Developing New Methods For Quantifying Antitumor Drug Concentrations And Screening Biomarkers For Early Diagnosis Of Colorectal Cancer

The present study was constituted of two parts:firstly,ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)methods were developed for quantifying epithelial growth factor receptor-tyrosine kinase inhibitor(FCN-411)and PD1 monoclonal antibody(toripalimab)concentrations in human plasma.The two methods provided a basis for analyzing clinical pharmacokinetic(PK),pharmacodynamic(PD)study and monitoring of therapeutic drug concentration(TDM)of the two drugs;secondly,the high-throughput protein microarray was applied to detect plasma autoantibody levels in colorectal cancer(CRC),benign colorectal tumor and healthy controls,then screen and validate these autoantibodies as promising biomarkers for early diagnosis of colorectal cancer and differential diagnosis of benign and malignant colorectal tumors.Section 1:Developing methods to detect FCN-411 and toripalimab levelsChapter 1:Developing and validating a UPLC-MS/MS method to quantify FCN-411 in human plasma.Protein precipitation procedure was used to pretreat plasma samples.The method was validated in accordance with the guidelines for bioanalytical method validation.It yielded a good linearity in the range of 2-500 ng/mL;the intra and inter accuracy and precision were 9.18%～17.46%and-17.50%～7.14%for the low limit of quantification samples(LLOQ),and 4.24%～14.35%and 2.00%～11.89%for the high,medium and low quality controls(QCs),respectively;the mean values of the internal standard normalized matrix factors of the low and high QCs were 101.67%and 94.74%,with the coefficients of variation of 4.69%and 2.27%,respectively;no significant matrix difference was observed;the relative extraction recoveries of FCN-411 in low and high QCs were both between 85%and 100%,whose coefficients of variation were 5.78%and 1.23%,respectively;plasma samples were stable when preserved under the following conditions:ambient temperature for 2 h,-80℃ for 7 days,three freeze-thaw cycles,and extracts in 10℃ autosampler for 26 h.Besides,FCN-411 and internal standard in stock solution stored in-20℃ were both stable for 4.8 months.This method was successfully used to detect a clinical plasma sample from a patient administrated 4 mg dose in a phase I clinical trial,which demonstrated a good clinical application.The method was ready to be applied to measure FCN-411 in subsequent clinical pharmacokinetic study.Chapter 2:Development and validation of a UPLC-MS/MS method for detecting toripalimab in human plasma,in comparison with electrochemiluminescence immunoassay(ECLIA).A unique,sensitive and stable peptide was selected as a surrogate peptide of toripalimab to be monitored.Meanwhile,an extended stable isotope-labeled surrogate peptide was synthesized as internal standard.The method was fully validated and successfully applied to determine 77 plasma samples from 29 patients in a phase I clinical trial.Afterwards,method comparison was performed based on 56 samples,which had been previously measured by ECLIA.A good consistent trend observed between the two methods(Spearman correlation r=0.96),but constant and proportional biases were observed by Passing-Bablok regression and Bland-Altman analysis.This discrepancy could be mainly attributed to different forms of toripalimab determined:free and total forms of drug were quantified by ECLIA and UPLC-MS/MS,respectively.As a result,this UPLC-MS/MS method may be complementary to ECLIA to monitor different forms of toripalimab.Section 2:Study on plasma autoantibodies related to early detection of CRCChapter 1:Meta-analysis of anti-p53 autoantibodies for exploring the diagnostic value on colorectal cancer.The PubMed,Cochrane Library,and Embase databases were searched to explore the diagnostic potential of autoantibodies on CRC.A total of 145 autoantibodies published in 80 articles was reviewed.About 70%of the articles reported a sensitivity lower than 30%,and about 80%showed the specificity more than 90%.Of these,anti-p53 antibody was the most commonly studied,thus we performed a meta-analysis on anti-p53 antibody in 27 studies from 24 articles.According to the different cut-off difinitions,we divided the included studies into five groups.Owing to the presence of threshold effect and non-threshold effect in the other four groups,pooled analysis was focused on group 3 using a fixed-effect model.Group 3 was comprised of five articles including 733 patients with CRC and 172 controls(126 healthy individuals and 46 benign diseases).The pooled sensitivity,specificity,positive and negative likelihood ratios,diagnostic odds ratio,and summary area under the receiver operating characteristic curves were 0.21,0.99,12.26,0.80,15.46,and 0.87,respectively.Serum anti-p53 autoantibody may potentially help distinguish CRC from healthy controls or benign diseases.Additionally,further study was needed to explore optimized combination with other markers due to the low sensitivity.Chapter 2:Screening autoantibody for early diagnosis of CRC based on high-throughput protein microarray.In the discovery phase,35 patients with CRC(TNM stage Ⅰ-Ⅱ),25 patients with benign colorectal tumors and 20 healthy controls were included.And 198 differential autoantibodies were screened.In the validation phase,305 patients with CRC(stage Ⅰ-Ⅱ),61 patients with benign colorectal tumors and 240 healthy controls were included to validate these differential autoantibodies.Firstly,we assessed 4 positive cut-off definitions based on data from early CRC and healthy controls,and found Youden index+at least 90%specificity method could ensure high specificity and sensitivity.Thus,this method was used to assess diagnostic efficacies of autoantibodies.The results were as follows:(1)11 autoantibodies related to CRC early detection were obtained,and the positive rates of single autoantibody were between 12.46%and 20.00%,of which p53,GATA6,MAP3K5,SOX9-AS1_frag and TIMM8A were selected in a Logistic regression model with the sensitivity of 51.47%and specificity of 75.42%;(2)13 autoantibodies for distinguishing early CRC from benign colorectal tumors were obtained,and the positive rates were between 14.10%and 28.20%,of which HIST1H1C,OSGIN1,CRY2,PRR7,REG3 A and SLC43 A2 were included in a Logistic regression model with the AUC of 0.78,sensitivity of 74.10%and specificity of 70.49%;(3)13 autoantibodies related to benign colorectal tumors were obtained,and the positive rates were between 1.64%and 24.59%,of which p53,RXFP2 and UROS were selected in a Logistic regression model with the sensitivity of 34.43%and specificity of 86.67%;(4)compared with healthy controls and benign colorectal tumors,early CRC mainly expressed≥2 positive autoantibodies.Therefore,the specificity would be significantly improved when the individuals expressing≥2 positive autoantibodies were defined as positive individuals,and its diagnostic value was comparable to above Logistic regression model.In summary,several plasma autoantibodies were significantly increased in patients with CRC and benign tumors than normal controls,with greater potential for early diagnosis of CRC and differential diagnosis of benign and malignant tumors.Further study with a larger cohort is needed to explore the optimal combination of joint detection for identifying early CRC patients.