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Lovelace Respiratory Research Institute, Lung Cancer Program, Albuquerque, New Mexico 87185 [W. A. P., K. K. D., S. A. B.]; St. Mary’s Hospital, Grand Junction, Colorado 81501 [G. S.]; Department of Preventive Medicine, Division of Occupational and Environmental Health, University of Southern California, Los Angeles, California 90033 [F. D. G.]; and Johns Hopkins University Medical Institutions, The Johns Hopkins Comprehensive Cancer Center, Baltimore, Maryland 21231 [S. B. B., J. G. H.]
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Because current and even former cigarette smokers have increased bronchial secretions that contain exfoliated cells from the bronchial tree, the analysis of sputum from these individuals has been an active area of research for marker development (2) . The late Dr. Geno Saccomanno, pioneer of sputum cytology, demonstrated that premalignant cytological changes could be detected several years before a clinical diagnosis of lung cancer in high-risk subjects (3) . Unfortunately, these studies were difficult to replicate, most likely because of the skills required for identifying subtle nuclear changes in cells that often comprise <5% of the sputum slide. Subsequent studies suggested that molecular assays could be used to enhance the predictive value of sputum samples. Mutations within the K-ras gene have been detected in sputum specimens collected before tumor resection (4) ; identical microsatellite alterations have been detected in primary tumors and corresponding sputum samples (4 , 5) . However, methods to detect both alterations lack sensitivity, and the overall prevalence of these changes in NSCLC3 is <25%.
We have pursued another approach for developing DNA-based assays to aid in the early diagnosis of lung and other cancers. Our approach involves the detection of gene promoter regions that are aberrantly hypermethylated in human tumors. This change is associated with an epigenetically mediated gene silencing that constitutes an alternative to coding region mutations for loss of tumor suppressor gene function in cancer (6) . Recent studies (7) in our laboratory demonstrated that aberrant promoter methylation of the p16 tumor suppressor gene, which plays a key role in cell cycle regulation, is an early and very frequent event in SCC of the lung. Another gene frequently inactivated by aberrant promoter methylation in NSCLC (8) is MGMT. MGMT is a DNA repair enzyme that protects cells from the carcinogenic effects of alkylating agents by removing adducts from the O6 position of guanine. Thus, the p16 and MGMT genes are strong candidate biomarkers for early detection of lung cancer.
Our recent observations that aberrant methylation of the p16 promoter region can be detected in DNA from exfoliated cells in sputum of patients with this disease further support this approach in the early diagnosis of cancer. In that study, aberrant methylation of the p16 gene promoter was detected in sputum from 3 of 7 patients with lung cancer, and 5 of 26 cancer-free individuals at high-risk (7) . We have modified the sensitive MSP assay (9) used initially, to achieve an even higher detection efficiency. This new modification has now been used not only to extend analyses of the p16 promoter but also to study the methylation status of a similar region of MGMT. Aberrant methylation of one of these two promoter regions was detected in the sputum of 100% of patients with proven SCC, not only at the time of diagnosis, but also in all sputum samples taken from patients 5–35 months before clinical tumor detection. Furthermore, these sputum markers were detected in a subset of cancer-free individuals with very high risk for developing lung tumors. Our results suggest that detection of aberrant promoter region methylation constitutes a promising approach for using DNA-based markers for the early detection of lung and other common human cancers.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Microdissection of Tumors and DNA Isolation.
SCC tumors were obtained as frozen or formalin-fixed specimens. Frozen
tumors were not microdissected. Sequential sections (5 µm) were
prepared from tumors or biopsies, deparafinized, and stained with
toluidine blue to facilitate dissection. A 25-gauge needle attached to
a tuberculin syringe was used to remove the lesions under a dissecting
microscope. Because some SCCs are extensively contaminated with normal
tissue or very small in size (the situation for the biopsy), it was
essential to include normal-appearing cells to ensure that, after
bisulfite modification and column clean-up of the DNA template, enough
sample remained to conduct the MSP assay as described below. Thus,
because the goal of the study was to determine whether p16
methylation was present and not to quantitate methylation levels,
microdissection was used to enrich the samples examined. DNA was
isolated from frozen tumors, microdissected tumors, and sputum by
digestion with Pronase in SDS (1%), followed by standard
phenol-chloroform extraction and ethanol precipitation.
MSP.
