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.

Transthyretin amyloidosis (serine 44) with headache, hearing loss, and peripheral neuropathy.

A 32-year-old man of Irish descent presented with severe progressive headache and sensorineural hearing loss. MRI/magnetic resonance angiography head scans were normal. A length-dependent sensorimotor peripheral neuropathy with autonomic dysfunction predated these symptoms. Systemic organ involvement and transthyretin (TTR) amyloid immunostaining of bone marrow and fat aspirate were documented. Direct DNA sequencing revealed both the normal TTT (phenylalanine) and a new variant TCT (serine) at position 44 of the TTR gene. This case expands the genotypic and phenotypic variability within TTR amyloidosis.

Immunosuppression and infection in multiple myeloma.

Patients with multiple myeloma are at increased risk of severe bacterial infection. A variety of immune deficits has been described in such patients, including a decreased primary antibody response and defects in complement and granulocyte function. The depressed humoral response appears to result primarily from the activity of suppressor monocytes. Pneumovax (Merck Sharp & Dohme, West Point, Penn) should be administered to patients with myeloma, although its effectiveness in this population has not been proven. The role of other potential modalities of treatment and prophylaxis, such as IV gamma globulin, requires further study.

Novel transthyretin missense mutation (Thr34) in an Italian family with hereditary amyloidosis.

We report on the genetic and molecular characterisation of an Italian family with a late-onset, autosomal dominant transthyretin amyloidosis. The transthyretin gene was analysed by polymerase chain reaction (PCR), restriction generating PCR, and sequencing, allowing us to discover in one allele a novel point mutation. It consists of a G to C transversion at position 1692 of the genomic sequence, leading to a Thr for Arg substitution at the position 34 of the polypeptidic chain. This mutation is associated with a severe sensory-motor peripheral neuropathy and a restrictive cardiomyopathy.

Iris angiography and aqueous photofluorometry in normal subjects.

Iris angiography and photofluorometry of the aqueous were performed on 44 normal subjects equally distributed between 15 and 65 years of age. Hemoglobin AIc determinations were performed in 20 of these persons. Leakage from the pupillary margin was present in 20% of the eyes studied. In all cases of leakage, the dye faded quickly after the initial peak concentration. No leakage was observed from radial iris vessels. Photofluorometric readings of the aqueous 60 minutes after injection increased significantly with age. Hemoglobin AIc measurements were observed to correlate significantly with aqueous fluorescein concentration. Both these observations may reflect an altered permeability of biologic membranes associated with aging.

The effect of pan-retinal photo-coagulation on rubeosis iridis.

Fifteen eyes of 11 patients with rubeosis iridis and angle neovascularization associated with retinal vascular disorders were treated with pan-retinal photocoagulation. In seven of the 15 eyes, the new vessels on the surface of the iris and in the angle regressed after pan-retinal photocoagulation therapy for disk neovascularization; five of the remaining eight eyes that were treated prospectively demonstrated similar involution of the rubeosis iridis. In three of the five preexisting peripheral anterior synechiae regressed and angle structures previously obscured became visible. Three to 36 months after therapy, three eyes developed a few new abnormal iris and angle vessels.

Graves' disease with ophthalmopathy following radiotherapy for Hodgkin's disease.

The number of patients achieving long-term survival following neck irradiation for Hodgkin's disease and other malignancies is increasing. Paralleling this increase in survivors is the development of late complications of the therapy itself. Eleven patients have previously been reported who developed Graves' ophthalmopathy 18 months to seven years after receiving neck radiotherapy for nonthyroidal malignancies. The seven patients who had HLA typing were all HLA-B8 negative, despite the reported association of the HLA-B8 antigen with Graves' disease. A patient who is HLA-B8 positive who developed Graves' ophthalmopathy and hyperthyroidism nine years after receiving mantle radiotherapy for Hodgkin's disease is reported. It is recommended that Graves' disease be included among the thyroid diseases that receive consideration during follow-up of patients who have received mantle radiotherapy.

Transthyretin Ile 122 and cardiac amyloidosis in African-Americans. 2 case reports.

