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Background: Companion diagnostic tests play a crucial role in guiding treatment decisions for patients with non‐small cell lung cancer (NSCLC). The Oncomine Dx Target Test (ODxTT) Multi‐CDx System has emerged as a prominent companion diagnostic method. However, its efficacy in detecting driver gene mutations, particularly rare mutations, warrants investigation. Aims: This study aimed to assess the performance of the ODxTT in detecting driver gene mutations in NSCLC patients. Specifically, we aimed to evaluate its sensitivity in detecting epidermal growth factor receptor (EGFR) mutations, a key determinant of treatment selection in NSCLC. Materials and Methods: We conducted a retrospective analysis of NSCLC patients who underwent testing with the ODxTT at Keio University Hospital between May 2020 and March 2022. Patient samples were subjected to both DNA and RNA tests. Driver gene mutation status was assessed, and instances of missed mutations were meticulously examined. Results: Of the 90 patients, five had nucleic acid quality problems, while 85 underwent both DNA and RNA tests. Driver gene mutations were detected in 56/90 (62.2%) patients. Of the 34 patient specimens, driver mutations were not detected using the ODxTT; however, epidermal growth factor receptor (EGFR) mutations were detected using polymerase chain reaction‐based testing in two patients, and a KRAS mutation was detected by careful examination of the sequence data obtained using the ODxTT in one patient. For the above three cases, carefully examining the gene sequence information obtained using the ODxTT could identify driver mutations that were not mentioned in the returned test results. Additionally, we confirmed comparable instances of overlook results for EGFR mutations in the dataset from South Korea, implying that this type of oversight could occur in other countries using the ODxTT. EGFR mutation was missed in ODxTT in Japan (6.3%, 2/32), South Korea (2.0%, 1/49), and overall (3.7%, 3/81). Conclusion: Even if sufficient tumor samples are obtained, rare EGFR mutations (which are excluded from the ODxTT's genetic mutation list) might not be detected using the current ODxTT system due to the program used for sequence analysis. However, such rare EGFR mutations can still be accurately detected on ODxTT's sequence data using next‐generation sequencing. [ABSTRACT FROM AUTHOR]

Abstract Background Companion diagnostic tests play a crucial role in guiding treatment decisions for patients with non‐small cell lung cancer (NSCLC). The Oncomine Dx Target Test (ODxTT) Multi‐CDx System has emerged as a prominent companion diagnostic method. However, its efficacy in detecting driver gene mutations, particularly rare mutations, warrants investigation. Aims This study aimed to assess the performance of the ODxTT in detecting driver gene mutations in NSCLC patients. Specifically, we aimed to evaluate its sensitivity in detecting epidermal growth factor receptor (EGFR) mutations, a key determinant of treatment selection in NSCLC. Materials and Methods We conducted a retrospective analysis of NSCLC patients who underwent testing with the ODxTT at Keio University Hospital between May 2020 and March 2022. Patient samples were subjected to both DNA and RNA tests. Driver gene mutation status was assessed, and instances of missed mutations were meticulously examined. Results Of the 90 patients, five had nucleic acid quality problems, while 85 underwent both DNA and RNA tests. Driver gene mutations were detected in 56/90 (62.2%) patients. Of the 34 patient specimens, driver mutations were not detected using the ODxTT; however, epidermal growth factor receptor (EGFR) mutations were detected using polymerase chain reaction‐based testing in two patients, and a KRAS mutation was detected by careful examination of the sequence data obtained using the ODxTT in one patient. For the above three cases, carefully examining the gene sequence information obtained using the ODxTT could identify driver mutations that were not mentioned in the returned test results. Additionally, we confirmed comparable instances of overlook results for EGFR mutations in the dataset from South Korea, implying that this type of oversight could occur in other countries using the ODxTT. EGFR mutation was missed in ODxTT in Japan (6.3%, 2/32), South Korea (2.0%, 1/49), and overall (3.7%, 3/81). Conclusion Even if sufficient tumor samples are obtained, rare EGFR mutations (which are excluded from the ODxTT's genetic mutation list) might not be detected using the current ODxTT system due to the program used for sequence analysis. However, such rare EGFR mutations can still be accurately detected on ODxTT's sequence data using next‐generation sequencing.

