The Base And Clinical Study Of A New Dye Fluorescent Tracer For Sentinel Lymph Node In Breast Cancer

Objective:The sentinel lymph node biopsy is a mini-invasive technique for axillary node staging in breast cancer.The accuracy and safety of this technique has been validated by some clinical trials.Currently,the clinical practice guideline of breast cancer recommends that patients with negative sentinel lymph node in axilla should perform sentinel lymph node biopsy to replace axillary lymph node dissection,furthermore,patients with one to two positive sentinel lymph nodes undergoing breast-conserving surgery and whole breast irradiation could omit axillary lymph nodes dissection.The sentinel lymph node is the first station in the metastatic process of carcinomas,which receives the lymphatic drainage from the primary tumor and the entire breast organ.The status of sentinel lymph node could help to identify the metastasis in axilla.Due to the difference between the sentinel lymph node and surrounding tissues is not clear,we should use the tracer of sentinel lymph node to identify the location of sentinel lymph node and complete the sentinel lymph node biopsy.In the clinical practice,the tracers of sentinel lymph node include non-specific tracer, such as blue dye,radiotracer,and fluorescence tracer,and specific tracer,such as Lymphoseek,^99mTc-rituximab,and rituximab-indocyanine green.However,the disadvantages of these tracers limited the using in the clinical practice of sentinel lymph node biopsy.The blue dye guiding sentinel lymph node biopsy need a smart anatomy skill of a surgeon and a serious learning-curve training,which still has a high false negative rate of sentinel lymph node biopsy.The radiotracer guiding sentinel lymph node biopsy cannot avoid the radiation injury of patients,doctors,and nurses,which need the nuclear medicine department and expensive equipment to produce radiotracer.The fluorescence tracer guiding sentinel lymph node biopsy also need a smart anatomy skill of a surgeon and a serious learning-curve training,but the fluorescence tracer leaking out could pollute the surround tissue and lead to miss the location of sentinel lymph node.Although the fluorescence specific tracer could target the location of sentinel lymph node,the fluorescence light intensity is slight which cannot throughout the depth tissue.The aim of the study was to produce a new polymethine dye Cyanine 754 by the chemical synthesis method which would improve the water solubility of Cyanine 754,bathochromic shift the largest absorption and emission wavelengths,increase the optical stability,the quantum yield,and the Stokes shift.The largest absorption and emission wavelengths of Cyanine 754 would be tested,and the safety and the fluorescence light intensity also would be tested.Then,the new polymethine dye Cyanine 754 would be combined directly with the monoclonal antibody rituximab to produce a new fluorescence targeting tracer rituximab-Cyanine 754.The molecular integrity,immunological activity,and Cyanine 754 labelling rate of protein in the new fluorescence targeting tracer rituximab-Cyanine 754 would be tested.After testing the safety of the rituximab-Cyanine 754,the new fluorescence targeting tracer would be tested to identify the location of sentinel lymph node in the Animal model of sentinel lymph node.Finally,the rituximab-Cyanine 754would be compared with combination methods of sentinel lymph node biopsy in the clinical validation study,which could analyze the agreement and correlation of these methods in the identification of sentinel lymph node.Methods:The new polymethine dye Cyanine 754 was produced by the chemical synthesis method.The largest absorption and emission wavelengths of Cyanine 754 were tested by spectrophotometer.The sample of Cyanine 754 was incubated in cell culture mediums to identify no bacteria composition.The bacterial endotoxin in the sample of Cyanine 754 was tested by limulus test.The acute toxicity of the sample of Cyanine 754 was tested with high dose,median dose,and low dose in the animal experiment,which need to identify the living conditions,the liver function and renal function,and the drug residue in organs.The comparison of the fluorescence light intensity and the fluorescence tissue penetration were tested between Cyanine 754 and indocyanine green under the same energy excitation pulsed laser.The new polymethine dye Cyanine 754 was combined directly with the monoclonal antibody rituximab to produce the new fluorescence targeting tracer rituximab-Cyanine 754.The Cyanine 754 labelling rate of protein was analyzed by instant thin-layer chromatography-silica gel.The molecular integrity of rituximab was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The immunological activity of rituximab was tested by enzyme linked immune sorbent assay.The sample of rituximab-Cyanine 754was incubated in cell culture mediums to identify no bacteria