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Willey and colleagues describe a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method
using standardized mixtures of competitive templates to
identify mRNA for specific genes expressed in a variety of
cells (1). In this technique, dual amplification is performed
with PCR primers for native product and an internal competitive template. Amplified products are compared with
the similarly amplified housekeeping gene (
-actin or glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). Digital analysis is used to compare pixel quantity of amplified
product. This method appears to be a highly accurate, reproducible technological advance; its simplicity will be
documented by the number of other laboratories that adopt
it as routine. It is currently simple to amplify an unknown
gene and compare the quantity of -actin or GAPDH as a
semiquantitative assay. The gold standard continues to be
Northern blot analysis, which reviewers and editors readily
accept. This technique is labor intensive and requires a large
amount of sample material. Moreover, it is difficult to assess
expression of multiple genes. The quantitative RT-PCR
methods described in this issue of the journal begin to address this problem. The technique works well for cells grown
in tissue culture where pure populations can be propagated;
however, because some genes are differentially expressed in the cell cycle, upon confluence in tissue culture or in response to immortalization, care must be taken in the interpretation of the results. Comparison with fresh, native tissue to assess changes wrought by culture or immortalization
is required. Here is where the technique has an important
Achilles heel: Human samples usually are not pure samples of cells but mixtures of cell types. Tumors can have fibrous stroma, and premalignant dysplastic lesions are surrounded by normal tissues in biopsies. Microdissection has alleviated much of this pitfall since relatively pure samples of cells can be obtained; however, contamination with more
than 20% of normal cells will frequently invalidate quantitative PCR results or make them difficult to interpret or
reproduce. Although the same criticism applies to Northern blot analyses, this may be more problematic for quantitative PCR, which can amplify multiple genes from small
samples simultaneously.
Gene expression data will give us tables of data, as Willey and colleagues readily demonstrated (1). How are we to take all of this is in and digest it? As Willey and coworkers (1) suggest, certain themes are worth evaluating, for example, cell-cycle and apoptosis genes in the malignant phenotype. As expected, the dominant oncogene c-myc was overexpressed in small-cell carcinoma cell lines. The Proliferating Cell Nuclear Antigen, a measure of cell proliferation capability, was also overexpressed in small-cell lung carcinoma (SCLC). The cell cycle brake, p21, was significantly suppressed in squamous-cell carcinoma cell lines. Cyclin E and cyclin-dependent kinase 4 were both increased in SCLC lines and squamous-cell carcinoma lines but not in adenocarcinoma. p21 is generally associated with checkpoint control at G1/S, but we recently demonstrated that phorbol myristate acetate stimulation inhibits cell proliferation of Calu 1 cells in G2/M by 24 to 48 h (2). This was associated with a truncated p21 molecule that had lost carboxy terminus amino acids (2). In a second paper, DeMuth and colleagues developed a gene expression index that performed well in differentiating malignant from normal bronchial epithelial cell types (3).
PCR techniques have been very useful in understanding the genetic changes that accompany dysplasia of the bronchial epithelium induced by cigarette smoking. Auerbach's classic microscopic studies of airway epithelium documented an orderly progression of changes from mild and moderate dysplasia to severe dysplasia, carcinoma-in-situ, and invasive cancer (4). Immunohistochemistry of these lesions demonstrates extensive immunostaining for various proliferation markers concomitant with rare immunostaining for p21. Bronchial epithelial cell dysplasia is considered a pre-invasive stage of lung cancer, especially squamous-cell carcinoma. Comparison of dysplastic lesions from airways of resected lung cancer specimens has shown existence of genetic abnormalities in the preneoplastic lesions as well as the cancer, consistent with the concept that the genetic damage was progressive and sequential (5). PCR has been used to detect loss of heterozygosity (LOH); for example, Hung and colleagues evaluated LOH on chromosome 3 (3p14, 3p21.3, 3p25) from seven lung specimens of non-small-cell lung carcinoma (NSCLC), and observed six of seven to lose heterozygosity at one or more 3p sites (6). In the accompanying preneoplastic lesions, LOH was detected in none of two normal bronchioles, 13 of 17 hyperplasias, six of seven dysplasias, and all four noninvasive cancers. In 18 of 23 preneoplastic lesions, the specific alleles lost were identical to those lost in the corresponding carcinomas.
Two recent reports in the Journal of the National Cancer Institute highlighted multiple bronchial biopsies from
almost 100 long-term smokers without lung cancer evaluated for LOH by PCR (7, 8). LOH
that is, loss of DNA
sequences from one member of a chromosome pairwas
detected in 75%, 57%, and 18% of informative subjects at
chromosomes 3p14, 9p21, and 17p13, respectively (7). There was an association between increasing metaplasia
index and LOH. Only one of five nonsmokers had LOH at
3p14, and none were detected at 9p21. Multiple LOH lesions were found more frequently in carcinoma-in-situ lesions, and ex-smokers had similar results to those of smokers, suggesting that these changes were persistent. In an
analysis of seven lung resections for adenocarcinoma, 52 microdissected lesions were assayed by PCR for LOH at
chromosome 9p (p16 or cyclin dependent kinase N2) (9).