In advanced lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutation, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have an excellent and long-lasting therapeutic response; however, virtually all patients eventually develop drug resistance and experience disease progression. The use of immunotherapy after EGFR-TKIs may be a successful therapeutic option for individuals who are resistant to them. It is still unclear if EGFR-TKIs can be administered again after immunotherapy has failed. We describe a case of a 37-year-old woman who was found to have T4N3M1a stage IVa lung adenocarcinoma. Amplification refractory mutation system PCR (ARMS-PCR) genetic testing suggested EGFR exon 19 deletion. The patient was initially treated with a regimen of icotinib (125 mg tid) combined with anlotinib (8 mg qd d1-d14) with an optimal efficacy rating of partial response (PR) and was granted a PFS of 7 months. In second-line treatment, the patient received three cycles of a KN046 (KN046 is a bispecific antibody inhibitor of PD-L1 and CTLA-4) 295 mg d1, pemetrexed 800 mg d1, plus carboplatin 750 mg d1 regimen, with an optimal efficacy rating of stable disease (SD) on CT. The third-line therapy was chosen to be afatinib with docetaxel, and the patient was evaluated for PR on CT. Up to 15 August 2022, the patient had a progression free survival (PFS) of 14 months. The successful treatment of this patient is a reminder that EGFR-TKI rechallenge in EGFR exon 19 deletion patients with EGFR-TKI resistance, in which immunotherapy has failed, may be effective.

Abstract Background Epidermal growth factor receptor (EGFR) activating mutations are usually associated with DNA damage repair (DDR) deficiency. However, the precise mechanism has remained elusive. In this study, we aimed to investigate whether EGFR exon 19 deletion mutation downstream signals contributed to DDR deficiency by downregulation of excision repair cross‐complementation group‐1 (ERCC1), a key factor in DDR, expression and function. Methods We first measured cell survival, DNA damage (γ‐H2AX foci formation) and damage repair (ERCC1 and RAD51 foci formation) ability in response to DNA cross‐linking drug in EGFR exon 19 deletion and EGFR wild‐type cells separately. We then investigated the involvement of EGFR downstream signals in regulating ERCC1 expression and function in EGFR exon 19 deletion cells as compared with EGFR wild‐type ones. Results We observed increased γ‐H2AX, but impaired ERCC1 and RAD51 nuclear foci formation in EGFR exon 19 deletion cells as compared with EGFR wild‐type ones treated with DNA cross‐linker. In addition, we identified that inhibition of EGFR exon 19 deletion signals increased ERCC1 expression, whereas blocked wild‐type EGFR signals decreased ERCC1 expression, on both mRNA and protein levels. Furthermore, EGFR exon 19 deletion downstream signals not only inhibited ERCC1 expression but also influenced ERCC1 foci formation in response to DNA cross‐linker. Conclusion Our findings indicated that the aberrant EGFR exon 19 deletion signals were not only associated with decreased expression of ERCC1 but were also involved in impaired ERCC1 recruitment in response to DNA cross‐link damage, thereby providing us with more evidence for exploring the mechanism of DDR deficiency in EGFR mutant NSCLC.

