Molecular damage in the bronchial epithelium of current and former smokers.

BACKGROUND: Most lung cancers are attributed to smoking. These cancers have been associated with multiple genetic alterations and with the presence of preneoplastic bronchial lesions. In view of such associations, we evaluated the status of specific chromosomal loci in histologically normal and abnormal bronchial biopsy specimens from current and former smokers and specimens from nonsmokers. METHODS: Multiple biopsy specimens were obtained from 18 current smokers, 24 former smokers, and 21 nonsmokers. Polymerase chain reaction-based assays involving 15 polymorphic microsatellite DNA markers were used to examine eight chromosomal regions for genetic changes (loss of heterozygosity [LOH] and microsatellite alterations). RESULTS: LOH and microsatellite alterations were observed in biopsy specimens from both current and former smokers, but no statistically significant differences were observed between the two groups. Among individuals with a history of smoking, 86% demonstrated LOH in one or more biopsy specimens, and 24% showed LOH in all biopsy specimens. About half of the histologically normal specimens from smokers showed LOH, but the frequency of LOH and the severity of histologic change did not correspond until the carcinoma in situ stage. A subset of biopsy specimens from smokers that exhibited either normal or preneoplastic histology showed LOH at multiple chromosomal sites, a phenomenon frequently observed in carcinoma in situ and invasive cancer. LOH on chromosomes 3p and 9p was more frequent than LOH on chromosomes 5q, 17p (17p13; TP53 gene), and 13q (13q14; retinoblastoma gene). Microsatellite alterations were detected in 64% of the smokers. No genetic alterations were detected in nonsmokers. CONCLUSIONS: Genetic changes similar to those found in lung cancers can be detected in the nonmalignant bronchial epithelium of current and former smokers and may persist for many years after smoking cessation.

Sex-related differences in bronchial epithelial changes associated with tobacco smoking.

BACKGROUND: Lung cancer is the most common cause of cancer death in North American women. Because smoking-related changes in the bronchial epithelium and in lung function have not been studied in detail in women, we used fluorescence bronchoscopy-directed biopsy to determine the prevalence of high-grade preinvasive lesions in former and current smokers of both sexes. METHODS: Spirometry, white-light bronchoscopy, and fluorescence bronchoscopy were performed in 189 women and 212 men older than 40 years of age who had smoked 20 pack-years or more (pack-years = number of packs of cigarettes smoked per day x number of years of smoking). RESULTS: Carcinoma in situ was found in 1.8% of the subjects, severe dysplasia was found in 6.5%, and moderate dysplasia was found in 14% (all preinvasive lesions). Compared with men, women had a lower prevalence of high-grade preinvasive lesions in the observed airways (14% versus 31%; odds ratio = 0.18; 95% confidence interval = 0.04-0.88), and women with preinvasive lesions had fewer such lesions (two-sided P = .048). The prevalence of preinvasive lesions did not change substantially for more than 10 years after cessation of smoking. Lung function was associated with the prevalence of preinvasive lesions, but the association was weaker in women than in men. If the presence of airflow obstruction was defined by an FEV1/FVC (forced expiratory volume in 1 second/forced vital capacity) value of 70% or less, only 56% of the men and 44% of the women with preinvasive lesions had abnormal lung function. CONCLUSION: In developing strategies for chemoprevention or early detection of lung cancer in high-risk populations, it is important to consider the effect of sex and arbitrarily chosen lung function values on the prevalence of preinvasive airway lesions.

Evidence of cumulative gene losses with progression of premalignant epithelial lesions to carcinoma of the bronchus.

