Detection of K-ras Oncogene Mutations in Bronchoalveolar Lavage Fluid for Lung Cancer Diagnosis (original) (raw)

ARTICLES Molecular Detection of Tumor Cells in Bronchoalveolar Lavage Fluid From Patients With Early Stage Lung Cancer

1999

Background: Conventional cytologic analysis of sputum is an insensitive test for the diagnosis of non-small-cell lung cancer (NSCLC). We have recently demonstrated that polymerase chain reaction (PCR)-based molecular methods are more sensitive than cytologic analysis in diagnosing bladder cancer. In this study, we examined whether molecular assays could identify cancer cells in bronchoalveolar lavage BAL) fluid. Methods: Tumor-specific oncogene mutations, CpGisland methylation status, and microsatellite alterations in the DNA of cells in BAL fluid from 50 consecutive patients with resectable (stages I through IIIa) NSCLC were assessed by use of four PCR-based techniques. Results: Of 50 tumors, 28 contained a p53 mutation, and the identical mutation was detected with a plaque hybridization assay in the BAL fluid of 39% (11 of 28) of the corresponding patients. Eight of 19 adenocarcinomas contained a K-ras mutation, and the identical mutation was detected with a mutation ligation assay in the BAL fluid of 50% (four of eight) of the corresponding patients. The p16 gene was methylated in 19 of 50 tumors, and methylated p16 alleles were detected in the BAL fluid of 63% (12 of 19) of the corresponding patients. Microsatellite instability in at least one marker was detected with a panel of 15 markers frequently altered in NSCLC in 23 of 50 tumors; the identical alteration was detected in the BAL fluid of 14% (three of 22) of the corresponding patients. When all four techniques were used, mutations or microsatellite instability was detected in the paired BAL fluid of 23 (53%) of the 43 patients with tumors carrying a genetic alteration. Conclusion: Although still limited by sensitivity, molecular diagnostic strategies can detect the presence of neoplastic cells in the proximal airway of patients with surgically resectable NSCLC. [J Natl Cancer Inst 1999;91:332-9].

Molecular Detection of Tumor Cells in Bronchoalveolar Lavage Fluid From Patients With Early Stage Lung Cancer

JNCI Journal of the National Cancer Institute, 1999

Background: Conventional cytologic analysis of sputum is an insensitive test for the diagnosis of non-small-cell lung cancer (NSCLC). We have recently demonstrated that polymerase chain reaction (PCR)-based molecular methods are more sensitive than cytologic analysis in diagnosing bladder cancer. In this study, we examined whether molecular assays could identify cancer cells in bronchoalveolar lavage BAL) fluid. Methods: Tumor-specific oncogene mutations, CpGisland methylation status, and microsatellite alterations in the DNA of cells in BAL fluid from 50 consecutive patients with resectable (stages I through IIIa) NSCLC were assessed by use of four PCR-based techniques. Results: Of 50 tumors, 28 contained a p53 mutation, and the identical mutation was detected with a plaque hybridization assay in the BAL fluid of 39% (11 of 28) of the corresponding patients. Eight of 19 adenocarcinomas contained a K-ras mutation, and the identical mutation was detected with a mutation ligation assay in the BAL fluid of 50% (four of eight) of the corresponding patients. The p16 gene was methylated in 19 of 50 tumors, and methylated p16 alleles were detected in the BAL fluid of 63% (12 of 19) of the corresponding patients. Microsatellite instability in at least one marker was detected with a panel of 15 markers frequently altered in NSCLC in 23 of 50 tumors; the identical alteration was detected in the BAL fluid of 14% (three of 22) of the corresponding patients. When all four techniques were used, mutations or microsatellite instability was detected in the paired BAL fluid of 23 (53%) of the 43 patients with tumors carrying a genetic alteration. Conclusion: Although still limited by sensitivity, molecular diagnostic strategies can detect the presence of neoplastic cells in the proximal airway of patients with surgically resectable NSCLC. [J Natl Cancer Inst 1999;91:332-9].

Adenocarcinoma of the lung

Journal of Cancer Research and Clinical Oncology, 1987

The reaction patterns in 80 adenocarcinomas of the lung were examined with P A P-m e t h o d using a m o n o c l o n a l a n t i b o d y against keratin a n d one against c a r c i n o e m b r y o n i c antigen (CEA) a n d a polyclonal a n t i s e r u m against CEA. A l m o s t all tumors showed a positive reaction to the antibodies which, however, varied quantitatively. Even t h o u g h a reliable correlation of positive i m m u n o h i s t o c h e m i c a l reaction with the different light microscopical types was n o t possible according to W H O subtypes a n d degrees of differentiation, specific localization of the reaction within the t u m o r cells was seen with increasing differentiation. There was no correlation between the i m m u n o h i s t o c h e m i c a l reactions a n d 14 clinically measured plasma C E A levels. The plasma C E A level n o t only depends o n C E A p r o d u c t i o n by the t u m o r b u t also on other factors.