Background: Epidermal growth factor receptor (EGFR) activating mutations are usually associated with DNA damage repair (DDR) deficiency. However, the precise mechanism has remained elusive. In this study, we aimed to investigate whether EGFR exon 19 deletion mutation downstream signals contributed to DDR deficiency by downregulation of excision repair cross‐complementation group‐1 (ERCC1), a key factor in DDR, expression and function. Methods: We first measured cell survival, DNA damage (γ‐H2AX foci formation) and damage repair (ERCC1 and RAD51 foci formation) ability in response to DNA cross‐linking drug in EGFR exon 19 deletion and EGFR wild‐type cells separately. We then investigated the involvement of EGFR downstream signals in regulating ERCC1 expression and function in EGFR exon 19 deletion cells as compared with EGFR wild‐type ones. Results: We observed increased γ‐H2AX, but impaired ERCC1 and RAD51 nuclear foci formation in EGFR exon 19 deletion cells as compared with EGFR wild‐type ones treated with DNA cross‐linker. In addition, we identified that inhibition of EGFR exon 19 deletion signals increased ERCC1 expression, whereas blocked wild‐type EGFR signals decreased ERCC1 expression, on both mRNA and protein levels. Furthermore, EGFR exon 19 deletion downstream signals not only inhibited ERCC1 expression but also influenced ERCC1 foci formation in response to DNA cross‐linker. Conclusion: Our findings indicated that the aberrant EGFR exon 19 deletion signals were not only associated with decreased expression of ERCC1 but were also involved in impaired ERCC1 recruitment in response to DNA cross‐link damage, thereby providing us with more evidence for exploring the mechanism of DDR deficiency in EGFR mutant NSCLC. [ABSTRACT FROM AUTHOR]

Abstract Background Epidermal growth factor receptor (EGFR) mutations, including a known exon 19 deletion (19 del) and exon 21 L858R point mutation (L858R mutation), are strong predictors of the response to EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment in lung adenocarcinoma. However, whether patients carrying EGFR 19 del and L858R mutations exhibit different responsiveness to EGFR-TKIs and what are the potential mechanism for this difference remain controversial. This study aimed to investigate the clinical outcomes of EGFR-TKI treatment in patients with EGFR 19 del and L858R mutations and explore the genetic heterogeneity of tumors with the two mutation subtypes. Methods Of 1127 patients with advanced lung adenocarcinoma harboring EGFR 19 del or L858R mutations, 532 received EGFR-TKI treatment and were included in this study. EGFR 19 del and L858R mutations were detected by using denaturing high-performance liquid chromatography (DHPLC). T790M mutation, which is a common resistant mutation on exon 20 of EGFR, was detected by amplification refractory mutation system (ARMS). Next-generation sequencing (NGS) was used to explore the genetic heterogeneity of tumors with EGFR 19 del and L858R mutations. Results Of the 532 patients, 319 (60.0%) had EGFR 19 del, and 213 (40.0%) had L858R mutations. The patients with EGFR 19 del presented a significantly higher overall response rate (ORR) for EGFR-TKI treatment (55.2% vs. 43.7%, P = 0.017) and had a longer progression-free survival (PFS) after first-line EGFR-TKI treatment (14.4 vs. 11.4 months, P = 0.034) compared with those with L858R mutations. However, no statistically significant difference in overall survival (OS) was observed between the two groups of patients. T790M mutation status was analyzed in 88 patients before EGFR-TKI treatment and 134 after EGFR-TKI treatment, and there was no significant difference in the co-existence of T790M mutation with EGFR 19 del and L858R mutations before EGFR-TKI treatment (5.6% vs. 8.8%, P = 0.554) or after treatment (24.4% vs. 35.4%, P = 0.176). In addition, 24 patients with EGFR 19 del and 19 with L858R mutations were analyzed by NGS, and no significant difference in the presence of multiple somatic mutations was observed between the two genotypes. Conclusions Patients with EGFR 19 del exhibit longer PFS and higher ORR compared with those with L858R mutations. Whether the heterogeneity of tumors with EGFR 19 del and L858R mutations contribute to a therapeutic response difference needs further investigation.

