外周血液中循环肿瘤细胞和循环游离DNA检测在乳腺癌患者中的应用

Application of peripheral blood circulating tumor cells and circulating-free DNA in diagnosis of breast cancer

目的探究外周血液中循环肿瘤细胞(CTCs)和循环游离DNA(cfDNA)检测在乳腺癌患者中的应用效果。方法收集本院2018-02—2019-02收治的94例乳腺癌患者、48例乳腺良性疾病患者及56例健康体检者外周血液标本,采用基于尺寸的高通量微流控芯片捕获CTCs、基于Alu序列的实时荧光定量PCR检测游离DNA长、短片段(247 bp、115 bp),并以长、短片段扩增产物比值作为DNA完整性指标。分析CTCs和cfDNA与乳腺癌临床参数的关系,并评估其在乳腺癌诊断中的作用。结果乳腺癌患者在CTCs、血浆cfDNA的基于Alu序列的Alu 247/115的比值上显著比健康体检者、乳腺良性疾病者高(P<0.05),乳腺癌患者外周血中CTCs的数量与血浆cfDNA的基于Alu序列的Alu 247/115的比值在年龄、病理分期、淋巴结转移、分子分型、远处转移、PR有一定的相关性(P<0.05),以病理学结果作为金标准,以约登指数最大点对应的临界值,以乳腺良性疾病患者和健康体检者作对照,当CTCs cut-off值为7.68个/ml,灵敏度为80.4%,特异度为96.4%,ROC曲线下面积为0.885(95%CI 0.805~0.965);Alu 247/115的cut-off值为0.431时,特异度为71.4%,ROC曲线下面积为0.727(95%CI 0.608~0.847),灵敏度为71.7%。结论 CTCs与cfDNA可能是乳腺癌辅助诊断的潜在生物学指标,CTCs检测的是肿瘤细胞,cfDNA检测的是肿瘤的DNA片段,可作为判断乳腺癌患者的病情变化和诊断的良好辅助指标,此方法值得应用与推广。

Objective To study the applied value of peripheral blood circulating tumor cells(CTCs)and circulating-free DNA(cfDNA)in diagnosis of breast cancer.Methods From February 2018 to February 2019,peripheral blood samples from 94 breast cancer patients,48 benign breast disease patients and 56 healthy check-ups were obtained.High-throughout micro-fluid chip was applied for detection of CTCs;based on Alu sequence,real time quantitative PCR was used to detect long and short DNA segment(247 bp,115 bp)while the ratio of PCR product based on long and short segment was used to determine the integrity of DNA.The diagnostic value of CTCs and cfDNA was assessed.Results Number of CTCs and Alu 247/115 of cfDNA in breast cancer patients were significantly higher than healthy check-ups and benign breast disease patients(P<0.05);number of CTCs and Alu 247/115 of cfDNA was related to patient’s age,grade,lymphatic metastasis,molecular subtyping,distant metastasis and PR(P<0.05);pathological results were regarded as gold standards,Youden index at maximum point was used as critical value;a control study was conducted among breast cancer patients,benign breast disease patients and healthy check-ups;when cut-off value was 7.68 CTCs/ml,the diagnostic sensitivity and specificity were 80.4%and 96.4%,area under the curve was 0.885(95%CI:0.805~0.965);when cutoff value of Alu 247/115 was 0.431,the diagnostic specificity and sensitivity were 71.4%and 71.7%,area under the curve was 0.727(95%CI:0.608~0.847).Conclusion CTCs and cfDNA can be used as the potential biological indicators for auxiliary diagnosis of breast cancer;CTCs are applicable for detection of tumor cells and cfDNA for DNA segment of tumor.Therefore,CTCs and cfDNA are conductive to analyzing the illness states and taking the auxiliary diagnosis of breast cancer.