| Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among females in 2018 which has a significant impact on women's health and quality of life in this decade.More and more breast cancer patients are able to eliminate the systemic micrometastases and degrade the primary lesions by receiving neoadjuvant chemotherapy treatment for surgery.For HER2 positive breast cancer,preoperative neoadjuvant chemotherapy treatment with trastuzumab will be the priority.The therapeutic response is evaluated by postoperative pathologic results.However,this method depends on the surgical sampling with a time lag which is not repeatable.So we need a simple,real-time and dynamic method to evaluate the patients' response.In recent years,circulating tumor DNA(ctDNA)is the research focus and has been widely used in clinical as one of the booming technologies-liquid biopsy.It comes from the tumor DNA in the blood circulation of patients,and it is able to be used for early diagnosis of tumors,disease monitoring,response evaluation,prognosis prediction with good reproducibility,less invasiveness,high sensitivity,powerful dynamic which has created a new way for individualized treatment of breast cancer.A large number of studies have indicated that the levels of total ctDNA and HER2 copy number in patients with advanced HER2 positive tumors are higher than the normal,and their fluctuations are able to reflect tumor progression and evaluate treatment response dynamically.However,in patients with neoadjuvant treatment,the tumor has not progressed to the advanced stage with low level of tumor burden and systemic metastasis.There were few studies on plasma ctDNA and HER2 copy number during this period,and the study concluded that there was no difference in total ctDNA and HER2 copy number between HER2 positive patients and healthy control group during neoadjuvant chemotherapy and HER2 copy number after treatment was significantly higher than the baseline level.This is controversial with previous conclusions on advanced tumors.Detection method of plasma HER2 copy number may effect the results at that time.So we will improve the detection technology and use the droplet digital PCR to detect the plasma HER2 copy number in an absolute quantitative way.Objective:Our study will apply the ddPCR to quantify plasma total ctDNA and HER2 copy number in HER2 positive breast cancer patients during neoadjuvant chemotherapy treatment with trastuzumab,and explore the capacity of the baseline level and changes of total ctDNA and HER2 copy number in breast cancer to diagnosis and evaluate treatment response,and the potential capacity of plasma HER2 amplification dectection to supplement tissue HER2 amplification dectection with the concordance of HER2 amplication in plasma and FFPE tissue.Method:1.Difference between plasma total ctDNA and HER2 copy number between HER2 positive breast cancer patients and the healthy control(1)43 HER2 positive breast cancer patients who were selected with pathological result of HER2 positive from June 2017 to February 2019 in the Department of Vascular Endocrime Surgery,Xijing Hospital,and 40 healthy volunteers were enrolled in the medical examination clinic of Xijing Hospital as the control group.(2)5 ml blood collected in 43 HER2 positive breast cancer patients before treatment(baseline)and 5 ml blood collected in 40 healthy volunteers during medical examination add up to 83 samples.(3)Quantify the total ctDNA in plasma and detect HER2 copy number in plasma by ddPCR.(4)Compare total ctDNA and HER2 copy number between HER2 positive breast cancer and the control group.2.Changes in plasma total ctDNA and HER2 copy number before and after treatment in HER2 positive breast cancer(1)30 HER2 positive breast cancer patients who have recieved the whole neoadjuvant chemotherapy treatment with trastuzumab were included in the study,and 13 patients were excluded from the 43 patients of part one.(2)30 samples of 5ml blood after treatment(preoperative)were collected.(3)Quantify the total ctDNA in these samples and detect HER2 copy number in plasma by ddPCR.(4)Compare plasma total ctDNA and HER2 copy number before and after treatment in HER2 positive breast cancer.3.Concordance of HER2 amplification in FFPE tissue and plasma in HER2 positive breast cancer(1)Collect 30 FFPE tissues which were paired the above 30 baseline plasma samples in HER2 positive breast cancer from the Department of Pathology of Xijing Hospital.(2)Extract DNA from FFPE tissue.(3)Detect HER2 amplification in FFPE tissue by ddPCR.(4)Analyze the concordance of HER2 amplification between FFPE tissue and baseline plasma by ddPCR.Result:1.43 female patients with HER2-positive breast cancer and 40 healthy women were included.Clinical characters: age,hormone receptor,tumor size,lymph node metastasis were not correlated with baseline total ctDNA(P values =0.43,0.16,0.70,0.38 respectively)and the baseline level of HER2 copy number(P values = 0.95,0.75,0.94,0.79,respectively).The median total ctDNA of breast cancer patients was 434.0 ng/ml,95% CI 465.2-859.7 ng/ml,and the median total ctDNA of healthy people was 356.0 ng/ml,95% CI 334.0-475.7ng/ml.There was a significant difference between the two parameters(P?0.05).The median HER2 copy number of breast cancer patients was 1.3,95% CI 1.2-1.36,and the median HER2 copy number of healthy control was 0.98,95% CI 0.95-1.03,and there was a significant difference between the two parameters(P?0.05).2.After neoadjuvant chemotherapy treatment with trastuzumab,13 patients were excluded and 30 patients were included.Clinical features: age,hormone receptor,tumor size,lymph node metastasis,chemotherapy programs were neither correlated with total ctDNA before and after treatment nor plasma HER2 copy number before and after treatment(P>0.05).There was no significant difference in the total ctDNA before and after treatment(P?0.05),but there was a significant difference in HER2 plasma copy number before and after treatment(P?0.05)with a decline result.The software calculated a ddPCR ratio cutoff of 1.25 which was used to divide “amplified” from “non-amplified” in HER2 copy number.10 patients obtained pCR which included 7 baseline HER2 copy number amplified and 3 baseline HER2 copy number non-amplified,and 20 patients didn't obtained pCR which included 11 baseline HER2 copy number amplified and 9 baseline HER2 copy number non-amplified.There was no significant difference in baseline HER2 copy number amplification between patients with or without pCR.There was also no significant difference in baseline HER2 copy number between patients with or without pCR.3.The ratio cutoff of HER2 copy number by ddPCR in FFPE tissue is 2.1.Kappa test analysis showed that the Kappa value of HER2 amplification in FFPE tissue and HER2 amplification in plasma at baseline was 0.12,indicating that the concordance of the two methods was low.Conclusion:1.The total ctDNA at baseline in HER2 positive breast cancer patients is higher than the control(P?0.05).HER2 copy number in HER2 positive breast cancer patients was also significantly higher than the control(P?0.05),and it will help to diagnose HER2 positive breast cancer.Clinical features: Age,hormone receptor,tumor size,lymph node metastasis were not correlated with baseline total ctDNA and HER2 copy number.2.There was no significant difference in total ctDNA between HER2 positive breast cancer patients before and after treatment,while HER2 copy number decreased significantly which has reflect the treatment response.However,there was no difference in the baseline HER2 copy number between the patients with pCR and the patients without pCR,indicating that single plasma HER2 copy number could not be used to predict the patient's treatment response.3.The concordance between plasma baseline HER2 copy number and FFPE tissue HER2 copy number was low.Plasma HER2 detection by ddPCR is not a supplement to tissue HER2 detection by ddPCR. |
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