Clinical And Basic Research Of Intrabeam Intraoperative Radiotherapy In Nipple-Sparing Mastectomy For Breast Cancer

Operation methods for breast cancer include mastectomy and breast conserving surgery.Nipple-sparing mastectomy(NSM)preserves nipple areola complex(NAC)on the basis of traditional mastectomy.For patients who need total breast resection,NSM combined with breast reconstruction can better improve the aesthetics quality.However,the indications of NSM is still controversial.Radiation therapy for the NAC may play some roles in the oncologic safety of NSM.Intrabeam device is the most flexible intraoperative radiotherapy(IORT)device so far.Some studies have preliminarily proved its effectiveness in the local control for breast conserving surgery.The study aims to broaden the application of Intrabeam device for breast cancer,investigating the safety and feasibility of Intrabeam IORT in NSM with breast reconstruction,by means of clinical and basic research.Part ?.Clinical Study of Intrabeam Intraoperative Radiotherapy in Nipple-Sparing Mastectomy for Breast CancerThe study aims to broaden the application of Intrabeam device for breast cancer,investigating the safety and feasibility of Intrabeam IORT in NSM with breast reconstruction.From October 2015 to August 2018,34 female patients were enrolled in the study.All of them were admitted to the breast surgery department of the first affiliated hospital of Guangzhou Medical University.After NSM,Intrabeam IORT for the NAC was carried out,with a single dose of 16 Gy,followed by breast reconstruction.The primary endpoints were recurrence-free survival rate and NAC conditions.The secondary endpoints included:wound and prosthesis conditions,aesthetics quality,acute and chronic radiation injury,metastasis-free survival rate,and overall survival rate.The median age of the patients was 41 years old.In the 34 cases,the median intraoperative radiation time was 13 min 35 sec,no NAC was removed intraoperatively owing to the poor local bleeding.28 cases(82.4%)appeared mild-medium congestion or insufficiency of blood supply to the NAC in the first month postoperatively.Among which two cases(5.9%)appeared partial necrosis of the NAC,but neither needed NAC removal.The contours of the breasts(including the NAC)were satisfactory.The median follow-up time was 17 months.In all 34 cases,partial or total decrease of the NAC sensitivity were noted at the last visit,with "none","poor" and "medium"sensitivity scores for 20.6%,70.6%,and 8.8%of the cases,respectively.No recurrences or metastases were identified,however a breast-cancer mortality occurred in one patient.And no acute(<3 months)or chronic(>1 year)radiation injury to the heart,lung,or hematological system was observed.Additionally,paraffin pathological examination showed positive results in the retroareolar tissues in two cases.Although their NACs were still preserved,no recurrence or metastasis occurred with follow-ups of 25 months and 29 months,respectively.In short,Intrabeam IORT may be a feasible and safety approach for NSM with breast reconstruction in the selected patients with breast cancer.PART ?.A Mouse Model of Subclinical Breast CancerThe two major animal models for breast cancer research are 1)applying interventions after a solid tumor has formed and 2)inoculating tumor cells with an ex vivo manipulation.Both have limitations in simulating subclinical breast cancer.This study aimed to construct a mouse model of subclinical breast cancer.First,EMT6 cells were subcutaneously injected into the bilateral 4th mammary fat pads of six female BALB/c mice,using a modified inoculation method.Accurate measurements of the bilateral tumors were carried out.Then,another 24 mice were bilaterally inoculated EMT6 cells with different cell numbers.Unilateral resections of the 4th nipple and mammary fat pad were undertaken in each group in turn,with an interval of 24 hr.Serial sectioning was used for pathological assessments,and the growth status of EMT6 cells on the non-surgical side was used to simulate the surgical side(if resection had not been applied).We found that the modified inoculation method by subcutaneous injection was simple,safe and manifested a good consistency in bilateral tumor growth rates during the early stages of tumor formation.The differences in the physical characteristics of the bilateral tumors varied from 75%to 120%for each mouse.Additionally,by varying the number of injected cells,the times for forming palpable solid tumors of approximately 5 mm in maximum length were 5?6 days or 6?8 days after inoculation;for these models,the ideal time points representing subclinical stages were 24 hr and 72 hr after inoculation,respectively.Our study provides a novel and feasible mouse model of subclinical breast cancer.Part ?.Basic Research of Intrabeam Intraoperative Radiotherapy in Nipple-Sparing Mastectomy for Breast CancerThe study aims to manifest the histological changes of breast cancer cells and normal tissues after Intrabeam IORT in NSM,and evaluate the irradiation dose distributions.Additionally,the effects of Intrabeam irradiation on the biological functions and genetic changes of MCF-7 cells will also be investigated.The tissues from the bottom of the NAC flap and the breast tumor were examined by both optical microscopy and transmission electron microscopy.The irradiation dose distributions were simulated by the Intrabeam computer and measured by the thermoluminescence materials.The MCF-7 cells were used for in vitro study.Colony formation assay for the irradiated cells with single radiation doses ranged from 0-16 Gy was employed.Moreover,after irradiated with single radiation doses ranged from 0-6 Gy,cells were kept being cultured.24 hours later,the cell cycle distribution and apoptotic rate were measured using flow cytometry assay.About 4 weeks later,scratch test,transwell test,CCK-8 test and TUNEL staining were used to measure the migration,invasion,proliferation and apoptosis abilities;high-throughput sequencings were employed to detect the genetic changes(mRNA,miRNA,long non-coding RNA)in the 6 Gy group.Morphological changes in tumor cells and normal tissues were observed after a single-dose irradiation(16 Gy)immediately.Comparing to the NAC,the viscera beneath the chest wall received much lower irradiation doses.Colony formation assay revealed that irradiations with a single dose of 6 Gy or more could effectively kill and inhibit the MCF-7 cells.According to the results of flow cytometry assay,scratch test,transwell test,CCK-8 test and TUNEL staining,irradiations with a single dose of 2-6 Gy could inhibit proliferation and promote apoptosis and necrosis for the MCF-7 cells in the early stage after the irradiation.Additionally,the inhibition effects on migration,invasiveness and proliferation,and promotion effect on apoptosis existed till the late stage after single-dose irradiations;the larger irradiation dose it employed,the greater effect it appeared.The expressions of some mRNAs,miRNAs and long non-coding RNAs changed a lot 4 weeks after a single-dose(6 Gy)irradiation.In conclusion,the effect of single-dose(2-6 Gy)irradiations by the Intrabeam device for the MCF-7 cells can last for a long time.A single-dose irradiation(16 Gy)by the Intrabeam device can effectively kill and inhibit the MCF-7 cells.Additionally,even if a single irradiation dose of 16 Gy is delievered to the NAC intraoperatively,the irradiation doses at the viscera beneath the chest wall are very low.