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(Chest. 2003;123:545-550.)
© 2003 American College of Chest Physicians

Detection of Loss of Heterozygosity by High-Resolution Fluorescent System in Non-small Cell Lung Cancer*

Association of Loss of Heterozygosity With Smoking and Tumor Progression

Ichiro Yoshino, MD, PhD; Seiichi Fukuyama, MD; Toshifumi Kameyama, MD; Yasunori Shikada, MD; Shinya Oda, MD, PhD; Yoshihiko Maehara, MD, PhD and Keizo Sugimachi, MD, PhD

* From the Department of Surgery and Science (Drs. Yoshino, Fukuyama, Kameyama, Shikada, Maehara, and Sugimachi), Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; and the Pathological Research Laboratory (Dr. Oda), National Kyushu Cancer Center, Fukuoka, Japan.

Correspondence to: Ichiro Yoshino, MD, PhD, Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan; e-mail: iyoshino{at}surg2.med.kyushu-u.ac.jp


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: We recently developed a novel system for detecting microsatellite alteration, which is an important process in carcinogenesis. In patients with non-small cell lung cancer (NSCLC), loss of heterozygosity (LOH) is frequently observed and causes functional disorders of tumor suppressor genes.

Patients and methods: In a consecutive series of 51 patients with NSCLC who had undergone a surgical resection, microsatellite instability (MSI) and LOH in tumors were analyzed by polymerase chain reaction using five fluorescence-labeled dinucleotide markers (D2S123, D5S107, D10S197, D11SS904, and D13S175) and an autosequencer.

Results: MSI was detected in only one patient (2.0%) with only one marker. LOH was detected in at least one chromosomal region that was tested in 39 patients (76%). The mean (± SD) number of LOHs detected by each marker was 1.74 ± 1.40, with 1 LOH detected in 10 patients, 2 LOHs detected in 15 patients, 10 LOHs detected in 3 patients, 1 LOH detected in 4 patients, and 3 LOHs detected in 5 patients. The number of LOHs detected in each patient was significantly associated with the pack-year index ({rho} = 0.501; p = 0.0004), although there was no relationship with having a history of multiple cancers and familial cancer. Patients with stage IA disease showed a significantly lower number of LOHs than did patients with other stages of disease (1.15 vs 2.38, respectively; p = 0.0013).

Conclusion: LOH is very common in patients with NSCLC, and the number of LOHs increases with increases in smoking, suggesting the presence of an important event in lung carcinogenesis.

Key Words: loss of heterozygosity • microsatellite instability • microsatellite marker • non-small cell lung cancer • smoking habit


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The incidence and mortality associated with lung cancer have both increased worldwide during the last several decades.1 2 The main reason for the increase in mortality due to lung cancer lies in an increase in the number of tobacco smokers.3 4 5 It has been reported that a loss of heterozygosity (LOH) at chromosomes 3p14, 9p21, and 17p13 has been observed frequently in the bronchial epithelia of smokers6 and that the frequency of LOH was significantly associated with the incidence of carcinoma in situ.7 LOH also has been considered to be a basic genetic disorder causing allelic losses at specific chromosomal loci of several recessive oncogenes (eg, the FHIT gene at 3p14,8 the p53 gene at 17p13,9 10 and the CDKN2 gene at 9p21).11 Therefore, the risk of lung cancer incidence is likely to be predicted by the frequency of LOH in bronchial epithelial cells.

