| Objective:1. To evaluate the diagnostic value of multicolor fluorescence in situhybridization (M-FISH) combined with cytomorphology of bronchial brushing in the detection of lung cancer.2. To validate the feasibility of cytology samples for detecting EGFR and k-ras mutations in NSCLC.Materials and methods:1. Centromeric enumeration probes (CEPs) for chromosomes7,8and17were analyzed using M-FISH combined with cytomorphology of69cases of bronchial brushing specimens from Department of Cytology, Cancer Hospital, Chinese Academy of Medical Sciences (CHCAMS).2. Polymerase chain reaction (PCR) was carried out to amplify EGFR exons18-21and k-ras codons12,13by using cytological samples including fine-needle aspiration (FNA), pleural effusion (PLE) and bronchial brushing from CHCAMS, and then the PCR products sequencing and analysis were performed.Results:1. M-FISH combined with cytomorphology of bronchial brushing(1) The positive rates of CEP7, CEP8and CEP17in carcinoma cases diagnosed by cytology were50.0%(16/32),80.8%(21/26) and65.4%(17/26), respectively. It was significant higher for CEP8than for CEP7(P=0.015), but no statistic differences between CEP8and CEP17, as well as between CEP7and CEP17(P>0.05). In suspicious carcinoma cases, the positive rates of CEP7, CEP8and CEP17were46.6%(7/15),66.7%(10/15) and58.8%(7/12). While in atypical cases, the positive rates of the three probes were20.0%(1/5),33.3(2/6) and25.0%(1/4), respectively.There were no significant differences between the two groups (P>0.05). The statistical differences were also not found between carcinoma cases and suspicious cases (P>0.05). No chromosome aberrations were found in normal cases diagnosed by cytology.(2) The positive rates of all the three probes were higher in adenocarcinoma (ADC) than in squamous carcinoma, as well as small cell carcinoma. But the statistical differences were only found in CEP8(P=0.044).(3) If combining cytology with M-FISH (CEP7, CEP8and CEP17), the sensitivity was80.3%(49/61), which was significantly higher than that of cytology (54.1%,33/61) alone, whereas the specificity was not affected (100.0%).2. EGFR and k-ras gene mutations analysis(1) Among50cytological samples, the DNA of43samples were extracted successfully, and42samples with specific PCR amplification products sequencing and analysis were performed.(2) EGFR mutations were detected in14of42specimens (33.3%), and the frequencies of EGFR mutations in exon19,20,21were16.7%(7/42),4.8%(2/42)and11.9%(5/42) respectively, no mutations were found in exon18. Higher frequencies of EGFR mutations were detected in exons19and21(85.7%) than in exon20(P=0.03)(3) EGFR Mutations were more frequently detected in women (47.8%,11/23) than in men (15.8%,3/19)(P=0.028),and in non-smokers (53.3%,8/15) than in smokers (22.2%,6/27)(P=0.040). Mutations were identified in38.7%(12/31) cases of ADC and none in non-ADC.(4) k-ras mutations were found in2of42specimens (4.8%), and they all occurred in12codon.(5) EGFR and k-ras mutations were not found simultaneously in the same cases.Conclusions:1. M-FISH combined with cytomorphology of bronchial brushing could be established successfully in the specimens of liquid-based cytology. The aneuploidy of chromosmes7,8and17can be detected in carcinoma, suspicious carcinoma and atypia of bronchial brushing specimens. M-FISH combined with cytomorphology of bronchial brushing could become an effective method in detecting lung cancer.2. Cytology samples were validated for detecting EGFR and k-ras mutations, especially in patients who can't get histological materials.
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