| Part 1The role of systemic inflammatory and coagulatory markers as surrogate biomarkers of response and prognosis in neoadjuvant chemotherapy of breast cancerBackground and Objective:Neoadjuvant chemotherapy(NAC)is the standard treatment in locally advanced breast cancer.Meanwhile,it significantly improves the rate of breast conservation and reduces the postoperative recurrence risk.Inflammation,immune system and coagulation participate the process of cancer initiation,invasion,metastasis,etc.Recently,systemic inflammation markers such as neutrophil-to-lymphocyte ratio(NLR),platelet-to-lymphocyte ratio(PLR)and coagulatory markers such as fibrinogen(FBG)showed predictive value in therapeutic response and prognosis of NAC in breast cancer and other solid tumors.However,due to the small amount,the results were not consistent.In the present study,we aimed to evaluate the therapeutic and prognostic significance of baseline level and change of NLR,PLR,FBG in a large cohort of breast cancer patients who underwent NAC.Additionally,another goal was to build up a prognostic score system combining systemic inflammatory and coagulatory biomarkers to determine whether to reinforce the adjuvant therapy or not.Methods:This was a retrospective study of breast cancer patients who underwent NAC and subsequent surgery between January 2000 and October 2017 in Cancer Hospital Chinese Academy of Medical Sciences.Totally 635 patients who had pre-NAC complete blood count(CBC)were included in the study of NLR and PLR,377 patients who had the results of pre-NAC and pre-operation FBG were enrolled in the study of fibrinogen.We evaluated baseline NLR,PLR,FBG and their changes with clinicopathological parameters,therapeutic response,relapse-free survival(RFS),breast specific cancer survival(BCSS)and overall survival(OS).Both univariate and multivariate analysis were performed to evaluate the independent prognostic significance of NLR,PLR and FBG change.Results:1)Baseline NLR,PLR was observed no effect for pCR and overall response either as a continuous or categorical parameter(p>0.05);Decreased PLR after NAC appeared to have lower likelihood of achieving pCR than increased PLR(21.2%vs.10.9%,p=0.001).2)The optimal cutoff for NLR was 2.3.Compared to high NLR group(NLR≥2.3),low NLR group had more patients with histological Grade 1/2(53.3%vs.36.2%,p=0.041);The optimal cutoff for PLR is 118.5,though not significant.Compared to high PLR group(PLR≥118.5),low NLR group(PLR<118.5)have more patients>50 years old(53.3%vs.36.2%,p<0.001),menopausal women(51.1%vs.34.7%,p<0.001),histological grade G1/2(57.6%vs.45,1%,p＝0.010).3)Low NLR group was comfirmed to have better RFS(p=0.007),BCSS(p＝0.003)and OS(p=0.013)than high NLR group;Multivariate analysis revealed that NLR<2.3 was the independent favourable factor of RFS(p=0.046,HR 1.375[95%CI 1.006-1.880])and BCSS(p=0.049,HR 1.467[95%Cl 1.002-2.148]),but not OS.Low NLR was associated with significant favourable prognosis in ER/PgR negative subgroup and non-pCR group(p<0.05),according to univariate analysis.There was no statistical significance between low PLR and high PLR group in RFS,BCSS and OS(p>0.05).However,low PLR group had longer RFS(p=0.009)and BCSS(p=0.010)in TNBC subgroup.4)The baseline FBG value was higher in patients ≥50 years old,menopausal women,BMI≥24,histological grade G3、ER/PgR negative patients and patients who could achieve pCR(p<0.05),Logistic univariate analysis revealed that higher baseline FBG was related with higher pCR rate when evaluated as a continuous variable(p= 0.030,OR 1.520[95%CI 1.040-2.220]),though not remained statistically significant when adjusted ER/PgR status and histological grade(p=0.332).5)It is found that decreased FBG group(FBG decreased≥0.2g/L)had a higher pCR rate than increased or stable group(20.7%vs.11.9%,p=0.039),but was not independent predictive factor of pCR in multivariate Logistic analysis(p=0.117).However,it is shown that FBG decreased>0.2g/L was the independent predictive factor of achieving pCR after adjusting other variates(p=0.030,OR 5.877[95%CI 1.187-29.087])in TNBC;Deceased FBG group had a favourable RFS(p=0.025,HR 1.819[95%CI 1.079-3.066])and BCSS(p=0.022,HR 2.507[95%CI 1.236-5.084])in multivariate analysis,and decreased FBG had significantly favorable RFS(p=0.013)and