We developed a novel system by which to investigate microsatellite disorder in genomic DNA12 13 using fluorescence-labeled markers. As regards detecting such microsatellite instability (MSI), this system has the high-resolution capacity to identify easily and consistently minimal disorder within fewer than six bases. Using this system, we observed the following two types of MSIs: type A was a disorder of fewer than six bases; and type B involved more than eight base pairs. More recently, we reported the unique MSI profile of a clinical colorectal cancer that had been analyzed by this system, in which rectal cancer was determined to be type B-dominant, whereas distal and proximal colon cancers showed heterogeneous patterns. These results suggested that there is a differential molecular background in colorectal cancers.14

In this study, microsatellite disorders of non-small cell lung cancer (NSCLC) were investigated using high resolution fluorescent microsatellite analysis and 5 dinucleotide markers; these markers were not matched to the chromosomal locations of known recessive oncogenes. The study was conducted in order to elucidate the relationships between the frequency of LOH and clinicopathologic factors such as smoking history, familial cancer and multiple cancers.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
During the period between April 2000 and September 2001, a consecutive series of 51 patients with NSCLC underwent surgical resection at the Second Department of Surgery, Kyushu University Hospital. The histologic diagnosis of the tumors was based on the criteria of the World Health Organization,15 and the TNM stage was determined according to the criteria revised in 1997.16 The age range of the patients was 43 to 80 years (mean [± SD], 64.0 ± 10.1 years). The patient group included 29 men and 22 women with 35 adenocarcinomas, 13 squamous cell carcinomas, and 3 large cell carcinomas. Written informed consent was obtained from each patient for study of the tissue excised from surgical specimens. The institutional review board of our university gave approval.

Extraction of Genomic DNA From Specimens
Cancer tissues and the corresponding normal lung tissues were obtained from surgical specimen immediately after resection. Specimens were placed in liquid nitrogen and used for the analysis. The remaining specimens were routinely processed for histologic examination. Frozen tissues were broken up in liquid nitrogen and lysed in a digestion buffer (10 mM Tris-Cl [pH 8.0]; 0.1 M ethylenediaminetetraacetic acid [EDTA] [pH 8.0]; 0.5% sodium dodecyl sulfate; and 20 µg/mL paracreatic RNase). After treatment with proteinase K and extraction with phenol, the DNA was precipitated with ethanol and then was dissolved in 1 x Tris buffer (10 mM Tris-Cl [pH 7.5] and 1 mM EDTA).

High-Resolution Fluorescent Microsatellite Analysis
Five dinucleotide microsatellites, D2S123, D5S107, D10S197, D11S904, and D13S175, were used as markers for the analysis of MSI and LOH.12 Using genomic DNA derived from tissue specimens, the five microsatellite sequences were amplified by polymerase chain reaction (PCR). Oligonucleotide primers corresponding to the microsatellite sequences12 were synthesized and purified by high-performance liquid chromatography. 5' primers were labeled with the fluorescent compounds 6-carboxy-x-rhodamine or 6-carboxy-2',4',7',4,7-hexachloro-fluorescein. PCR reactions were performed using Taq reagent kits (Takara Co, Ltd; Tokyo, Japan) and were run using a PCR system (GeneAmp PCR system 9600 or 2400; Perkin-Elmer; Norwalk, CT). Each 50 µL reaction mixture contained 1 times the reaction buffer, 350 µM each deoxynucleoside triphosphate, 10 pmol each primer, 2.5 U polymerase, and 25 µg genomic DNA. The thermal conditions of the system were as follows: one cycle at 95°C for 4 min; 35 cycles at 95°C for 0.5 min, 55°C for 0.5 min, and 72°C for 0.5 min; and 1 cycle at 72°C for 10 min. Then 0.5 U T4DNA polymerase was added to the mixture, followed by incubation at 37°C for 10 min. Each 1.5 µL product was mixed with 0.5 µL loading buffer (ie, blue dextran and 25 mM EDTA), 2.5 µL formamide, and 0.5 µL dH2O. To compare the electrophoretic profiles of two samples, 1.2 mL 6-carboxy-x-rhodamine-labeled product and 0.3 mL 6-carboxy-2',4',7',4,7-hexachloro-fluorescein-labeled product were mixed. Samples were denatured and loaded onto a sequencer (model ABI 373A; Applied Biosystems; Foster City, CA). In each case, a size marker labeled with N, N, N', N'-tetramethyl-6-carboxyrhodamine always underwent electrophoresis in each lane in order to standardize the mobility of the sample. The running conditions were 1,500 V, 20 mA, and 30 W for 5.5 h. The data were processed using computer software (ABI GeneScan; Applied Biosystems).

