Comparative Liquid Biopsy Testing for KRAS Mutations From Plasma Cell-Free DNA (cfDNA) and Extracellular Vesicles in Lung Adenocarcinoma.
Author(s): Zahra CJ, Chee TM, Stephens EKH, Keir EJ, Parris BA, O'Farrell HE, Goldsworthy AF, Bowman RV, Yang IA, Fong KM
Publication: Cancer Rep (Hoboken), 2026, Vol. 9, Page e70517
PubMed ID: 41810889 PubMed Review Paper? No
Purpose of Paper
This paper compared protein expression, particle size and concentration, DNA yield, and copies of wildtype and mutant KRAS in centrifugation pellets (1600, 20,000 and 100,000 g) and the 20,000 g supernatant from plasma collected from patients with early- and late-stage lung adenocarcinoma (LUAC).
Conclusion of Paper
Flotillin-1, albumin and CD-9 were expressed in pellets from all three centrifugation speeds, but flotillin-1 expression was much lower and CD-9 was not detected in the plasma supernatant, indicating that while extracellular vesicles (EVs) were present in all pellets they were more diluted in the plasma supernatant. The concentration of particles was slightly higher in the 20,000 g pellet (abbreviated P2) than the 1,600 g pellet (P1) or the 100,000 g pellet (P3) (2.18E+08 versus 1.26E+08 and 1.4 E+10 particles/ml, respectively). Mean particle sizes were comparable between P1 and P2 but larger than in P3 at 200 Pa (367 ± 177.2 nm, 370 ± 125.4 nm and 99 ± 42.1 nm, respectively), 400 Pa (394 ± 195.3 nm, 388 ± 124.1 nm and 107 ± 41.6 nm, respectively) and 600 Pa (424 ± 208.4 nm, 429 ± 142.9 and 117 ± 48.8 nm, respectively). Quantifiable DNA was isolated from all 58 plasma supernatants but was only detected in 18 of the 58 P1 (8 early stage and 10 late stage), 17 of the 58 P2 (7 early-stage and 10 late-stage) and 14 of the 58 P3 (4 early stage and 10 late stage) specimens. KRAS (mutant or wildtype) was detected in the majority of supernatants, P1, P2 and P3 specimens from the 30 patients with early-stage LUAD (30, 27, 24 and 27, respectively) and the 28 patients with late-stage LUAD (28, 24, 26 and 25, respectively). Importantly, the mean number of detected wildtype and mutant KRAS events was higher in supernatant than pellet specimens, both from patients with early-stage (596.4 and 417.9 versus 2.9-186.5 and 11.5-275.3, respectively) and late-stage (591.9 and 1246.4 versus 13.6-88.4 and 20.4-69.7, respectively) LUAD. While mean levels of wildtype KRAS were comparable in the supernatants of specimens from patients with early-stage and late-stage LUAD (596.4 and 591.9, respectively), there was a trend toward higher mean detection of mutant KRAS in specimens from patients with late-stage than early-stage LUAD (1246.4 versus 417.9 events, not significant). Similarly, among P2 and P3 specimens there was a trend toward higher mutant and wildtype KRAS in specimens from patients with late-stage than early-stage LUAD, but the differences were very small and not significant. When broken down by patient, ≥3 KRAS mutation positive events were detected in 13 of 14 late-stage supernatants and 1 of 15 early-stage supernatants from patients known to harbor a KRAS mutation, but ≥3 KRAS mutation positive events were detected in none of the P1 specimens, 0 of 15 early- and 2 of 14 late-stage P2 specimens and 1 of 15 early- and 2 of 13 late-stage P3 specimens from these same patients. Additionally, ≥3 KRAS mutation positive events were detected in a P2 specimen from a patient with early-stage LUAD and no known KRAS mutation in the tumor and in the supernatant, P1, and P2 of a patient with late-stage LUAD and no known KRAS mutation in the tumor. While the sensitivity for detection of KRAS mutations was low in early-stage LUAD specimens (0% for P1 and P2 and 6.7% for P3 and supernatants), the specificity was high (93.8% for P2 and 100% for others). In contrast, the sensitivity and specificity for the detection of KRAS mutations were high in late-stage LUAD supernatants (92.9%, both). The sensitivity for detection of KRAS mutations remained low with high specificity in late-stage LUAD pellets (0% and 92.9%, respectively, for P1; 14.3% and 92.9%, respectively, for P2; and 15.4% and 100%, respectively, for P3). Finally, in pellets and supernatants from patients with early-stage LUAD, the concordance in KRAS mutation status with the known tumor status was not significant, but in supernatants from patients with late-stage LUAD, there was a strong concordance with the tumor status (ĸ = 0.857, P<0.0001).