Cytodifferentiation of atypical adenomatous hyperplasia and bronchioloalveolar lung carcinoma: immunohistochemical and ultrastructural studies

Virchows Archiv, 1997

We used immunohistochemistry and electron microscopy to evaluate the differentiation of cells comprising atypical adenomatous hyperplasia (AAH; n = 26), early bronchioloalveolar lung carcinoma (BAC; n = 11), and overt BAC (n = 16), which are assumed to constitute a continuous spectrum of developmental steps of BAC. Surfactant apoprotein (SAP), a marker for type 2 alveolar cells, was expressed in cells from all the lesions of AAH, early BAC, and overt BAC. However, the proportion of SAP-positive cells decreased and their distribution became more heterogeneous with advancing lesion grade. Urine protein 1, which is identical to the Clara cell-specific 10 kDa protein, was expressed in 70% of overt BAC, whereas only 20% of early BAC showed weak reactivity and none of AAH lesions showed any reactivity at all. Ultrastructurally, type 2 alveolar cell differentiation was predominant among cells from AAH and early BAC. Our results suggest that precursor cells of BAC differentiate predominantly towards type 2 alveolar cells. Cells comprising overt BAC retain this differentiation phenotype, but to a reduced extent. In contrast, concomitantly with progression, cells with Clara cell differentiation emerge and their proportion increases. Such phenotypic changes may reflect metaplasia occurring in tumour cells during the development of BAC.

Biochemical and cytogenetic studies of human lung cancers

The Journal of Thoracic and Cardiovascular Surgery, 1988

In ongoing studies, we have tested resected lung cancers from 41 men and 49 women; of those with primary lung cancer, 46 patients are free of disease and 35 have died of cancer or have persistent disease. Measurements andstudies were as foUows: total ceUular deoxyribonucleic acid content by image analysis (n = 77); total genomic deoxyribonucleic acid methylation state and banding patterns from probed Southern blots (n = 36); radioimmunoassay for motilin, bombesin, gastrin, vasoactive intestinal peptide, and cholecystokinin (n = 18); and cytogenetic analysis (n = 39). All lung cancers were hyperploid. Adenocarcinomas andepidermoid carcinomas were generaUy hexaploid to nearly septaploid; comparisons by stage and histologic features suggested potential prognostic correlations. There was general hypomethylation of deoxyribonucleic acid (p < 0.(01). Deoxyribonucleic acid digests from restriction endonuclease Hpa II, when probed with deoxyribonucleic acid homologous to KPN, showed banding patterns that separated histologicaUy indistinguishable primary adenocarcinomas and metastatic adenocarcinomas from one another. Cancers studied with radioimmunoassay were aU negative for polypeptide hormones. Five cancers grew adequately in vitro to permit study of 190 detailed karyotypes (20 to 50 per tumor). Chromosome modal numbers ranged from 49 to 109. There were from 4 to 20 clearly abnormal marker chromosomes per tumor; abnormality derived from chromosome 1 was prevalent. Ten of 19 tumors xenotransplanted to nude mice were carried through two to five transplant generations without a change in histologic patterns.

Cytopathology of Lung Cancer, Abstract 159–165, Symposium

Pathology - Research and Practice, 2003

Isolating 30-40 vessel profiles a comparable amount of cells was achieved. The cells were resuspended in Hepes/Triton buffer, the lysate immobilized on a SAX ProteinChip ® (strong anionic exchanger) and the protein profile measured by mass spectrometry. Additionally, the samples were lysed in a thiourea buffer, applied on a WCX ProteinChip ® (weak cationic exchanger) and measured. Results: All applied samples (n = 12) could generate protein spectra. After Hepes/Triton lysis, proteins up to 20 kDa were detectable; after thiourea lysis, spectra up to 50 kDa could be found. SAX and WCX spectra differed remarkably. In AS after 1 d hypoxia, new peaks appeared at 7.69 und 7.95 kDa. After 21 d hypoxia up-/downregulated proteins as well as new peaks were noticed. In IV cells we found i.e. an additional peak at 11.4 kDa after 21d hypoxia. Conclusion: Laser-microdissection and SELDI-TOF mass spectrometry generate a compartment specific proteome analysis, that allowed to find hypoxia induced expression changes in IV and AS.