| Objective:Accurate selection of treatment option and timely monitoring of efficacy in patients with platinum-resistant ovarian cancer(PROC)are of great importance.However,traditional imaging and CA125 examination show certain limitations,especially in the treatment of new drugs such as targeted drugs.We integrated both copy number variation(CNV)and TP53 mutation in circulating tumor DNA(ctDNA)testing to assess whether the quantitative percent change in ctDNA could monitor treatment response and predict progression free survival(PFS)in PROC patients.Content:185 dynamic blood samples and 53 primary surgical tumor tissues from 79 PROC patients,respectively,were performed by ctDNA testing and whole exome sequencing(WES).All plasma samples underwent the tumor uninformed analysis(TUCA)strategy,which included shallow whole genome sequencing(sWGS)for CNV and targeted enrichment sequencing for TP53 mutation.Dynamic(two or more)blood samples matched with tumor tissues were also performed for tumor informed cfDNA assay(TICA)surveillance strategy based the specific mutations detected by WES.Through TICA data training the TUCA algorithm,we established and optimized ctDNA evaluation criteria,which were used to evaluate therapeutic efficacy and predict survival outcomes in PROC patients.Method:In this study,we designed individualized ctDNA testing kits for 43 patients with tumor tissues and dynamic blood samples according to the mutations found by WES,and 123 plasma samples were tested for TICA and TUCA at the same time.These 43 patients were obtained as the TUCA training set and their TICA results were applied to train the TUCA algorithm.57 plasma samples from 36 patients without tumor tissues were tested as a validation set for the TUCA algorithm.The results of serum CA125 were also collected.Based on patients’ symptom benefit and RECIST results,we tentatively developed the ctDNA evaluation criteria,and then evaluated and compared the sensitivity and specificity between TUCA and TICA,CA125 for monitoring therapeutic efficacy and predicting survival outcomes of PROC patients.Results:In TUCA,we found that ctDNA benefit(decreased≥80%over baseline or remained negative throughout the treatment)before three cycles predicted the clinical benefit(RECIST-PR:partial response,or RECIST-SDa:decrease in lesion but without PR)and no clinical benefit(RECIST-PD:progressive disease,or RECIST-SDb:increase in lesion but without PD)in training set with 81.82%sensitivity,76.92%specificity and AUC=0.82,which is comparable to TICA(77.27%sensitivity,85.71%specificity,AUC=0.81).When combining TUCA and CA125,the sensitivity and specificity were respectively increased to 90.91%and 84.62%.In validation set,the sensitivity and specificity of ctDNA benefit before three cycles predicting clinical benefit were 100%and 83.33%.TUCA combined with CA125 increased both sensitivity and specificity by 100%.A long median lead time to clinical drug resistance of 75.5 days in TUCA compared to RECIST.Kaplan-meier curves showed that ctDNA benefit before three cycles in TUCA had a median PFS of 267 days versus 109 days(HR:2.92;95%CI 1.57-5.42;Log-rank p=0.0004).Multivariate Cox regression analysis showed that ctDNA benefit before three cycles was an independent prognostic factor for PFS(HR:4.22;95%CI:1.48-12.00;p=0.007).Conclusions:The TUCA strategy may be a highly sensitive,broadly applicable and feasible ctDNA testing method that can predict treatment response and prognosis in PROC patients.TUCA combined with CA125 could better benefit more PROC patients with limited treatment options and help guide the personalized management.Objective:Due to the extremely low incidence rate and high heterogeneity,the prognosis of metastatic neuroendocrine neoplasms(mNENs)is poor,and the available treatment options are still limited.Therefore,it is of great importance to select appropriate treatment options for patients with mNENs and monitor of treatment efficacy timely and accurately.At present,the monitoring of therapeutic efficacy of mNENs patients mainly relies on Response Evaluation Criteria in Solid Tumors(RECIST1.1)and some NENs molecular markers,but traditional imaging examination is limited and many latent lesions in patients cannot be comprehensively evaluated.The sensitivity and specificity of NENs biomarkers,such as chromogranulocin A(CgA),are not ideal due to the limitation of biological characteristics.Based on tumor informed analysis(TICA)and copy number variation(CNV)analysis of circulating tumor DNA(ctDNA)for detection of minimal residual lesions(MRD),this study explored whether the ctDNA quantitative changes could monitor therapeutic efficacy and predict progression free survival(PFS).Content:A total of 128 blood samples and 29 primary or metastatic tissues were collected during the treatment of 29 patients with mNENs for ctDNA detection and whole exon sequencing(WES)respectively.Each patient had more than two blood samples and then tumor informed analysis(TICA)and copy number variation analysis(CNV)were performed.At the same time,each plasma sample was tested for CgA.Due to the low amount of ctDNA in plasma of mNEN s and few related reports,in the preliminary stage of this study,we first explored the optimal experimental conditions for cfDNA extraction,library construction and TICA detection in plasma samples with mNENs,and evaluated the proportion of ctDNA in cfDNA,the feasibility of TICA and CNV for monitoring the therapeutic efficacy of mNENs.Finally,we tried to develop the ctDNA criteria for monitoring the treatment response and predicting the prognosis of mNENs patients.Method:In this study,personalized ctDNA test kit was customized for each patient based on the WES data of tumor tissues,and the multiple PCR targeted enrichment sequencing technology(Raceseq)was used to conduct TICA detection in 128 plasmas.Copy number variation of all plasma samples were detected by shallow Whole Genome Sequencing(sWGS)and analyzed by Wisecondor X software.CgA in plasma was detected by enzyme linked immunosorbent assay(ELISA).According to the clinical information and RECIST results of patients,the reliability of TICA experimental conditions was firstly evaluated.Referring to the normal range of CgA level of healthy people provided by the kit and the levels of CgA in plasma samples of healthy people detected in our laboratory conditions,we trained the reliability of CgA.Then,we compared the sensitivity and specificity of ctDNA and CgA for monitoring therapeutic efficacy and predicting the survival outcomes of mNENs patients.Results:In this study,the average cfDNA concentration of 128 blood samples from 29 mNENs patients was 21.14(3.02-291.67)ng/mL,and the average plasma volume used for extraction of cfDNA was 3.20(1.50-5.00)mL,the median CF%value was 0.1%,indicating the need for an ultra-sensitive assay to quantitatively analyze ctDNA levels in patients with mNENs.90.48%(19/21)of the patients showed MRD positive before treatment based on ctDNA test;84.62%(22/26)of the patients remained MRD positive during and the end of the treatment,and ctDNA was not cleared,which was in line with the concept of "survival with tumor" management for mNENs patients.Four patients showed transient MRD negative during treatment,suggesting that transient clearance of ctDNA during treatment may predict clinical benefit(RECIST-PR,partial response,or RECIST-SDa,decrease in lesion but without PR)of mNENs patients.Analysis of a typical case suggested that ctDNA testing can detect signals of disease progression in patients with mNENs simultaneously or even earlier than RECIST.Conclusions:In this study,we tentatively found that ctDNA testing can be used to monitor the therapeutic efficacy of mNENs patients,providing a foundation for the later research.This study is expected to develop a widely applicable and feasible ctDNA evaluation standard to help clinicians accurately evaluate the efficacy and make treatment decisions. |
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