A Novel Approach For Detection Of Circulating Tumor Cells In Head And Neck Cancer

Objective The spread of circulating tumor cells(CTCs)in the blood plays a major role in the initiation of tumor metastases and recurrence after surgery.Therefore,To test the amount of CTCs in the blood from the patients with cancer has been considered to be important clinical significance in the early diagnosis,individualized treatment,disease assessment and prognosis.However,the the technology in isolation and characterization of CTCs become limited because these cells are extremely rare and mixed with normal blood components.In our previous study,the telomerase-specific replication-selective oncolytic adenovirus is a genetically modified virus,also we developed that it can expresses a green fluorescent protein(GFP)gene under telomerase promoter.The virus was used to trap the circulating tumor cells in this study to establish a simple,noninvasive and repeatable method.,as well as the reliability of the test method and the potential value of clinical application were tested.MethodsFirst,the experiment needs to be done to evaluate if this virus could effectively capture CTCs.10,50,and 100 A549 or SKBR3 cell lines were spiked into 1-ml blood sample from healthy volunteers,respectively.AD11-5ETel-GFP virus were added into peripheral blood to observed its expression in normal white blood cells and cancer cell lines.Next,we labelled CD45 molecules by cell immunostaining method to distinguish white blood cells from CTCs.The fresh blood samples were collected from 48 patients with head and neck malignant tumor.Red blood cells were lysed in NH4 Cl buffer,subsequently,the cell pellets were infected with AD11-5ETel-GFP at the indicated concentrations after incubation for 24 h at 37 ?.Finally we analyzed the CTCs' number by counting Green fluorescent protein-expressing cells under the fluorescent microscope in the peripheral blood on the base of the different clinical parameters.Results 1.The telomerase-specific replication-selective oncolytic adenovirus rarely infect normal white blood cells.2.The detection rate results of tumor cell lines A549,SKBR3 tumor,respectively were 77.5%,84.0%,80.25%;80.0%,82.5%,81.25%.Compared differences between the two groups by Pearson chi-square test,the detection rate was no significant difference by Ad11-5ETel-GFP method,namely the method of this experiment study on the tumor cell lines was sensitive and recovery rate was higher.Through cell immunostaining experiments,also we further verified the capture of cells to tumor cells.3.In the clinical specimens of 48 cases of head and neck malignant tumors,19 patients were detected CTCs positive,with the detection rate of 39.6%.And the detection rate had no statistical association with gender,age,tumor stage,location of the diseases,lymph node metastasis.It significantly correlated with tumor differentiation degree.The lower of the tumor differentiation,the higher of CTCs detection rate.The detection rates of of CTCs was different due to different location.Conclusions This study established a novel approach for detection of circulating tumor cells in head and neck cancer.Preliminarily we determined the standard of CTCs,and researched reliability of the test method and the value of clinical application.The method is simple,noninvasive and repeatable,it can trace circulating tumor cells specificity and has great potential clinical value.