Human bronchial carcinoma is thought to develop through progressive stages from basal cell hyperplasia to squamous metaplasia, dysplasia, carcinoma in situ, and finally invasive cancer. In this study, we used tissue microdissection to examine loss of heterozygosity of chromosomes 3p21, 5q21, and 9p21 at each stage of the epithelial progression to invasive cancers. Forty-eight premalignant/malignant bronchial sites were biopsied from 13 patients (including 9 subjects without cancer) using fluorescence bronchoscopy. Eighteen sites with moderate/severe dysplasia in 6 patients were subjected to bronchoscopic and molecular follow-up during a 3-month to 2-year period. Seven separate cases of advanced non-small cell bronchial cancers were also analyzed. From the baseline biopsies, the prevalence of 3p and 9p deletions increased significantly from no deletion in the hyperplasia/metaplasia samples (n = 9) to 37 and 31% of the informative cases, respectively, in the dysplasia samples (n = 29), to 100 and 83%, respectively, for the carcinomas in situ (n = 6), and 100% in the invasive cancers (n = 11). Chromosome 5q deletion was significantly more frequent in invasive cancers (70% of the informative cases) as compared to carcinoma in situ (40%), dysplasias (33%), and hyperplasia/metaplasia samples (11%). The number of chromosome alterations also increased significantly from the lowest to the highest grade lesions, showing evidence of accumulation of genetic damage from one group to another. The molecular follow-up analysis showed that the same genomic alteration can persist in a given dysplastic bronchial area for several months or years, and that the persistence or the regression of the molecular abnormality is well correlated with the evolution of the disease on follow-up. Our results suggest that molecular analysis of bronchial biopsies obtained by fluorescence bronchoscopy may be a very useful means to study the natural history of preinvasive bronchial lesions and the outcome of interventions, such as with chemopreventive treatment.

Genetic analysis of second primary lung cancers in patients surviving small cell lung cancer.

We performed genetic analysis on 12 second primary non-small cell lung cancers in patients surviving small cell lung cancer to assess the potential contribution of smoking to the development of these tumors. Mutations of TP53 were found in three (25%) tumors, KRAS2 in three (25%) tumors, and CDKN2 in two (18%) tumors. Four (50%) mutations (one each in TP53 and CDKN2 and two in KRAS2) were G:C to T:A transversions on the coding strand, a mutation accounting for approximately one-third of mutations in smoking-related tumors but uncommonly found in lung cancers not associated with smoking. The genetic changes in these second lung cancers are more representative of smoking-associated malignancies than lung cancers arising in patients occupationally exposed to irradiation and atomic bomb survivors.

Electron paramagnetic resonance imaging for real-time monitoring of Li-ion batteries.

Batteries for electrical storage are central to any future alternative energy paradigm. The ability to probe the redox mechanisms occurring at electrodes during their operation is essential to improve battery performances. Here we present the first report on Electron Paramagnetic Resonance operando spectroscopy and in situ imaging of a Li-ion battery using Li2Ru0.75Sn0.25O3, a high-capacity (>270 mAh g(-1)) Li-rich layered oxide, as positive electrode. By monitoring operando the electron paramagnetic resonance signals of Ru(5+) and paramagnetic oxygen species, we unambiguously prove the formation of reversible (O2)(n-) species that contribute to their high capacity. In addition, we visualize by imaging with micrometric resolution the plating/stripping of Li at the negative electrode and highlight the zones of nucleation and growth of Ru(5+)/oxygen species at the positive electrode. This efficient way to locate 'electron'-related phenomena opens a new area in the field of battery characterization that should enable future breakthroughs in battery research.

Molecular follow-up of a preinvasive bronchial lesion treated by 13-cis-retinoic acid.

We report the result of the follow-up molecular analysis of a bronchial carcinoma in situ treated by 13-cis-retinoic acid, which relapsed 9 months after cessation of drug therapy. Loss of heterozygosity at 3p21 and 9p22 genomic sequences were assessed by polymerase chain reaction (PCR) after microdissection of the dysplastic epithelia. Despite a transient regression of the lesion to a lower grade with treatment, molecular analysis showed the persistence of a 3p and 9p deletion in all the bronchial biopsies taken in the same area during a 1 year follow-up, preceding by 9 months the recurrence of the carcinoma in situ. Our findings suggest that molecular follow-up analysis can help to assess the persistence of a malignant clone within a bronchial epithelium that displays a more benign phenotype under retinoid treatment on follow-up. Molecular analysis may be of great importance to evaluate the effects of chemoprevention and to determine the duration of such intervention in responder patients.

