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Ann Thorac Surg 1999;67:810-814
© 1999 The Society of Thoracic Surgeons

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Original Articles

MUC1 mucin mRNA expression in stage I lung adenocarcinoma and its association with early recurrence

^a Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan
^b Department of Surgery II, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan

Accepted for publication August 14, 1998.

Address reprint requests to Dr Ohgami, Department of Environmental Health Engineering, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
e-mail: gamisan{at}med.uoeh-u.ac.jp

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. MUC1 is a membrane-bound mucin with an extensively O-glycosylated core protein and is developmentally regulated and aberrantly expressed by carcinomas. A high level of MUC1 mucin expression and secretion is associated with high metastatic potential and a poor prognosis. We studied the expression of MUC1 messenger ribonucleic acid (mRNA) in stage I lung adenocarcinoma by reverse transcriptase–polymerase chain reaction and examined its correlation with early recurrence.

Methods. The expression of MUC1 mRNA, in surgical specimens from 33 patients with stage I lung adenocarcinoma was determined by reverse transcriptase–polymerase chain reaction. The MUC1 and ß-actin sequences were subsequently coamplified to analyze the semiquantitative determination by polymerase chain reaction. The ratio of MUC1 to ß-actin product was used for further analysis.

Results. An analysis of the disease-free survival (median follow-up, 33.4 months) revealed that a high expression of MUC1 was associated with early recurrence (p = 0.0191). Six of the 33 patients had recurrence within 2 years after operation. The recurrence sites suggested hematogenic metastasis.

Conclusions. Our results indicate that MUC1 mRNA level may be useful as a marker of early recurrence in stage I lung adenocarcinoma.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Mucin is a large glycoprotein (>10⁶ daltons) consisting of more than 50% carbohydrates [1]. Nine different human mucin genes (MUC1–4, 5AC, 5B, 6–8) have been identified from several types of epithelial tissue [2–7]. MUC1 is a membrane-bound mucin with an extensively O-glycosylated core protein, and the mature glycosylated forms appear to be distinct [1]. Previous studies [8–13] have reported that MUC1 mucin is developmentally regulated and aberrantly expressed by carcinomas (breast, pancreatic, ovarian, gastric, and colorectal cancer), and a high level of MUC1 messenger ribonucleic acid (mRNA) expression in adenocarcinoma is associated with a poor prognosis. Our preliminary data [14] showed it likely that MUC1 mucin expression is a marker of the malignant potential of tumor cells.

The functional role of MUC1 mucin is unknown. Some reports have indicated that MUC1 mucin inhibits the E-cadherin–mediated cell-cell adhesion [15] or that MUC1 interferes with cellular adhesion by steric hindrance from the rigid ectodomain [16]. Recently, Agrawal and associates [17] reported that cancer-associated MUC1 mucin, affinity-purified from ascites of cancer patients, and synthetic tandem repeats of MUC1 mucin core peptide can suppress T-cell proliferation, which can be reversed by exogenous interleukin-2. It is more likely that MUC1 mucin plays an important role in forming a "malignant potential" of tumor cells.

In lung cancer, even after a complete resection for stage I disease, about 30% of all patients have recurrence and eventually die of the disease [18, 19]. Therefore, it is important to evaluate the malignant potential of tumor cells for a more precise evaluation of the prognosis of patients with early-stage lung cancer. However, in lung cancer (especially adenocarcinoma), little is known about the clinical relationship between its metastatic potential and the expression of MUC1 mucin. Therefore, the identification of patients with increased metastatic potential can help determine the prognosis and eventually allow the addition of effective adjuvant therapy.

To determine the correlation between the expression of MUC1 mRNA and various clinical and pathologic factors, we analyzed the semiquantitative determination of MUC1 mRNA expression in resected specimens from patients with stage I lung adenocarcinoma by reverse transcriptase–polymerase chain reaction (RT–PCR).

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Thirty-three consecutive patients with pathologic stage I lung adenocarcinoma were entered into this study. Each had undergone a complete pulmonary resection between September 1991 and February 1995. There were 14 men and 19 women with a mean age of 65.6 years. Preoperatively, all patients underwent extensive diagnostic procedures, including chest roentgenography computed tomography of the chest and brain, ultrasonography of the abdomen, and bone scanning to determine the disease stage according to the TNM classification of the International Union Against Cancer as modified in 1987.

For the postoperative follow-up, the patients were requested to visit our clinic to undergo chest roentgenography and tumor marker examinations every month for the first year, every other month for the second year, and every 3 months thereafter. Both computed tomography and bone scanning were routinely performed every 6 months postoperatively when there was no indication of recurrence. If a sign of recurrence was observed, occasional examinations were added. The date of first recurrence was considered to be the day when evidence of distant metastatic disease was initially observed at these examinations.