The methylation status of the p16 and MGMT genes
was determined by the method of MSP (9
, 10)
with the
following modifications. We developed a nested, two-stage PCR approach,
which improved the sensitivity to detect methylated alleles by
>50-fold over the original method (one methylated allele in >50,000
unmethylated alleles). DNAs were subjected to bisulfite modification,
and PCR was performed to amplify a 280-bp and 289-bp fragment of the
p16 and MGMT genes, respectively, including a
portion of their CpG-rich promoter region. The primers recognize the
bisulfite-modified template but do not discriminate between methylated
and unmethylated alleles. The stage-1 PCR products were diluted
50-fold, and 5 µl was subjected to a stage-2 PCR in which primers
specific to methylated or unmethylated template were used. Primer
sequences used in the stage 1 amplification of the p16 and
MGMT genes are as follows: p16-Forward,
5'-GAAGAAAGAGGAGGGGTTGG-3'; p16-Reverse, 5'-CTACAAACCCTCTAC
CCACC-3'; MGMT-Forward, 5'-GGATATGTTG GGATAGTT-3'; and
MGMT-Reverse, 5'-CCAAAAACCCCAAACCC-3'. Taq Gold polymerase
(Perkin-Elmer) in a 50-µl volume was used in all of the PCRs. The PCR
amplification protocol for stage 1 was as follows: 95°C for 10 min,
then denature at 95°C for 30 s, anneal at 60°C
(p16) or 52°C (MGMT) for 30 s, extension
at 72°C for 30 s for 40 cycles followed by a 10-min final
extension. Primers used to selectively amplify unmethylated or
methylated alleles of the p16 and MGMT genes in
the stage 2 PCR have been described previously (8
, 9) .
Annealing temperatures were increased to 70°C (p16) and
62°C (MGMT), and all of the cycling times were reduced to
15 s for a total of 40 cycles. Normal human tissue collected from
autopsy of never smokers and cell lines positive for p16
(Calu6) and MGMT (SkuLu1) methylation served as negative and
positive controls. All of the assays were conducted in at least
duplicate. Sensitivity for detecting methylated alleles was determined
by mixing DNA isolated from either Calu6 or SkuLu1 cells with DNA
isolated from lung tissue of a never smoker to achieve dilutions up to
1 in 100,000. The mixed DNA sample was then subjected to bisulfite
modification and subsequent analysis by the two-stage MSP approach.
Sputum samples that gave positive methylation products were also analyzed by a second method using restriction enzyme digestion of the resulting PCR product. Second-stage PCRs were performed in duplicate for each sample. Then one of each sample pair was incubated with the restriction enzyme FNU4HI that cuts (G/CGGCG) at two sites within the amplified region of the methylated p16 gene promoter. Thus, this restriction enzyme will only cut template that is methylated at the two CpG sites because the unmethylated cytosines would be modified by the bisulfite treatment to uracils. Because FNU4HI cuts at two different sites within the promoter, we confirmed that four CpGs were methylated, which verified the methylation status of the sample. An identical approach was used for the MGMT gene using the TaqI and BstU1 restriction enzymes to assay three different CpG sites. All of the sputum samples that scored as positive for methylation were confirmed by both procedures.
Data Analysis.
Statistical comparisons were done by Fisher’s exact test.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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-particle-emitting decay products polonium-214 and -218 cause an
excess of lung cancers in smokers who mined uranium (11)
.
Sputum was collected from 10 of the above 21 individuals at the time of
diagnosis of SCC; however, only four samples were diagnostic of cancer
by cytological criteria (Table 1)
. In marked contrast to cytology findings, one or both of the gene
promoters tested were abnormally methylated in all of these sputum
samples. Abnormal p16 gene methylation was present in sputum
from all eight patients whose tumors were also positive for this
marker, but not in sputum from the two individuals whose tumors were
negative for this change (Table 1)
. Four of the six patients with
abnormal methylation of MGMT in their tumors also had this
change detected in their sputum, including the two patients whose
tumors lacked the p16 change (Table 1)
. Methylation of
p16 was present in sputum of the two individuals whose
sputum was negative for MGMT (Table 1
, cases 4 and 5).
Aberrant MGMT methylation was not detected in sputum or
tumor from three cases. In case 8 (Table 1)
, MGMT
methylation was detected in the sputum, but not in the tumor.
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. In only one patient (Table 2
, case 3) was the sputum sample thought
to have unequivocal signs of cancer by cytological criteria. However,
abnormal methylation of the p16 promoter region was detected
in DNA from sputum of all 11 subjects, with the longest time to tumor
diagnosis being 35 months. In those cases in which multiple sputum
samples were available either as replicate specimens (case 2) or as
temporal samples (e. g., case 6), methylation of
p16 was always present (Table 2)
. A 90% concordance was
also observed between p16 methylation in the primary SCC and
paired sputum samples (Table 2
; Fig. 1
). Sputum from 7 of 11 cases (Table 1
; Fig. 1
) also showed methylation
of the MGMT gene. A 78% concordance was noted between
MGMT methylation in the primary SCC and the paired sputum
sample. For the two discordant samples, MGMT methylation was
detected in the tumor but not in the single sputum sample obtained 34
or 35 months before cancer (Table 2)
.