BCSS(p=0.013)in ER/PgR negative breast cancer.It was observed more significantly different in RFS(p=0.016)and BCSS(p=0.019)if FBG decreased>0.5g/L was set as the cutoff point.6)There were more patients of Ki67>14%in △FBG-NLR score 1 and 2 than those in score 0(66.1%.60.2%vs.51.1%,p=0.008).It was observed that △FBG-NLR score 0,1,2 had significantly different RFS(p<0.001)and OS(p<0.001).Multivariate analysis showed that score 2 was the independent factor of inferior RFS(p = 0.002)and BCSS(p = 0.003),which is meaningful in different subgroup.Conclusion:These results indicated that baseline NLR,PLR,FBG were not correlated to pCR and overall response.However,it was more likely to achieve pCR when PLR was decreased after NAC.FBG decreased>0.2g/L was the independent predictive factor of achieving pCR in TNBC subtype.Baseline NLR<2.3 and decreased FBG group revealed better prognosis in patients who underwent NAC.It was promising that the combination of baseline NLR and △FBG as a useful prognostic classification tool in neoadjuvant chemotherapy of breast cancer could be helpful in decision-making of adjuvant settings.Part 2High-throughput sequencing of plasma circulating tumor DNA in response to neoadjuvant therapy in triple negative breast cancerBackground and Objective:Triple negative breast cancer(TNBC)is a heterogeneous and progressive subtype of breast cancer,lack of expression of estrogen receptor(ER)and progesterone receptor(PgR),and lack of overexpression or amplification of human epidermal growth receptor-2(HER-2)expression.TNBC represents a highly relevant subgroup given that patients with the disease do not benefit from endocrine or anti-HER2 therapy,currently,chemotherapy is the only therapeutic option.Pathologic complete response(pCR)after neoadjuvant chemotherapy correlated with improved prognosis.Circulating tumor DNA(ctDNA)oringinates from primary tumor,circulating tumor cells and micrometastasis,carrying genetic and epigenetic information.ctDNA testing is a kind of non-invasive,repeatable technique.This study aimed to reveal response related genomic characteristics in plasma and monitor the dynamic change in levels of ctDNA in order to identify potential predictive biomarkers in neoadjuvant chemotherapy.Methods:We enrolled 9 TNBC patients treated with neoadjuvant chemotherapy from a registered study.All patients received paclitaxel and carboplatin in combination.Baseline plasma and plasma after treatment were sampled.Next-generation sequencing was performed to identify genomic alteration.Results:TP53 mutation was the most frequently mutated genes in plasma in 55.56%(5/9)of patients.MYC,MET,PIK3C2B,SDHA,DNMT3A,ASXL1 and SETD2 mutations were only detected in pCR/CR group or SD/PD group at least twice.In the pathway richment analysis,the highest rich rate is the PI3K-AKT pathway,7 of 11 genes in this pathway is related to SD/PD;TP53 mutations were detected in 3 patients in pCR/CR group and 2 patients in SD/PD group respectively,however,only 2 patients in pCR/CR group were detected with frameshift mutations.The mutations with highest allele frequency(AF)at baseline in pCR/CR group were all turned to negative after treatment,but it was still positive for 2 patients in SD/PD group(2/4).TP53 mutation dynamics showed all 3 patients in pCR/CR group with TP53 mutations turned to negative when tested at last and there was 1 of 2 patients in SD/PD group with TP53 mutation positive after treatment.We found two patients with pathogenic BRCA1 germline mutation were both in pCR/CR groups.Conclusion:The most frequent mutated gene in plasma ctDNA was TP53 in TNBC.Gene MYC,MET,PIK3C2B,SDHA,DNMT3A,ASXL1,SETD2 could be candidate mutation in predicting treatment response,mutations in PI3K-AKT pathway could be associated with poor response of neoadjuvant chemotherapy.TP53 mutation status was not different in pCR/CR group and SD/PD group,though,TP53 frameshift mutations which lead to change of protein function might associated with pCR;In analysis of ctDNA dynamics,the highest allele frequency(AF)mutations at baseline or TP53 mutations ctDNA remaining positive may indicate a poor response of neoadjuvant chemotherapy.BRCA1 germline mutations played an important role in predicting pCR. |
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