Effect of filtration and concentration on the composition of bronchoalveolar lavage fluid.

The effects of filtration and concentration on the cellular and protein composition of bronchoalveolar lavage fluids were examined in ten normal subjects and 11 patients with asthma. Filtration of lavage fluid preferentially removed bronchial epithelial cells, resulting in a relative increase in the proportion of alveolar macrophages. Concentration of the lavage fluid results in a significant loss of proteins, with a greater loss of large molecular weight proteins. A similar loss was not observed when diluted sera form the same subjects were concentrated in the same manner. Our results suggest that when studying the cellular composition of bronchoalveolar lavage fluid, the aspirated fluid should not be filtered through cotton gauze if changes in the bronchial epithelium may be of importance because it will remove a significant proportion of these cells. Proteins in lavage fluids should be quantitated in unconcentrated lavage fluid whenever possible.

Aspiration biopsy in the diagnosis of pulmonary disease.

We reviewed the results of 146 aspiration lung biopsies (ALB) performed on 140 patients over a five-year period. A negative fiberoptic bronchoscopy in patients with a pulmonary mass lesion or infiltrate was the major indication for ALB in this group. Seventy-two patients had various malignant chest lesions, 63 had benign or inflammatory pulmonary disease. A definite diagnosis was not obtained in the remaining five patients. The diagnostic accuracy of ALB was 73.6 percent in malignant disease and 17.5 percent in benign disease with no false positive results. Of 50 patients ultimately proven to have unresectable cancer, 46 (92.6 percent) were spared the necessity of exploratory thoracotomy for diagnosis by prior ALB. Complications included pneumothorax in 30 percent necessitating chest tube drainage in 14.3 percent. Minor hemoptysis occurred in 3.4 percent, hemothorax in 0.68 percent and subcutaneous emphysema in 1.36 percent. There were no deaths directly attributable to the procedure. We conclude that ALB is a valuable procedure in the diagnosis of malignant chest lesions, sparing exploratory thoracotomy for histologic diagnosis in many patients.

Effect of bronchial lavage volume on cellular and protein recovery.

To investigate the optimal lavage technique to study the airway response in patients with asthma, differential volume lavage was carried out in 22 normal subjects and 18 patients with red cedar asthma. Ten ml of fluid was instilled into a main-stem bronchus followed by 5, 10, 20, 50 and 100 ml into different segmental bronchi. Small volume lavage (less than 20 ml) in a main stem bronchus or a segmental bronchus recovered more epithelial cells and neutrophils while a lavage volume of 20-100 ml in a segmental bronchus recovered more lymphocytes, alveolar macrophages, albumin, IgG and alpha 1-antitrypsin. In patients with asthma, a higher proportion of epithelial cells was observed in the 20 ml segmental bronchus lavage 24 to 48 hours after bronchial challenge with plicatic acid, the chemical compound responsible for red cedar asthma. There was an increase in eosinophils and IgG with all volume lavages, but the changes were most marked in the 10 ml main bronchus lavage and the 10 and 20 ml segmental bronchus lavage. The results show the importance of using a systematic lavage protocol to characterize the cellular and protein changes in the airways of patients with asthma. A single volume lavage in one site may not encompass all the changes observed.

Detection of dysplasia and carcinoma in situ with a lung imaging fluorescence endoscope device.

The performance of a novel bronchoscopic fluorescence imaging system was compared with conventional white light bronchoscopy with a data base of 328 biopsy-confirmed sites from 53 patients and 41 volunteers. The two methods were found to have the same specificity (94%); however, the sensitivity of the fluorescence system (72.5%) was found to be 50% greater than that of the white light bronchoscopy (48.4%) in detecting dysplasia and carcinoma in situ. The fluorescence system uses a nonlinear discriminant function combining the red and green image intensity values to form a pseudoimage that, when displayed on an RGB monitor, allows the detection and delineation of abnormal areas. In 15% of the patients with lung cancer, synchronous carcinoma in situ was found in addition to the large invasive cancer. Of the current smokers in this study, 40% had moderate dysplasia and 12% had severe dysplasia. For the ex-smokers 25% had moderate dysplasia, 6% had severe dysplasia, and 13% had carcinoma in situ. Fluorescence imaging may become an important adjunct to conventional bronchoscopic examination to improve our ability to diagnose and stage lung cancer more accurately.

