanning data, records of surgery were evaluated without knowledge of the p53 status with immunohistochemical examination. Postoperative follow-up surveillance study was conducted by the use of outpatient records and telephone or letter inquiries. The day of the thoracotomy was regarded as the starting day for counting the postoperative survival days.

Clinical characteristics of the patients
There were 170 men and 66 women (Table 1). Mean age at thoracotomy was 62.4 ± 9.74 years (mean ± standard deviation, range 17 to 83 years). Performance status (PS) was 0 or 1 for most patients. With respect to the histologic type, adenocarcinoma was most frequent (130 patients, 55.1%) and squamous cell carcinoma (85 patients, 36.0%), was the next most frequent. As for the postoperative p-stage, the number of stage 1 patients was the highest (138 patients, accounting for 58.5%).

Antihuman p53 MoAb (DO-7, mouse IgG2b, kappa, 250 µg/mL, (Dako) was used as primary antibody for immunostaining against p53. The slides were incubated with the diluted antibody (1:50) after microwave heating for 10 min to retrieve the antigenicity.

Stained tissue slides were evaluated by three of the authors (F.T., Y.O., and R.M.) independently without knowing their clinical data. Tumor cells were judged as positive staining only when the cytoplasm was faintly stained. A total of 1,000 tumor cells were counted for positive staining and the percentage of positive cells were determined. The degree of Le^Y expression was graded according to the percentage of Le^Y-positive-staining cells as follows: (-) if the percentage was 5% or less than 5%, (+) if the percentage was over 5% and under 10%, (2+) if the percentage was over 10% and under 25%, (3+) if the percentage was over 25% and under 50%, (4+) if the percentage was over 50% and under 75%, and (5+) if the percentage was over 75%. Aberrant expression of p53 was judged when the percentage of the positive-staining cells exceeded 5%.

Statistical methods
Counts were compared by ² test. Continuous data were compared by Student’s t test if the distribution of samples was normal or by Mann-Whitney U-test if the sample distribution was asymmetrical. Postoperative survival rate was analyzed by Kaplan-Meier method, while the differences in survival rates were assessed by log-rank test. Multivariate analysis of the prognostic factors was performed by Cox regression model. The difference was considered significant if p value was less than 0.05. All statistical manipulations were performed using SPSS for Windows software system (SPSS Inc, Chicago, IL).

	Results

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Expression of Le^Y
The number of patients for whom the grade of Le^Y expression was judged (-), (+), (2+), (3+), (4+), or (5+) was 57 (24.2%), 17 (7.2%), 42 (17.8%), 51 (21.6%), 31 (13.1%), and 38 (16.1%), respectively. The 5-year survival rates were 50.2% for (-) group, 79.3% for (1+) group, 80.0% for (2+) group, 74.4% for (3+) group, 76.0% for (4+) group, and 77.6% for (5+) group. The Le^Y expression (-) group showed significantly worse postoperative prognosis than the other (+), (2+), (3+), (4+), and (5+) groups. No significant difference in the postoperative prognosis was observed among the (+), (2+), (3+), (4+), and (5+) groups. As a consequence, the (-) patient group was determined to be the Le^Y expression negative and the other patients groups, (+), (2+), (3+), (4+), or (5+), were determined to be the Le^Y expression positive, and the assessment shown below was carried out. One hundred seventy-nine (75.8%) of 236 patients were judged to be Le^Y expression positive, and the other 57 patients (24.2%) Le^Y-expression negative.

Le^Y expression and clinical characteristic (table 1)
Increased positivity for Le^Y expression was seen in adenocarcinoma (84.6%) versus squamous cell carcinoma (70.6%, p = 0.001). Le^Y expression was seen correlated with cancer differentiation; Le^Y expression was positive in 138 (81.2%) of 170 patients with moderately to well-differentiated cancer whereas in only 39 (67.2%) of 58 patients with poorly differentiated cancer (poorly differentiated squamous cell carcinoma and adenocarcinoma, and large cell carcinoma) (p = 0.028).

