Detection of K-ras oncogene mutations in bronchoalveolar lavage fluid for lung cancer diagnosis.

BACKGROUND: Lung cancer is the leading cause of cancer deaths in the United States. A long-standing goal of cancer researchers has been to develop tests that would facilitate earlier diagnosis and treatment of lung cancer and thereby decrease mortality from this disease. Because cancer results from the accumulation of a variety of genetic events (e.g., mutations, rearrangements, and deletions) in genes controlling cell growth and differentiation, these changes might serve as diagnostically useful molecular markers. Activation of the K-ras oncogene by point mutations in codon 12, which occurs in many cases of lung adenocarcinoma, may serve as one such clinically useful molecular marker. For detection of K-ras point mutations in bronchoalveolar lavage fluid, in which small numbers of malignant cells are mixed with a population of predominantly genetically normal cells, the sensitivity of commonly used assays for ras mutations risks false-negative results. PURPOSE: By applying a highly sensitive assay, we investigated whether detection of K-ras codon 12 mutations in samples of bronchoalveolar lavage fluid could be clinically useful in diagnosing lung cancer. METHODS: We developed a highly sensitive assay for detecting K-ras codon 12 mutations based on an enriched polymerase chain reaction (PCR) technique. This technique was applied to 87 specimens of bronchoalveolar lavage fluid specimens that were obtained from 86 patients, and associated tumor biopsy specimens obtained from 35 of these patients who underwent diagnostic bronchoscopy for clinically suspected lung cancer. Statistical comparisons were performed by using the two-tailed Fisher's exact test [corrected]. RESULTS: Of 52 patients with confirmed lung cancer, samples of bronchoalveolar lavage fluid from 16 patients contained K-ras codon 12 mutations, including 14 (56%) of 25 patients with lung adenocarcinomas, one (33%) of three with bronchoalveolar carcinomas, one (20%) of five with large-cell carcinomas, and none of the 14 with squamous cell carcinomas. Mutations were detected in four additional cases in which cancer was suspected but had not been histologically confirmed. Tissue samples from 35 of the patients all yielded the identical K-ras codon 12 genotype found in the corresponding samples of bronchoalveolar lavage fluid. No mutation was found in any sample from 30 patients with diagnoses other than non-small-cell lung cancer. Thus, for those cases in which tissue was available and tested, the sensitivity and specificity of detecting K-ras mutations in bronchoalveolar lavage fluid for diagnosing K-ras mutation-positive lung cancer were both 100%. For nine patients, K-ras mutations were detected in bronchoalveolar lavage fluid obtained during otherwise nondiagnostic bronchoscopies. CONCLUSIONS: Our data demonstrate that sensitive detection of K-ras codon 12 mutations can serve as an important adjunct to cytology in the diagnosis of lung cancer. IMPLICATIONS: Detection of these mutations could lead to earlier cancer diagnosis and less need for invasive diagnostic procedures.

Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma.

Reported estimates of ras mutation prevalence in lung adenocarcinoma of 15-24% may be underestimates because of the insensitivity of the assays used. We have devised a rapid, non-radioactive assay for ras mutations, which detects 1 mutant allele/10(3) normal alleles and have used it to study DNA isolated from 53 lung tumor samples (including 28 adenocarcinomas) previously analyzed by PCR/allele specific oligonucleotide hybridization, which is less sensitive. We detected mutations in 13 of 28 samples, including 7 not detected by PCR/allele specific oligonucleotide hybridization. We also found ras mutations in 14 of 25 previously unstudied samples (56%). Our results indicate that the prevalence of K-ras codon 12 mutations in lung adenocarcinoma is higher than previously reported; thus, ras mutations may be more clinically useful as molecular markers for lung cancer than has been appreciated.

A highly sensitive assay for mutant ras genes and its application to the study of presentation and relapse genotypes in acute leukemia.

