| Background:Endometrial cancer(EC)is the most common gynecological malignant tumor in developed countries,and the incidence rate in China is second only to cervical cancer.In recent years,the incidence rate has been increasing and showing a younger trend,which has brought a heavy burden to individuals and society.Endometrioid endometrial carcinoma(EEC)is the most common histological type,accounting for about 75%-80%of all endometrial cancer,and is associated with long-term unantagonized estrogen stimulation.Endometrial intraepithelial neoplasia(EIN)is considered to be the precursor lesion of EEC.If untreated,approximately 20%of EIN will progress to EC,but the molecular evolution underlying this progression is still unclear,and the identification of high-risk factors contributing to this progress has important clinical value.In recent years,researchers have paid more attention to hereditary endometrial carcinoma.Lynch syndrome-associated endometrial cancer is the prototypical hereditary cancer syndrome,which is caused by germline mutations of mismatch repair genes.The role of other cancer predisposition genes in the development of endometrial cancer is obscure.Advances in next-generation sequencing(NGS)technology allow researchers to dig deeper into the human genome.Target region deep sequencing can be used to explore the risk and susceptibility of tumor patients by sequencing all exons of a few to hundreds of targeted genes.The sequencing depth is deeper compared to whole genome sequencing(WGS)and whole exome sequencing(WES),which provides a convenient and economical method for genomics research.Objectives:To explore the key pathogenic genetic variants which may be involved in the development and progression of endometrial cancer and to determine the prevalence of cancer predisposition genes in an unselected endometrial cancer patient cohort.Methods:1.Peripheral blood and frozen tumor tissues were collected from 36 patients with EIN and 79 patients with EEC who underwent gynecological surgery in Peking Union Medical College Hospital from September 2017 to September 2018.2.We applied a comprehensive cancer gene panel(363 cancer-related genes)to capture the exomes of tumor-related genes.Results:1.The median tumor mutation burden(TMB)was significantly higher in the EEC group compared to EIN group(5.49 vs 2.96 mutations/Mb,P<0.001).The high-mutation load can be attributed to mutations in the DNA damage repair genes.2.The most frequently mutated genes in all cases were PTEN(53.9%),PIK3CA(46.1%),CTNNB1(29.6%),PIK3R1(29.6%),ARID1A(28.7%),KRAS(11.5%),CTCF(13.0%),FGFR2(12.2%),and ARID5B(11.3%).The high frequency mutations of the two groups were mostly consistent,but the mutation frequencies of PTEN,PIK3R1,ARID1A,KRAS,CTCF,ARHGAP35 and KDR in EC group were significantly higher than those in EIN group(P<0.05).In addition,mutations of CTCF,ARHGAP35,NF1,KDR,and TP53 gene only presented in EC group.3.Copy number variations(CNVs)were presented in 41.7%samples,but the overall CNV load is relatively low(median CNV is 2,range:0-37).The vast majority of CNVs were gene amplification,of which amplification of VEGF-B gene occurred most frequently(17.4%),and the frequency of VEGF-B amplification in EEC group was significantly higher than that of EIN group(P=0.008).4.Through bioinformatic analysis,44 genes were predicted to be driver genes in EEC development.PTEN,PIK3CA,PIK3R1,CTNNB1,ARID1A,KRAS,CTCF,FGFR2,ARID5B,TP53 and FOXA2 are predicted to be more likely to be driver genes compared to others(P<10-7).In addition,four candidate endometrial cancer-associated genes(CTCF,ARHGAP35,NF1 and KDR)were newly discovered.5.The results of germline mutation detection showed that 2 patients harbored pathogenic germline mutations in Lynch syndrome-related genes(MLH1 and MLH2),and 8 patients carried pathogenic or suspected pathogenic germline mutations in 6 non-Lynch syndrome genes(MUTYH,GALNT12,POLE,MPL,ATM and ERCC4).Conclusions:1.Our study,for the first time,portrayed the mutational spectra of both EEC and its precursor lesions in Chinese population.2.EEC has a higher mutation burden than its precancerous lesions,which is mainly due to the disturbance of DNA damage repair system.3.EIN and EEC share genetic similarity,but EEC harbors unique genetic mutations(CTCF,ARHGAP35,NF1,KDR and TP53)and VEGF-B gene amplification.These genetic changes may be involved in the development of endometrial cancer.In the future,further researches are need to explore the predictive value of these genetic variants on the response to targeted drugs and prognosis of endometrial cancer.4.In addition to mutations in Lynch syndrome-related genes,there may be additional predisposition variants that may increase the genetic susceptibility of endometrial cancer.Future researches from a larger population are needed to testify.Background:Endometrial cancer(EC)is one of the most common female reproductive malignancies.In developed countries and some economically developed cities in China,the incidence rate has ranked first overtaking that of other gynecological malignancies.Generally,endometrial cancer is considered to have a high cure rate and good