Ninety-nine (41.9%) patients were judged to show aberrant expression of p53. Only 68.7% of patients with aberrant p53 expression showed positive Le^Y expression, whereas 81.0% of those without aberrant p53 expression showed positive Le^Y expression (p = 0.029).

Sex, performance status, or p-stage was not correlated with Le^Y expression.

Le^Y expression and postoperative prognosis (table 2)
Five-year survival rate of Le^Y-positive patients was 77.9%, which was significantly higher than that of Le^Y-negative patients (50.2%, p = 0.001) (Fig 1). In patients with squamous cell carcinoma, 5-year survival rate of Le^Y-positive patients was 75.1%, which was significantly higher than that of Le^Y-negative patients (46.4%, p = 0.028). In patients with adenocarcinoma as well, prognosis of Le^Y-positive patients tended to be better than that of Le^Y-negative patients, although showing no significant difference (p = 0.059).

View larger version (13K): [in this window] [in a new window]   Fig 1. Survival after complete tumor resection with lymph node dissection for non–small cell lung cancer during 1985–1990 at Kyoto University. Comparison between that for Lewis Y antigen (LeYLeY) expression positive disease and that for LeY expression negative disease.

View larger version (13K): [in this window] [in a new window]   Fig 2. Survival after complete tumor resection with lymph node dissection for non–small cell lung cancer during 1985–1990 at Kyoto University. Comparison between that for Lewis Y antigen (LeY) expression positive disease and that for LeY expression negative disease.

View larger version (19K): [in this window] [in a new window]   Fig 3. Comparison of postoperative survival between LeY-positive and p53 aberrant expression negative disease (A), that for LeY-positive and p53 aberrant expression positive disease (B), that for LeY-negative and p53 aberrant expression negative disease (C), and that for LeY-negative and p53 aberrant expression positive disease (D).

View larger version (18K): [in this window] [in a new window]   Fig 4. Comparison of postoperative survival for p-stage 1, NSCLC between LeYpositive and p53 aberrant expression negative disease (A), that for LeY-positive and p53 aberrant expression positive disease (B), that for LeY-negative and p53 aberrant expression negative disease (C), and that for LeY-negative and p53 aberrant expression positive disease (D).

	Discussion

Top Abstract Introduction Material and methods Results Discussion Acknowledgments References

 
During neoplastic transformation, glycosylation may revert to an earlier development form with lung cancer cells reexpressing antigens found on immature embryonic lung cells. The tumor-associated Le^Y is the focus of the present study and is correlated with postoperative survival.

Several findings have been reported with respect to Le^Y expression in normal tissue and in cancer tissue since the 1980s, when various kinds of MoAbs against Le^Y were generated. As for Le^Y expression in lung cancer, only two results have been reported. Miyake and coworkers conducted an immunohistologic study on NSCLC using a MoAb, MIA-15-5 [12]. MIA-15-5 recognizes sugar chains having a structure of Fuc1 2Galß1 R (R: type 1 or type 2 chain), and react with not only Le^Y but also H-antigen (Fuc1 2Gal) and Lewis b antigen (Le^b, Fuc1 2Galß1 3[Fuc1 4]GlcNAc). Positive expression was judged when more than 5% of tumor cells were stained, and MIA-15-5-positive was seen in 91 (61.1%) of 149 patients. Postoperative prognosis of MIA-15-5-positive patients proved to be significantly worse as compared with that of MIA-15-5-negative patients, which did not agree with our findings that Le^Y-positive patients had a better prognosis. However, because MIA-15-5 recognizes not only Le^Y but also H-antigen and Le^b, the disagreement with our results obtained by using MoAb, BM-1/JIMRO recognizing only Le^Y could be understandable.