Most studies of ras oncogene activation use assays for ras mutations based on the polymerase chain reaction (PCR) of DNA segments containing ras exons 1 and 2, followed by allele-specific oligonucleotide (ASO) hybridization or direct sequencing, which require that to be detectable, a mutation must be present in at least 3-25% of ras alleles. Thus, studies of tissues in which only a fraction of cells contains a ras mutation risk false negative results. To minimize this risk, we have developed a highly sensitive, non-radioactive assay for ras mutations. Ras genes were PCR-amplified using mismatched primers, to introduce restriction sites into products derived from normal alleles. Repeated restriction digestion and PCR enriched for mutant alleles, visualized by agarose gel electrophoresis. Serially diluted DNA samples containing ras mutations demonstrated detection of 1 mutant/10(6) normal alleles (four orders of magnitude more sensitive than PCR/ASO hybridization). This assay was applied to DNA from four patients with relapsed acute leukemia in whom ras mutations present at diagnosis were not detectable by PCR/ASO hybridization at relapse. In one case, the mutation present at diagnosis was demonstrated at relapse. In the others, loss of the mutation was confirmed, at a greatly increased sensitivity. This method is widely applicable to detection of mutant ras alleles admixed with larger numbers of normal alleles.

Effects of hypoxia on embryonic development in two Ambystoma and two Rana species.

Oxygen available to amphibian embryos fluctuates widely and is often very low. We investigated the effects of oxygen partial pressure (1. 3-16.9 kPa) on embryonic development and hatching of two salamander (Ambystoma) and two frog (Rana) species. In Ambystoma, chronic hypoxia resulted in slowed development, delayed hatching, and embryos that were less developed at the time of hatching. Although hypoxia was not lethal to embryos, temporary developmental abnormalities were observed in Ambystoma at oxygen partial pressures of 3.8 kPa and below. Posthatching survival decreased below 3.3 kPa. In Rana, hypoxia did not affect developmental rate, presumably because hatching occurs at a very early stage of development relative to Ambystoma. However, Rana embryos hatched sooner in hypoxia than in normoxia, resulting in less developed embryos at the time of hatching. The results suggest that embryonic hypoxia may negatively affect survival and fitness in these species.

Effects of hypoxia on egg capsule conductance in Ambystoma (Class Amphibia, Order Caudata).

Aquatic amphibian eggs frequently encounter hypoxic conditions that have the potential to limit oxygen uptake and thereby slow embryonic development and hatching. Oxygen limitation might be avoided if egg capsule surface area and oxygen conductance increased in response to hypoxia. We investigated this possibility in two salamander species, Ambystoma annulatum and Ambystoma talpoideum. The effective surface area of egg capsules increased in response to hypoxia, which increased the conductance for oxygen and enhanced oxygen transport. The ability of amphibian eggs to adjust their conductance in response to oxygen availability may increase survival in hypoxic environments.

Molecular genetic tumor markers in the early diagnosis and screening of non-small-cell lung cancer.

BACKGROUND: Little progress has been made in decreasing lung cancer mortality by applying conventional methods to early diagnosis and screening. Recent advances in molecular oncology, however, have provided tools which may be of use in this area. Many genes involved in controlling cell growth and differentiation are abnormal in lung cancer cells. Such genes include K-ras, p53, rb, myc, her2/neu, and probably one or more tumor suppressor genes on chromosome 3p. The involvement of these genes in lung cancer is reviewed. The K-ras oncogene contains a mutation in codon 12 in many cases of non-small-cell lung cancer, particularly adenocarcinoma, and is thus a potentially useful lung cancer tumor marker. DESIGN; We have developed a highly sensitive, simple assay for ras mutations, and applied it to bronchoalveolar lavage fluid obtained from patients undergoing evaluation for suspected lung cancer. RESULTS: In many cases, the ras assay was more sensitive than routine cytology and histopathology, demonstrating that this is a potentially clinically useful assay. CONCLUSION: Molecular genetic tumor markers, including mutations in ras and other genes, and/or immunohistochemical tumor markers, may provide tools which can be applied to bronchoalveolar lavage fluid or sputum, for use in diagnostic tests and in screening programs. The use of such markers may lead to decreased lung cancer mortality.

B-cell lymphoma presenting as infiltrative renal disease.

Acute renal failure is rarely the presenting manifestation of non-Hodgkin's lymphoma. Of the reported cases of renal insufficiency secondary to diffuse renal infiltration with lymphoma, few have presented with acute renal failure. We present a patient with acute renal failure secondary to diffuse bilateral renal infiltration by a B-cell non-Hodgkin's lymphoma. The findings of an elevated serum lactate dehydrogenase (LDH), lymphopenia, and homogenous bilateral renal enlargement on computed tomographic (CT) imaging were important in suggesting the diagnosis of primary renal lymphoma. Renal biopsy with immunohistochemical and ultrastructural analysis was instrumental in confirming this diagnosis.