Utilization of Archived Plasma to Detect Epidermal Growth Factor Receptor Mutation in Non-Small Cell Lung Cancer Patients.
Author(s): Oh AC, Lee JK, Kim JY, Jin HO, Jung JW, Chang YH, Hong YJ
Publication: Biopreserv Biobank, 2019, Vol. 17, Page 319-325
PubMed ID: 30888199 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of patient gender, diagnosis, chemotherapy treatment status, delay to centrifugation, frozen storage duration of plasma, and cell-free DNA (cfDNA) extraction kit used on determination of epidermal growth factor receptor (EGFR) mutation status of non-small cell lung cancer (NSCLC) patients and compared results of cfDNA analysis to those obtained from tissue or cytology specimens.
Conclusion of Paper
The EGFR mutation detection rate was higher in women than men and in patients who had undergone chemotherapy than those who had not and varied significantly according to stage with Stage IV specimens showing the highest mutation burden. However, there was no difference in the EGFR mutation detection rate in plasma based on histologic type. The cfDNA concentration was higher when extracted with the Cobas cfDNA Sample Preparation Kit than the Maxwell ccfDNA Plasma Kit, but there was no difference in the detection rate for EGFR mutations between the two kits. A lower percentage of plasma specimens were found to have EGFR mutations than matched tissue/cytology specimens (19% versus 53.4%, concordance rate of 58.6%). There were no differences in EGFR mutation detection rate or cfDNA concentration in plasma due to a delay to centrifugation (≤4 h versus >4 h) or storage duration (≤12 months, >12 months).
Studies
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Study Purpose
This study investigated the effects of patient gender, diagnosis, chemotherapy treatment status, delay to centrifugation, frozen storage duration of plasma, and cfDNA) extraction kit used on EGFR mutation status analysis of cfDNA from NSCLC patients and compared results to those obtained from tissue or cytology specimens. Blood was collected in K2EDTA tubes from 116 NSCLC patients (median age=65 y; 61 males, 55 females; 86 adenocarcinomas, 1 adenosquamous carcinoma, 1 bronchioalveolar carcinoma, 1 large cell carcinoma, 27 squamous cell carcinomas) diagnosed as Stage I (n=35), II (n=9), III (n=22), or IV (n=50) undergoing chemotherapy treatment (n=50) or not receiving chemotherapy treatment (n=66). Plasma was obtained by centrifugation for 10 minutes at 2000 x g at 4°C within 0-172.7 h of collection. Plasma was immediately stored at -70°C until analysis (average 9.6 – 25.0 mos). cfDNA was extracted using the Cobas cfDNA Sample Preparation Kit or the Maxwell RSC ccfDNA Plasma Kit. cfDNA concentration was measured by fluorometry using the Qubit dsDNA HS Assay Kit. EGFR mutation was detected by real-time PCR using the Cobas EGFR Mutation Test v2. Results were compared with those previously obtained in the tissue or cytology EGFR mutation test (details not provided).
Summary of Findings:
The EGFR mutation detection rate was higher in women than men (P=0.035) and in patients who had undergone chemotherapy than those who had not (P=0.030) and varied significantly according to stage (P=0.015) with Stage IV specimens showing the highest mutation burden. However, the EGFR mutational burden did not significantly differ among the different histologic types. The cfDNA concentration was higher when extracted from plasma with the Cobas cfDNA Sample Preparation Kit than the Maxwell ccfDNA Plasma Kit (P<0.001), but there was no difference between the two kits in the detection rate for EGFR mutations. The sensitivity and specificity of the plasma test for mutations identified in matched tissue/cytologic specimens was 33.9% and 98.1%, respectively. A total of 26 EGFR mutations were observed in 22 plasma specimens (19%, 4 specimens with multiple mutations) compared to 65 mutations in 62 tissue/cytology specimens (53.4%, three specimens with multiple mutations) with a concordance rate of 58.6%. The exon 19 deletion was the most frequent mutation observed in the plasma and tissue/cytology specimens (9.5% and 30.2%, respectively) followed by L858R (6.9% and 20.7%, respectively). The T790M mutation was detected in five (4.3%) of the plasma specimens and two tissue/cytology specimens (1.7%) but was positive in both tests for specimens from only one patient. There were no differences in EGFR mutation detection or cfDNA concentration in plasma due to a delay to centrifugation (≤4 h versus >4 h) or storage duration (≤12 months, >12 months).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient gender Female
Male
Preaquisition Diagnosis/ patient condition adenocarcinoma
adenosquamous carcinoma
bronchioalveolar carcinoma
large cell carcinoma
squamous cell carcinoma
Preaquisition Other drugs Received chemotherapy
No chemotherapy
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage duration ≤12 months
>12 months
Analyte Extraction and Purification Analyte isolation method Cobas cfDNA Sample Preparation Kit
Maxwell RSC ccfDNA Plasma Kit
Real-time qPCR Specific Targeted nucleic acid EGFR mutation
Preaquisition Prognostic factor Stage I
Stage II
Stage III
Stage IV