Ogawa and coworkers also conducted a study on Le^Y expression in NSCLC [13]. They examined Le^Y expression in patients with p-stage 1 disease using BM-1/JIMRO, the same MoAb used in our study. They judged Le^Y expression was positive when more than 50% of tumor cells were stained, and reported that 84 (63.2%) of 133 patients were Le^Y positive. They also reported that postoperative prognosis of Le^Y-positive patients was poor as compared with that of Le^Y-negative patients, which was not in agreement with the result of our study which showed that the prognosis of Le^Y-positive patients was favorable. They used a 50% positive cell count rate as the cut-off value because the mean percentage of Le^Y-positive cells for all patients was 56%. They determined that patients with a positive cell count rate of 50% or higher were Le^Y positive and that those with a positive cell count rate of lower than 50% were Le^Y negative, and Le^Y-positive patients accounted for 63.2% of the total patients. If a cut-off value of the 50% positive cell rate were applied to our patients, Le^Y-positive patients would account for 29.2% of the total patients (69 of 236 patients). Thus, the Le^Y-positive cell count rate reported by Ogawa and coworkers appears too high in comparison with our findings. Moreover, in a study reported by Miyake and coworkers using MIA-15-5 recognizing H antigen and Le^b in addition to Le^Y, a cut-off value of 5% was also used as in our study, and 91 (61.1%) of 149 patients exhibited positive cell count rates of over 5% [12]. Studies on hepatocellular cancer [10] and breast cancer [11], where patients showing only one positively stained cell were determined to be positive, also showed that positive patients accounted for 43.5% and for 66.9%, respectively. Therefore, the cut-off value at 50% as stated by Ogawa and coworkers might be questionable.

Our study suggests that Le^Y-negative patients had poor prognosis because more patients had poorly differentiated cancer. Conversely, in poorly differentiated cancer, decreased Le^Y antigen expression on the surface of tumor cells may result in a poor prognosis. Another reason why Le^Y-negative patients had poor prognosis contrary to other studies can be related to apoptosis. It has recently been reported that Le^Y expression is closely related to apoptosis; Le^Y expression is a phenotypic marker of apoptosis [16]. In the present study, the relationship between Le^Y expression and p53 status was also investigated. It was demonstrated that Le^Y expression decreased in patients with aberrant p53 expression, and aberrant p53 expression was increased in Le^Y-negative patients. Aberrant p53 expression detected by immunohistologic staining usually reflects abnormal accumulation of gene product caused by p53 gene mutation, and immunohistological staining is widely used as a simple and easy method to detect p53 gene mutation [17]. p53 Gene is a tumor suppression gene, which can control the cell cycle and induce apoptosis [18]. Apoptosis plays essential roles in normal development and maintenance of homeostasis, and also plays important roles in inhibition of growth of cancer cells. When mutation of p53 occurs, apoptosis can not occur easily in tumor cells, and inhibition of tumor growth becomes difficult. It has recently been reported that Le^Y expression is closely related to apoptosis [16]. Taking this concept into consideration, in patients showing aberrant p53 expression, apoptosis may not occur easily due to the p53 gene mutation, and as a consequence, Le^Y expression might decrease. Further basic studies are needed to disclose relationships between p53 disorder and Le^Y expression.

Aberrant p53 expression detected by immunohistological staining has been generally reported to be an independent prognostic factor to predict poor prognosis in lung cancer patients [19], as shown in the present study as well. Combined with the p53 status, the significance of Le^Y expression as a prognostic factor proved to be marked. If a limited population with poor prognosis after operation for lung cancer can be identified, it would be clinically useful because patients who require postoperative adjuvant therapy can be identified. From the point of view, assessment of Le^Y expression along with aberrant p53 expression proved to be clinically very important.

	Acknowledgments

Top Abstract Introduction Material and methods Results Discussion Acknowledgments References

 
We thank Dr Nobuyuki Hamajima, Division of Epidemiology, Aichi Cancer Institute, Aichi, Japan, for his helpful comment and critical reading of the statistical section of the manuscript.

	References

Top Abstract Introduction Material and methods Results Discussion Acknowledgments References

 

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