Localization of bronchial intraepithelial neoplastic lesions by fluorescence bronchoscopy.

BACKGROUND: In the treatment of lung cancer, the best outcome is achieved when the lesion is discovered in the intraepithelial (preinvasive) stage. However, intraepithelial neoplastic lesions are difficult to localize by conventional white-light bronchoscopy (WLB). OBJECTIVE: To determine if autofluorescence bronchoscopy, when used as an adjunct to WLB, could improve the bronchoscopist's ability to locate and remove biopsy specimens from areas suspicious of intraepithelial neoplasia as compared with WLB alone. METHOD: A multicenter clinical trial was conducted in seven institutions in the United States and Canada. WLB followed by fluorescence examination with the light-induced fluorescence endoscopy (LIFE) device was performed in 173 subjects known or suspected to have lung cancer. Biopsy specimens were taken from all areas suspicious of moderate dysplasia or worse on WLB and/or LIFE examination. In addition, random biopsy specimens were also taken from other parts of the bronchial tree. RESULTS: The relative sensitivity of WLB + LIFE vs WLB alone was 6.3 for intraepithelial neoplastic lesions and 2.71 when invasive carcinomas were also included. The positive predictive value was 0.33 and 0.39 and the negative predictive value was 0.89 and 0.83, respectively, for WLB+LIFE and WLB alone. CONCLUSION: Autofluorescence bronchoscopy, when used as an adjunct to standard WLB, enhances the bronchoscopist's ability to localize small neoplastic lesions, especially intraepithelial lesions that may have significant implication in the management of lung cancer in the future.

Malignancy associated changes in bronchial epithelial cells and clinical application as a biomarker.

A total of 74 bronchial brushing specimens, 24 from patients with advanced stage cancer, eight from patients with CIS, 31 from patients with atypical metaplasia and 11 from normal subjects were examined for the existence of malignancy associated changes (MAC). Conventional fiberoptic bronchoscopy and fluorescence endoscopy was carried out on every case. Each case was classified according to the highest grade of abnormality diagnosed by bronchial biopsy of the suspect areas. During the endoscopy examination, a bronchial brushing specimen was obtained from a visually normal area very remote from the abnormal area as possible such as the opposite lung or another lobe. The bronchial brushing specimens were fixed, mounted and stained by a DNA specific method and approximately 1500 images of individual nuclei per case were captured by an automated high resolution image cytometry. For each of these images, more than 100 nuclear features such as size, shape and chromatin spatial organization were calculated. Discriminant function analysis revealed nuclear features which differentiated between normal bronchial cell nuclei from the normal subjects and ostensively normal nuclei (MAC cell nuclei) from the lung cancer patients. The best discrimination was achieved when the frequency of individual cells expressing MAC was 50% or greater. With this threshold, 75% of the patients with invasive cancer and CIS were correctly classified. Fifty percent of those with severe or moderate atypia and 35% with mild atypia were also MAC positive. The frequency of cells expressing MAC also increased as the degree of abnormality of the groups increased. MAC may be a useful criterium to determine biological behavior of the intra-epithelial (pre-invasive) neoplasia.

Ovarian carcinoma: a multivariate analysis of prognostic factors.

Five hundred and fifty six women with invasive epithelial ovarian carcinoma were assessed for postoperative treatment between 1966 and 1976. The records of this group were reviewed retrospectively. Sixteen characteristics of the patient and tumor were analyzed for prognostic significance by univariate and multivariate techniques. Tumor grade, the presence of residual disease, and patient performance status are identified as stage-specific independent prognostic factors. These independent factors define patient subsets with good, intermediate, and poor prognosis within each stage. The results show that more effective treatment strategies are required for patients in poor prognosis subsets. Estimates of tumor grade, extent of residual disease, and performance status should be included in reports of treatment outcome.

