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Molecular Oncology, Markers, Clinical Correlates |
The Pulmonary Center and the Department of Medicine, Boston Veterans Administration Medical Center, and Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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85%, a rate that has been relatively unchanged over the past 3 decades (1)
. Previous attempts to improve survival have focused on the early detection of tumors by aggressive screening of high-risk individuals with sputum cytology and chest X-rays, but these methods have not improved mortality (2, 3, 4)
. Recent research has been directed to evaluating molecular diagnostic strategies to improve the early diagnosis of carcinoma. For example, nucleic acid-based methods, such as analysis of LOH3
in DNA extracted from shed urothelial cells, show great promise for the early detection of tumors such as bladder carcinoma (5)
. These methods, when applied to cells microdissected from lung tumors and from preneoplastic bronchial epithelium, have demonstrated LOH at multiple chromosomal loci in tumors and have shown that LOH is an early event in the progression from normal to neoplastic cells (6
, 7)
. These observations have encouraged the development of diagnostic tests based on the detection of LOH in readily obtainable clinical specimens (8)
. However, two recent studies of LOH in bronchial biopsies of smokers without apparent lung cancer have raised questions about the diagnostic significance of LOH (9
, 10)
. Both studies found evidence of LOH at one or more chromosomal sites in histologically normal or minimally abnormal cells, casting doubt on the diagnostic potential of LOH as an early indicator of lung cancer. Although the presence of LOH is nonspecific for lung cancer, there are data to suggest that LOH accumulates as the epithelium undergoes neoplastic transformation (10 , 11) . We undertook the present study to determine whether measuring the extent of LOH in the airway epithelial cells would be a useful diagnostic test for lung cancer. We first developed and validated a method for measuring and quantitating LOH in a mixed population of normal and abnormal cells obtained from bronchial brushings of airways at bronchoscopy. Using this method, we measured the presence and extent of LOH at 22 chromosomal loci in epithelial cells obtained from the lung bearing the suspect cancer and from the contralateral lung of a cohort of 55 adult patients undergoing diagnostic bronchoscopy for suspected lung cancer. The aims of this study were to assess the feasibility of measuring extent of allelic loss in a heterogeneous population of lung epithelial cells and to assess the sensitivity and specificity of LOH as a diagnostic marker for lung cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bronchoscopic Collection of Cells for LOH Analysis.
At bronchoscopy, the normal lung was evaluated first and brushings were obtained under direct vision from the lower lobe bronchus. Brushings were obtained with a cytology brush using two series of 10 strokes. The brushings were vortexed into normal saline and divided into aliquots for DNA analysis and for routine cytology analysis. Samples were then obtained with a new brush in the same fashion from the lung with the radiographic lesion. These brushings were directed toward the involved bronchial segment, either by direct vision if there was an endobronchial abnormality (12 subjects), or by brushing the bronchial segment that was abnormal on a chest radiograph or computerized tomography scan. Brushings were obtained first for routine diagnostic testing, and an additional brushing from the same area was then obtained for DNA analysis. Whole blood (2 ml) was collected in heparinized tubes from each patient.
DNA Analysis.
