Ann Thorac Surg 2003;75:1597-1600
© 2003 The Society of Thoracic Surgeons
Original article: general thoracic
Loss of heterozygosity on the long arm of chromosome 21 in non–small cell lung cancer
Eung Bae Lee, MDa*,
Tae In Park, MD, PhDb,
Sun Ha Park, MScd,
Jae Yong Park, MD, PhDc
a Department of Thoracic and Cardiovascular Surgery, Kyungpook National University Hospital, Jung-gu, Daegu, South Korea
b Department of Anatomical Pathology, Kyungpook National University Hospital, Jung-gu, Daegu, South Korea
c Department of Internal Medicine, Kyungpook National University Hospital, Jung-gu, Daegu, South Korea
d Department of Department of Biochemistry, School of Medicine, Kyungpook National University, Jung-gu, Daegu, South Korea
Accepted for publication November 25, 2002.
* Address reprint requests to Dr Lee, Department of Thoracic and Cardiovascular Surgery, Kyungpook National University Hospital, 50 Samdeok 2-ga, Jung-gu, Daegu 700-721, South Korea.
e-mail: bay{at}knu.ac.kr
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Abstract
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BACKGROUND: In Down syndrome, the incidence of solid tumors including lung cancer is considerably lower than that of the general population. The low risk of lung cancer in individuals with Down syndrome may be related to the gene-dosage effect of the extra chromosome 21. It may suggest that tumor suppressor genes playing a role in the pathogenesis of lung cancer may be present on chromosome 21.
METHODS: A total of 39 surgically resected non–small cell lung cancers were analyzed using nine microsatellite markers for 21q. Loss of heterozygosity was considered to be present when the signal intensity of the allele in tumor DNA was less than 50% of that in the corresponding normal DNA.
RESULTS: Loss of heterozygosity for at least one locus was detected in 22 of 39 tumors (56.4%). Allelic loss was frequently detected at three distinct regions: at the locus D21S1432 on 21q21.1, the region between D21S1435 and D21S1442 on 21q21.2 to 21.3, and the region between D21S1270 and D21S1445 on 21q22.1.
CONCLUSIONS: These results indicate that loss of heterozygosity on 21q may play an important role in the pathogenesis of non–small cell lung cancer.
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Introduction
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Lung cancer is a predominant cause of cancer deaths throughout the world [1]. Recent advances in molecular genetics have revealed that multiple tumor suppressor genes (TSGs) are involved in the genesis or progression of lung cancer [2, 3]. Unlike oncogenes, in which an abnormality in one of the two alleles is sufficient to lead to an abnormal function, TSGs are inactivated by two mutational events. The paradigms of such events are a mutation of one allele and a loss of the other allele [4, 5]. Thus, loss of heterozygosity (LOH) is a landmark of chromosomal regions that may harbor TSGs [6, 7]. In non–small cell lung cancer (NSCLC), frequent LOH has been demonstrated on several chromosome arms such as 3p, 5q, 8p, 9p, 11p, 13q, 17p, and 22q [8–13]. Studies based on the somatic LOH have led to the discovery of several TSGs such as RB, p53, and FHIT [14–17].
In Down syndrome with trisomy 21, the incidence of solid tumors including lung cancer is considerably lower than that of general population [18, 19]. The low risk of lung cancer in individuals with Down syndrome may be related to the gene-dosage effect of the extrachromosome 21. This suggests that TSGs playing a role in the pathogenesis of lung cancer may be present on chromosome 21. To determine the frequency of LOH on the long arm of chromosome 21 with the aim of identifying potential TSG loci, we examined 39 NSCLCs using nine microsatellite markers for this chromosome arm. In addition, we looked for correlations between LOH on 21q and clinicopathological factors.