Preparation of RNA, complementary deoxyribonucleic acid synthesis, and PCR
Total RNA was extracted from resected specimens by the acid guanidinium thiocyanate/phenol-chloroform method and quantified by spectrophotometry [20]. The specimens were snap-frozen as soon as possible and stored at -80°C before use. The properties of RNA (2.5 µg) were evaluated by electrophoresis on a 1% agarose-formamide gel. Twenty micrograms of total RNA was processed with 1,000 U Moloney murine leukemia virus reverse transcriptase (USB, Cleveland, OH), 10 ng random hexanucleotide primers (Takara, Japan), 10 mmol/L deoxynucleoside-triphosphate (Takara), and 100 U RNase inhibitor (USB) in a total volume of 20 µL.

The MUC1 was amplified by PCR using a 0.5 µg complementary deoxyribonucleic acid template, 0.5 U recombinant Taq DNA polymerase (Toyobo, Japan), and 1 µmol sense and antisense primers in a total volume of 50 µL. ß-Actin was subsequently coamplified as an internal (endogenous) standard to quantify the PCR amplification of mRNA. The number of thermocycles used allowed quantification without saturation. The primers were synthesized according to the published sequences of ß-actin and the extracellular domain of MUC1 [2]. The PCR was carried out in a DNA thermocycler (Astech, Japan) under the following conditions: 94°C for denaturation for 45 seconds, 60°C for annealing for 45 seconds, and 72°C for extension for 2 minutes. The control PCR reactions were incubated in the absence of complementary deoxyribonucleic acid.

Detection of the fragments amplified by the PCR was done by electrophoresis on a 2% agarose gel containing ethidium bromide. The gels were photographed with Polaroid Type 665 positive/negative film (Polaroid Corporation, Cambridge, MA) over ultraviolet light at the same exposure and developing time. The bands of the positive film were scanned, and the density of each PCR product was measured using the public domain National Institutes of Health image 1.56: (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/) for an Apple computer.

Quantitative analysis
We quantified the transcriptional level of MUC1 mRNA as follows: MUC1 samples were coamplified with ß-actin–specific primers as an internal standard. Aliquots containing complementary deoxyribonucleic acid were subjected to 26, 29, 32, 35, or 38 cycles of amplification under the same conditions as in the protocols. The density recovered from excised bands were plotted as a function of the number of the cycles. The rates of amplification were exponential between 29 and 35 cycles for both templates. We determined that a cycle in which the slope of the sigmoid curve of both genes was almost straight was appropriate. The data were normalized to represent the equivalent RNA loading based on the density of ß-actin at the appropriate cycle of both genes [21]. The ratio of specific gene product to ß-actin product was used for further analysis. The reproducibility of RT–PCR was demonstrated by repeat calculations.

Statistical analysis
A comparison of the proportion was performed with the Mann-Whitney U test. The Kaplan-Meier method was used to estimate the probability of the disease-free intervals.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
MUC1 mRNA expression in stage I adenocarcinoma
The mean value of the MUC1 to ß-actin expression ratios was significantly higher in the 33 patients with adenocarcinoma (1.29 ± 1.00) than in 9 patients with normal lung tissue (0.22 ± 0.35; p = 0.0004). MUC1 expression in 88% (29/33) of the tumors was higher than the mean ratio in normal lung. The relationship between the mean MUC1 to ß-actin ratio and various clinical variables in the 33 patients is shown in Table 1. These ratios were not significant in regard to age, sex, or tumor size.

MUC1 mRNA expression and recurrence in stage I adenocarcinoma
Follow-up of the 33 patients ranged from 603 to 1,721 days (median follow-up, 33.4 months). Six patients had systemic metastases within 2 years. Our study of the recurrence sites indicated that the pattern of recurrence seemed to be hematogenic. Two patients had pulmonary metastases, and 1 patient each had liver metastasis, bone metastasis, brain metastasis, and multiple sites of metastasis (brain, bone, and lung) (Table 2). No lymph node metastasis was observed in this study. Figure 1 shows the pattern of recurrence by the MUC1 to ß-actin ratio.

View larger version (16K): [in this window] [in a new window]   Fig 1. Relationship between MUC1 to ß-actin ratio and recurrence for 33 patients with stage I lung adenocarcinoma. The recurrences were observed within 2 years after the primary surgical procedure.

