Influencing Factors And Predictors Of Effect Of Pathological Complete Response Of HER2-positive Breast Cancer Treated With Neoadjuvant Treatment And Mechanism Of Drug Resistance

In 2020,the number of new cases of breast cancer in the world reached2.26 million,making it the cancer with the highest incidence.Human epidermal growth factor receptor 2(HER2)positive breast cancer accounts for15%-25%of all breast cancers.It has the characteristics of strong invasiveness and poor prognosis.Targeted drugs improve the survival of patients with HER2-positive breast cancer.Neoadjuvant therapy can improve the tumor resection rate and breast preservation rate.Studies have shown that achieving pathologic complete response(p CR)will achieve longer survival,and p CR has also become a reliable prognostic indicator.Trastuzumab and pertuzumab combined with chemotherapy can increase the p CR rate of HER2-positive breast cancer treated with neoadjuvant therapy and has become a standard treatment.Pertuzumab started to clinical application in March 2019 in our country.In our real world,double targeted combined with chemotherapy research data is less.This study was to explore the p CR rate of double target in the real world,evaluate curative effect and adverse reactions,and analyze the related factors influencing the p CR.Neoadjuvant therapy of breast cancer had a higher control to the axillary lymph nodes.For part of patients with clinical axillary lymph node positive before the neoadjuvant therapy transferred to negative after neoadjuvant therapy,sentinel lymph node biopsy(SLNB)in eligible patients can replace axillary lymph node dissection(ALND).Axillary pathological complete response(apCR)help doctors to judge prognosis and guide the follow-up whether rising ladder intensive treatment.In this study,we study the related factors in HER2-positive lymph node positive breast cancer affecting apCR.At the same time,we analyzed the ap CR rate of patients with c T1~3 N1 and influencing factors of ap CR,provide data support for the patients with SLNB.As the population ages,elderly patients with breast cancer increase corres-pondingly.Because elderly patients are under-represented in clinical trials,so the available data are limited.We analyzed the efficacy and safety of neoad-juvant therapy of dual target combined chemotherapy in elderly aged 60 and above patients with HER2-positive breast cancer and analyze the impact on the tp CR.Tumor associated inflammatory mediators produced by tumor cells affect the antitumor activity of the drug.Peripheral blood lymphocytes can response the body immune state,assessment of peripheral immune cells,also can to a certain extent,reflects the curative effect of neoadjuvant therapy.Study found that tumor infiltrating lymphocytes(TILs)can predict the curative effect of neoadjuvant therapy and longer long-term survival in HER2-positive and Triple negative type of patients with breast cancer,but the related research is still less.In order to further explore the predictors of p CR in HER2-positive breast cancer treated with neoadjuvant therapy,we studied the effect of the 13kinds of the expression of inflammatory cytokines in peripheral blood and immune cells subgroup and TILs in tumor tissues on p CR.Trastuzumab resistance is an important factor that leading to non-p CR of neoadjuvant treatment in HER2-positive breast cancer.Tumor microenvironment in breast cancer metastasis plays an important role.In this study,we use single-cell sequencing to study tumor microenvironment of HER2-positive breast cancer and explore the mechanisms of trastuzumab resistance.This study is divided into four parts to study the influence factors of pathologic complete response in HER2-positive breast cancer treated with neoadjuvant therapy,predictors of curative effect and mechanism of drug resistance.Part One Real-world study of trastuzumab and pertuzumab combined with chemotherapy in neoadjuvant treatment for patients with HER2-positive breast cancerObjective:To analyze the influencing factors of trastuzumab(H)and pertuzumab(P)combined with chemotherapy in neoadjuvant therapy for human epidermal growth factor receptor 2(HER2)positive breast cancer and observe the efficacy and safety in the real world.Methods:Clinical data from HER2-positive breast cancer patients who received HP combined with chemotherapy as neoadjuvant therapy at the Breast Cancer Center of the Fourth Hospital of Hebei Medical University from March 2019 to February 2022 were retrospectively analyzed.Adverse reac-tions were evaluated using CTCAE version 5.0.The primary endpoint was total pathologic complete response(tp CR;yp T0/isyp N0),and the secondary endpoints were breast pathologic complete response(bp CR;yp T0/is)and axillary