TERC Gene Detection Of Liquid-Based Brushing Cytologic Specimens On The Significance Of Early Diagnosis Of Lung Cancer

IntroductionThere are lots of mucus,blood,inflammatory cells and necrotic material in the pick and smear slides,resulting in a low detection rate.Liquid-based cytologic test (LCT) has been applied for cervical cytology diagnosis successfully and widely, however it is few reported yet for brushing cytology diagnosis at present.More and more techniques were use for the study of oncology genetics.The Fluorescence In Situ Hybridization tech was accepted as an important methods to study the change of tunor chromosome,because its safe,fast,lasting,sensitive and other advantages.Using LCT and FISH,we aim to evaluate clinical diagnosis value of Liquid-based Cytologic Test and Fluorescence In Situ Hybridization in 142 liquid-based brushing cytologic specimens of lung cancer of our study.Materials and methods1,Objects and specimen collectionThe present study was conducted in accordance with the regulations of the institutional review boards at China Medical University and performed between January and October 2008 at The First Affiliated Hospital,China Medical University. One hundred and two were diagnosed with lung cancer on the basis of histopathology of bronchoscope and post-operation.These 102 patients,as well as 40 randomly selected patients without lung cancer.2,Sample preparation for LCTTwo smears were prepared from brushing and immediately fixed in 95%ethanol and stained with Papanicolaou.Residual brushing was transferred to a small bottle with CytoRich liquid.One millilitre of the mucolytic agent(Tripath Imaging) was added to the small bottle with CytoRich liquid,incubated at room temperature for 30 min and vortexed for 10s.Additional mucolytic agent was added to the mixture until the mucus was completely lysed.The mixed liquid was then transferred to a 50-ml tube and centrifuged at 2000 g for 10min.The supernatant was removed and the pellet was resuspended in 7.5ml of distilled water.This suspension was vortexed again and centrifuged at 2000 g for 5min. The supernatant was removed and the pellet was vortexed and transferred to the AutoCyte PREP system(Tripath Imaging),in which slides were automatically prepared and stained.Two slides were prepared from each tube and were stained with Papanicolaou.3,Sample preparation for FISHFor this study,each specimen was fixed with FISH fixative(3:1 methanol:acetic acid) for 10 min,centrifuged at 2000rpm for 10 min and repeated it again.A slide was prepared using the AutoCyte PREP system.The probe panel(China Medical Technologies,Inc.,Beijing,China),which consists of two probes:TERC(red) and the control,centromeric chromosome 3(green).The slides were placed twice in 2×sodium saline citrate(SSC;pH 7.0) for 5 min,digested in 0.1 g pepsin powder/0.01 tool HCL 40ml for 10 min at 37℃,placed twice in 2×SSC for 5 min again.After dehydration of the slides in a graded series of concentrations of ethanol,10μl of probe and slides were denatured in 70%formamide,for 5 minutes at 77℃.After overnight hybridization at 42℃,the coverslips were removed gently,slides washed three times in 50%formamide/2×SSC at 46℃for 10 minutes,followed by washes in 2×SSC at 46℃for 10 minutes and 0.1%NP40/2×SSC at 46℃for 5 minutes,and fixed in 70%EtOH for 3 minutes.After air-drying(out of direct light),the slides were counterstained with 15μl of DAPI/anti-fade solution and coverslipped.4,Judgement of FISH signal The slides were scanned using a Olympus fluorescent microscope BX-51 (Olympus,Japan) equipped with a X-cite 120 mercury lamp.100 cells were enumerated per slide.The signals were evaluated by screening the entire slide visually for TERC and centromeric chromosome 3 probe and recorded on a scoring sheet.Cells that could not be evaluated(because of insufficient hybridization or cell clumps) were excluded from further analysis.To avoid counting split signals as two signals,the distance between any two signals had to be at least the diameter of one signal for them to be counted as separate signals.A cell was scored positive for amplification of TERC if more than two red signals and not less than two green signals were detected.To index the chromosomal instability,we calculated abnormal hybridization patterns of cells in each diagnostic group.5,Statistical analysisAll comutations were carried out SPSS 11.5.Apply X~2 test,Test criterionα=0.05.Result1,Sensitivity comparison of LCT,PS and LCT+PS methodsResulting in a low detection rate,there are lot s of mucus,blood,inflammatory cells and necrotic