Five of seven tumors had LOH at 9p and four of these also
revealed LOH at preneoplastic foci. In the doubly informative cases, LOH was detected in 38% foci of hyperplasia, 80% of dysplasia, and 100% of carcinoma-in-situ lesions. The identical alleles were lost from both the preneoplastic lesions and the tumors. PCR followed by denaturing gradient gel electrophoresis has been used to catalogue all types (missense, non-sense, splicing, deletions) of
p53 mutations from exons 3 to 9. Murakami and colleagues showed that p53 mutations were found in 41% of
cytopathology and biopsy specimens, ranging from 58% in
SCLC to 45% in squamous-cell carcinoma to 32% in adenocarcinoma of the lung (10). Immunostaining of bronchial mucosa also shows increasing frequency of immunopositive cells in dysplastic lesions peaking at 60% in
carcinoma-in-situ and 80% in invasive lesions (11). p53
overexpression can also be detected in sputum cytology specimens, and p53 immunostaining in numbers of cells correlated with malignant and atypical cytologic changes (12).
p53 protein is increased in adenocarcinoma cell lines
treated with benzo(a)pyrene diol-epoxide (BPDE) in vitro,
and BPDE forms DNA adducts at guanine positions in
codons 157, 248, and 273 (13, 14). Interestingly, these are
the same positions that are the major mutational hot spots
in human lung cancers. The K-ras oncogene is mutated in half of lung adenocarcinomas, which is a late effect in the
sequence of genetic changes occurring in preneoplastic lesions. Point mutations (nearly always in codon 12, although 13 and 61 can occur) confer transforming properties on the ras genes by yielding proteins with constitutive
activation that generate continuous signals to proliferate.
We have used polymerase chain reaction-primer introduced
restriction with enrichment for mutant alleles (PCR-PIREMA) to identify K-ras codon 12 mutations in bronchoalveolar lavage fluid (BALF) cells obtained from patients
being evaluated for lung cancer (15). PCR-PIREMA was
performed by using a mismatched primer that introduced
a BstNI restriction site into PCR products derived from
normal but not mutant alleles. BstNI digestion of the PCR
products left only PCR products derived from mutant alleles intact ("enrichment"), after which further PCR selectively amplified the mutant PCR products. BALF from 16 of 52 patients with confirmed lung cancer, including 14 (56%) of 25 patients with lung adenocarcinomas, contained
K-ras codon 12 mutations. No mutation was found in any sample from 30 patients with diagnoses other than NSCLC.
Tissue samples from 35 of the patients all yielded the identical K-ras codon 12 genotype found in the corresponding
BALF.
A novel mechanism for field carcinogenesis is widely dispersed but identical mutations in the respiratory epithelium. In a case report of a 66-yr-old smoker (50 pack-years) who unexpectedly died after surgery, DNA was extracted from microdissected bronchial epithelial cells from ten sites in both lungs (16). PCR products were analyzed with single-strand conformation polymorphism and direct sequencing. A single point mutation consisting of a G:C to T:A transversion in codon 245 of the p53 gene was found in seven of ten sites, consistent with the concept that a single bronchial epithelial clone may expand to populate broad areas of the bronchial mucosa. The same group used fluorescence in situ hybridization to analyze 3p or 9p loss in bronchial brushings, finding loss at 3p21 in 10 of 10, and hemizygous allelic loss at 9p in 7 of 10 smokers with mild obstructive airways disease and moderate dysplasia or worse on sputum cytology (17). Because the bronchial brushings had come from multiple sites and had similar chromosomal loss patterns for 3p and 9p, this has been considered further support for the clonal origin of field carcinogenesis. Use of quantitative competitive RT-PCR could further evaluate expression of multiple cancer-related genes at multiple sites of the bronchial epithelium of high-risk smokers.
Cells obtained by BAL in patients with interstitial lung
disease are "activated" in that they spontaneously release
increased quantities of peptide growth factors, cytokines,
chemokines, matrix proteins, and oxidants. Quantitative
competitive RT-PCR can assess these multiple genes simultaneously and compare them with normal control cells.
Even better, this technique can be used to assess experimental therapies longitudinally with multiple BAL samples. Gene expression studies of detoxifying or activating
enzymes could also assess sensitivity to environmental toxins (18). Quantitative competitive RT-PCR can also be
used to detect differentiation characteristics in other cell
types. As peripheral blood monocytes differentiate for 7 to
14 days in vitro, they develop macrophage characteristics,
including increased tumor necrosis factor-
gene expression and reduced interleukin-1 production. An example
by DeMuth and colleagues linked the loss of the terminal
differentiation marker spr1 and malignant change in bronchial epithelial cells (19). THP-1 myelomonocytic cells differentiated in vitro with phorbol myristate acetate will express different transcription factors when subsequently
activated.
The quantitative competitive RT-PCR technique should prove useful in assessing many genes simultaneously in small samples to study the lung cell processes of activation, differentiation, and carcinogenesis. Its judicious use in the right situations will prove invaluable.
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Footnotes |
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Address correspondence to: William N. Rom, M.D., M.P.H., Bellevue Chest Service, NYU Medical Center, Division of Pulmonary & Critical Care Medicine, 550 First Avenue, New York, NY 10016.
(Received in original form May 19, 1998).
Abbreviations: bronchoalveolar lavage fluid, BALF; loss of heterozygosity, LOH; reverse transcription-polymerase chain reaction, RT-PCR; small-cell lung carcinoma, SCLC.Acknowledgments: This article was supported by NIH grant No. M01 00096.
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