View larger version (13K): [in this window] [in a new window]   Fig 2. Kaplan-Meier survival curves for 33 patients with stage I lung adenocarcinoma classified by MUC1 to ß-actin ratio. Patients with a high MUC1/ß-actin ratio ( 1.0) had a significantly shorter disease-free period than patients with a low ratio (< 1.0) (p = 0.0191).

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
We have shown in this study that a high MUC1 mRNA expression estimated using the RT–PCR technique may indicate a high risk for recurrence in patients with stage I disease. Mucin mRNA levels are difficult to quantify by Northern blot analysis because of the polydisperse signal of the transcripts [23]. Using endogenous standards such as ß-actin and normalizing the quantity of the ß-actin levels would allow for a correction resulting from differences in RNA loading, potential RNA degeneration, or inaccuracies in the RNA estimation by densitometry [24]. This semiquantitative RT–PCR technique of measurement has successfully been used to show the effects of mineral fibers on the expression of several genes [25, 26]. The reproducibility of this method was confirmed by repeated calculations. We measured the ratios at least twice by this method, and each time they demonstrated reproducibility. Although the ratio can be affected by some conditions (eg, gel concentration, electrophoresis duration, and length of exposure), the method is relatively easy and useful.

In lung cancer, even after a complete resection for stage I disease, about 30% of all patients have recurrence and eventually die of the disease [18, 19]. One of our previous studies [27] showed that micrometastatic tumor cells (cytokeratin 18–positive cells) were observed in the bone marrow of about 40% of patients with lung cancer who had undergone complete resection. Micrometastatic cancer cells in the bone marrow may indicate that disseminated tumor cells are circulating systemically. Whether or not these cells will metastasize to other organs may depend on the "malignant potential" of the tumor cell itself. Therefore, it is important to evaluate the malignant potential of tumor cells at an early stage for a more precise evaluation of the prognosis for patients with lung cancer.

In this study, although there was a range in the ratio of MUC1 to ß-actin in patients with lung adenocarcinoma, the early recurrences were seen in patients with a ratio of 1.0 or higher. This may imply that various malignant potentials exist in lung adenocarcinoma. Using the method previously described [27] to detect occult micrometastases in bone marrow, we found that 2 of the 3 patients examined were cytokeratin 18–positive in bone marrow, and the MUC1 to ß-actin ratio in these 2 patients was higher than 1.0. Further analysis is needed to confirm this finding, but it may be a partial explanation of the relationship between aberrant expression of MUC1 mucin and metastasis.

It is known that the metastatic process consists of several different stages, all of which are indispensable for the development of clinically overt metastases. MUC1 mucin inhibits the E-cadherin–mediated cell-cell adhesion [15], which implies that MUC1 mucin enables adenocarcinoma cells to be detached easily from the primary tissue. The MUC1 polypeptide is associated with cell surface glycolipids such as sialyl-Lewis^a and sialyl-Lewis^x [28]. They have been implicated in tumor cell binding to the endothelial cell adhesion molecule E-selectin and in cellular extravasation during metastasis [28]. Thus the expression of MUC1 mucin may play a particular role in adhesion and extravasation during the metastatic process. However, there was no correlation between the serum level of Sialyl-Lewis^x and the MUC1 to ß-actin ratio in this study (data not shown).

Concerning the differentiation status of adenocarcinoma and MUC1 mRNA expression, the MUC1 to ß-actin ratio tended to correlate with the degree of histologic differentiation (low in poorly differentiated adenocarcinoma and high in well-differentiated adenocarcinoma). These findings may indicate that the expression of MUC1 mRNA in the early stage of adenocarcinoma is closely related to the differentiation of the tumor. This is consistent with the report by Yu and coauthors [29] that the poorly differentiated lung adenocarcinoma cell lines have a relatively lower level of MUC1 expression than the well-differentiated cell lines. The prognosis regarding differentiation did not show a significant difference in our study (data not shown). Because the number of poorly differentiated adenocarcinomas was low (n = 4), we cannot deny that the prognosis may be different if the population changes. Generally, poorly undifferentiated adenocarcinomas are thought to have a greater malignant potential than well-differentiated adenocarcinomas. We hypothesize that the malignant potential raised by MUC1 expression is more characteristic of well-differentiated lung adenocarcinomas.

In conclusion, our findings suggest that MUC1 mRNA expression estimated using the RT–PCR method may help predict early recurrence and may be a marker of malignant potential in stage I lung adenocarcinoma.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
This work was supported by a grant for New Medicine from the Kanae Foundation of Research for New Medicine, the Kaibara Morikazu Medical Science Promotion Foundation, the Fukuoka Cancer Society, and grants-in-aid for scientific research 05770413 and 06670636 from the Ministry of Education, Science, Sports and Culture, Japan.

	References

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