The yield of the bronchoscopy brushings was determined by hemocytometer to be 100,000- 500,000 cells of which 90% were histologically classified as epithelial cells. The cells were washed twice with PBS, and 0.2–1 µg of genomic DNA was extracted with proteinase K (0.1 mg/ml) at 55°C for 12 h using standard procedures (12)
. Normal, nonlung (control) DNA was obtained from peripheral leukocytes and prepared in the same manner as the bronchial epithelial cells. DNA samples were examined for LOH by PCR-based microsatellite analysis using four-color fluorescent primers, robotics, and capillary electrophoresis to facilitate precise quantitation and automation. All DNA templates were amplified with a panel of fluorescent PCR primers from chromosomal regions 3p, 9p, 11q, and 17p that were obtained from Research Genetics (Huntsville, AL) and PE Applied Biosystems (Foster City, CA). The primers were selected to amplify chromosomal regions that had been reported to frequently demonstrate LOH in lung tumors (6
, 7
, 13
, 14)
. The microsatellite markers and cytogenetic locations were as follows: D3S2387 (3p26.3), D3S2409 (3p21.31), D3S1300 (3p14.3), D3S1285 (3p14.1), D3S1233 (3p13), D3S2406 (3p12.2); GATA62FO3 (9p24), D9S925 (9p22.3), D9S169 (9p21.3), D9S741 (9p21), D9S1118 (9p13); D11S2371 (11q13.3), D11S2002 (11q21), D11S2000 (11q22.3), D11S1986 (11q22.3), D11S1998 (11q23.2), D11S925 (11q23.3–24); D17S513 (17p12), D17S969 (17p12), D17S1303 (17p12), D17S520 (17p12), and D17S122 (17p11.2). All PCR reactions were performed on the automated 877 Integrated Thermal Cycler from PE Applied Biosystems. Each 5-µl reaction consisted of 10 ng of DNA, 1.5 mM MgCl2, 0.5 µM of each primer, 0.125 units of AmpliTaq Gold Taq polymerase from PE Applied Biosystems with GeneAmp PCR Buffer II. Amplification was done at 95°C for 10 min, followed by 10 cycles of 95°C for 15 s, 55°C for 30 s, 72°C for 60 s, and 20 cycles of 89°C for 15 s, 55°C for 30 s, 72°C for 60 s, with a final extension at 72°C for 10 min. Several water blank controls were included in each PCR. From each of the six different reaction products, 1 µl was pooled. All products in the pooled sample were amplified from the same template and distinguished by allele size and emission wavelength. Reactions were then analyzed by capillary electrophoresis on the PE Applied Biosystems 310 Genetic Analyzer with Genescan 2.1 and Genotyper 2.0 software. All DNA templates were coded such that investigators were unaware of the cytological and clinical data from patients until the PCR data analysis was complete. Only primers that demonstrated heterozygosity in leukocyte DNA were considered informative. Most DNA templates were amplified with a panel of 22 primers, with the mean number of informative primers being 14 ± 4 per patient. There was no difference in the mean number of informative primers between the patients with or without cancer nor between patients with or without LOH.
Determination of Allelic Loss.
Allelic loss was calculated using a normalized allele ratio (LOH ratio) equation in which R = (A1)(N2)/(A2)(N1) where A1 and A2 are the heights of the alleles from lung and N1 and N2 are the heights of the alleles from the DNA from blood (Refs. 15
and 16
; Fig. 1
). For informative primers, based on our mixing experiments (see below), LOH was scored as a 20% decrease in the height ratio of an allele in the lung compared with height ratio of the same allele in leukocyte DNA. Therefore, cases in which the LOH ratio was 
0.8 or 
1.25 were scored as LOH. For the purposes of subsequent data analyses, the LOH ratio was represented by R or by 1/R if R was >1. All instances of LOH were verified by repetition, and the mean LOH ratio was used for data analysis.
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. These studies demonstrate that the proportion of DNA with allelic loss and, therefore, the percentage of cells with allelic loss corresponded to the LOH ratio. Repetition of this mixing experiment revealed the SE of the LOH ratio to be 9%, validating the precision of the method and confirming that the LOH ratio cutoff value of 20% was outside the 95% confidence interval of the method. The mixing experiment was duplicated using DNA templates from two lung cancer cell lines (NCIH446 and NCI441) amplified with primer D9S1118 (data not shown).
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To define a value of the extent of LOH that had the greatest sensitivity while retaining specificity, we incorporated measurements of FAL and severity [the percentage of DNA (cells) with LOH] into a LOH score. The total LOH score was obtained by assigning a value of 1 for all LOH sites with ratios between and a value of 2 for all LOH ratios of 45% or more (ratios of <0.55). The total LOH score was calculated as the sum of the above LOH values divided by the maximum potential LOH score [2 x (number of informative primers) x 100]. This total LOH score may range from 0–100%.
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Comparisons between groups were performed using either the Mann-Whitney U Test for independent samples or the Wilcoxon test for matched samples. The t test was used to compare the FAL between airways containing lung cancer and the contralateral airways of the patients with lung cancer. The reported Ps are two-sided. The 95% confidence limit for the proportion of alleles demonstrating allele-specific loss was computed as described by Zar (17) .