Studies
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Study Purpose
This study compared protein expression, particle size and concentration, DNA yield and copies of wildtype and mutant KRAS in pellets from different centrifugation speeds (1,600, 20,000 and 100,000 g) and the 20,000 g supernatant from plasma of patients with early- and late-stage lung adenocarcinoma (LUAC). K2EDTA blood was collected from 58 patients with lung adenocarcinoma (30 early stage and 28 late stage) and a known tumor KRAS mutational status (29 with KRAS G12/G13 mutations and 29 wildtype for KRAS G12/G13). Plasma was obtained by centrifugation at 1,200 g for 10 min at room temperature. Plasma was recentrifuged at 1,600 g for 10 min at room temperature, followed by 20,000 g for 40 min at 4°C and 100,000 g for 100 min at 4°C. The pellets from the 1,600 (P1) and 20,000 g (P2) centrifugation and an aliquot of the plasma supernatant from the 20,000 g centrifugation were stored at -80°C, whereas the pellet of the 100,000 g (P3) was used immediately for DNA extraction. All pellets were resuspended in phosphate buffered saline before analysis. The particle concentration and size distribution were measured by Tunable Resistive Pulse Sensing (TRPS) on an IZON Science's Exoid system. EVs were visualized by transmission electron microscopy (TEM). Levels of albumin, flotillin-a and CD9 were quantified in the pellets and supernatant by Western blot analysis. DNA was extracted using the Norgen Plasma/Serum cfc-DNA/cfc-RNA Advanced Fractionation Kit and purified with the Monarch PCR and DNA Cleanup Kit. DNA was quantified with the Qubit High Sensitivity DNA Kit and KRAS mutations were detected using the droplet digital PCR (ddPCR) KRAS G12/G13 Screening Kit.
Summary of Findings:
Flotillin-1, albumin and CD-9 were expressed in pellets from all three centrifugation speeds, but flotillin-1 expression was much lower and CD-9 was not detected in the plasma supernatant, indicating that while EVs were present in all three pellets, they were more diluted in the plasma supernatant. The concentration of particles was slightly higher in the 20,000 g pellet (P2) than the 1,600 g pellet (P1) or the 100,000 g pellet (P3) (2.18E+08 versus 1.26E+08 and 1.4 E+10 particles/ml, respectively). The mean particle sizes were comparable between P1 and P2, which were larger than in P3 at 200 Pa (367 ± 177.2 nm, 370 ± 125.4 nm and 99 ± 42.1 nm, respectively), 400 Pa (394 ± 195.3 nm, 388 ± 124.1 nm and 107 ± 41.6 nm, respectively) and 600 Pa (424 ± 208.4 nm, 429 ± 142.9 and 117 ± 48.8 nm, respectively). Quantifiable DNA was isolated from all 58 plasma supernatants but was only detected in 18 of the 58 P1 (8 early-stage and 10 late-stage), 17 of the 58 P2 (7 early-stage and 10 late-stage) and 14 of the 58 P3 (4 early-stage and 10 late-stage) specimens. KRAS (mutant or wildtype) was detected in the majority of supernatant, P1, P2 and P3 specimens from the 30 patients with early-stage LUAD (30, 27, 24 and 27, respectively) and the 28 patients with late-stage LUAD (28, 24, 26 and 25, respectively). Importantly, the mean number of detected wildtype and mutant KRAS events was higher in supernatant than pellet specimens, both from patients with early-stage (596.4 and 417.9 versus 2.9-186.5 and 11.5-275.3, respectively) and late-stage (591.9 and 1246.4 versus 13.6-88.4 and 20.4-69.7, respectively) LUAD. While mean levels of wildtype KRAS were comparable in the supernatants of specimens from patients with early-stage and late-stage LUAD (596.4 and 591.9, respectively), there was a trend toward higher mean detection of mutant KRAS in specimens from patients with late-stage than early-stage LUAD (1246.4 versus 417.9 events, not significant). Similarly, among P2 and P3 specimens, there was a trend toward higher mutant and wildtype KRAS in specimens from patients with late-stage than early-stage LUAD, but the differences were very small and not significant. When broken down by patient, ≥3 KRAS mutation positive events were detected in 13 of 14 late-stage supernatants and 1 of 15 early-stage supernatants from patients known to harbor a KRAS mutation, but ≥3 KRAS mutation positive events were detected in none of the P1 specimens, 0 of 15 early- and 2 of 14 late-stage P2 specimens and 1 of 15 early- and 2 of 13 late-stage P3 specimens from these same patients. Additionally, ≥3 KRAS mutation positive events were detected in P2 specimens from a patient with early-stage LUAD and no known KRAS mutation in the tumor or in the supernatant, P1 and P2 of a patient with late-stage LUAD and no known KRAS mutation in the tumor. While the sensitivity for detection of KRAS mutations was low in early-stage LUAD specimens (0% for P1 and P2 and 6.7% for P3 and supernatants), the specificity was high (93.8% for P2 and 100% for all others). In contrast, the sensitivity and specificity of KRAS mutation detection was high in late-stage LUAD supernatants (92.9%, both). The sensitivity for KRAS mutation detection remained low, with high specificity in late-stage LUAD pellets (0% and 92.9%, respectively, for P1; 14.3% and 92.9%, respectively, for P2; and 15.4% and 100%, respectively, for P3). Finally, in pellets and supernatants from patients with early-stage LUAD, the concordance in KRAS mutation status with the known tumor status was not significant, but in supernatants from patients with late-stage LUAD, there was a strong concordance with the tumor status (ĸ = 0.857, P<0.0001).
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Fluorometry Protein Western blot Morphology Electron microscopy DNA Digital PCR Cell count/volume Coulter counter Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Early stage LUAD
Late stage LUAD
Biospecimen Aliquots and Components Blood and blood products Plasma
Plasma pellet
Plasma vesicles
Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared