Macrocytosis, a new predictor for esophageal squamous cell carcinoma in Japanese alcoholic men

Akira Yokoyama1,6, Tetsuji Yokoyama2, Taro Muramatsu3, Tai Omori5, Sachio Matsushita1, Susumu Higuchi1, Katsuya Maruyama1 and Hiromasa Ishii4

1 National Institute on Alcoholism, Kurihama National Hospital, Yokosuka, Kanagawa 239-0841, 2 Department of Technology Assessment and Biostatistics, National Institute of Public Health, Wako, Saitama 351-0197, 3 Department of Neuropsychiatry and 4 Department of Internal Medicine, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582 and 5 Departments of Gastroenterology and Surgery, Kawasaki Municipal Hospital, Kawasaki, Kanagawa 210-0013, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Early esophageal squamous cell carcinoma detected by esophageal iodine staining can be easily treated by endoscopic mucosectomy, and identifying its predictors is important in better selecting candidates to screen for this high-mortality cancer. The common etiologies of elevated mean corpuscular volume (MCV) and esophageal cancer, including folate deficiency, smoking, drinking and high acetaldehyde exposure, suggest testing MCV as such a predictor. Japanese alcoholic men with (n = 65) and without (n = 206) esophageal squamous cell carcinomas, excluding those with liver cirrhosis, were assessed for MCV within 7 days of their last drink, alone or in combination with findings from either the alcohol flushing questionnaire or genotyping to identify inactive aldehyde dehydrogenase-2 (ALDH2*1/2*2) and the less-active form of alcohol dehydrogenase-2 (ADH2*1/2*1), which pose risks for esophageal squamous cell carcinoma. MCV was higher in cancer patients than in the control group. MCV was higher in both groups in those who were heavier smokers, had lower body mass index (BMI), experienced alcohol flushing, and had ALDH2*1/2*2. After adjusting for age, drinking and smoking habits, BMI and ALDH2/ADH2 genotypes, macrocytosis of MCV >=106 fl was associated with increased risk for esophageal cancer (OR = 2.75). Men with both MCV >=106 fl and alcohol flushing had an even higher cancer risk (OR = 5.51). The combinations of MCV >=106 fl with ALDH2*1/2*2 or ADH2*1/2*1 alone, and both ALDH2*1/2*2 and ADH2*1/2*1 (ORs = 11.44, 21.22 and 319.7, respectively) showed consistently higher risk than the corresponding group with MCV <106 fl (ORs = 7.24, 4.71 and 27.01, respectively). In conclusion, MCV measurement, alone or in combination with the markers of alcohol sensitivity, provides a new means of predicting risk for esophageal squamous cell carcinoma in Japanese alcoholic men.

Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; BMI, body mass index; LSM, least square mean; MCV, mean corpuscular volume; OR, odds ratio; ROC, receiver operating characteristic


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Diagnostic and therapeutic technologies, including esophageal iodine staining and endoscopic mucosectomy, for early squamous cell carcinoma of the esophagus have dramatically improved and have become widespread in Japan (1,2). Iodine staining greatly increases the detection of very early esophageal cancer: in Japanese male alcoholics 40 years of age or older, 0.7% by endoscopy alone (3) versus 3.3–3.8% by endoscopy combined with esophageal iodine staining (1,4). The 5-year survival rate after endoscopic mucosectomy for intraepithelial or mucosal cancer is nearly 100%, whereas the majority of symptomatic patients who present at an advanced stage have a poor prognosis (2). A recent European survey reported that even patients in whom esophageal cancer was completely resected at the time of surgery had a 5-year survival of 38.5%. (5) Thus, very early detection of this high-mortality cancer by screening is extremely important, and a biomarker or predictor should be useful in better selection of individuals for screening.

Elevated mean corpuscular volume (MCV) has long been recognized as a biomarker for alcohol abuse and alcoholism (6,7), but the nature of the relationship between high MCV and heavy drinking is not yet clear. Although alcoholics with inadequate diets have shown lower levels of folate in their serum and red cells, folate status is not the sole determinant for macrocytosis in alcoholics (6). For example, recent studies have reported that male Japanese heavy drinkers who have inactive aldehyde dehydrogenase-2 (ALDH2) encoded by ALDH2*1/2*2 have higher MCV than those with active ALDH2 (8,9), and that MCV served as a marker for moderate-to-heavy drinkers with inactive heterozygous ALDH2 in male Japanese workers (10). ALDH2 is the key enzyme in eliminating acetaldehyde after drinking alcohol, suggesting a role for acetaldehyde in the development of macrocytosis. Aging and smoking also independently contribute to the development of macrocytosis (11). Inasmuch as poor nutrition including folate deficiency, high acetaldehyde exposure, aging and smoking all enhance the risk for esophageal cancer (1214), we hypothesized that MCV could be used as a biomarker for this cancer in its earliest stage.