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. Smoking status was available for a subset (n = 35) of the uranium miners, and the methylation markers were
present in sputum from both current and former smokers, which is
consistent with the continued risk for lung cancer in former smokers.
Bisulfite sequencing of sputum samples (n = 5
for each gene) positive for p16 or MGMT revealed
methylation at all of the CpG sites between the PCR primers (not
shown). Direct comparison of sputum from current or former smokers to
never smokers is technically difficult because never smokers produce
very little sputum, and composition of the sample also differs from
that of a current or former smoker. If a low background for methylation
of this gene existed in normal lung tissue, it should have been
detected by our improved MSP assay in the majority of the sputum
samples, and this was not the case. Furthermore, we have examined DNA
isolated from the lungs of autopsy cases, bronchial epithelial cells
obtained by bronchoscopy, and lymphocytes (a component of the sputum)
from never smokers, and all have proven negative for the methylation
markers (Ref. 9
; not shown).
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25% for the detection of either of the sputum markers in the
cancer-free individuals is approximately equal to the known risk of
lung cancer development in the populations studied (11
, 12) . This indicates that these hypermethylation markers may
potentially identify those patients at high risk who are most likely to
actually get the disease. Exactly how high the risk status is for cancer-free patients who have the positive sputum markers remains to be determined. However, the follow-up on this cancer-free population, which now ranges from 2 to 6 years after sputum collection, is intriguing. To date, three lung cancers have been reported: two in former uranium miners who smoked and one in a smoker. Eight deaths have occurred not related to lung cancer. Two lung cancers were diagnosed as SCC, 1 and 3 years after sputum collection. The MGMT gene was methylated in sputum samples from both of these subjects. The other tumor was an adenocarcinoma. The incidence of p16 and MGMT hypermethylation is lower in this tumor type as opposed to SCC, and neither marker was present in the sputum sample collected 2 years before diagnosis.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Patients with stage I or -II disease have a 5-year cure rate of 60–80%, whereas stage III patients have a median survival of 13 months (15) . Lung cancer rates could be reduced through the development of a cost-effective screening approach. Key to this screening approach is the identification of markers of lung cancer that can be detected through sensitive and specific diagnostic assays. It is clear that mutation of the K-ras and p53 genes and microsatellite instability constitute alterations important for lung carcinogenesis (5 , 16 , 17) . However, a recent study examining the concordance of detecting these changes in bronchoalveolar lavage fluid from patients with early-stage lung cancer showed limited sensitivity (18) . For example, microsatellite instability was detected in 46% of tumors but in only 14% of the corresponding lavage fluid. Similarly, 56% of tumors contained a p53 mutation; however, this change was detected in 39% of the corresponding lavage fluid. Both of the genetic alterations are detected through assays that are either limiting in sensitivity (PCR for microsatellite instability) or labor intensive (plaque hybridization assay for p53). This study did examine methylation of p16 using the original assay developed by Herman et al. (9) that has a sensitivity for detecting one methylated allele in a background of 1000 unmethylated alleles. The p16 gene was the best marker, with 63% of the corresponding lavage fluids containing a methylated p16 gene.
One of the latest markers proposed for noninvasive detection of cancer is mitochondrial DNA mutation (19) . Although it is fascinating that mitochondria within a cell accumulate identical cellular mutations, these mutations are dispersed across 16,000 bp and do not show any pattern within tumor types. In fact, across this length of DNA no two tumors of any type appear to contain identical mutations. Furthermore, for lung cancer, mitochondrial mutation was seen in <50% of the 14 tumors examined. Thus, without prior knowledge of the mitochondrial mutation present in a given patient’s tumor, an efficient screening system is not possible, and this precludes current use of this marker system for early cancer detection. Our approach in contrast relies on assays that detect a very frequent and early DNA alteration in tumors that occurs in a genomic region identical for all patients. Thus, sputum analyses can be done in a cancer screening setting without knowledge of the methylation changes within the tumor.