Differentiation of thyroid neoplasms by evaluating epithelial membrane antigen, Leu-7 antigen, epidermal growth factor receptor, and DNA content.

The expression of epithelial membrane antigen (EMA), Leu-7 antigen, epidermal growth factor receptor (EGFr), and deoxyribonucleic acid (DNA) content in 40 thyroid nodules was investigated to identify those factors that might differentiate these lesions or correlate to their prognosis. There were 22 carcinomas (16 papillary, 4 follicular, 1 anaplastic, 1 medullary) and 18 benign lesions prospectively obtained between 1989 and 1993. Patients' charts were reviewed to establish a database of known clinical prognostic indicators. Expression of EMA and Leu-7 was significant in malignant lesions when compared to benign lesions (P < 0.02 and P < 0.001). EMA was expressed significantly more frequently by follicular carcinomas than by follicular adenomas (P < 0.03). Leu-7 antigen was expressed by all papillary carcinomas. Neither of these antigens showed any association with known clinical prognostic indicators. EGFr expression neither differentiated benign from malignant lesions nor correlated with prognostic factors. The presence of aneuploidy correlated with poor tumor differentiation (P < 0.02), but did not distinguish benign from malignant lesions. These results suggest that EMA expression may be useful for confirming malignancy in follicular neoplasms and that the expression of Leu-7 antigen can assist in distinguishing papillary carcinoma from benign lesions with pseudopapillae.

Carcinoma of the lung in patients under 40 years of age.

Bronchogenic carcinoma in the young population (40 years of age or less) is reported to present in an advanced stage and to have a virulent course. Between 1969 and 1979, 101 patients (65 men and 36 women) presented with cancer of the lung. Their mean age was 36.2 +/- 3.9 years (range 18 to 40 years). Eighty-seven percent had a history of cigarette smoking. Fifty percent of the patients had a strong familial history of malignancy of several organs. The interval between onset of symptoms and diagnosis was 4.01 +/- 3.48 months (3.56 +/- 3.34 for the surgically treated group and 4.16 +/- 3.53 for the nonoperated or unresectable group). Diagnosis was made at bronchoscopy in 32 patients, during thoracotomy in 30 patients, during nodal biopsy in 28 patients, and on cytologic examination of the sputum in 9 patients. The most common cell types were adenocarcinoma in 39 patients, squamous carcinoma in 29 patients, and oat cell carcinoma in 18 patients. Eighty-six patients (the majority) presented in stage III, whereas 9 were in stage I and 6 were in stage II. Twenty-seven patients (26.7 percent) underwent resection for cure, whereas 18 patients were inoperable at surgery. Eighteen of the surgical patients had adjuvant radiotherapy, and chemotherapy, immunotherapy, or both. The average length of survival for the nonresected patients was 7.12 +/- 5.9 months (range 1 to 36 months) and the actuarial survival was 1.5 percent at 36 months. The survival for the surgically managed patients was 56.1 +/- 52.6 months (range 3 to 168 months) or 48 percent at 36 months. At 46 to 168 months after treatment, the only survivors were 13 patients who were surgically managed. Stage III patients had longer survival after surgery (24.1 +/- 24.6 months to 7.09 +/- 5.90 months; range 3 to 74 months and 1 to 36 months, respectively). The survival at 5 years for patients with stage I disease was 78.8 percent, stage II disease 66.6 percent, and stage III disease, 3.6 percent. Early diagnosis and aggressive surgical management are necessary to improve the survival of patients with bronchogenic carcinoma under 40 years of age.

Prognostic value of DNA ploidy as assessed with flow cytometry in uveal melanoma.

