Canadian Neighbor Pharmacy: Quantitative Detection of Lung Cancer Cells by Fluorescence
Fifty consecutive patients who underwent cytologic examination for abnormal chest radiography or CT scan findings at Tokyo Medical University Hospital from July 2003 to January 2004 were enrolled in this prospective study. The patients included 32 men and 18 women, with an average age of 64 years. The final definitive diagnosis was made by histologic examination, as follows: 38 primary lung cancers (24 adenocarcinomas, 8 squamous cell carcinomas, 2 large cell carcinomas, and 4 small cell carcinomas); 1 metastatic renal cell carcinoma; and 11 benign lesions. All patients with lung cancer were staged according to the latest Union Internationale Centre le Cancer criteria. Cases included 10 tumors in stage IA, 5 in stage IB, 1 in stage IIA, 3 in stage IIB, 10 in stage IIIA, 6 in stage IIIB, and 3 in stage IV (Table 1).
Cells gathered from lung lesions were independently analyzed by conventional cytology and FISH. Informed consent for the cytologic examinations and genetic analyses of the specimens were obtained from all patients.
In this study, the following four types of cell specimens were analyzed: cells obtained by TB (n = 18) and transbronchial fine-needle aspiration (n = 5) using a fiberoptic bronchoscope under fluoroscopy, CT scan-guided PN using the 19-gauge Tokyo Medical University Needle (n = 17), and BWs (n = 10). On every examination, duplicate specimens were made for simultaneous analyses of conventional cytology and FISH.
For conventional cytology, cells were stained by the Papanicolaou method. Diagnosis was made by cytologists in the Department of Pathology at Tokyo Medical University Hospital. The various classes in conventional cytology are defined as follows: class I, absence of atypical or abnormal cells; class II, atypical cytology but no evidence of malignancy; class III, cytology suggestive of, but not conclusive for, malignancy (IIIa, mild dysplasia; IIIb, advanced dysplasia); class IV, cytology strongly suggestive of malignancy; and class V, cytology conclusive for malignancy. Breaking news about medicine and pharmacy are collected on Canadian Neighbor Pharmacy website.
For FISH, cells on glass slides were air-dried overnight and stored at — 80°C until they were used. Direct fluorochrome-labeled centromeric probes were used for the enumeration of different chromosomes. Spectrum-orange-labeled or Spectrum-green-labeled probes for the respective centromeric regions of chromosomes 3 and 17 were purchased (Vysis Inc; Downers Grove, IL), and dual-color FISH was performed. Slides were denatured by incubation with 70% formamide (two times the standard saline citrate [SSC] solution) at 74°C for 2 min in a water bath. Then, slides were dehydrated through a graded ethanol system (70% for 2 min, 85% for 2 min, and 100% for 2 min). A hybridization solution (10 |j,L) was applied to each slide, which was coverslipped and sealed with rubber cement. The hybridization solution contained 1 |j,L each DNA probe in 70% formamide (two times the SSC solution), and 10% dextran sulfate solution (cot I DNA). After incubation for 16 h at 37°C in a humidified chamber, slides were washed (two times SSC solution) for 3 min at 74°C. A di-amidinophenylindole antifade solution (8 |j,L) was applied to each spot and coverslipped. The slides were observed under a fluorescence microscope that was connected to a cooled charge-coupled device camera and an image analyzer system (CytoVision; Applied Imaging, Ltd; Newcastle, UK).
FISH signal analysis was performed as follows. All cells in a fluorescence microscopy field, except for those with damaged or overlapped nuclei, were evaluated. One hundred cells were counted, and the numbers of each centromeric signal were recorded. If there were 10%, we judged the lesion to be malignant.
Comparison of Conventional Cytology and FISH
FISH diagnoses were made without clinical information or the results of conventional cytology. The results of FISH analysis were not shown to the cytologists. Thus, both diagnoses were independently made in a blind fashion.
Differences in the number of countable cells according to the histology of the lung lesions or the cell-gathering methods used were analyzed by the Kruskal-Wallis test. A p value of < 0.05 was considered to be significant.
Table 1—Histology and Stage of Lung Cancer in This Series of Patients