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thirty-eight of the 55 patients were diagnosed with lung cancer of the following cell types: 12 adenocarcinomas, 10 squamous cell carcinomas, 9 large cell carcinomas, 5 SCLCs, and 2 NSCLCs of unspecified cell type. The remaining 17 patients were diagnosed with other aerodigestive tract tumors (primary esophageal and head and neck cancers), other malignancies (lymphoma and mesothelioma), chronic interstitial and granulomatous disease, and acute infections (bronchitis, pneumonia, tuberculosis). None of these 17 patients were determined to have pulmonary neoplasms, primary nor metastatic. Endobronchial abnormalities were visible in 12 (32%) of the 38 patients with lung cancer. Bronchial brushing cytology was positive in 14 (37%) of the patients with lung cancer, including 10 of 12 patients with visible endobronchial abnormalities.
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. Complete loss of one allele is demonstrated in Fig. 3a
. Cells from this patient came directly from a visible small cell carcinoma; microsatellite analysis revealed complete allelic loss at D3S2387 on chromosome 3p26 [LOH ratio (R) = 0]. More frequently, analysis revealed partial allelic loss as is seen in Fig. 3b
. This example demonstrates 37% allelic loss of the shorter allele in cells obtained from the same patient using primer D9S925. Complete allelic loss occurred in three patients, all at chromosome 3p. However, each patient with complete allelic loss also demonstrated partial allele loss (r = 0.30-0.60) at a different chromosomal region, such as 9p or 11q. This suggests that the cells within the tumor were genetically heterogeneous in terms of allelic loss, rather than being genetically clonal.
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and 3
). However, LOH was present in brushings from the contralateral lung in 20 (53%) of 38 patients. In 75% (P <0.05 versus random distribution of allele loss, i.e., 50%) of these instances, the same allele was affected, a finding that is consistent with the concept of allele-specific loss (Fig. 3c)
. There were five instances of allele-specific loss in four patients without lung cancer. Although the number of patients was small, there was no difference in the frequency of allele-specific loss among patients with different lung cancer cell types, nor was there a predilection for a specific chromosomal region to be affected. Thus, LOH at one or more sites was frequent and bilateral in the airways of smokers who have cancer. We did not observe microsatellite instability in our samples.
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showed that both the proportion of informative primer sites that demonstrated LOH (FAL) and the percentage of cells demonstrating allelic loss, expressed by the LOH score, was greater on the side of the tumor. The same was true for tumors with positive cytology versus negative cytology or when an airway abnormality was brushed. Thus, the extent of allelic loss increased as one moved closer to the tumor, as has been seen in studies of cells dissected from pathological specimens (10
, 11)
.
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. Using a LOH score >10, the sensitivity was 58%, which was greater than the sensitivity of cytology (37%). The LOH score was >10 in the contralateral lung in five (13%) of 38 patients. This LOH score of >10 was 100% specific in the 17 patients without lung cancer. Despite often having at least one LOH site in lesion or contralateral lung, none of these 17 patients had a positive LOH score (>10) in cells from either lung. The positive and negative predictive values of the LOH score were 100% and 52%, respectively. These values compare favorably with those of cytology, which demonstrated positive and negative predictive values of 100% and 41%, respectively. Combining positive cytology and LOH score >10 increased the sensitivity in all subjects with cancer to 66%. The sensitivity of cytology combined with the LOH score was 80% in SCLC versus 55% in NSCLC, but this difference was not statistically significant.