material in the pick and smear slides.We found 69 positive patients in 102(Se 67.6%),but 84 in 102(82.4%) using LCT(P＜0.05).And the combined sensitivity of LCT+PS improved the sensitivity to 88.2%over PS method(P＜0.01).2,FISH and LCTThere was no significant difference between FISH and LCT(86.3%Vs 82.4%,P＞0.05).FISH detected 88 positive in 102 lung cancer specimen,and 84 by LCT. There is no significant deviation between two groups(x~2=0.59,P＞0.05).3,FISH and BiospyFISH is more sensitive than biopsy for patients with lung cancer(88/102 86.3%Vs 65/102,63.7%,P＜0.01).4,PS,LCT,BIOPSY,FISH and postop Histology102 of 142 brushing cytologic specimens of our study diagnose to lung cancer by histology,40 patients were diagnose to benign lesion.69 positive patients were detect by histology.84 patients were positive by histology in 90 were detect by LCT.65 positive specimens were were detect by biopsy.88 patients were positive by histology in 96 were detect by FISH.In our study,the sensitivity and specific of the four merhods were demonstrate:PS SE 67.6%,SP 100%,AC 76.8%;LCT SE 82.4%,SP 85.0%AC 83.1%;biopsy SE 63.7%,SP 100%,AC 73.9%;FISH SE 86.3%,SP 80.0%,AC 84.5%%.DisscussionSensitivity of LCT was higher than PS method in brushing cytologic specimens.So we can use LCT brushing specimen to diagnose early stage lung cancer.If only use LCT,Of the 17 positive AC cases as determined by LCT,seven were negative by the PS method and 10 were positive by either LCT or the PS method. Of the 12 positive AC cases as determined by the PS method,two was positive by the PS method but negative by LCT,and 10 were positive by either LCT or the PS method. Although there was no significant difference in diagnostic sensitivity between the two methods(P＞0.05),when LCT and the PS method were combined,the diagnostic sensitivity for AC was 88.2%,significantly higher than that of the PS method alone (67.6%,P＜0.05).LCT method demonstrated a number of advantages.First,screening time was reduced and screening efficiency was increased due to the decreased areas of cell monolayers.Second,the slides had a clearer background due to the dissolution of mucous material,the destruction of most of the red blood cells and significantly reduced numbers of inflammatory cells.Thus,abnormal and tumor cells were more readily discernible.Regardless of the cell type,the cells were clearly stained and their morphology was well preserved.The results of this study demonstrate that FISH is more sensitive than conventional cytology for detecting lung cancer in bronchial brushing specimens;when combined with cytology,FISH can improve the diagnostic sensitivity of detecting malignancy in bronchial brushing specimens.The brushing data show that FISH was able to increase the diagnostic sensitivity of detecting peripheral tumors over routine cytology.The data also suggests that FISH and cytology brushing results combined can improve the detection of early stage lung cancers over cytology alone.FISH has an ability to detect small populations of tumor cells that are distant from the primary tumor.We also find sample preparation for LCT,it can depress the FISH cost.The results of this study demonstrate FISH is sensitive for detect Squamous Cell Lesions of the Lung,the sensitivity is 98.1%(P＜0.01).there was no significant difference between AC and SCLC.Chromsome changes have been found in the 88 maligant specimens,which are obtained by bronchoscopic brushing biopy,through FISH analysis on total 102 cases. And among the 8 specimens that have been diagnosized as the malignant with FISH method but as the benign with Histology,5 cases are of moderate-to-severe epithelial dysplasia suggesting precancerous lung lesion that may happen at gene level before the morphological changes emerging,2 cases are of tuberculosis and 1 is the inflammatory pseudotumor.In the negative LCT and biopsy specimens,the aneuploid,marker of malignancy,can also be found through FISH.For the patients with FISH-positive but cytology and(or) biopsy negative results,a close follow-up should be carried out.In conclusion,our data support the theory that LCT and FISH are more sensitivities than tradition cytology detection.it may be useful methods for diagnose lung cancer,to improve the efficiency of the diagnose,meanwhile FISH can predict lung cancer.Conclusion1,LCT is more sensitive than conventional cytology for detecting SCLC in bronchial brushing specimens;combined sensitivity of the two methods is significantly higher than using cytologic only;2,FISH is sensitive for detect SCC;3,FISH can predict the carcinogenesis of lung.