| BackgroundCancer has become an important cause of morbidity and mortality in every region of the world,posing a serious threat to human health.Among them,the incidence and mortality of lung cancer still ranked first,showing an increasing trend year by year.With the continuous development of tumor immunology,tumor immunotherapy has become the new hot spot after molecular targeted therapy.including the PD-1 / PD and L1 signaling pathways has been one of the research hotspots of tumor immunotherapy in recent years.Its blockers anti-PD-1 and anti-PD-L1 antibodies can reverse the tumor escape by blocking negative immune regulation signalsand kill tumor.It has been clinically proven that they show better curative effects on a variety types of tumor,especially PD-L1..Currently,the method of detecting PD-L1 is still mainly IHC for tissues.However,tissue detection has some shortcomings,such as collecting tissue specimens from tumor difficultly,invasive and possibly causing iatrogenic implantation.Therefore,can a simple,effective and non-invasive method be found to detect the expression of PD-L1?Previous studies have shown that the mRNA level of PD-L1 in tumor tissues is consistent with the protein level.Furthermore,it is speculated that the mRNA level of PD-L1 in plasma may represent the expression level of PD-L1 mRNA in tumor tissues.Quantitative real-time PCR(RT-qPCR),as one of the important methods for Quantitative detection of nucleic acid,has been widely used and has significant advantages.However,due to the various steps in the experiment process has different efficiency,and affected by factors such as the amount of the starting sample and the mass of the model version,,the number of cycles(CT values)experienced when the fluorescent signal in the reaction tube reaches the set domain value cannot truly reflect the absolute amount of the template in the sample.Therefore,we need a stably expressed reference gene to normalize the starting amount of different samples.Currently,?-actin,GAPDH and U6 are commonly used as internal references in tissues and cells,but there is no standard reference for gene detection in plasma.Therefore,we intend to design an external reference for the determination of nucleic acid content in plasma and name it PLACON.By comparing the external reference PLACON with internal reference ?-actin,GAPDH and U6 in the plasma samples and the the culture supernatant of cancer cell lines,it was verified that it was more advantageous for nucleic acid detection.And it further explores whether the mRNA of PD-L1 in plasma is consistent with the protein level of PD-L1 in tissues,and its relationship with the efficacy and clinical characteristics.PurposesTo verify whether the mRNA level of PD-L1 in plasma is consistent with the protein level of PD-L1 in tissues,and to explore the relationship between the mRNA level of PD-L1 in plasma and the efficacy and clinical characteristics of immunotherapy.Methods1.The outer reference sequence(PLACON)was selected from the genome of Caenorhabditis elegans(t27e9.1d.39),a lower class species far from the genome of human species.The PLACON sequence was selected by PCR amplification using a forward primer(5'-AGTGCAGGGTCCGAGGTATT-3')and a reverse primer(5'-CGACTCTACAACGA CCGTGA-3'): 5'-cucgcuaacgacucuacaacgaccgugaauucaagcgccgcuuggauguccgc-3'.Its base number was 53,GC content was 56.6% and Tm value was 87.1?.Sequence similarity was searched by using the online tool BLAST(website:https://www.ncbi.nlm.nih.gov/).2.RT-qPCR was used to compare the miRNA and mRNA levels of external reference and internal reference ?-actin,GAPDH and U6 in the plasma samples and cell culture supernatants of lung cancer patients,verifying that the application of external reference(PLACON)for nucleic acid detection was more advantageous.3.Tumor tissues and corresponding plasma samples and their clinical characteristics of malignant tumor patients with different tumor species were collected.Plasma samples of tumor patients before immunotherapy and immunotherapy for 2 months were collected.The protein expression level of pd-l1(tpd-l1)within the tissue of the patients was detected by IHC method,and the mRNA expression level of pd-l1 within the plasma was detected by qrt-pcr method.To explore whether the expression level of pd-l1 protein in tissues is