Statistical Analysis
The number of LOHs detected among the five markers was compared among subgroups, which were divided according to clinicopathologic factors, and the differences were analyzed using a t test. The correlation between the number of LOHs and the pack-year index (PYI) was analyzed by Spearman test. The data were considered to be significant when the p value did not exceed 0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of MSI and LOH
The method of detecting MSI and LOH used here has been described previously.12 Briefly, for the detection of LOH, fluorescence of a peak decreased more than did that of the normal control when the LOH occurred in the amplified region of the genomic DNA derived from tumor tissue (Fig 1 ). The number and proportion of positive MSI and LOH was summarized in Table 1 . MSI was detected in only one product amplified by D13S175 in one 64-year-old female patient with adenocarcinoma who also had a chondromatous hamartoma growing in the same lobe of the lung. On the contrary, LOH was frequently detected by every microsatellite marker that was used. The number of patients with positive LOHs were 20 (39%), 20 (39%), 15 (30%), 19 (38%), and 19 (38%), respectively, in the regions detected by D2S123, D5S107, D10S197, D11S904, and D13S175, respectively. The number of patients with at least one LOH was 39 (76%). Among these patients, 28 (56%) had multiple (ie, two or more) LOHs. The mean number of positive LOHs was 1.74 ± 1.40.



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  		Figure 1. Detection of LOH. The fluorescence of two peaks at base pairs 212 and 214 decreased more than that of normal tissues, as regards the amplified product derived from the genomic DNA of tumor tissue.

 

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  		Table 1. The Proportion and Number of Positive MSIs and LOHs

 
Frequency of LOH and Clinicopathologic Factors
The mean numbers of LOH for subgroups divided by clinicopathologic factors, including age, gender, cell type, pathologic stage, presence of multiple cancers, presence of cancer in the family, and smoking status, were compared (Table 2 ). Among them, patients with pathologic stage IA disease had significantly more infrequent LOH (1.15 ± 1.26 LOHs) than did patients with disease in pathologic stages IIB to IV (2.38 ± 1.19 LOHs; p = 0.0013). Smokers exhibited significantly more frequent LOH (2.17 ± 1.34) than did nonsmokers (1.14 ± 1.28 LOHs; p = 0.0087). There was no statistical significance found in the comparisons between the presence or absence of familial cancer or multiple cancer.


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  		Table 2. Relationship Between Number of LOHs and Clinicopathologic Factors

 
PYI and Frequency of LOH
The relationship between smoking habits and frequency of LOH was further investigated. Three subgroups were classified by the number of LOHs (0, 1 to 2, and 3 to 5), and the PYI of each subgroup was compared (Fig 2 ). The mean PYIs were as follows: group with 0 LOHs, 12; group with 1 to 2 LOHs, 14; and group with 3 to 5 LOHs, 44. The subgroup with 3 to 5 LOHs showed a significantly higher number of LOHs. The PYI and the number of LOHs were found to be positively correlated by analyzing with the Spearman test ({rho} = 0.501; p = 0.0004).



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  		Figure 2. Relationship between PYI and the frequency of LOH. In the three subgroups classified by LOH frequency, the PYI of each individual is shown. The mean number of PYIs were as follows: group with 0 LOHs, 12; group with 1 to 2 LOHs, 14; and group with 3 to 5 LOHs, 44.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have evaluated MSI and LOH in patients with NSCLC by a unique automated system using fluorescent labeling and laser scanning. The precise analysis is presented here and elsewhere.12 13 As compared with analysis by electrophoresis, a higher resolution analysis was achieved by the present system in that amplicons of different length with a few base pairs were clearly distinguished, and that detection of the fluorescence was quantitatively evaluable, leading to reproducible results and precise assessment. Moreover, as this method is not as time-consuming, such analysis can be performed routinely.