prognosis.However,in recent years,the mortality rate of endometrial cancer has increased year by year,which may be contributable to the advanced stage at diagnosis and poor differentiation.Currently,the treatment plan is mainly based on clinicopathologic stage and morphological diagnosis.For patients with early stage,surgery alone can obtain a better prognosis;while,for patients with advanced stage,a comprehensive treatment which includes surgery,chemotherapy,radiotherapy and even targeted therapy or immunotherapy may be needed.Therefore,the early diagnosis of endometrial cancer is of critical significance to improve the survival rate and prognosis of patients.Currently,the diagnostic methods for endometrial cancer which are commonly used in clinical practice include transvaginal ultrasound,plasma CA125 detection,diagnostic curettage,hysteroscopy,and so on.But each of these examinations has certain limitations,which may affect the diagnostic accuracy rate of endometrial cancer.In recent years,liquid biopsy,as an emerging non-invasive detection technology,has received increasing attention due to its advantages of easy accessibility,non-invasiveness,and reproducibility.It can acquire tumor genetic information by detecting molecular markers in the body fluid of patients.Tumor-derived DNA is present in peripheral blood and pap smear of patients with endometrial cancer,which contains tumor-related genetic information.Detection of tumor-derived DNA and related genetic mutations from pap smear and peripheral blood by highly sensitive DNA detection technology can help to diagnose tumor types in patients,dynamically monitor disease burden and select optimal treatment regimen.Objectives:1.To explore the relationship between cell-free DNA level and the clinicopathological features of endometrial cancer.2.To explore the consistency of somatic mutations presented in peripheral blood,pap smear and tissue of endometrial cancer and precancerous lesions.3.To explore the relationship between the detection of tumor-derived DNA in cell-free DNA of plasma and pap smear and the clinicopathological features of endometrial cancer.Methods:1.Peripheral blood,pap smear and frozen tumor tissues were collected from 84 patients with EIN or EC who underwent surgery in Peking Union Medical College Hospital from September 2017 to September 2018.2.Using a 363 cancer-related gene panel which based on cSMART technology,next-generation sequencing was performed on cell-free DNA extracted from peripheral blood and pap smear at the time of diagnosis and matching tumor DNA from 84 patients.Results:1.Among the 84 patients included in the study,the median cfDNA yield extracted from plasma and pap smear samples were 6.8 ng/ml(range,3.0-44.7 ng/ml)and 19.2ng/ml(range,1.5-32.2ng/ml)respectively.And there was no significant difference between the cell-free DNA level of EIN group and EC group(P>0.05).Plasma cfDNA level was significantly associated with histopathologic grading(P=0.004)and lymph node metastasis(P=0.03).Plasma cfDNA level tended to be higher in patients with advanced FIGO stage(P=0.06)and lymphatic vascular involvement(P=0.05).There was no significant association between cell-free DNA level of pap smear and clinicopathological characteristics(P>0.05).2.Based on the 363 gene panel,42.9%(36/84)of the plasma cfDNA was detected with consistent somatic mutations in tumor,covering 115 sites of 73 genes.77.4%(56/84)of the pap smear cfDNA was detected with consistent somatic mutations in tumor,covering 701 mutation sites of 235 genes.The detection rate of consistent somatic mutations in pap smear was significantly higher than that of the plasma(P=0.002).3.The detection rate of consistent mutation site in plasma of the EC group was significantly higher than that in the EIN group(53.4%vs 15.4%,P<0.001).Detection of a consistent mutation in plasma was significantly associated with histopathologic grading(P=0.01),lymph node metastasis(P=0.047)and lymphatic vascular involvement(P=0.03),and it had no significant correlation with FIGO stage,depth of myometrial invasion,CA125 level at diagnosis,and plasma cfDNA level(P>0.05).4.The detection rate of consistent mutation site in pap smear of the EC group was significantly higher than that in the EIN group(94.8%vs 38.5%,P<0.001).FIGO stage,histopathological grade,myometrial invasion depth,lymphatic vascular infiltration,lymph node metastasis,CA125 level at diagnosis,and pap smear cfDNA level had no significant influence on the detection of a consistent mutation in pap smear(P>0.05).Conclusions:1.Multi-gene panel based on cSMART technology can detect the presence of tumor-derived DNA in pap smear and plasma of patients with endometrial cancer and precancerous lesions.2.Compared with peripheral blood,it is easier to detect a consistent mutation site with tissue in pap smear sample,which indicates that pap smear may be more suitable as a liquid biopsy method for endometrial cancer.3.In the future,liquid biopsy can be used as a supplement to tissue biopsy,which may play a guiding role in the early diagnosis and risk stratification of endometrial cancer. |
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