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Material and methods
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Tumor tissue was obtained from 39 patients with NSCLC at the time of surgical resection. These included 23 cases of squamous cell carcinomas and 16 adenocarcinomas. None of the patients had received chemotherapy or radiotherapy before surgery. The pathologic stages of the patients were as follows; 21 stage I, 7 stage II, 8 stage IIIA, and 3 stage IIIB. Patients’ peripheral lymphocytes were used as the source for the normal DNA. Tumor cells were selectively procured from hematoxylin and eosin–stained slides using a 30 G1/2 hypodermic needle (Beckton Dickinson, Franklin Lake, NJ) affixed to a microdissection device.
Nine microsatellite markers along 21q were used to study LOH: 21q21.1 (D21S1432, D21S1437, D21S1436, and D21S1994); 21q21.2 to 21.3 (D21S1435 and D21S1442); 21q22.1 (D21S1270 and D21S1445); and 21q22.2 to 22.3 (D21S266). The primer sequences were obtained from the Human Genome Database. Deoxyribonucleic acid from frozen tissues and peripheral blood lymphocytes were extracted using standard methods using proteinase K digestion and phenol/chloroform extraction, and amplified by nonradioactive polymerase chain reaction at microsatellite markers. A quantity of 10 µL of the polymerase chain reaction products was analyzed in 6% polyacrylamide-8 urea gels and silver stained. We considered LOH to be present when the signal intensity of the allele in tumor DNA was less than 50% of that in the corresponding normal DNA. The fractional allelic loss (FAL) in a tumor was calculated as the ratio of the number of markers showing LOH to the number of informative markers. Fisher’s exact test and Student’s t test were used for statistical analysis. A value of p less than 0.05 was considered to be statistically significant.
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Results
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Loss of hererozygosity was detected in 22 of 39 tumors (56.4%), which were informative for at least one of loci analyzed. Among the 22 tumors with LOH, six (27.3%) showed LOH at almost all informative loci. The other 16 showed partial or interstitial deletions. The frequencies of LOH detected at nine microsatellite markers are summarized in Figure 1,
and representative autoradiograms of a case showing interstitial deletions are shown in Figure 2. We found that LOH was frequently detectable at three distinct legions: the D21S1432 locus on 21q21.1; the region between D21S1435 and D21S1442 on 21q21.2 to 21.3; and the region between D21S1270 and D21S1445 on 21q22.1 (Fig 1).
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Fig 1. Frequencies of loss of heterozygosity (LOH) detected at nine microsatellite markers on 21q. Open boxes indicate retention; diagonally striped boxes indicate noninformative cases; filled boxes indicate LOH. a = incidence is expressed by number of cases with LOH per number of informative cases.
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Fig 2. Representative autoradiograms of a case showing interstitial deletions. Loss of heterozygosity at markers D21S 1432, D21S1437, and D21S1435 and retention of heterozygosity at other markers. Arrows indicate allelic loss in tumor DNA. Numbers at bottom of radiograms represent microsatellite markers. (L = lymphocyte DNA; T = tumor DNA.)
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There was no significant correlation of LOH with age, sex, smoking status, and histologic type and stage of the disease (Table 1). Although statistically not significant, however, LOH was found more frequently in squamous cell carcinomas (15 of 23, 65.2%) than in adenocarcinomas (seven of 16, 43.2%), and in squamous cell carcinomas the frequency of LOH was higher in stage II to III (80.0%) than in stage I (53.8%).
We compared the FAL value, considered to be a marker for accumulation of allelic loss in tumors, with clinicopathologic factors (Table 2). The FAL value in squamous cell carcinomas (0.446 ± 0.399) was statistically higher than that in adenocarcinomas (0.206 ± 0.274). In squamous cell carcinomas, the FAL value in stage II to III (0.574 ± 0.397) was higher than that in stage I (0.347 ± 0.387), although the difference was not statistically significant. The FAL value was significantly higher in cases with more than 40 pack-years of smoking (0.566 ± 0.365) than in cases with 40 or fewer pack-years of smoking (0.257 ± 0.334) among patients with squamous cell carcinomas.
