Basic And Clinical Research Of Circulating Tumor DNA As The Marker For Breast Cancer Progression

Breast cancer is the malignant tumor with the highest incidence in the world.Thanks to the advanced diagnosis and treatment technology,the survival of breast cancer patients extended.The treatment of breast cancer nowadays targets to precision and personalization.Circulating tumor DNA（ct DNA）is derived from tumor cells with characteristics of non-invasive detection and continuous long-term monitoring.At present,most of the reported studies on the correlation between ct DNA and breast cancer are clinical studies.The value of clinical research is important while with lots of limitations,therefore,we can avoid the unfavorable factors and complete the research goals by designing experiments in animal.Therefore,the research studied on the molecular characteristics of ct DNA and its correlation with breast cancer progression on genetic,animal and clinical levels,we aim to expound the scientific nature of the application of ct DNA as a marker in the diagnosis and treatment of breast cancer patients,and to provide a scientific basis for the clinical application of ct DNA in the diagnosis and treatment of breast cancer.First,we studied the relationship between ct DNA and breast cancer treatment.Using the mouse breast cancer cell 4T-1,we successfully established the in situ mouse breast cancer model with competent immune function.The tumor-bearing mice were treated with chemotherapeutic drugs and surgical treatments,and the changes in ct DNA content were monitored,and the survival of the mice was observed.The results showed that the breast cancer mice whose ct DNA turned negative after treatment had a longer survival period,indicating that the positive detection of ct DNA after treatment was an indication of poor prognosis.At the same time,we found that the ct DNA of mice bearing breast cancer and clinical breast cancer patients became negative after surgery,while the ct DNA of mice with residual lesions remained positive after surgery,which indicates that if ct DNA is detected in the blood after surgery,It should be highly suspected of residual lesions after surgery.At the same time,in order to verify the role of ct DNA in monitoring recurrence and metastasis in mice,we performed CT scan and ct DNA detection on mice after surgery.The data showed that the detection rate of ct DNA for metastases was 100%and the specificity was 67%.These data indicate that ct DNA can be used as an effective diagnostic marker for breast cancer recurrence and metastasis after surgery.At the same time,we recommend combining imaging examinations in clinical applications to avoid false positive results.Next,in order to study whether ct DNA can be used to diagnose the status of pathological complete response（p CR）after neoadjuvant chemotherapy（Na C）,we performed CT scans on the mice whose tumors disappeared after Na C,and then took blood from the mice to detect ct DNA.We also took the skin and subcutaneous tissues at the primary site of the tumor for pathological examination.We found that these mice had achieved clinical complete response（c CR）by imaging examination,their ct DNA test was negative,and the pathological examination also confirmed that they had reached p CR.These data indicate that ct DNA can be used as an effective marker to determine whether breast cancer mice have reached p CR after Na C.While conducting animal-level research,we have also carried out clinical research.In order to verify the consistency of the ct DNA gene mutation profile of clinical patients with tumor tissues,we used Next-generation sequencing（NGS）method to sequence and compare the ct DNA,tumor tissues and normal tissues of newly diagnosed breast cancer patients.The results showed that the detection rate of blood ct DNA is 100%,and the gene mutation profile of ct DNA is highly consistent with tumor tissue（97%-100%）,indicating that ct DNA can be used as a clinical diagnostic marker for breast cancer.Furthermore,Based on the sequencing results,we found that the mutations of AKAP9^R1276Qand AKAP9^M1400Twere highly correlated with triple negative breast cancer（TNBC）.AKAP9 gene is highly expressed in TNBC cell lines.Knockdown of AKAP9 can inhibit the proliferation and migration of TNBC cells MDA-MB-231.Through TCGA database analysis and enlarged patient sample sequencing,we confirmed that the AKAP9 mutation is a high-frequency mutation of TNBC,but the effect of the mutation on the progression of TNBC and its mechanism need to be further studied and verified.In summary,our results show that ct DNA can monitor tumor burden,tumor residual,and tumor recurrence during the entire course of breast cancer treatment.It is a highly accurate breast cancer liquid biopsy diagnosis and prognostic marker.