A review of case-control studies on the effect of ALDH2 and ADH2 genotypes showed consistently positive associations between inactive ALDH2 (ALDH2*1/2*2) and the low- activity form of alcohol dehydrogenase-2 (ADH2*1/2*1) genotypes and the risk for esophageal cancer in East Asian heavy drinkers (13). The ALDH2*1/2*2 and ADH2*1/2*1 gene combination enhanced the risk for esophageal cancer in a multiplicative fashion (4,14).

This knowledge emphasizes the importance of developing screening tests for inactive ALDH2 on the basis of alcohol flushing, which may offer alternatives to the expensive ALDH2 genotyping. After light drinking, individuals with inactive ALDH2 exhibit facial flushing due to severe acetaldehydemia. It diminishes in intensity in men with long or heavy drinking histories, suggesting the development of tolerance to acetaldehydemia (4,15). For that reason, we have designed a questionnaire to detect changes in flushing responses over time by asking about both current and past facial flushing (4,15). When individuals who reported current or past flushing were considered to have inactive ALDH2, it yielded a very high sensitivity for detecting inactive ALDH2 in Japanese elderly men (15), but less sensitivity in Japanese alcoholic men with esophageal cancer, among whom the ‘at risk’ combination of ALDH2*1/2*2 and ADH2*1/2*1 is prevalent (4). The flushing response may be triggered by an initially steep rise in blood or cutaneous acetaldehyde after drinking. This dramatic change may not occur in persons with ADH2*1/2*1 in spite of the presence of ALDH2*1/2*2. As a result of the weak intensity of the aversive flushing, individuals with this gene combination are likely to become heavy drinkers and be exposed to higher levels of acetaldehyde.

In the present study, we evaluated the reliability of MCV testing alone and in combination with the alcohol flushing questionnaire or genotyping of ALDH2 and ADH2 in predicting the risk of esophageal cancer among Japanese alcoholic men.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
The participants in this study were Japanese alcoholic men (>=40 years of age) who visited the National Institute on Alcoholism, Japan, for treatment of alcoholism within 7 days of taking their last alcoholic drink. They had no history of upper aerodigestive tract cancer, gastrectomy or recent bleeding. Between January 1993 and July 2002, with an initial endoscopic screening using esophageal iodine staining (1), we consecutively diagnosed 77 patients as having esophageal squamous cell carcinoma. Between January 2000 and July 2002, using the same method, we consecutively diagnosed another 256 as esophageal cancer free. After the exclusion of those diagnosed as having liver cirrhosis on the basis of clinical features, laboratory data, endoscopic confirmation of esophageal varices and systematic examination using abdominal ultrasound, abdominal computed tomography scan and liver scintiscans, we enrolled the remaining 65 cancer patients and 206 cancer-free patients in this study. All but one of the cancer patients had superficial esophageal cancer, which was confined within the epithelium in 20 patients and had invaded to the proper mucosal layer in 29, to the submucosa in 12, and to the proper muscle layer in three.

The proposed study was reviewed and approved by the ethics committee of the Institute and informed consent was obtained from all participating patients.

Drinking and smoking habits
Information on the patients' drinking and smoking habits, including the date of taking their last alcoholic drink, was obtained from the patients themselves and, when available, their partners. All of the alcoholics met the DSM-III-R criteria for alcohol dependence (16). Daily alcohol consumption during the preceding year was expressed in grams of ethanol per day, using a standard conversion for alcohol beverages. Beer was considered to be 5% ethanol (v/v); wine, 12%; sake, 16%; shochu, 25%; and whiskey, 40%.

MCV testing
Blood was obtained from each patient's antecubital vein and placed in a tube containing EDTA. The procedure was performed the first day they visited the Institute for treatment of alcoholism, which was within 7 days of taking their last alcoholic drink. MCV was measured within 4 h of collection by the electrical impedance method using an autoanalyzer (CELL-DYN 3500, Abbott, North Chicago, IL).

Flushing questionnaire
We uniformly asked all of the patients the following flushing questions: (i) do you always flush in the face immediately after drinking a glass of beer? (ii) Did you always flush in the face immediately after drinking a glass of beer during the first to second year after you started drinking? The designation ‘current flushing’ was applied to individuals who answered ‘yes’ to question (i), ‘former flushing’ to those who answered ‘no’ to question (i) and ‘yes’ to question (ii), and ‘never flushing’ to those who answered ‘no’ to both questions.

ALDH2 and ADH2 genotyping
The methods of Xu et al. (17) and slight modifications of the methods of Harada and Zhang (18) were used for polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) for ADH2 and ALDH2 genotyping of lymphocyte DNA samples from the patients' blood. The modifications for ALDH2 genotyping were as follows: briefly, 100–200 ng genomic DNA was mixed with 5 pmol of each primer (5'-CAAATTACAGGGTCAACTGCT-3' sense; 5'-CCACACTCACAGTTTTCTCTT-3' antisense) in a total volume of 50 µl containing 50 µM concentration of each dNTP, 1.5 mM MgCl2, and 1 U Taq DNA polymerase (Promega, Madison, WI). Thirty-five cycles of PCR (denaturation at 94°C for 15 s, annealing at 58°C for 1.5 min and polymerization at 72°C for 30 s) were performed in a Perkin Elmer Cetus GeneAmp PCR System 9600. After purification, each PCR product was digested with MboII, electrophoresed on 20% polyacrylamide gel, stained with ethidium bromide and viewed.

Statistical analysis
The deviation of the genotype distribution from the Hardy–Weinberg equilibrium was evaluated by the exact test. To compare the basic clinical characteristics in cases and controls, we used Fisher's exact test, Cochran–Mantel–Haenszel statistics, and analysis of covariance (ANCOVA) where appropriate. The direct method was used to compare age-adjusted percentage values between cases and controls by using the age distribution of the cases as the standard population. Associations of MCV with other factors were examined by ANCOVA, in which we calculated the age-adjusted least square means (LSMs). We employed a receiver operating characteristic (ROC) curve to determine the MCV cut-off point that could most effectively discriminate between esophageal cancer cases and controls. Using various cut-off points of MCV, sensitivity (percentage of alcoholic men whose MCV >= the cut-off point among esophageal cancer cases) and false positive rates (100% - specificity: percentage of alcoholic men whose MCV >= the cut-off point among cancer-free controls) were calculated. A ROC curve was drawn by plotting the results. The risk of esophageal cancer associated with MCV and other factors was expressed in terms of the odds ratio (OR) and 95% confidence interval (CI) using a multiple logistic regression model. A test for trend through multiple categories was conducted by applying the median value in each category. All analyses were done with the Statistical Analysis System (version 8.2; SAS Institute, Cary, NC).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There were no significant differences in mean age, daily alcohol consumption, or daily cigarette smoking between the cancer patients and the controls (Table I, left column). The body mass index (BMI) in the cancer patients was significantly lower than in the controls. The cancer patients had a significantly higher prevalence of current or former flushing, and of the ALDH2*1/2*2, and ADH2*1/2*1 genotypes than the controls. The frequency of MCV >=106 fl (highest quartile) was markedly higher in the cancer patients than in the controls (age-adjusted prevalences = 43.1 and 17.3%, respectively). MCV was significantly higher among smokers, those with lower BMI, and men with either the ALDH2*1/2*2 genotype or self-reported current/former flushing among both cancer patients and controls (Table I, right column). The presence of the ADH2*2 allele was significantly related to higher MCV in only the patients with cancer.


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Table I. Comparison of the basic characteristics of Japanese alcoholic men with and without esophageal squamous cell carcinoma, and relationship between MCV and these factors

 
Figure 1 graphically illustrates the ability of MCV measurement to discriminate between esophageal cancer cases and controls in terms of sensitivity (y-axis) and false positive rate (x-axis) at various cut-off values (shown along the curve) using a ROC curve. Simultaneously higher sensitivity and lower false positive rate means a greater ability to discriminate between esophageal cancer cases and controls. The sensitivity of this possible predictor increased with a relatively small increase in the false-positive rate above the cut-off point of 106 fl, at which the sensitivity was 43.1% and the false- positive rate was 17.0%. However, below the cut-off point of 105 fl, the increment of false-positive rate became prominent. We therefore employed MCV >=106 fl to define macrocytosis in selecting individuals with a high risk for esophageal cancer and used this cut-off point in the following analyses.