Two cases of cardiac amyloidosis resulting from deposition of the Ile 122 variant of transthyretin in African-Americans are presented. These cases illustrate several typical features of this disorder, including electrocardiographic abnormalities and digoxin toxicity. Transthyretin Ile 122 is a common amyloidogenic variant in African-Americans (present as a heterozygous variant in 4% of this population); therefore, the diagnosis of transthyretin Ile 122 cardiac amyloidosis should be considered in African-Americans with unexplained restrictive cardiomyopathy or arrhythmias.

Marked systemic amyloid angiopathy in patients with val 107 transthyretin mutation.

We report three non-inbred patients with Val 107 transthyretin (TTR) amyloidosis. Clinical features were remarkable by the combination of peripheral polyneuropathy, carpal tunnel syndrome, cardiomyopathy, and epilepsy. Pathologic examination disclosed unusual striking systemic amyloid angiopathy in all studied tissues including nerve, muscle, gut, lung, salivary glands, and synovial membrane. It appears that the rare TTR Val 107 variant causes a peculiar familial amyloid syndrome characterized by both widespread systemic TTR amyloidosis and central nervous system deposition sufficient to cause seizures, pointing out the extent of TTR amyloidosis phenotypic heterogeneity.

A novel application for 222Rn emanation standards: radon-cryptophane host chemistry.

In collaboration with the University of Pennsylvania, a (222)Rn emanation source was used for the determination of the binding affinity of radon to a cryptophane molecular host. This source was similar to a (222)Rn emanation standard that was developed and disseminated by the National Institute of Standards and Technology (NIST). The novel experimental design involved performing the reactions at femtomole levels, developing exacting gravimetric sampling methods and making precise (222)Rn assays by liquid scintillation counting. A cryptophane-radon association constant was determined, K(A)=(49,000±12,000) L mol(-1) at 293 K, which was the first measurement of radon binding to a molecular host.

A specific test for transthyretin 122 (Val----Ile), based on PCR-primer-introduced restriction analysis (PCR-PIRA): confirmation of the gene frequency in blacks.

The variant transthyretin (TTR) allele, TTR (122 Val----Ile), associated with cardiac amyloidosis in blacks, is caused by a G----A transition which destroys a MaeIII site. This variant has previously been detected by PCR around codon 122, followed by MaeIII digestion, but this test is not specific: any of 12 mutations in the MaeIII recognition site, each of which yields a different amino acid change, would also destroy this site. A modification of PCR, termed "PCR-primer-introduced restriction analysis," was used to introduce a new FokI site into the PCR products derived from the variant (122 Ile) but not wild-type (122 Val) allele. This test demonstrated that each of six previously identified MaeIII(-) alleles had lost its MaeIII site because of a G----A transition encoding TTR (122 Val----Ile), confirming that the same TTR variant was present both in 4/177 healthy black individuals and as a homozygous variant in an individual with cardiac amyloidosis.

Radiographic exposure calculator and mammographic dose calculator.

A set of dose calculators has been designed to facilitate quick and easy calculation of surface exposure (or skin dose) for routine radiography and average glandular dose in mammographic studies. Acceptance by technologists has been good, and in two inspections by the Joint Commission for Accreditation of Health Care Organizations, the calculators were deemed adequate to satisfy the diagnostic radiology standards that doses be monitored.

A double-variant transthyretin allele (Ser 6, Ile 33) in the Israeli patient "SKO" with familial amyloidotic polyneuropathy.

Transthyretin (TTR) isolated from amyloid fibrils from an Israeli patient ("SKO") with familial amyloidotic polyneuropathy has been studied by two groups of investigators. Originally, a position 49 Thr-->Gly substitution was reported; subsequently, a position 33 Phe-->Ile substitution was found instead. We have studied DNA from this patient by single strand conformation polymorphism analysis, restriction analysis, and DNA sequencing. On one allele, exon 2 contained both a T-->A transversion at the first position of codon 33, encoding the previously described Phe-->Ile substitution, and a G-->A transition at the first position of codon 6, encoding a Gly-->Ser substitution. The originally reported position 49 mutation was not encoded in the genomic DNA. This is the first report of a TTR double-variant allele in a patient with TTR amyloidosis.