ABSTRACT
Objective To establish a novel method for detecting circulating tumor cells (CTCs) in phripheral blood of lung cancer patients with high sensitivity and specificity.Methods Experimental study.42 cases of initial treatment patient who underwent resection and diagnosed to be non-small cell lung cancer by biopsy were studied,including 7 patients at stage Ⅰ,9 patients at stage Ⅱ,16 patients at stage Ⅲ and 10 patients at stage Ⅳ.As a control group,20 cases of healthy volunteers were selected.A series of experiments was conducted to determine the efficiency of tumor cells isolation,in which varied concentration (50,100,200,500,1000 cells) of A549 cells spiked into 2 ml peripheral blood drawn from healthy donors.The blood was removed of unwanted erythrocytes by lysis buffer,and made the rest of nucleated cells incubate with anti-EpCAM magnetic beads,then separated and enriched by a specific detector.All epithelia cells were retained on a slide because of a magnetic force and identified by H&E staining protocol.On the basis of cell recovery rate we calculated the sensitivity of tumor cells isolation.20 blood samples taken from healthy individuals were also detected to validate the specificity of this method.Samples of 42 patients with lung cancer were assayed for CTCs detection by above method.The correction of CTCs quantity with the patients' clinical features,for example,ages,gender,clinical stage,tumor size was analyzed in lung cancer patients by chi-square statistics.The correction of recovery cells with the spiked cells were assayed by linear correlation.Results The recovery rate was ranging from 68％ to 82％ by spiking varying numbers of A549 lung cancer cells into 2ml blood samples of healthy volunteers.Regression analysis of number of recovered vs.spiked A549 cells yielded a regression equation of Y =0.6419X + 8.8875.The number of CTCs detected has signification correlate with the cells spiked (R2 =0.9916,P ＜ 0.05),Eighteen of the 42 patients (43％) were found have CTCs in peripheral blood.The detection rate of lung cancer cells was 0 at stage Ⅰ,the detection rate of lung cancer cells was 11.1％ at stage Ⅱ,the detection rate of lung cancer cells was 62.5％ at stage Ⅲ and the detection rate of lung cancer cells was 70％ at stage Ⅳ.The positive rate of CTCs has no signification correlate with ages and gender of patients and tumor size (P ＞ 0.05),has signification with the clinical stage (P ＜ 0.05).None of the peripheral blood samples of the 20 healthy subjects analyzed was found to have CTCs.Conclusions This novel immunomagnetic separation technology is a sensitive and specific method,which provides a new tool allowing for feasible and specific detection of CTCs in lung cancer patients.The level of CTCs increases with the clinical stage and tumor size increased,which has important value to discover the early stage micrometastasis and redefine the clinical stage.But further multicenter and large sample clinical research are needed to confirm its clinical value.
ABSTRACT
Objective To investigate the correlation between the expression level of BLM and its clinical significance in leukemia.Methods 125 bone marrow specimens of inpatients and outpatients with leukemia were collected in Anhui provincial hospital from January 2011 to December 2011.125 leukemia patients were diagnosed and classified into acute leukemia (AL,n =66) and chronic myelogenous leukemia (CML,n =59) by Morphologic and Immunologic criteria,5 non-tumor individuals were included as control group.The BLM mRNA expressions were by reverse transcription-polymerase chain reaction(RT-PCR).The specimens were devided into groups according to the age,gender,leukemia type,peripheral blood leukocyte counts,hepatomegalia and(or) splenomegaly,fusion gene,chromosome karyotype,whether first visit and transplantation.The expression of BLM gene in each group and the correlation with above factors were retrospectively analysed.The statistical methods such as chi-square test,single factor variance analysis,t test and Pearson correlation test were mainly used.Results BLM mRNA was detected in leukemia.In bone marrow cells,the BLM gene expression was positive in 71 patients and negative in 54 patients.But none of 5 non-tumor bone marrow cells expressed BLM gene.The difference of BLM expression between patients and controls was statistically significant in two groups,i.e.peripheral blood leukocyte counts and fusion gene (x2 =14.730,22.399 ; P ＜ 0.05),but there is no statistical significant differences in other groups.The expressions of BLM mRNA in leukemia patients who had been treated with chemotherapy were lower than those newly diagnosed (0.1788 ± 0.1091 vs 0.3276 ± 0.2016 ; P ＜ 0.05).Moreover,BLM mRNA level in post-bone marrow transplant patients was lower than those not treated (0.1271 ± 0.1009 vs 0.2902 ±0.2034 ; P ＜ 0.05).Pearson correlation analysis showed that higher BLM mRNA expression positively correlated with fusion gene (r =0.357,P ＜ 0.01) and chromosome abnormality (r =0.279,P ＜ 0.05).Conclusion The BLM mRNA expression level of measurement can be used as judgment for leukemia patients disease severity and the index of prognosis,testing the level may provide a basis for clinical and curative effect judgment.