Patients with non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations (exon 19 deletions and L858R) benefit from EGFR tyrosine kinase inhibitors (TKIs). However, some researchers have reported that responses to TKIs differ by subtypes of EGFR exon 19 mutations. We retrospectively analyzed EGFR exon 19 deletion subtypes and their correlation with clinical outcomes of treatment with TKIs. A cohort of 2664 consecutive patients with NSCLC was enrolled. A total of 440 EGFR exon 19 deletions were defined as 39 subtypes. Among them, 158 patients with advanced lung adenocarcinoma with EGFR exon 19 deletion mutations received EGFR-TKIs. There were no significant differences in progression-free survival or overall survival among patients with non-LRE deletions, delE746, or delL747 ( P = 0.463 and P = 0.464, respectively). Furthermore, two patients with EGFR exon19 insertion had durable response to EGFR-TKIs. In conclusion, EGFR exon 19 is highly fragile, resulting in many different deletion and insertion subtypes. There were no significant differences in clinical outcomes after TKI treatment across the different subtypes. It is necessary to attempt to identify all patients with exon 19 deletions so that they can be offered TKI treatment., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest. This work was presented as a poster in part at the 19th Annual Meeting of Chinese Society of Clinical Oncology (2016, CSCO); Sept. 21–24, Xiamen, China.

Detection of exon 19 deletion mutation in the epidermal growth factor receptor (EGFR) gene, which results in increased and sustained phosphorylation of EGFR, is important for diagnosis and treatment guidelines in non-small-cell lung cancer. Here, we have developed a simple and convenient detection system using the interaction between G-quadruplex and fluorophore thioflavin T (ThT) for discriminating EGFR exon 19 deletion mutant DNA from wild type without a label and quencher. In the presence of exon 19 deletion mutant DNA, the probe DNAs annealed to the target sequences were transformed into G-quadruplex structure. Subsequent intercalation of ThT into the G-quadruplex resulted in a light-up fluorescence signal, which reflects the amount of mutant DNA. Due to stark differences in fluorescence intensity between mutant and wild-type DNA, we suggest that the induced G-quadruplex structure in the probe DNA can report the presence of cancer-causing deletion mutant DNAs with high sensitivity.

Background: Epidermal growth factor receptor (EGFR) mutations, including a known exon 19 deletion (19 del) and exon 21 L858R point mutation (L858R mutation), are strong predictors of the response to EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment in lung adenocarcinoma. However, whether patients carrying EGFR 19 del and L858R mutations exhibit different responsiveness to EGFR-TKIs and what are the potential mechanism for this difference remain controversial. This study aimed to investigate the clinical outcomes of EGFR-TKI treatment in patients with EGFR 19 del and L858R mutations and explore the genetic heterogeneity of tumors with the two mutation subtypes., Methods: Of 1127 patients with advanced lung adenocarcinoma harboring EGFR 19 del or L858R mutations, 532 received EGFR-TKI treatment and were included in this study. EGFR 19 del and L858R mutations were detected by using denaturing high-performance liquid chromatography (DHPLC). T790M mutation, which is a common resistant mutation on exon 20 of EGFR, was detected by amplification refractory mutation system (ARMS). Next-generation sequencing (NGS) was used to explore the genetic heterogeneity of tumors with EGFR 19 del and L858R mutations., Results: Of the 532 patients, 319 (60.0%) had EGFR 19 del, and 213 (40.0%) had L858R mutations. The patients with EGFR 19 del presented a significantly higher overall response rate (ORR) for EGFR-TKI treatment (55.2% vs. 43.7%, P = 0.017) and had a longer progression-free survival (PFS) after first-line EGFR-TKI treatment (14.4 vs. 11.4 months, P = 0.034) compared with those with L858R mutations. However, no statistically significant difference in overall survival (OS) was observed between the two groups of patients. T790M mutation status was analyzed in 88 patients before EGFR-TKI treatment and 134 after EGFR-TKI treatment, and there was no significant difference in the co-existence of T790M mutation with EGFR 19 del and L858R mutations before EGFR-TKI treatment (5.6% vs. 8.8%, P = 0.554) or after treatment (24.4% vs. 35.4%, P = 0.176). In addition, 24 patients with EGFR 19 del and 19 with L858R mutations were analyzed by NGS, and no significant difference in the presence of multiple somatic mutations was observed between the two genotypes., Conclusions: Patients with EGFR 19 del exhibit longer PFS and higher ORR compared with those with L858R mutations. Whether the heterogeneity of tumors with EGFR 19 del and L858R mutations contribute to a therapeutic response difference needs further investigation.