Study Of Near-infrared (NIR) Heptamethine Dye IR-80 For Distinguishing Benign Neoplasms From Malignant Thyroid Nodules

Objective:To observe different dye uptake of near-infrared(NIR)heptamethine dye IR-80 in normal breast epithelial cell line(MCF-10A)and breast cancer cell lines(MCF-7 and MDA-MB231)firstly,and to optimize experimental parameters including the concentration of dye,and the parameters of shooting for distinguishing benign from malignant cells.Then the optimal experimental parameters were determined to detect the fluorescence intensity of IR-80 dye between thyroid cancer and benign thyroid nodule.The capability of IR-80 staining technique to distinguish benign neoplasms from malignant thyroid nodules was evaluated by compared with cytological analysis.Methods:Cell lines experiment:IR-80 was diluted in RPMI-1640 medium with the final concentrations of 5,2.5,1.25,0.625μM.Then breast cancer cell lines(MCF-7 and MDA-MB231)and breast epithelial cell line(MCF-10A)were incubated with IR-80 at the different concentration.Fluorescence intensity between normal and cancer cells were observed by using a fluorescence microscope at the exposure time of 0.5s,1s and 2s.Mean fluorescence intensity was calculated by Image J.IR-80 dye for preferential accumulation capability in live cancer cells was evaluated and optimize experimental parameters were determined to distinguish benign from malignant cells.Study on fine needle aspiration(FNA)specimens of thyroid gland:120 patients undergoing FNA were recruited in the study at Jinling Hospital from September 2015 to October 2016.The results of cytological diagnosis were positive in 59 cases(thyroid cancer in 59 cases),negative results in 61 cases(benign nodules).The optimal experimental parameters determined from cell lines experiment were used to observe the different cellular uptake of IR-80 dye between thyroid cancer and noncancer cells.The sensitivity and specificity of IR-80 staining method were calculated by using cytological analysis as the diagnostic gold standard.ROC curve was used to evaluated the diagnostic value of IR-80 staining method.Results:Cell lines experiment:The fluorescence intensity decreased as the concentration of IR-80 decreased in both breast cancer cells and breast epithelial cells when the exposure time was certain.There was no significant increase in fluorescence intensity varying from 0.5s to 2s in each cell line.The NIR fluorescence was more intense in MCF-7 and MDA-MB231 than in MCF-10A when the concentration of IR-80 and exposure time were certain.Increased dye uptake by tumor cells was observed while normal cells did not take up dye at the point of 1.25μM.An optimal experimental parameters with incubation concentration of 1.25μM and exposure time of 1s were determined for identification of malignant cells.Study on FNA specimens of thyroid gland:NIR fluorescence was detected specifically in the thyroid cancer specimens in fairly contrast to the noncancer samples,in which the benign cells(including benign thyroid follicular cells,macrophages and lymphocyte)were not stained.The dye uptake by thyroid cancer cells was clear and well-distributed.IR-80 staining was used to detect 120 cases of FNA thyroid specimens,and 65 cases showed positive results,55 cases showed negative results.The sensitivity was 100%(54/59),the specificity was 90.1%(55/61);the area under the ROC curve was 0.949 and the area of the standard error was 0.023.IR-80 staining method for distinguishing benign from malignant cells had significant difference(P=0.000).Conclusion:The NIR fluorescence is more intense in breast cancer cells than normal breast epithelial cells by using NIR heptamethine dye IR-80.An optimal experimental parameters with incubation concentration of 1.25μM and exposure time of 1s are able to distinguish malignant from nonmalignant cells.NIR fluorescence is detected specifically in the thyroid cancers while benign nodules are not stained,indicating the preferential accumulation capability of IR-80 in cancer and IR-80 can be used to distinguish benign neoplasms from malignant thyroid nodules.The sensitivity and specificity of IR-80 staining method are high and ROC curve showed its high diagnostic value,IR-80 staining technique shows promise in the application of this staining technique as new adjuvant diagnosis tool for clinical thyroid FNA specimens.