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Fig. 1. ROC curve for the detection of esophageal cancer by various cut-off points of MCV (fl), which are shown along the ROC curve. The percentages of alcoholic men whose MCV >= the indicated cut-off point among esophageal cancer cases and controls can be read on the y-axis (sensitivity) and x-axis (false positive rate), respectively. For example, 43.1% (see y-axis) of esophageal cancer cases, whereas only 17.1% (see x-axis) of controls, have MCV >=106 fl.

 
Assessing the risk for esophageal cancer according to MCV and other factors (Table II), we found, using age-adjusted univariate analysis, the risk for esophageal cancer was 3.68-fold higher in men with MCV >=106 fl than in those with MCV<106 fl. Other significant risk factors were current/ former flushing, ALDH2*1/2*2, ADH2*1/2*1 and lower BMI. Notably, the increased risk due to macrocytosis (MCV >=106 fl) remained significant (OR = 2.75), even after adjusting for age, daily alcohol consumption, daily cigarette smoking, current/former flushing, ALDH2 and ADH2 genotypes and BMI, where current/former flushing was no longer significant.


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Table II. Risk of esophageal cancer according to MCV, flushing and ALDH2 and ADH2 genotypes among Japanese alcoholic men

 
Alcoholic men with the combination of current/former flushing and MCV >=106 fl were at a higher risk for esophageal cancer (Table III; OR = 5.51). The combinations of MCV >=106 fl with ALDH2*1/2*2 alone, ADH2*1/2*1 alone, and both ALDH2*1/2*2 and ADH2*1/2*1 (Table III; ORs = 11.44, 21.22 and 319.69, respectively) showed consistently higher risk than the corresponding genotype groups having MCV<106 fl (ORs = 7.24, 4.71 and 27.01, respectively).


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Table III. Risk for esophageal cancer in Japanese alcoholic men, by selected combinations of MCV, flushing, and ALDH2 and ADH2 genotypes

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This is the first study to show that MCV alone could serve as a predictor of esophageal squamous cell carcinoma in alcoholics. Thus, MCV, which is a traditional marker of alcoholism, could potentially be used as a marker to identify super-high risk persons in the general population because heavy drinking is a strong risk factor for esophageal cancer. The predictive power of MCV increased when combined with ALDH2 and ADH2 genotypes. Nevertheless, macrocytosis enhanced cancer risks across all combinations of the ALDH2/ADH2 genotypes, suggesting that MCV-associated cancer risk applies to alcoholics regardless of ethnicity.

Alcoholics with macrocytosis may behave in such a manner as to increase both cancer risk and MCV. Macrocytosis has many possible causes, including aging, poor nutritional status, and smoking as well as drinking. We observed lower BMI in patients with macrocytosis and patients with cancer. The present study confirmed a reported inverse relationship between BMI and risk for esophageal squamous cell carcinoma (19). Whereas a large majority of the cancers we diagnosed were at early stages and without symptoms, it is probable that the lower BMI was not a consequence of cancer-related weight loss, but rather was associated with dietary aspects of the alcoholics' lifestyle.

There is substantial evidence that poor intake of vegetables and fruit enhance the risk for esophageal cancer (20). Folate deficiency leads to alterations in DNA methylation and disruption of DNA integrity and repair (21) and is associated with increased risk for several cancers, including esophageal squamous cell carcinoma (12). It is also a major cause of macrocytosis in alcoholics (6). Independent of inadequate diet, heavy drinking itself is associated with deterioration of folate absorption and metabolism (22). Acetaldehyde increases folate catabolism in vitro (23). MCV-associated cancer risks may provide new insight about the relationship between nutritional status including folate deficiency and cancer risk.

Inactive ALDH2 in both our cancer patients and controls and superactive ADH2 in the cancer patients were associated with higher MCV levels, in part because of high acetaldehyde exposure due to its rapid production and slow elimination. In a laboratory study, the presence of low concentrations of acetaldehyde induced macrocytosis in several human hematological disorder-derived cell lines, including an erythroleukemia cell line (24). The formation of acetaldehyde–erythrocyte adducts has been reported in the blood and bone marrow of patients with macrocytosis and excessive alcohol consumption (25). A much stronger correlation of acetaldehyde–hemoglobin adduct levels with alcohol drinking was observed in carriers of inactive ALDH2 than in those with active ALDH2 (26).