pathologic complete response(ap CR;yp N0).Factors influencing tp CR and bp CR were also analyzed.Correlation analysis was performed to explore the factors affecting the ap CR rate of node-positive patients.Results:1.A total of 302 patients were included in the analysis,of which 145were treated with H+P+taxane+carboplatin(Tcb HP),94 with H+P+taxane(THP)and 63 with sequential anthracycline and cyclophosphamide,followed by H+P+taxane(AC-THP).The overall tp CR rate was 64.9%,and those of Tcb HP,THP,and AC-THP were 73.1%,52.1%,and 65.1%,respectively,P=0.004,and was statistically significant.The bp CR rate was73.5%,those of Tcb HP,THP,and AC-THP were 79.3%,60.6%,and 79.4%,respectively,P=0.003,and was statistically significant.The tp CR rate of the hormone receptor(HR)negative group(80.3%)was higher than that of the HR positive group(52.1%).The overall bp CR rate was 73.5%and the ap CR rate was 75.8%.2.In the univariate analysis,HR,HER2 status and treatment regimen were related factors that affected tp CR and bp CR.In the multivariate analysis,HR,HER2 status and treatment regimen were independent predictors of tp CR(P(27)0.001,P(27)0.001 and P=0.009)and bp CR(P(27)0.001,P(27)0.001 and P=0.003).3.Among 281 patients with node-positive,HER2-positive breast cancer,the overall ap CR rate was 74.4%,the bp CR rate was 73.0%and the tp CR rate was 63.7%.In the univariate analysis,hormone receptor(x~2=17.078,P<0.001),HER2 status(x~2=44.016,P<0.001)and bp CR(x~2=66.595,P<0.001)were statistically significant correlated factors that affected ap CR.In the multivariate analysis,HER2 status(OR=4.439,95%CI 1.798-10.959,P=0.001)and bp CR(OR=6.136,95%CI 3.096-12.160,P<0.001)were indepen-dent predictors of ap CR.4.The ap CR rate of 126 T1-3N1 patients was 73%(92/126).The ap CR rate of HER2 IHC(3+)patients(79.8%)was higher than that of IHC(2+)/FISH(+)patients(29.4%),x~2=18.964,P<0.001.The ap CR rate of patients with bp CR(89.7%)was higher than that of patients without bp CR(35.9%),x~2=39.498,P<0.001.5.Most the toxicities were level 1 and 2.The levels 3 and 4 toxicities rates of Tcb HP were slightly higher than those of THP and AC-THP.Conclusions:1.HP combined with chemotherapy has achieved a high p CR rate.The Tcb HP regimen has the highest p CR.HR-negative tumors demonstrate a higher p CR.2.HR,HER2 status and treatment regimen are independent predictors of tp CR and bp CR.3.HER2 status and bp CR are independent predictors of ap CR in node-positive patients.4.T1-3N1 stage breast cancer patients have higher ap CR rate and are suitable for sentinel lymph node biopsy instead of axillary lymph node disse-ction.5.The adverse reactions are controllable.Part Two Curative Effect Observation and Influencing Factors Anal-ysis of Elderly HER2-positive Breast Cancer Treated with Trastuzumab and Pertuzumab combined with Chemothe-rapy as Neoadjuvant TreatmentObjective:To observe the curative effect of elderly HER2-positive breast cancer treated with trastuzumab and pertuzumab(HP)combined with chemotherapy as neoadjuvant therapy and analyze the influencing factors of tp CR.Methods:The clinical data of 63 patients aged 60 and above with HER2-positive breast cancer who received HP combined with chemotherapy as neoadjuvant therapy and completed surgery in the Breast Center of the Fourth Hospital of Hebei Medical University from March 2019 to February2022 were retrospectively analyzed.We observe the curative effect and analyze the relationship between clinical pathological characteristics of breast cancer patients and their tp CR rate.Results:1.Among 63 patients,22 cases(34.9%)were treated with H+P+taxane+carboplatin(Tcb HP),29 cases(46.0%)with H+P+taxane(THP)and 12cases(19.0%)with sequential anthracycline and cyclophosphamide,followed by H+P+taxane(AC-THP).2.There were 34 cases(54.0%)with CR,22 cases(34.9%)with PR,7cases(11.1%)with SD,no case with PD and the total effective rate(CR+PR)was 88.9%.The tp CR rate was 58.7%,the bp CR rate was 68.3%and the ap CR was 73.0%.3.In the univariate analysis,age(x~2=7.102,P=0.008),hormone receptor(x~2=4.926,P=0.026)and HER2 status(x~2=8.590,P=0.003)were statistically significant correlated factors that affected tp CR.In the multivariate analysis,age(OR=4.612,95%CI:1.357-15.668,P=0.014)and HER2 status(OR=8.103,95%CI 1.681-39.068,P=0.009)were independent predic-tors of tp CR.4.Most of the adverse reactions related with neoadjuvant therapy in elderly HER2-positive breast cancer were level 1 and 2 grade.Conclusions:1.HP combined with chemotherapy has achieved a high tp CR rate in elderly HER2-positive breast cancer patients.2.Age and HER2 status are independent predictors of tp CR.3.The adverse reactions in elderly patients are controllable.Part There The study of predictors of effect on pathological