The current improvement of the MSP procedure incorporates a nested, two-stage PCR approach, which is more sensitive (1 methylated allele in >50,000 unmethylated alleles). The improvement of the MSP procedure resulted in detecting the methylation of the p16 and MGMT genes at frequencies higher than previously reported (7 , 8) . Our higher frequency for methylation of p16 actually corroborates immunohistochemical studies (20 , 21) that have reported loss of p16 protein in 60–80% of SCCs. Because of the inherent difficulties (e.g., DNA degradation) of assaying for methylation in DNA recovered from fixed tissues, previous reports most likely underestimated the actual frequency of methylation. Our previous studies (7 , 8) have also shown a strong correlation between the loss of expression of both p16 and MGMT and methylation.
This improvement almost assuredly also accounts for detecting at least
one methylation marker in both the sputum and the corresponding tumor
from every patient at the time of tumor diagnosis or within 3 years of
tumor diagnosis. This is in contrast to our previous study
(7)
, in which, with the one-stage PCR approach,
p16 methylation was detected in sputum from 43% of lung
cancer cases. There were a few instances in which there were discordant
findings for one of the two markers. In each case, the other marker
allowed for a positive sputum change to correlate with presence of a
cancer, or the development of one, within 3 years or less. There are at
least three likely explanations for the two instances in which a
positive marker was found in the sputum but not in the corresponding
tumor. First, the presence of promoter hypermethylation for a given
gene can be heterogeneous within a tumor, and sampling would then
determine whether it is detected or not. Second, the cancer may arise
within a widespread process of bronchial epithelial changes, or
so-called "field cancerization," in which cells have a very high
risk for malignant transformation. Our studies have detected
p16 methylation in nonmalignant bronchial epithelial cells
from different lung lobes of the same patient with a defined lung
cancer, and such high-risk cell populations could contribute exfoliated
cells that contain a hypermethylation marker to the
sputum.4
Finally, the finding that MGMT methylation was not present
in two sputum samples collected 3 years before clinical diagnosis
(cases 10 and 11, Table 2
) could be related to the timing for
inactivation of this gene in those tumors. Similarly, MGMT
methylation was not seen in the sputum sample collected in case 6
(Table 1)
15 months before cancer diagnosis but was present at 9
months before diagnosis.
Our data emphasize the functional implications that the loss of gene function associated with promoter hypermethylation has for early stages of lung and other cancers. Hypermethylation-associated inactivation of p16 is an early and frequent event in NSCLCs (SCC, 60–80%; adenocarcinoma, 30–45%) and other cancers (6 , 7 , 20) . In experimental systems, such loss appears to act as a "gatekeeper" in permitting cells to pass through early steps of cellular immortalization (22 , 23) . These findings could profoundly affect the area of chemoprevention in which markers are needed to identify high-risk subjects, evaluate efficacy of preventive agents, and identify steps for achieving the prevention itself.
Findings from our present study also strongly support the implementation of longitudinal studies in subjects at high risk for developing lung cancer but also emphasize the need for the development of additional hypermethylation markers for lung and other common human cancers. Although detecting methylation of p16 and/or MGMT methylation in sputum most likely confers a higher risk for lung cancer, the time to tumor is quite variable, presumably because of the necessity for acquiring additional genetic alterations that promote tumor progression. This is quite evident within our cohort of cancer-free subjects in which p16 methylation was detected in some sputum samples collected up to 6 years ago. Supporting this premise is a marked difference between the prevalence of both markers in sputum from lung cancer cases versus cancer-free subjects. Inactivation of genes by methylation is a major mechanism in cancer (6) , and new candidate hypermethylation markers will continue to emerge. Thus, longitudinal studies with these methylation markers should facilitate the development of more accurate risk models to incorporate time to tumor and the relationship to multiplicity of biomarkers in the sputum. Our studies have focused on detecting SCC. However, gene promoter hypermethylation changes are now well defined in breast, prostate, colorectal, and other common human cancers (6) , and these sensitive biomarkers should prove as useful for these tumors as our results now indicate they are for lung cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants P50 CA58184 and R01
CA70190 and by the Office of Biological and Environmental Research,
United States Department of Energy under cooperative agreement
DE-FC04–96AL76406. 
2 To whom requests for reprints should be
addressed, at Lovelace Respiratory Research Institute, P. O. Box 5890,
Albuquerque, NM 87185. Phone: (505) 845-1165; Fax: (505) 845-1198;
E-mail: sbelinsk{at}LRRI.org
3 The abbreviations used are: NSCLC, non-small
cell lung cancer; MSP, methylation-specific PCR; SCC, squamous cell
carcinoma; p16, p16INK4a; MGMT,
O6-methylguanine-DNA methyltransferase.
4 S. A. Belinsky, unpublished
observations.
Received 6/ 5/00. Accepted 9/19/00.
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