There is ample evidence of the carcinogenicity of acetaldehyde in experimental animals (27), where acetaldehyde interacts covalently with DNA to form DNA adducts. In fact, the levels of acetaldehyde adduct in leukocyte DNA have been found to be much higher in alcoholics than in healthy controls (28). Multiple studies showing that inactive ALDH2 enhances the risk for esophageal cancer in alcoholics (4,13,29), lend support to the concept that acetaldehyde plays a common role in increasing MCV and developing esophageal cancer.

Smoking was a probable contributor to macrocytosis in the cancer patients and controls in the present study. It is speculated that one of the mechanisms of the effect of smoking on macrocytosis is a compensatory response to reduced oxygen-carrying capacity, caused by either the persistent presence of carboxyhemoglobin or chronic lung disease due to smoking (11). As one of the major chemical constituents of tobacco smoke (30), acetaldehyde, or other ingredients inhaled with tobacco smoke, may have some effect(s) on macrocytosis. Further investigation is needed in this area.

Although our data demonstrate that high MCV alone could serve as a biomarker for esophageal cancer, other risk factors, including drinking and smoking habits, BMI and ALDH2/ADH2 genotypes, substantially increase cancer risk. After adjusting for these factors, the magnitude of risk attributable to macrocytosis decreased slightly, from an OR of 3.68 to 2.75, suggesting that these factors only partially explain MCV-associated risk. We speculate that unidentified factors in the alcoholic lifestyle or hereditary traits affecting cancer risk are strongly reflected in the MCV of the patient who develops esophageal cancer.

Another possible explanation of MCV-associated risk is related to unexplained individual variations in acetaldehyde exposure. An alcohol challenge test showed wide variations in the blood acetaldehyde levels of individuals with identical ALDH2 genotypes and amounts of alcohol consumption per body weight (31). Alcoholics, who tend to be exposed to higher levels of acetaldehyde regardless of ALDH2 genotype, may have greater increases in MCV and susceptibility to the development of esophageal cancer.

High MCV and both current and former flushing, as revealed by the flushing questionnaire, were reliable predictors for esophageal cancer in this study. Furthermore, the combination of inexpensive MCV testing and the questionnaire provided even more information. Individuals with both predictors were at the greatest risk for esophageal cancer, but the presence of either predictor alone also indicated high risk. Its high cost has prohibited using ALDH2/ADH2 genotyping as a predictive or screening tool for esophageal cancer in sizable East Asian populations. One of the biggest advantages of using inexpensive MCV testing and the flushing questionnaire in lieu of genotyping is that at present they can be easily applied in both public education and screening. In this study, we used PCR–RFLP for genotyping ALDH2/ADH2. Although this method is relatively laborious and expensive, new single nucleotide polymorphism (SNP) genotyping methodologies (for example, DNA chip-based microarray, fluorescent dye-based genotyping, etc.) enabling cost-effective and high-throughput genotyping are emerging (32). Therefore, SNP analysis using these technologies might be effective alternatives to PCR–RFLP as our previous study has shown (33).

Esophageal iodine staining markedly improves the detection rate of superficial cancers that can be treated by endoscopic mucosectomy (1,2). This procedure safely and easily serves a therapeutic purpose because cancerous involvement of lymph nodes, lymphatics and vessels is exceedingly rare prior to the cancer's encroachment into the muscularis mucosae (2). Therefore, clinicians might well focus on identifying high-risk individuals and persuading them to be screened by esophageal iodine staining. When confirmed in future large studies, high MCV, revealed in this study as a predictor for esophageal squamous cell carcinoma, has the potential to serve as a crucial complement in early intervention strategies to control this high-mortality cancer.


    Notes
 
6 To whom correspondence should be addressed Email: shiguchi{at}wa2.so-net.ne.jp Back


    Acknowledgments
 
We wish to thank Mrs Haruko Yoshimizu for her expert technical assistance. This work was supported by a Grant-in-Aid for Cancer Research 12-12 from the Ministry of Health, Labor and Welfare of Japan.


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 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 12, 2003; revised June 20, 2003; accepted August 4, 2003.





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