| Objective: To evaluated the prognostic potential of cf DNA concentration,integrity,single nucleotide variation(SNV),copy number variation(CNV)in breast cancer.Methods: Ninety-two patients with diagnosed breast cancer were recruited in the Taizhou People’s Hospital,between August 2018 and August 2019.DNA was purified according to the manufacturer’s instructions.Quantitative polymerase chain reaction(q PCR)was performed to measure the concentration(LINE1-97)and the integrity(LINE1-223/LINE1-97)of plasma cf DNA.cf DNA was used to generate libraries according to the manufacturer’s instructions.Library circularization and MGI sequencing were performed to detect CNV and SNV.Results: A total of 92 breast cancer patients with a median age of 50 years were enrolled.Patients with positive HER2 status,negative HER2 status and triple-negative breast cancer(TNBC)were 65(70.6%),17(18.5%),10(10.9%)。BC patients were divided into <2cm,2-5cm and >5 cm three groups,with 37(40.2%),49(53.3%)and 6(6.5%),respectively.Forty-two cases(45.6%)were diagnosed with stage I/II and 50cases(54.4%)with stage III/IV at the first visit.Of the whole cohort,38(41.3%)patients were not metastatic,while 54(58.7%)were at the beginning of the study.Lymph node(48/92,52.2%),bone(18/92,19.6%),liver(7/92,7.6%),lung(10/92,10.9%),chest wall(3/92,3.3%),brain(5/92,5.4%),heart(2/92,2.2%)were the most frequent metastatic sites.Serum CA15-3 levels were elevated(>31.3 U/ml)in 24patients(27.2%)at the time of blood sampling,but 68 patients(73.9%)not.The concentration of CA15-3 tends to be higher in patients with tumor size of ≥2cm compared to those with <2 cm(77.05 ± 22.02 vs 15.91 ± 2.31).The concentration,integrity index and CNV index were all associated with the tumor stage.cf DNA concentration [12.77 ng/ml(8.66 ng/ml,18.54 ng/ml)vs 32.72 ng/ml(21.20 ng/ml,46.30 ng/ml)],CNV index [1.03(0.89,1.18)vs 1.20(0.95,1.94)] and CA15-3concentration [9.25 U/ml(6.92 U/ml,14.18 U/ml)vs 24.35 U/ml(11.82 U/ml,53.80U/ml)] were significantly increased in patients with stage III/IV compared to patients with stage I/II(P < 0.05).Compared with stage I / II patients,the integrity of cfdna in stage III / IV patients was significantly lower[0.37±0.05 vs 0.23±0.03].The difference was statistically significant(P < 0.05).Those metastatic patients at the beginning of the study had significant higher cf DNA concentration [14.78 ng/ml(9.71 ng/ml,21.53 ng/ml)vs 26.16 ng/ml(15.25ng/ml,42.83 ng/ml)],CNV index [1.01(0.87,1.14)vs 1.22(0.97,1.75)] and CA15-3concentration [10.85 U/ml(7.35 U/ml,18.18 U/ml)vs 15.10 U/ml(9.55 U/ml,50.90U/ml)],and lower cf DNA integrity index [0.26(0.14,0.55)vs 0.19(0.07,0.33)](P <0.05).Interestingly,patients with distant metastatic had significant higher cf DNA concentration [15.10 ng/ml(9.71 ng/ml,24.36 ng/ml)vs 33.95 ng/ml(21.41 ng/ml,49.47 ng/ml)],CNV index [1.02(0.90,1.18)vs 1.44(1.03,1.98)] and CA15-3concentration [10.65 U/ml(7.35 U/ml,14.50 U/ml)vs 38.40 U/ml(14.10 U/ml,76.40U/ml)],and lower cf DNA integrity index [0.35 ± 0.04 vs 0.20 ± 0.03](P < 0.05).Nineteen of interest 50 genes were found appearing SNV.The total of 19 mutant genes in enrolled breast cancer patients were TP53,PIK3 CA,KRAS,EGFR,NRAS,CDKN2 A,CTNNB1,KIT,SMAD4,GNAS,FGFR3,MET,STK11,VHL,MLH1,HRAS,FBXW7,SMO and RB1.As expected,known tumor suppressor or oncogenic genes such as TP53(27/92,29.3%),PIK3CA(14/92,15.2%)and KRAS(9/92,9.8%)were found top 3 mutated in our cohort.Chi-square analysis revealed that SNV and TP53 mutation were closely related to tumor stage(P < 0.05).And the TP53 mutation patients was more in distant metastasis group than non-distant metastasis group(P <0.05).High concentration of cf DNA was associated with a poor PFS,and BC patients with TP53 mutation showed shorter PFS compared with those non mutation patients(P < 0.05).Conclusion: cf DNA variables,in terms of integrity,CNV and SNV patterns,can be as liquid-biopsy biomaterial carrying important clinical information for breast cancer prognosis and monitoring. |
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