Predicting osimertinib‐treatment outcomes through EGFR mutant‐fraction monitoring in the circulating tumor DNA of EGFR T790M‐positive patients with non‐small cell lung cancer (WJOG8815L)

The WJOG8815L phase II clinical study involves patients with non‐small cell lung cancer (NSCLC) that harbored the EGFR T790M mutation, which confers resistance to EGFR tyrosine kinase inhibitors (TKIs). The purpose of this study was to assess the predictive value of monitoring EGFR genomic alterations in circulating tumor DNA (ctDNA) from patients with NSCLC that undergo treatment with the third‐generation EGFR‐TKI osimertinib. Plasma samples of 52 patients harboring the EGFR T790M mutation were obtained pretreatment (Pre), on day 1 of treatment cycle 4 (C4) or cycle 9 (C9), and at diagnosis of disease progression or treatment discontinuation (PD/stop). CtDNA was screened for EGFR‐TKI‐sensitizing mutations, the EGFR T790M mutation, and other genomic alterations using the cobas EGFR Mutation Test v2 (cobas), droplet digital PCR (ddPCR), and targeted deep sequencing. Analysis of the sensitizing—and T790M—EGFR mutant fractions (MFs) was used to determine tumor mutational burden. Both MFs were found to decrease during treatment, whereas rebound of the sensitizing EGFR MF was observed at PD/stop, suggesting that osimertinib targeted both T790M mutation‐positive tumors and tumors with sensitizing EGFR mutations. Significant differences in the response rates and progression‐free survival were observed between the sensitizing EGFR MF‐high and sensitizing EGFR MF‐low groups (cutoff: median) at C4. In conclusion, ctDNA monitoring for sensitizing EGFR mutations at C4 is suitable for predicting the treatment outcomes in NSCLC patients receiving osimertinib (Clinical Trial Registration No.: UMIN000022076).

Abbreviations

CIs

confidence intervals

ctDNA

circulating tumor DNA

ddPCR

droplet digital PCR

EGFR

epidermal growth factor receptor

MFs

mutant fractions

NGS

next‐generation sequencing

NSCLC

non‐small cell lung cancer

ORR

overall response rate

OS

overall survival

PD

progressive disease

PFS

progression‐free survival

PR

partial response

SD

stable disease

TKI

tyrosine kinase inhibitor

     

Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73

Purpose: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.

Methods: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72.

Results: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37?°C after heat treatment at 44?°C for 15?min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3?h of incubation at 37?°C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3?h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment.

Conclusions: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.