composition.The bacterial endotoxin in the sample of rituximab-Cyanine 754 was tested by limulus test.The acute toxicity of the sample of rituximab-Cyanine 754 was tested with high dose,median dose,and low dose in the animal experiment,which need to identify the living conditions,the liver function and renal function,and the drug residue in organs.The comparison of the fluorescence light intensity and the fluorescence tissue penetration were tested between rituximab-Cyanine 754 and rituximab-indocyanine green under the same energy excitation pulsed laser.The animal model of sentinel lymph node in the rat hind limb was established by the blue dye with time window.The comparison of the identify in sentinel lymph node between Cyanine 754,rituximab-Cyanine 754,and rituximab-indocyanine green was tested by the animal model of sentinel lymph node.In the clinical validation study of rituximab-Cyanine754,12 patients with breast cancer would be enrolling（Shandong Cancer Hospital Research Ethics Board:No.SDTHEC201603008）.The rituximab-Cyanine 754 was compared with combination methods of sentinel lymph node biopsy and analyzed the agreement and correlation of these methods in the identification of sentinel lymph node.Results:The new polymethine dye Cyanine 754 was blue black and water-soluble powder.The aqueous solution color of Cyanine 754 was indigo,which had a max absorption wavelength in 754 nm and a max emission wavelength in 795 nm.After one week observation,the culture mediums of Cyanine 754 were not found color changing and bacterial growth.The Limulus tests results of Cyanine 754 were negative which means no bacterial endotoxin in the sample.The results of bacterial endotoxin test meet the standard of Chinese Pharmacopoeia 2015 Edition.The results of acute toxicity showed no animal dead in the group of high dose,median dose,and low dose.The results of liver function and renal function were not abnormal.The drug residue in organs was not found in heart,liver,kidney,and lung.Using the same energy excitation pulsed laser,the fluorescence light intensity and tissue penetration of Cyanine 754 was better than indocyanine green.The aqueous solution color of rituximab-Cyanine 754 was dark blue.In the rituximab-Cyanine 754,the Cyanine 754 labelling rate of protein was more than 99%,the rituximab kept the immune activity and immunological activity.After observation,the culture mediums of rituximab-Cyanine 754 were not found color changing and bacterial growth.The Limulus tests results of rituximab-Cyanine 754 were negative which means no bacterial endotoxin in the sample.The results of bacterial endotoxin test meet the standard of Chinese Pharmacopoeia 2015 Edition.The results of acute toxicity showed no animal dead in the group of high dose,median dose,and low dose.The results of liver function and renal function were not abnormal.The drug residue in organs was not found in heart,liver,kidney,and lung.Using the same energy excitation pulsed laser,the fluorescence light intensity and tissue penetration of rituximab-Cyanine 754 was better than rituximab-indocyanine green.The popliteal lymph node was identified as sentinel lymph node in the rat hind limb model of sentinel lymph node.In the animal model of sentinel lymph node,only made lymphatic vessel dyeing and imaging but not lymph node after Cyanine 754 injection,sentinel lymph node could be identified and biopsy could be completed guiding by the fluorescence imaging system after rituximab-Cyanine 754 injection.The tissue penetration of rituximab-Cyanine 754 was better than rituximab-indocyanine green in the hind limb model of sentinel lymph node.There were 12 cases with breast cancer involving in the clinical validation study,11 of them were identified the location of sentinel lymph node by the rituximab-Cyanine 754 and performed sentinel lymph node biopsy guiding by the fluorescence imaging system.was compared with combination methods of sentinel lymph node biopsy and analyzed the agreement and correlation of these methods in the identification of sentinel lymph node.The rituximab-Cyanine 754 has strong correlation and good agreement with combination methods in the identification of sentinel lymph node（Case-base,r_s=0.782,P=0.01;kappa=0.76,P<0.01）.Conclusions:1.The new polymethine dye Cyanine 754 takes the advantages for high water solubility,good optical stability,and high quantum yield.The fluorescence light intensity and the tissue penetration of Cyanine 754 are better than indocyanine green.2.In the rituximab-Cyanine 754,the Cyanine 754 labelling rate of protein is high,the rituximab keeps the immune activity and immunological activity.The fluorescence light intensity and the tissue penetration of rituximab-Cyanine 754 are better than rituximab-indocyanine green.3.The rituximab-Cyanine 754 is similar with the combination methods in the identification of sentinel lymph node and could be used to guide sentinel lymph node biopsy.