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. The microsatellites affected most frequently were in the regions of 3p21 and 3p14, which is consistent with data derived from microdissection of lung tumors (18)
. The chromosomal distribution of microsatellites with LOH was not useful in distinguishing small cell carcinoma from NSCLC.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20%. This value is identical to that reported by Wang et al. (19)
using fluorescent primers and capillary electrophoresis to analyze LOH in bladder cells. Our cutoff value, which was determined by the data presented in Fig. 2
, is also in the same general range as those chosen by other investigators using radio-isotope labeling to evaluate LOH. (20
, 21)
. In our study, the procedures following DNA extraction (PCR reaction preparation, data collection, and data analysis) are automated, making the method suitable for screening large patient populations. We found, as have others, that LOH at any of the examined chromosomal loci was frequent in the airway epithelial cells of smokers. It was present in the contralateral lung of subjects with cancer and in both lungs of patients who had lesions on chest radiograph that proved not to be cancer. Thus, the presence of LOH is a nonspecific indicator of chromosomal DNA damage and it does not in and of itself indicate the presence of cancer. Brushings with positive cytology tended to have LOH at several microsatellites, often with an allele ratio <0.45. Brushings with negative cytology often demonstrated LOH, but the number of microsatellites involved was less, and the incidence of allele ratio <0.45 was less than in brushings with positive cytology. We, therefore, constructed a measure of the extent of LOH (LOH score) that incorporated the proportion of microsatellites with LOH or FAL and the percentage of cells with allelic loss or the LOH ratio. A positive LOH score >10 was determined by analysis of sensitivity and specificity of the score. The LOH score was positive (>10) in 55% of brushings from the tumor side and was negative in brushings from both lungs in the 17 patients who did not have lung cancer. The LOH score was >10 in brushings from four (11%) contralateral lungs in patients with cancer. It is unclear if a positive LOH score from the contralateral lung represented an early malignant lesion in the contralateral lung. Although none of the patients developed clinically evident cancer over the year of follow-up, second cancers do appear after treatment at a rate of 1–2% per year for NSCLC and 6% per year for SCLC (22) .
Our results are consistent with prior studies in lung cancer that have measured LOH in cells dissected from tumors and from adjacent normal lung and in bronchial biopsies taken from smokers and former smokers who did not have lung cancer. These studies have shown that LOH occurs in current and former smokers without lung cancer, both in atypical and cytologically normal cells (9 , 10) and that LOH accumulates as carcinogenesis progresses. Analysis of premalignant lesions in the airways of healthy smokers has demonstrated that the extent of LOH, as measured by fractional regional loss (fraction of chromosomal arms examined with LOH), accumulates as the histological stage progressed from dysplasia to carcinoma in situ (10) . Analysis of squamous cell carcinomas has demonstrated that the extent of LOH, as measured by FAL, was higher in clinically advanced stage tumors than in small tumors that were radiographically occult (11) . This concept of step-wise accumulation of DNA damage from "normal" to tumor tissue serves as the basis of the tumor progression models first put forth by Fearon and Vogelstein (23) and validated in lung cancer by Gazdar and coworkers (6 , 7 , 24) . In these models, LOH, subsequent inactivating mutations at the sites of growth suppression genes, and activating mutations in oncogenes all accumulate as cells progress from premalignant to transformed phenotypes.
Allelic loss in the contralateral lung of 53% of the cancer patients is consistent with the concept of field cancerization and is confirmed by two recent studies of bronchial biopsies in smokers without cancer (9 , 10) . In our study, 80% of the microsatellites that demonstrated LOH in the contralateral lung were also involved in the lung containing the tumor, involving the same allele 75% of the time. Furthermore, each instance of contralateral LOH in the patients without lung cancer involved the same allele. This allele-specific loss suggests the presence of genetically determined allelic susceptibility to LOH. Allele-specific loss has been found in several previous studies, and several explanations have been proposed (7 , 25) . Clonal proliferation of a progenitor cell with a somatic mutation that confers growth advantage may explain the widespread appearance of the same mutation in lung epithelial cells. Alternately, allele-specific mutations may result from inherited differences that predispose specific alleles to DNA damage. It is known that allelic methylation of genes results in genetic imprinting during embryonic development (26) . These specific methylated alleles may be targets of carcinogen-induced mutations, as demonstrated by benzo(a)pyrene adducts formation clustering preferentially at specific methylated codons on p53 (27) and for loss of imprinting of insulin-like growth factor II in lung cancer (28) . Although the mechanisms for allele-specific loss remains to be determined, its presence in clinical samples may provide insights into genetic factors that determine susceptibility to genetic damage and lung cancer.