OBJECTIVE: To investigate the prognostic value of tumour cell DNA content, as determined with flow cytometry, in formalin-fixed paraffin-embedded tissue from patients with uveal melanoma. DESIGN: Case series. SETTING: Pathology Laboratory, Vancouver General Hospital. PATIENTS: Ninety patients with primary uveal (choroid) malignant melanoma diagnosed between 1975 and 1984 followed for at least 60 months. OUTCOME MEASURES: Tumour cell DNA content, 5-year and 10-year disease-specific survival rates. RESULTS: The DNA histograms were classifiable as diploid (67 cases) or aneuploid (18 cases) in 85 cases. On univariate analysis DNA content, age at diagnosis, histologic type of tumour, largest single tumour dimension and tumour volume were predictors of 10-year survival. Multivariate analysis with the stepwise Cox proportional hazards regression model did not confirm cellular DNA content as an independent prognostic factor. However, age at diagnosis, largest tumour dimension and presence of spindle vs. mixed or epithelioid cell pattern were significant predictors of death from uveal melanoma. CONCLUSIONS: DNA ploidy of uveal melanoma is not a statistically significant predictor of survival.

An automated image cytometry system for monitoring DNA ploidy and other cell features of radiotherapy and chemotherapy patients.

DNA content and distribution in cell nuclei were studied in samples of fine-needle aspiration (FNA) from 27 locally advanced breast and head and neck cancers in two going randomized trials that compared accelerated fractionation to standard fractionation radiation in locally advanced breast cancer and head and neck cancer. Two image cytometry methods were compared: a new, fully automated DNA image cytometry system (AIC) and a conventional image cytometry (CIC) system with manual selection, focusing, and segmentation of cells. The results of both techniques were compared on the basis of DNA histogram parameters including DNA index (DI), mean DNA values (MDV), and Auer's DNA histogram patterns. An excellent correlation was achieved between the two imaging techniques in terms of DI (r=0.985, p<0.001) and MDV (r=0.951, p<0.001) as well as between Auer's histogram patterns, where both methods agreed completely. It was concluded in these analyses that the two image cytometry methods were equivalent. However, the AIC offered an advantage by scanning samples in a fully automated way, which represented significant time saving for cytopathologists working with the system, as well as a larger number of cells used in the automated analysis. With the automated image cytometer, 500 relevant cells were collected and analyzed in about 10 minutes, where with the interactive (manual) method, it took typically an hour to collect and analyze only about 250 cells. Seventeen samples were sufficient for flow analysis. Image cytometry and flow cytometry showed good agreement in DI determination; however, three cases reported as diploid by flow cytometry were found to be aneuploid by image cytometry techniques.

Comparison of cellular and protein changes in bronchial lavage fluid of symptomatic and asymptomatic patients with red cedar asthma on follow-up examination.

Seventeen patients with occupational asthma due to western red cedar had bronchial lavage during follow-up examination after removal from exposure for at least 1 year. Seven patients were asymptomatic while ten continued to have symptoms of asthma requiring treatment. Symptomatic patients had evidence of airway inflammation, as reflected by a significantly higher total cell count, neutrophils and eosinophils, as well as an increase in protein and albumin in their bronchial lavage fluid compared to those without symptoms. Asymptomatic patients had no evidence of airway inflammation in the lavage fluid. There was no correlation between the degree of non-specific bronchial hyperresponsiveness and the number or percentage of inflammatory cells to suggest that cellular infiltration is the sole cause of persistent bronchial hyperresponsiveness.

Cellular and protein changes in bronchial lavage fluid after late asthmatic reaction in patients with red cedar asthma.

To investigate the sequence of cellular and protein changes after a late asthmatic reaction (LAR), bronchial lavage was carried out in 44 patients with red cedar asthma at different time intervals after bronchial challenge with plicatic acid. The results were compared to five patients with red cedar asthma who became asymptomatic after removal from exposure to red cedar for more than 2 months and 31 healthy subjects without asthma. The LAR was found to be associated with an increase in eosinophils in the lavage fluid, an increase in sloughing of bronchial epithelial cells, and an increase in degenerated cells consisting mainly of degenerated epithelial cells and alveolar macrophages. There was an increase in vascular permeability as reflected by an increase in albumin in the lavage fluid. Although there was a slight but significant increase in neutrophils 48 hours after bronchial challenge, neutrophil infiltration was not a prominent feature earlier. The potential role of loss of epithelial cells to account for an increase in nonspecific bronchial hyperresponsiveness after an LAR was discussed.