complete response of HER2 positive breast cancer treated with carboplatin-based neoadjuvant therapyObjective:To explore the predicting role of plasma inflammatory cyto-kines,peripheral blood immune cell subsets and tumor-infiltrating lympho-cytes(TILs)in tumor tissues on pathological complete response(p CR)of patients with HER2-positive breast cancer treated with docetaxel-carboplatin-based neoadjuvant therapy.Methods:13 cytokines(including IL-1β,IFN-ɑ,IFN-γ,TNF-ɑ,CCL-2,IL-6,IL-8,IL-10,IL-12p70,IL-17A,IL-18,IL-23 and IL-33)in plasma and peripheral blood immune cells populations(total lymphocytes,CD3~+T cells,B cells,NK cells,NK-T cells,CD4~+T cells,CD8~+T cells,Treg,CD3~+PD-1~+T cells,CD4~+PD-1~+T cells,CD8~+PD-1~+T cells,CD4~+CD8~+T cells,CD4~-CD8~-T cells),as well as TILs in tumor tissues were evaluated in HER2-positive cases treated with 6 cycles of TCb H(docetaxel,carboplatin and trastuzumab)neoadjuvant therapy.Pearson chi-square test or Fisher’s exact test were used to assess the ratio of the difference between two groups,Multivariate analysis used Logistic regression model.Correlations among biomarkers and p CR were analyzed.Results:1.Totally 42 patients were enrolled and 18 cases achieved p CR at 42.9%(18/42)after 6 cycles of TCb H neoadjuvant therapy,with an ORR of 88.1%(37/42).Compared with non-p CR cases,patients achived p CR expressed significantly higher plasma level of TNF-ɑ(P=0.013),IL-6(P=0.025)and lower plasma level of IL-18(P=0.0004).Univariate analysis proved that IL-6(OR,3.429;95%CI,1.838-6.396;P=0.0001)was significantly associated with p CR.2.Peripheral blood immune cell populations of 13 subsets were also evaluated ahead of neoadjuvant therapy administration.Patients in p CR group displayed significantly higher level of NK-T cells population(P=0.009)and lower CD4~+T versus CD8~+T ratio(P=0.0014)before neoadjuvant therapy.Higher NK-T cells population(OR,0.204;95%CI,0.052-0.808;P=0.018),lower CD4~+T versus CD8~+T ratio(OR,10.500;95%CI,2.475-44.545;P=0.001)were found to be significantly associated with p CR by univariate analysis.3.TILs expression(OR,0.192;95%CI,0.051-0.731;P=0.013)in tumor tissues were found to be significantly associated with p CR by univariate analysis.Conclusions:1.Compared with non-p CR cases,patients achived p CR have signifi-cantly higher plasma level of TNF-ɑ,IL-6 and lower plasma level of IL-18.Univariate analysis proves that IL-6 is significantly associated with p CR.2.Higher level of NK-T cells population and lower CD4~+T versus CD8~+T ratio before neoadjuvant therapy are related with p CR of patients.3.TILs expression are significant predictors for TCb H neoadjuvant thera-py response in HER2-positive breast cancer patients.Part Four The study of drug resistance to trastuzumab by means of single-cell sequencing technologyObjective:To explore the mechanism of drug resistance to trastuzumab,we observe the tumor microenvironment in HER2-positive breast cancer by means of single-cell sequencing technology.Methods:In this study,totally 6 HER2-positive breast cancer patients who received standard neoadjuvant trastuzumab therapy were included.Acco-rding to the efficacy,these patients were divided into two groups:patients who responded to trastuzumab,including complete response and partial response,as well as patients who did not respond to trastuzumab,consisting of stable disease and progressive disease.TME in HER2-positive breast cancer patients were analyzed by means of single-cell sequencing technology and Gene set enrichment analysis.Results:1.Through the analysis of the differentially expressed genes(DEGs),12major cell types were found from those tumor specimens:breast cancer cells(express EPCAM),NK cells(express GNLY,XCL2,NKG7 and KLRD1),CAFs(express COL1A1,COL3A1,THY1 and FAP),adipocyte stem cells,B cells,monocytes,basophils,macrophages,CD4~+T cells,dendritic cells,CD8~+T cells,and endothelial cells.2.Gene Set Enrichment Analysis(GSEA)hinted the NK cells from the trastuzumab responders groups exhibit significantly higher cytotoxic activity than NK cells from the non-responders group.3.The expression of marker genes of CAFs showed that membrane protein PDPN was up-regulated in the trastuzumab non-responders group compared with that of the responders group,suggesting that the subset of CAFs expressing PDPN may participate in resistance to trastuzumab in patients with HER2-positive breast cancer.Conclusions:1.In this study,12 major cell types are found from those tumor specimens in HER2-positive breast cancer.2.The NK cells from the trastuzumab non-responders groups exhibit significantly lower cytotoxic activity.3.Podoplanin-positive(PDPN~+)cancer-associated fibroblasts(CAFs)subset is enriched in trastuzumab resistant tumor tissues.