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Comment
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We performed LOH analysis on the long arm of chromosome 21 in NSCLC to identify candidate regions of TSGs involved in the tumorigenesis of NSCLC. Our results strongly suggest that LOH on 21q may be involved in the development of NSCLC, and that putative TSG(s) contributing to the pathogenesis of this disease are present on 21q.
Allelotype studies have reported frequent LOH on 21q in several human cancers, including breast, gastric, and oral cancer [20–22]. Ohgaki and coworkers [20] reported that the region of 21q11 to 21 between loci D21S1432 and D21S1437 frequently deleted in breast cancer. Sakara and colleagues [21] and Yamamoto and associates [22] also reported that the proximal region of 21q was commonly deleted in stomach cancer and oral cancer, respectively. In our study, frequent LOHs were also found at 21q21.1 including the D21S1432 locus, suggesting that TSG for the development of a variety of human cancers including NSCLC may be harbored in this deleted region. Recently, abundant in neuroepithelium area (ANA) and ubiquitin-specific pretease (USP) genes were isolated from the region of 21q11 to 21 [23, 24]. However, it is unlikely that ANA or USP gene is a TSG inactivated by two mutational events, as mutation either in ANA or USP gene was not detected in the tumors with LOH at this locus. Thus, further studies are needed to rule out the presence of TSGs that could explain deletions in this region.
In the present study, two other distinct regions (one between D21S1435 and D21S1442 on 21q21.2 to 21.3 and the other between D21S1270 and D21S1445 on 21q22.1) also exhibited frequent LOH. In an allelotype analysis of breast cancer [20], LOH on loci D21S1435 was found only in 16 (17%) of 94 tumors. However, we detected LOH at D21S1435 in 13 (48.2%) of 27 NSCLCs. The high frequency of LOH at this locus in NSCLC but not in breast cancer suggests that it encodes a novel TSG that may be specific for the development of NSCLC. Yamamoto and colleagues [22] reported that 21q22.1 involving the region at the D21S1254 was commonly deleted in oral cancer. In our study, LOHs at D21S1270 and D21S1445 on 21q22.1 were detected in 14 (43.85%) of 32 and in 11 (39.3%) of 28 tumors, respectively. These loci include several genes such as T-lymphoma invasion and metastasis 1 (TIAM1) and interferon-
receptor (IFNAR) genes [25, 26]. These genes may be the targets of frequent LOH [27, 28].
Sato and colleagues [29] reported that the frequency of LOH on 21q was significantly higher in squamous cell carcinomas (seven of 14, 50%) than in adenocarcinomas of the lung (12 of 53, 23%), although failing to reach statistic significance in our study, LOH on 21q was also found more frequently in squamous cell carcinomas than in adenocarcinomas. In addition, the FAL value was significantly higher in squamous cell carcinomas than in adenocarcinomas. These results are consistent with previous studies [29, 30] reporting that more genetic changes accumulate during tumorigenesis in squamous cell carcinomas than in adenocarcinomas. Kohno and colleagues [23] reported that LOH on 21q11.1 to 21.1 was detected preferentially in advanced NSCLC. In the present study, there was no significant correlation between LOH on 21q and stage of the disease. In squamous cell carcinomas, however, the frequency of LOH and the FAL value were higher in stage II to III than in stage I, although the differences were not statistically significant. These findings suggest that the putative TSG on 21q may be related to progression of the disease in squamous cell carcinomas. In addition, the FAL value in squamous cell carcinomas was significantly higher in patients with more than 40 pack-years of smoking than in those with 40 or fewer pack-years of smoking. This result reflects the fact that inactivation of putative TSG on 21q may be due to tobacco smoking.
In conclusion, our findings indicate that TSG(s) contributing to the pathogenesis of NSCLC exist on 21q. It appears that the putative TSG(s) on 21q may be related to progression of the disease in squamous cell carcinomas. Because the number of cases analyzed is small, however, further studies are needed to evaluate the relationship between LOH on 21q and clinicopathologic features including prognosis of the disease.
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Acknowledgments
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This work was supported by Medical Research Institute grant, Kyungpook National University Hospital (2001).
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Abstract
Introduction