consistent with the expression level of pd-l1 mRNA in plasma,and to analyze the correlation between the expression level of pd-l1 mRNA and the efficacy and clinical characteristics of immunotherapy.Results1.Sequence of external reference(PLACON)has complete specificity and meets the ideal external reference standard.Rt-qpcr can detect the expression of PLACON in plasma with high specificity;2.The PLACON was diluted to a 10-fold gradient(0.005,0.05,0.5,5,50ng/ml),Rt-qpcr method was used to obtain the external reference CT value of plasma mRNA level(n=8),which was 23.57±0.29,21.36±1.22,17.14±0.66,10.70±0.36,6.95±0.27,respectively.There was an obvious linear relationship between the concentration of PLACON and the CT value,indicating the stability of PLACON amplification in plasma.When the concentration of PLACON is 0.5 ng/ml,the range of CT values between PLACON,?-actin and GAPDH groups is 2.87,8.36 and 6.44,respectively.The range of PLACON group is the smallest and the fluctuation range is small,indicating that the stability of PLACON in the amplification of plasma mRNA level is higher than that of ?-actin and GAPDH;3.The CT values of PLACON of plasma miRNA level and U6 in plasma samples of malignant tumor patients were obtained by rt-qpcr.The CT values of PLACON(16.20±0.93)were significantly lower than those of U6 group(19.49±1.01),and the difference was statistically significant.The range of CT values between PLACON and U6 groups was 3.01 and 2.95,respectively.There was no significant difference between PLACON and U6 range,and the fluctuation range was small.Although there is no obvious difference between the range of PLACON and U6,PLACON still has certain amplification advantages due to its higher amplification efficiency than U6;4.The PLACON was diluted to a 10-fold gradient(0.005,0.05,0.5,5 ng/ml),the CT value of PLACON in the cell supernatant was obtained by rt-qpcr(n=6),which was 23.99±0.24,17.95±0.20,15.83±0.07,7.86±0.24,respectively.The linear relationship between the concentration of PLACON and the CT value was obvious,which showed the stability,specificity and anti-interference of PLACON amplification in the cell supernatant.The range of CT values in the PLACON group,GAPDH group and ?-actin group was 3.42,8.02 and 7.76,respectively.The range of the PLACON group was still the smallest with the highest stability;5.For 51 patients,both TC3/IC3 group and TC1~2/IC1~2 group had higher PD-L1 mRNA expression than TC0/IC0 group.There also was a trend that TC3/IC3 group had higher PD-L1 mRNA expression than TC1~2/IC1~2 group.It suggests that pd-l1 mrna in plasma can represent the expression level of tpd-l1 protein,which may be a biomarker for predicting efficacy and guiding treatment;6.The age,gender,smoking status,primary tumor type and metastasis of 51 patients were collected,and the above clinical characteristics were not significantly correlated with pd-l1 expression in plasma;7.For the 21 patients receiving immunotherapy,patients with pd-l1 mRNA changes of more than 2.03 times(n = 11)during treatment had better progression-free survival(PFS)and overall survival(OS)than patients with changes of less than 2.03 times(n = 10),and these patients also had a higher best overall response(bOR)rate(45.45% vs 7.69%);Conclusions1.Compared with internal reference genes GAPDH,?-actin and U6,external reference PLACON has significant advantages of repeatability,uniformity,stability and high expression in plasma,so it is more suitable for external reference of nucleic acid detection;2.Compared with internal reference genes GAPDH and ?-actin,external reference PLACON has significant advantages of repeatability,uniformity,stability and high expression in the cell supernatant,so it is more suitable for external reference of nucleic acid detection;3.There is a significant correlation between pd-l1 protein level in tissues and pd-l1 mrna expression in plasma;4.There was no significant correlation between the expression of pd-l1 protein level in tissues and pd-l1 mrna in plasma with clinicopathological features;5.Patients with more than 2.03 times of changes in plasma pd-l1 mRNA had better progression-free survival and overall survival and higher best overall response rate than patients with less than 2.03 times of changes;... |
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