In a previous study analyzing MSI in patients with NSCLC by electrophoresis, the frequency of MSI data has been conflicting (2 to 34%).17 18 19 In our study, using a fluorescent system the incidence of MSI was very infrequent (2%). Since this system almost completely eliminated artifacts and was more precise than electrophoresis, our data appear to be significant. The frequency of LOH was detected by radioisotope labeling, and the electrophoresis method has been reported to be > 50%.19 In the specific analysis for 3p14 and 9p21, which are within the FHIT gene and are close to the p16 tumor suppressor gene, respectively, LOH was found in 63% and 60%, respectively.19 In our study, using five microsatellite markers that were nonspecific to such recessive oncogenes, the frequency of the LOH detected by each marker was 30 to 39%, whereas that of the LOH detected by at least one marker was 76%.

There have been several studies19 20 investigating the relationship between the incidence of LOH at selected chromosomal regions and patient prognosis. Zhou et al19 reported that the LOH at 10q24 was a strong prognostic indicator for patients with stage I NSCLC, whereas the MSI at the same region affected the prognosis for adenocarcinoma patients only. Fong et al20 reported that the LOH at 11p13 significantly correlated with advanced tumor stage and with poor prognosis, therefore they speculated that the chromosomal region harbors a tumor suppressor gene. As regards 3p14, where the FHIT gene is located, 63% of the tumoral DNA showed LOH,19 and this LOH was reported to be associated with poor prognosis.21 In the present study, the frequency of LOH was significantly lower in stage IA disease than in stage IIB to IV disease in patients with NSCLC, despite nonspecific markers of known tumor suppressor genes. This result might suggest that a condition such as high frequency of LOH was associated with the chance that critical LOH would affect tumor progression. The relationship between the specific LOH at 10q24, 3p14, or 11p13 and the frequency of LOH at nonspecific regions should be examined in this system.

The frequency of LOH in DNA from bronchial epithelial tissues has been associated with the amount of tobacco smoking.6 7 Wistuba et al7 reported that the LOH of at least one of 15 polymorphic microsatellite markers was observed in 86% of smokers. Although the incidence of bronchial dysplasia and the frequency of the LOH were not associated, patients in whom carcinoma in situ had been detected exhibited the highest frequency of LOH. Mao et al6 reported that 76% of smokers showed an LOH in the DNA of bronchial biopsy specimens, whereas only one subject among five nonsmokers showed LOH in an analysis of 3p14, 9p21, and 17p13, which correspond to the FHIT, the CDKN2, and the TP53 genes. The LOH of 3p53 was more frequently observed among current smokers (88%) than among former smokers (45%) and was associated with dysplasia of the bronchial epithelial cells. These results clearly indicated that an environmental factor such as smoking can affect the frequency of LOH, which is considered to be the most common genetic disorder underlining lung tumorigenesis. Therefore, the 30 smokers in this study might have very frequent LOH in their nontumoral DNA, however, our analysis was unable to detect microsatellite changes in normal lung DNA since our system included only a comparison between DNA from tumor tissues and that from normal lung tissues. Therefore, microsatellite changes were recognized by differences between these two tissue types as described in the "Materials and Methods" section. In other words, the LOH in our study was detected by the presence of additional microsatellite changes occurring in tumoral DNA in comparison with the DNA of normal tissues.

Our study indicated that there exists a correlation between LOH frequency and the smoking status. The study further suggested that the frequency of LOH might indicate an accumulation of genetic disorders. Our next study will investigate the relationship between the frequency of LOH in cases of NSCLC and the incidence of second primary NSCLCs. Moreover, the automated fluorescent system that has suggested here will be quite appropriate for the prospective study in terms of its accuracy, reproducibility, and brevity.


   Footnotes
 
Abbreviations: EDTA = ethylenediaminetetraacetic acid; LOH = loss of heterozygosity; MSI = microsatellite instability; NSCLC = non-small cell lung cancer; PCR = polymerase chain reaction; PYI = pack-year index

Received for publication March 18, 2002. Accepted for publication August 30, 2002.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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