There was no statistically significant correlation between cumulative cigarette dose (pack years) and extent of LOH in the contralateral lung of cancer patients. These results are similar to the biopsy studies of smokers without cancer that demonstrated no correlation between extent of LOH and amount of cigarette consumption. The absence of this correlation suggests that other genetic and environmental factors are important for the progression of DNA damage. The ultimate biological significance of allelic loss in bronchial epithelial cells remains to be determined. In a few instances, the detection of LOH clustering in chromosomal regions has led to the cloning or a tumor suppressor gene such as p53 or FHIT. However, given the frequency of allelic loss in nonmalignant epithelial cells, it is most unlikely that LOH always indicates deletion of a tumor suppressor gene. Alternately, LOH may be a biomarker of chromosomal instability that occurs as tumor suppressor genes, such as p53, are inactivated during carcinogenesis.
The LOH score calculation we devised is based, in part, on arbritray cutoff values that may theoretically introduce bias. However, the application of this method to our data illustrates the important principle that LOH is frequently detected in the epithelium of cigarette smokers, but extensive LOH is rare in the absence of cancer. Although the use of our measure of LOH in the diagnosis of lung cancer remains to be validated, the association of LOH score with lung cancer validates it as a potential biomarker (29) . Future studies measuring the extent of LOH may be directed to the genetic and epidemiological analysis of cohorts of cigarette smokers who demonstrate inter-individual differences in the extent of allelic loss, relative to cigarette exposure. These studies may provide insights into the genetic factors such as race, gender, polymorphism in metabolic enzymes, and the environmental factors such as ambient air pollution and diet that may be responsible for the accumulation of allelic loss within cells of the bronchial epithelium. Prospective studies of cigarette smokers may also determine whether the LOH score will be a useful biomarker to identify smokers who are at risk for developing lung cancer and to assess epithelial cell responses to chemoprevention and to smoking cessation.
Our study and the work of others indicate that LOH analysis may have clinical use in the diagnosis of lung cancer. The presence of LOH is nonspecific, but sensitive for the presence of cancer. These results indicate that molecular alterations associated with lung tumors are commonly detected in the epithelial cells of smokers without cancer. This paradigm similarly holds true for analyses of other genetic alterations including trisomy 7 (30) , trisomy 20 (31) , and p16 methylation (32) . The analysis of the extent of LOH demonstrates that a LOH score >10 is specific and sensitive for the diagnosis of lung cancer. Compared with cytology, the LOH score is more sensitive while retaining a positive predictive value of 100%. The LOH score and cytology were complementary, resulting in a combined test sensitivity of 66%. This suggests that the simultaneous analysis of bronchoscopy brushings for extent of LOH and for cytological malignant cells may increase the diagnostic yield of the procedure.
With the addition of new microsatellite primer pairs at other chromosomal loci shown to be involved in lung cancer, the sensitivity and the negative predictive value of the LOH assay should increase. The method and the panel of primers need to be validated in a prospective study of a larger population of patients that will have greater statistical power before this test can be considered to have clinical use. It is clear that the specific criteria used in defining a positive LOH score in this study may not be applicable to all studies of LOH. Presently, most allelic loss analysis is performed using microdissected tissues as the source of DNA templates that are amplified by P32-labeled PCR primers. The values of a positive LOH score will likely vary depending on the methods used in the study. Whether the LOH score will correlate with the presence of lung cancer, regardless of the specific methods, remains to be determined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grant T32-HL07035 (to C. A. P. and S. K.) and by the Boston University Evans Medical Foundation (to C. A. P.). 
2 To whom requests for reprints should be addressed, at Columbia University, 630 West 168th Street, Box 91, New York, NY 10032. Phone: (212) 305-0422; Fax: (212) 305-0089; E-mail: cap6{at}columbia.edu
3 The abbreviations used are: LOH, loss of heterozygosity; SCLC, small cell lung cancer; NSCLC, non-SCLC; FAL, fractional allelic loss.
Received 2/24/99; revised 5/ 3/99; accepted 5/10/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, samples from the contralateral lung; arrows, the median values for the groups. The LOH score was higher on the lesion versus contralateral side (P = 0.0006) in cancer patients and higher on the lesion side (P = 0.0002) cancer versus noncancer patients. However, there was no difference in LOH score (P = 0.24) between the contralateral lung of cancer versus noncancer patients. Dashed line, a LOH score of 10. None of the noncancer patients had a LOH score >10, whereas 58% of the cancer patients had a LOH score >10. b, LOH score from the side of the lesion in cancer patients with positive (•) and negative (