| ObjectiveTo explore the feasibility of sputum supernatant can be used as an effective surrogate sample for the detection of non-small cell lung cancer(NSCLC)clinical driver gene mutations.And establish a standard procotol for detection of sputum driver genes mutations.Materials and Methods 102 patients had been diagnosed with lung adenocarcinoma by histological or cytological specimens in Beijing Hospital from November 2018 to June 2019 were enrolled in this study.selected Sputum specimens were collected and frozen at-20 ℃,with sputum specimens from 20 non-tumor patients,including of pneumonia,chronic obstructive pulmonary disease,pulmonary heart disease.Baseline data of 102 patients were recorded,including gender,age,smoking history and clinical staging.(1)Two mucus solutions were selected to treat sputum and two supernatant were obtained to extract cf DNA,The concentration of cf DNA was measured by fluorescence staining and DNA fragments were analyzed with a high sensitivity measurement chip.The concentration and quality of cf DNA extracted from the supernatant of two sputum samples were compared to observe and evaluate the consistency with the results of matched specimens.(2)The cfDNA in sputum supernatant was extracted by high efficiency cracking buffer technique combined with silica gel membrane adsorption technique.The mutation status of EGFR gene was detected by super Amplification Refractory Mutation System.(3)CfDNA extracted from the supernatant of sputum samples from one to three generations of patients with suspected drug resistance to EGFR-TKIs were selected,The super-ARMS method was used to simultaneously detect the T790 M mutation of exon 20 and the original sensitive mutation type,The results of the paired specimens were compared and observed to be consistent.(4)Thirty sputum specimens were selected for cf DNA supernatant,followed by lung cancer-related 10 gene mutation detection by Next Genomic Sequencing(NGS),to observe the consistency of detection results with matched tumor specimen driver gene mutation,and to calculate the sensitivity and specificity,positive predictive value,and negative predictive value.Results(1)The sputum samples of 102 tumor patients included 48 males and 54 females,aged 29-89 years,with an average age of 65 years.There were 39 patients with clinical stage I-IIIA and 63 patients with clinical stage IIIB-IV.Through statistical analysis,there was no significant difference between EGFR mutant and EGFR wild-type patients in terms of age,gender,smoking status,paired tumor stage and paired tumor type.(2)By super-ARMS,the detection sensitivity of cf DNA in sputum specimens was46.15%(30/65),and the specificity was 100%(37/37).The positive predictive value was 100% and the negative predictive value was 51.39%.The detection sensitivity of clinical stage I-IIIA patients was 24%,and that of clinical stage IIIB-IV patients was65%;Combined with sputum cytology,the detection sensitivity of clinical stage I-IIIA patients was increased to 66.67%,and that of clinical stage IIIB-IV patients was increased to 100%.In the supernatant cf DNA of sputum specimens from non-tumor patients,sputum super-arms EGFR was negative with detection specificity of 100%(20/20).(3)Sputum supernatant treated with two mucus solutions,Mean value of 32.48ng/?l MS2 after processing DNA concentration,MS1 after processing the mean concentration of DNA is 2.44 ng/?l.The concentration of MS2 was significantly higher than that of MS1,and the difference was statistically significant.The detection specificity of cf DNA super-ARMS in sputum supernatant was 100% after treatment with the two mucus solutions,the detection sensitivity was 75% after MS2 treatment and 50% after MS1 treatment.The results showed that the detection sensitivity after MS2 treatment was higher than that after MS1 treatment,and the difference was statistically significant.(P<0.05)(4)The paired samples of 34 patients with suspected drug resistance to EGFR-TKIs all carried original EGFR-sensitive mutations,which were 11 cases of T790 M mutation and 23 cases of wild type.The detection rate of EGFR gene sensitive mutation in the supernatant cf DNA of sputum specimens was 55.9%(19/34),The detection rate of liquid-based cytology that was used to evaluate specimens with cancer cells was 100%(10/10),and the detection rate of qualified sputum without cancer cells was 52.9%(9/17),the sensitive mutations were consistent with the sensitive mutation types in the matched specimens;All samples with T790 M mutation were detected with original sensitive mutations,and the total T790 M mutation detection rate was 29.4%(10/34),The detection rate of liquid-based cytology that was used to evaluate specimens with cancer cells was 50%(5/10),and the detection rate of qualified sputum without cancer cells was 29.4%(5/17).Compared with the detection results of paired samples T790 M,the sensitivity was45.5%(5/11)and the specificity was 78.3%(18/23).For patients with EGFR-TKIs resistance and T790 M mutation,supplementation of sputum sample supernatant to extract cf DNA for detection can increase T790 M positivity by 14.7%(5/34).(5)Thirty sputum supernatant samples were collected from cf DNA samples by NGS,KRAS mutation was detected in 1 case and PIC3KA1 case respectively in 2non-tumor sputum samples;In the sputum specimens of 28 patients diagnosed with NSCLC,The detection results of driving gene in paired samples were: 10 cases of wild type and 18 cases of driving gene mutation,cf DNA was extracted from sputum supernatant for NGS detection,and the detection results of 14 cases were consistent with the results of driver gene mutation in paired samples.The detection sensitivity was 77.8%(14/18),and the specificity was 100%(10/10).The positive predictive value was 100% and the negative predictive value was 75%Conclusions(1)The supernatant of sputum specimens can be used as a specimen for the detection of NSCLC driver genes mutations through the treatment of mucolytic solutions.In addition,sputum is more suitable for detection in advanced clinical patients than in early and middle stage patients.Combined with the pathological diagnosis of sputum cytology,the supernatant extracted from sputum samples of tumor cells is more suitable for driver gene detection.(2)On the basis of keeping cytological morphology well in sputum specimens for pathological diagnosis,MS2 was superior to MS1 in terms of concentration,quality and consistency of detection results.(3)Sputum samples from patients with drug resistance to EGFR-TKIs can be used as an effective supplement to other tumor samples for clinical gene detection,which can guide the clinical adjustment of individualized medication.(4)The supernatant of sputum specimens can be used as a qualified sample for NGS detection under the condition of reasonable quality control,Meanwhile,under the premise of qualified sputum samples and DNA quality control,non-tumor patients can also meet the NGS detection requirements. |
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